{"text": "Potassium channels in the renal cortical collecting tubule (CCT) play an important role in potassium (K) secretion by the kidney and help to maintain the body's overall K balance. In this nephron segment, potassium secretion rates depend on both the apical membrane K permeability and the electrochemical driving forces for K across the apical membrane. The apical membrane K permeability relevant for K secretion appears to arise from a small conductance, mildly inward rectifying channel which has a high open probability (P-O) under normal conditions. However, the P-O of this channel is strongly dependent on intracellular pH. This largely explains the clinical observation that alkalosis is often associated with substantial K loss and hypokalemia. Recently two closely related renal K channels have been cloned from rat, designated ROMK1 and ROMK2. The physiological relevance of the ROMK family is its functional similarity to the small conductance apical K channels that are responsible for K secretion from mammalian CCT and K recycling in TALH. The predicted primary sequences and membrane topologies of ROMK are quite different from the voltage-gated K channels of excitable cells. However, both of these ROMK clones share significant homologies with the inward rectifier (IRK) family of channels that maintain resting potential near EK and permit long depolarizing responses in excitable cells. The present proposal exploits the structural and functional similarity between ROMK and IRK families to address some important issues regarding structure-function relations in ROMK2. Site directed mutagenesis will be used to determine the regions and specific residues of ROMK2 that are important for: (1) mild inward rectification, (2) strong pH dependence, (3) permeation path and selectivity characteristics, (4) site and affinity of barium block. The proposal also addresses structural issues involving the proximity of specific residues in the amino and carboxy termini to the putative pore region of the channel and to known \"inward rectifier\" sites in the second transmembrane spanning region. Interactions between K and barium within the conduction pathway will also be investigated as a method for characterizing the structure of the permeation path. The project will enhance our knowledge of basic processes underlying K homeostasis in health and disease."}
{"text": "The objectives of this proposal are to develop an understanding of the mechanisms by which protease activity in coagulation, fibrinolysis, and inflammation is regulated. To accomplish these objectives the interaction of proteases with the plasma protease inhibitor, a2-Macroglobulin (a2M), and the interaction of the resultant complexes with cell surface receptors will be characterized. Major areas of focus will be to elucidate the mechanism by which proteinases react with this inhibitor, to access the importance and role of a2M as a regulator of protease activity in vivo, and to isolate and characterize the a2M receptor from fibroblasts. Conformational changes occurring in a2M are central to the function of the inhibitor, and not only result in inhibition of protease activity, but also generate receptor determinants on the molecule. Studies are proposed to determine the relationship between conformational changes, protease inhibition, and the generation of the receptor binding sites on the inhibitor-protease complex. Specific probes for monitoring each process have been developed, and will be extensively utilized in these studies. The specific in vivo processes in which a2M plays a key role will be addressed by isolating complexes formed under physiological conditions and identifying which specific enzymes associate with the inhibitor. Complexes will be isolated by immunoaffinity chromatography using a monoclonal antibody that is specific for a2M-protease complexes. Studies are also proposed to characterize the interaction of the inhibitor-protease complex with specific receptors. To accomplish this goal the receptor determinants on the complex will be identified by preparing anti- bodies against synthetic peptides representing regions of the molecule. The receptor will also be isolated from fibroblasts, and the properties characterized. Studies will be initiated on the amino acid sequence of regions of the receptor. Monoclonal antibodies prepared against the purified receptor will assist in characterizing subunits with the goal of identifying regions that participate in receptor function, and will be useful for comparing the structure of the receptor from various cell types and species."}
{"text": "The purpose of this study is to determine the safety, tolerability and efficacy of pain relief of recombinant human nerve growth factor (rhNGF) for HIV-associated sensory neuropathy. Five patients were enrolled and active in this clinical trial during the above time period (recruitment started during 8/96). Patients can be randomized to either a low dose rhNGF, a high dose rhNGF or placebo. All five patients have tolerated the study medication well. Two of the patients have reported a benefit in their neuropathy while the other three patients have reported no change. Since 11/30/96, the recruitment for this study continues to grow. We now have 7 patients active in this study and more in the screening process. Nationwide, there are now 147 patients enrolled among 15 sites. At the current rate, the national recruitment goal of 180 participants will be reached by approximately 2/28/97. Due to the rapid accrual rate and to statistical reasons, the study is currently being amended to: 1) increase the number of participants in this study to 260 or 270 and 2) to provide a 48 week open-label phase of this study that would be open to all participants who successfully completed the initial double-blind phase of the study. Participants will either receive low dose or high dose rhNGF during this phase of the trial. No interim analysis has been conducted on the data collected thus far at this point in time."}
{"text": "This is a single-blind placebo controlled trial identifying patients with life-threatening ventricular arrhythmias."}
{"text": "Project Summary/Abstract Stress echocardiography is a clinically established, cost-effective technique for detecting and characterizing coronary artery disease by imaging the left ventricle (LV) of the heart at rest and then after either exercise or pharmacologically-induced stress to reveal ischemia. However, acquisitions are heavily operator dependent, two-dimensional (2D), and interpretation is generally based on qualitative assessment. While a variety of quan- titative 2D approaches have been proposed in the research literature, none have been shown to be superior to the still highly variable qualitative visual comparison of rest/stress echocardiographic image sequences for detecting ischemic disease. Here, we propose that the way forward must focus on a new computational im- age analysis paradigm for quantitative 4D (three spatial dimensions plus time) stress echocardiography. Our strategy integrates information derived from both radiofrequency (RF) and B-mode echocardiographic images acquired using a matrix array probe. The integrated analysis system will yield accurate and robust measures of strain and strain rate - at rest, stress and differentiallly between rest and stress - that will identify my- ocardial tissue at-risk after dobutamine-induced stress. This work will involve the development of novel (1) phase-sensitive, correlation-based RF ultrasound speckle tracking to estimate mid-wall displacements, (2) ma- chine learning techniques to localize the LV bounding surfaces and their displacements from B-mode data, (3) a meshless integration approach based on radial basis functions (RBFs) and Bayesian reasoning/sparse coding to estimate dense spatiotemporal parameters of strain and strain rate and (4) non-rigid registration of rest and stress image sequences to develop unique, 3D differential deformation parameters. The quantitative approach will be validated with implanted sonomicrometers and microsphere-derived flows using an acute canine model of stenosis. The ability of deformation and differential deformation derived from 4D stress echocardiography to detect new myocardial tissue at-risk in the presence of existing infarction will then be determined in a hybrid acute/chronic canine model of infarction with superimposed ischemia. The technique will be translated to hu- mans and evaluated by measuring the reproducibility of our deformation and differential deformation parameters in a small cohort of subjects. Three main collaborators will team on this work. A group led by Matthew O'Donnell from the University of Washington will develop the RF-based speckle tracking methods. An image analysis group led by the PI James Duncan at Yale University will develop methods for segmentation, shape tracking, dense displacement integration and strain computation. A cardiology/physiology group under Dr. Albert Sinusas at Yale will perform the acute and chronic canine studies and the human stress echo studies. A consultant from Philips Medical Systems will work with the entire team to bridge the ultrasound image acquisition technology."}
{"text": "Diffuse intrinsic pontine gliomas (DIPG) comprise 70-80% of all brainstem gliomas in children. Approximately 300 children develop brainstem gliomas per year. Prognosis for patients with DIPGs remains dismal with a median survival of less than 1 year and no improvement in survival has been realized in more than three decades. No effective chemotherapeutic regimens are currently available.Little is known about the biology of these lesions because they are typically not biopsied and surgical resection is impossible given their location.We are establishing an International Diffuse Intrinsic Pontine Glioma Registry to provide a comprehensive database of clinical, radiologic and pathologic data linked to a bioinformatics repository of molecular data of patients. This is a new project, currently under development. Two initial studies are planned."}
{"text": "Guidelines for providing quality care to patients with acute myocardial infarction (AMI) based on randomized controlled trial (RCT) evidence may not be appropriate in higher risk groups because of limited external validity imposed by stringent RCT exclusion criteria. Like the Agency for Healthcare Research and Quality (AHRQ) priority populations, RCT ineligible populations are often characterized by risk factors that typically indicate the need for additional or specialized care. Chronic conditions and comorbidities, disability, special health care needs and advanced age are all considered priority populations by AHRQ and are standard exclusion criteria for RCTs. The VA patient population shares a number of these risk factors, i.e. increased comorbidites and disability, in addition to high proportions of low income and rural patients also considered priority populations by AHRQ. This dilemma of less quality evidence for those in more need of care is amplified by the methodological challenges and ethical constraints of randomization in high risk groups. The overall objective of this project will use veterans as an example of a population typically excluded from RCTs to examine how they differ from the general population and to specifically examine outcomes for this population and as they relate to characteristics that make veterans ineligible for most RCTs. This objective is divided into three specific aims: 1) To describe the population of VA patients admitted with AMI and compare it to those patients represented in community based AMI registries using standard descriptive statistics; 2) to compare outcomes for VA patients with an ECG diagnosed ST-elevated myocardial infarction (STEMI) receiving reperfusion within guideline recommended time to those receiving later reperfusion using multivariable regression; 3) to describe the population of VA patients based on time from symptom onset to hospital arrival and evaluate outcomes for those presenting more than 12 hours from symptom onset using instrumental variable analysis. Because veterans often have one or more characteristics typically making them ineligible for RCTs, guidelines for treatment based on this evidence may not represent the best quality care for them. Prior to investing additional resources implementing quality improvement interventions to meet guideline recommendations, an indepth examination of characteristics and outcomes specific to the veteran population with AMI is important to better inform resource allocation and provide optimal individualized care. [unreadable] [unreadable] [unreadable]"}
{"text": "Colorectal Cancer (CRC) incidence and mortality rates are highest in African Americans (AA's) compared with all other ethnic groups. One factor that may contribute to this trend is the lower rate of participation in CRC screening among AAs, which is critical to the prevention and early detection of CRC. Recent data indicate that the removal of precancerous polyps (via colonoscopy) decreases CRC incidence by 75-90 percent. Despite the implementation of national policy changes to increase CRC screening (through Medicaid/Medicare reimbursement for CRC screening and easier \"open\" access to colonoscopy) adherence remains alarmingly low. Our preliminary data show that, even after implementation of standard patient navigation (SPN) (i.e., assisting patients with making/keeping their appointments), only 40 percent of low-income minorities followed-through on their physician recommendation. Guided by Cognitive-Behavioral Social Learning Theory as a conceptual framework and cultural targeting as an intervention strategy, the proposed randomized clinical trial will investigate integrating within SPN a targeted discussion of intrapersonal and cultural barriers to colonoscopy (i.e., fear, lack of knowledge, medical mistrust, fatalism and fear) prevalent with low-income AAs. Based on research on source credibility and reference group-based social identity theory, we will also explore navigator status as a peer on the impact of culturally targeted PN. Thus, we will compare three PN strategies: SPN carried out by a professional navigator, Culturally Targeted PN carried out by a professional (CTPN-Pro) and Culturally Targeted PN carried out by a peer who has undergone colonoscopy (CTPN-Peer). Specific Aims: Aim 1: Compare the efficacy of SPN, CTPN-Pro, and CTPN-Peer on adherence to colonoscopy CRC screening in average risk, low-income AAs who have a primary care physician referral for colonoscopy. Aim 2: Explore potential mechanisms (i.e., mediators) underlying the beneficial effects of CTPN-Pro and CTPN-Peer and to examine for whom the CTPN-Pro and CTPN-Peer are most effective (i.e., moderators). Aim 3: Compare the cost effectiveness of CTPN-Pro and CTPN-Peer. Cost effectiveness will be examined in terms of direct clinical costs of screening (i.e., savings associated with more efficient use of personnel, space, and equipment) and patient costs (i.e., costs of CRC treatment and the opportunity costs to the patient per life-year saved). Results from the proposed work will facilitate the broad dissemination of PN to reduce ethnic and racial health disparities in CRC incidence mortality and will advance our understanding of PN. [unreadable] [unreadable] [unreadable]"}
{"text": "The purpose of this project is to conduct two intervention trials using multiple vitamin-mineral supplements to evaluate the relationship between such supplements and esophageal cancer incidence and mortality. One trial is being conducted in patients diagnosed with esophageal dysplasia (n = 3400) and the other in the general population in a high-risk region (n = 30,000). The effect of these supplements on regression/progression of esophageal dysplasia and total cancer incidence, total cancer mortality, and total mortality will be evaluated. These two studies are being conducted in Linxian (Henan Province) in the People's Republic of China (PRC). Linxian, a rural country with population 800,000, was selected because it has the highest rate of esophageal cancer in the world (>100/100,000) and because there is suspicion that the population's chronic deficiencies of multiple nutrients may be etiologically involved. This study is being conducted jointly by the Biostatistics Branch of the Division of Cancer Etiology and the Cancer Prevention Studies Branch of the Division of Cancer Prevention and Control at the NCI in collaboration with the Cancer Institute of the Chinese Academy of Medical Sciences."}
{"text": "Research suggests that a substantial proportion of women who describe themselves as contraceptive users are using contraception ineffectively, thereby putting themselves at risk of unintended pregnancy. Numerous studies attest to the importance of women's beliefs about contraception and pregnancy in explaining ineffective contraceptive use. The relationship between beliefs and contraceptive use can be conceptualized in the Health Belief model, which posits that contraceptive behavior is a function of perceived susceptibility to pregnancy, perceived severity of pregnancy, and perceived benefits and barriers to contraceptive use. Due to time and personnel constraints, family planning providers have little opportunity to elicit and discuss these beliefs with their clients. This project designs and evaluates a method by which counselors can efficiently address those beliefs that may put clients at risk of unintended pregnancy. The method uses a questionnaire, filled out by clients waiting for services, as a risk assessment tool; counselors will be given access to this risk assessment tool and will be trained to use it to guide their counseling. Subsequent contraceptive behavior will be compared between three groups: 1) women who receive standard counseling according to the current protocol, 2) women whose counselors were trained to address \"risky\" beliefs but were not given access to the tool, and 3) women whose counselors had access to the tool. 525 women visiting Planned Parenthood of Metropolitan Washington who are seeking oral contraception but are not currently using it will be asked to participate in the study. The first 175~participants will receive standard counseling. The next 175 will receive standard counseling by specially trained counselors, and the final 175 will receive individualized counseling from the same trained counselors who will now have access to their risk assessment tools. All participants will complete the risk assessment tool, a client satisfaction questionnaire, and be called after three months to assess contraceptive behavior and changes in beliefs. Behavioral outcomes to be assessed include 1) continuation of oral contraceptive use, 2) consistency of oral contraceptive use, and 3) consistency of overall contraceptive use, including women who discontinue oral contraceptives or use them inconsistently. Staff perceptions of the interventions will be assessed, as will the efficiency and feasibility of the interventions. If the tool is successful, it will help family planning providers improve both the efficiency and the quality of services at a negligible cost."}
{"text": "The overall goal of this proposed work is to develop a system for delivering therapeutic ionizing xradiation along a flexible, curved path from an extracorporeal x-ray source to an internal body cavity or lumen and depositing this radiation into a desired volume of tissue. Such a device would operate in a manner comparable to the solid optical fiber methods used to transmit laser radiation into interior body cavities. It is indeed based upon similar physical principles, but utilizes reflection along the hollow fiber interior and hence is not affected by any material in contact with the capillary outside surface. A flexible conduit/catheter for transmitting x-rays is possible because of the phenomenon termed total surface specular x-ray reflection, which allows x-ray radiation incident at grazing angles along the interior surface of a hollow capillary to be transmitted without loss of energy. The possibility of using this phenomenon in a medical device does not appear to have been previously recognized. As the interior diameter of the hollow capillaries through which the x-rays are transmitted is reduced, increasingly large bending angles are permitted without loss of the transmitted intensity. Curvatures as sharp as 1 cm are possible using existing lead glasses. Such a conduit/catheter assembly can be used in conjunction with an endoscope or bronchoscope or other flexible assemblies. Radiation could also be delivered via an angiographic catheter or directly via a trochar needle into a lesion. Three specific aims are proposed: 1) to construct such an x-ray carrying conduit/catheter assembly; 2) to measure the deliverable dose rate as a function of x-ray photon energy, capillary diameter, bending radius, and the intensity of the extracorporeal x-ray source; 3) to assess quantitatively the feasibility of this device in clinical practice, particularly with existing endoscopes and bronchoscopes, and as a treatment modality generally."}
{"text": "ABSTRACT The current equipment supplement request is for an upgrade of the current mass spectrometry platform to enable nano-scale liquid chromatography. Active grants R01 GM099989 (PI: Lee), and R01s GM130810 and GM121603 (PI: Atkins) jointly request the funds for a Waters M-class HPLC with the corresponding NanoLockSpray ionization source. The upgrade will provide the enhancement in sensitivity to overcome the limits in sensitivity and material requirements that have hindered analytical characterization of numerous the proteins of interest. This will dramatically increase the range of systems and samples we can analyze. The equipment will be shared equally among three NIGMS-funded PIs while being managed and maintained by the School of Pharmacy Mass Spectrometry Facility."}
{"text": "The overall goal of this proposal is to understand how clostridial neurotoxins interact with their target cells and to use this knowledge to develop effective inhibitors of the toxin-cell interaction. Three botulinum toxins (A,B and E) will be studied to determine whether observations made with one toxin (botulinum toxin A) might have general applicability. If the botulinum toxins behave similarly, comparisons will be made to tetanus toxin to determine what similarities exist between the two classes of neurotoxins. Specifically, studies of the binding of botulinum toxins B and E to G1b gangliosides and derivatives thereof will be carried out. Results will be compared to those obtained for botulinum toxin A and tetanus toxin. Accumulating evidence suggests that gangliosides may not be the only cell surface receptor for these neurotoxins. In recent studies, we have found that botulinum toxin A, its heavy chain and the carboxy terminal half of the heavy chain bind to synaptosomal proteins. Tetanus toxin also adhered to synaptosomal proteins but it had to be preincubated with GT1b in order to bind to protein. The protein adhered to by both neurotoxins has an apparent molecular weight of approximately 77 kDa and is glycosylated. Experiments designed to purify and characterize the approximately kDa protein will be continued, and the site(s) adhered to by the toxin will be identified as will the portion of the neurotoxin responsible for the binding, and in in vitro experiments designed to identify the functional aspects of specific interactions."}
{"text": "The primary mammalian pacemaker, located in the suprachiasmatic nuclei (SCN) of the hypothalamus, drives circadian rhythms in physiology and behavior that are synchronized with the environment. In temperate climates, animals use annual changes in day length, or photoperiod, to adjust their physiological state with changes in season. The way in which the clock encodes photoperiod is unknown, but two coupled oscillators, one synchronized to morning (M) and the other synchronized to evening (E), whose phase relationship varies with day length would allow the SCN to compute seasonal changes in photoperiod. These dual oscillators may be embedded in the molecular transcriptional and translational feedback loops that participate in endogenous circadian rhythm generation in the SCN (4). The clock gene, Period 1 (Perl), is an important component of rhythm generation and also participates in the synchronization of the clock to light. The current proposal will investigate role of the isolated SCN in encoding day length in photoperiodic Perl reporter rats generated by crossing the photoperiodic Fischer 344 (F344) strain with transgenic Per1::luciferase (Per1::luc) rats that express luciferase under the control of the Perl promoter. The anatomical locations of putative M and E oscillators will be investigated by imaging real-time Perl promoter activity in subdivisions of the SCN in photoperiodic rats exposed to either short or long day lengths. Singlecell luminescence imaging will be used to examine photoperiod-dependent differences in Perl promoter activity within an individual SCN neuron. This proposal will also investigate the role of the molecular clock in encoding the critical photoperiod permissive for reproduction by comparing the Perl promoter activity profile of rats exposed to either inhibitory or permissive day lengths for reproduction. Furthermore, it will be determined whether photoperiod encoding in the SCN differs between photoperiodic F344 and nonphotoperiodic Wistar rats. Seasonal variations in photoperiod may also affect neurological processes in humans. PUBLIC HEALTH RELEVANCE Many patients diagnosed with seasonal affective disorder (SAD) experience depressive episodes only during the fall and winter, and treatment with early morning bright light corrects the delay in their circadian rhythms. Therefore, investigating the role of the SCN in encoding photoperiod may lead to the development of novel therapeutic treatments for SAD patients. [unreadable] [unreadable] [unreadable]"}
{"text": "SUPPRESSION OF ENDOCRINE FUNCTION IN HUMAN BREAST CANCER"}
{"text": "The long-term goals of this project are to elucidate the molecular mechanisms used by bacteriophage T4 to initiate DNA replication, and to understand the relationships between phage replication, recombination and transcription. As judged by striking similarities in the DNA metabolic machineries of T4 and eukaryotic cells, the phage T4 model system may be particularly relevant to studies of human chromosomal replication, recombination and repair, and thus to human health. Two well-characterized T4 replication origins contain a middle-mode promoter and a downstream segment of DNA that readily unwinds. The roles of the promoter and downstream region in initiation will be explored, using origin DNA mutations and T4 mutants that lack particular replication proteins. One interesting model is that the combination of a promoter and an unwinding region leads to a stable RNA-DNA hybrid in the unwinding region. This model will be directly tested in vitro, and the results could have important implications for both transcription and DNA replication. A new system of transposon mutagenesis will also be used to find additional genes involved in origin-dependent replication and in T4 recombinational processes. The mechanism of recombination-dependent T4 replication will be explored using a novel system in which DNA replication is triggered by site-specific double-strand breaks in the viral genome. Specific intermediates in recombination-dependent replication will be analyzed in wild type and various mutant infections, and the requirement for homologous DNA segments will be further explored. Direct studies of T4 recombination will also utilize site-specific double-strand breaks in the phage genome. A single-strand annealing model for genetic recombination has long been postulated for phage T4 and has recently emerged for recombination between repeated eukaryotic genes. This model will be tested by analyzing the recombination of DNA molecules that could provide complementary single-stranded regions adjacent to double-strand breaks. The mechanism of recombination at native T4 recombination hotspots will also be examined, particularly because these hotspots are triggered by the above-mentioned replication origins of the virus."}
{"text": "Hematopoietic stem cell (HCT) transplantation offers curative therapy for a variety of malignant and nonmalignant disorders. It is limited by donor availability, transplant related toxicity, graft vs. host disease (GVHD), malignant relapse, infections, and for some patients, reduction in their post-transplant quality of life. Continuing progress from the previous funding period, the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) proposes to develop and execute scientifically meritorious, prospective clinical trials addressing key issues in HCT. We will conduct multicenter Phase II and more importantly, prospective Phase III trials in six key areas including: alternative donors and graft sources; regimen related toxicity; GVHD; disease recurrence; infection and immune reconstitution; and late effects and quality of life. Due to the complexity of pediatric transplantation, especially in those with rare inherited disorders, which are complex and difficult to study, we will devote special attention and focused scientific expertise to using the coordinated strength of the Network to improve BMT outcomes for this unique population. The BMT CTN will also amplify and leverage Network resources through collaborative research and ancillary studies of biologic endpoints that will be integrated with and complement clinical endpoints. The Network will also seek active collaboration with other scientific bodies including NCI- funded Cancer Cooperative Groups to improve the efficiency of clinical transplant studies for the large number of patients who could benefit from BMT and to maximize our successful completion of high quality and high priority clinical trials. (End of abstract.)"}
{"text": "The proposal aims to define the molecular boundaries of size, shape, structure, and relevant physicochemical properties for a volatile organic compound (VOC) to act as an effective trigeminal (i.e., chemesthetic) stimulus in humans. Chemesthetic responses in the mucosae of the face to airborne chemicals include nasal pungency (i.e., irritation) and eye irritation, two frequently mentioned adverse symptoms arising from indoor (e.g., sick building syndrome) and occupational environments. This proposal is a follow-up of our previous work exploring the physicochemical basis for the production of these chemesthetic responses by VOCs. Systematic chemosensory testing of members of homologous chemical series has revealed that a homolog can be reached where the ability to evoke these trigeminal responses begins to fade, and finally disappears for all ensuing homologs, constituting a \"cut off\" effect. The proposal focuses on the two or three homologs from each series at the boundary of the cut off effect, and will measure the sensory responses of nasal pungency, nasal localization, and eye irritation. From each substance selected to be tested, we will obtain stimulus-response (psychometric) functions spanning the range from chance detection to virtually perfect detection in order to identify the precise homolog reaching the cut off point in each homologous series. By means of chemical modeling and by additional sensory testing of rigid chemical structures molded on the molecular parameters of the cut off homologs, we will define the molecular and physicochemical boundaries for any volatile compound to be able to act as an effective trigeminal chemosensory stimulus. Knowledge of such boundaries has important implications for both basic and applied aspects of trigeminal chemoreception. From a basic perspective it will contribute to the chemical characterization of the trigeminal reception process(es), from an applied perspective it will allow to understand and prevent adverse sensory reactions from air pollutants."}
{"text": "PROJECT SUMMARY/ABSTRACT Drug addiction is a chronic disease that produces significant costs to individuals and society. While almost one million Americans suffer from cocaine use disorders, there are currently no approved pharmacotherapies for this disease. Our laboratory has implemented animal models of cocaine abuse that analyze motivation to take cocaine (behavioral economics demand curve analysis) and the propensity for cocaine relapse (cued reinstatement of extinguished cocaine seeking). I will use those behavioral methods in conjunction with DREADD chemogenetics and morpholino antisense approaches to determine the roles of orexins (hypocretins) in nucleus accumbens shell (AcbSh) in cocaine abuse. Orexins are importantly involved in the motivation for cocaine, and orexin neurons provide direct projections to AcbSh. In addition, all drugs of abuse increase dopamine signaling in this brain region, and in vitro studies found that orexin in AcbSh increases dopamine concentration. These and other findings point to a role for orexin signaling in AcbSh in cocaine abuse. However, this possibility has received little examination. The hypothesis of this proposal is that orexin in AcbSh drives cocaine demand and is necessary for cued-reinstatement of cocaine seeking behavior. Aim 1 will use pharmacology to inactivate AcbSh orexin receptors OX1R and OX2R in behavioral economics (BE) and reinstatement paradigms. Previous work with BE found that OX1R signaling is associated with cocaine demand (motivation). OX2R is more abundant in AcbSh and has been recently implicated in drug seeking. I expect that inactivation of both receptors will decrease demand and cocaine cued-reinstatement. Aim 2 will use a disconnection approach with DREADDs and morpholino antisense to confirm results in Aim 1. A hM4Di DREADD will inactivate lateral or dorsomedial hypothalamic neurons projecting to AcbSh unilaterally while morpholino antisense microinjected into the lateral hypothalamus or dorsomedial hypothalamus will downregulate orexin on the contralateral side to the DREADDs during BE and cued-reinstatement. I predict that inactivation of AcbSh- projecting lateral hypothalamic neurons in rats that receive orexin morpholino antisense in LH will result in decreased cocaine demand during BE and increased lever pressing in cued-reinstatement. This would support previous suggestions that lateral hypothalamic orexin neurons mediate reward, whereas dorsomedial orexin neurons mediate arousal. The expected results would indicate that this lateral hypothalamic orexin-AcbSh circuit drives cocaine demand and reinstatement. Together, the proposed studies will increase our understanding of the orexin system, as well as the neural circuitry underlying cocaine addiction. This fellowship will train the applicant in orexin neurobiology and in several innovative techniques in systems and behavioral neuroscience, including chemogenetic and pharmacological approaches; self-administration and behavioral economics paradigms; cannula and catheter surgeries; morpholino knockdown; and immunohistochemical techniques. These skills will be critical for the applicant to become an independent investigator in the drug addiction field."}
{"text": "The NIAMS IRP Animal Program has oversight for 25 Animal Study Proposals (ASPs) using mouse, rat, fish, and rabbit models. At NIAMS, there is a great deal of research around arthritis, recombination/chromatin remodeling using AID transgenic mice, familial Mediterranean fever (FMF) and Tnf receptor associated periodic syndrome. There is also a great deal of research involving muscle and related diseases (experimentation with: N-RAP protein, glycogen storage disease Type II, SIRT1 gene, myofibril assembly, myositis including drug induced, and the voice generating muscle in Midshipman fish) and the study of sub-cellular organelles and cytoskeleton in skeletal muscle. Studies at NIAMS also include in vitro experiments studying retroviral transduction, bone marrow transplantation, and epidermal differentiation studies using fetal and adult skin tissues. Research involving allergic rhinitis and other transmit receptor-induced cellular responses is conducted at the institute. Many of the institutes studies involve apoptosis and autoimmunity. Also of interest within NIAMS are cartilage studies focused on tissue-engineered cartilage, chondrogenesis, GDF-F, COMP and osteoarthritis."}
{"text": "Musculoskeletal soft tissue injuries represent a large portion of sports- related injuries. The frequency of occurrence of these injuries has increased rapidly over the last decade as participation in athletic activities and physical conditioning have increased in the United States. The junction between skeletal muscle cells and tendon collagen fibers has been implicated in a common type of soft tissue injury which occurs when active muscle is passively extended. These injuries apparently occur most frequently in inadequately trained participants which suggests that training and, conversely, disuse can influence the structure or physical properties of these myotendinous junctions (MTJs). This proposed investigation is directed toward elucidating the effects of exercise and disuse on the structure, molecular composition and mechanical strength of MTJs using frog skeletal muscle as a model. These experimental animals were selected because: 1) they are unusual in that single cell mechanical testing can be easily performed, and 2) the molecular composition and quantitative ultrastructure of their MTJs has been studied previously. In the proposed investigation, these animals will be subjected to strength-training, increased-frequency exercise, reduced-frequency exercise or denervation atrophy. At selected times in their training, representative animals from each group will be sacrificed and hind limb muscles analyzed for changes in: 1) mass, 2) aerobic capacity, 3) anaerobic capacity, 4) quantitative MTJ structure, 5) concentration of structural proteins at the MTJ, or 6) breaking strength at the MTJ. These data will provide the first information on the response of MTJs to exercise and disuse. These data will also provide the basis of a comprehensive description of the relationships between modifications in muscle cell structure and molecular composition to load-bearing at MTJs in ways that can be related to clinically-observed defects in humans."}
{"text": "Voltage-activated Ca channels serve two critical functions: the regulation of cellular excitability and the regulation of Ca entry. Alterations in the density, or function, of L-type Ca channels are implicated in a variety of cardiovascular diseases. Thus, elaborating the basic mechanisms that regulate Ca channels is important for understanding both fundamental channel physiology and for therapeutic intervention. During the past funding period, we have identified the Rem GTPase as a novel modulator of I(Ca). It was originally thought that Rem association with CaVbeta- subunits chronically regulated I(Ca) by inhibiting channel trafficking. Our studies have disproved this hypothesis, demonstrating Rem-mediated Ca channel regulation without changes in surface density. Instead, Rem seems to modulate Ca channel activity through interactions with both CaVbeta and the proximal CaV1.2 C-terminus near the CB/IQ domain. Two of the most physiologically relevant controls of I(Ca) are PKA-modulation and calmodulin (CaM)-modulation. Our most recent studies suggest that Rem modulates I(Ca) responses to each of these signaling pathways. Thus, Rem appears to contribute to both beta-adrenergic and Ca-CaM control of I(Ca). The specific hypothesis to be tested is that Rem GTPase regulates Ca channel activity in cardiac muscle through interactions with both CaVb-subunits and the CaV1.2 C- terminus. Three hypothesis driven aims focus our studies and advance knowledge of this novel regulatory mechanism. Aim 1 will explore the nature of Rem-mediated channel regulation by examining the effect of Rem loss on Ca channel regulation. Initial characterization of Rem knockout mice indicates that i) Rem functions in vivo to regulate I(Ca);ii) contributes to the cardiac response to pressure-overload;and iii) contributes to cardiac myocyte growth/maturation homeostasis. Aim 2 will determine whether interaction of Rem with CaVbeta or CB/IQ is critical for modulation of I(Ca). Aim 3 will investigate how Ca- calmodulin modulates Rem-mediated channel blockade, and determine whether PKA phosphorylation alters Rem membrane trafficking or the interaction between Rem and its binding partners. RGK G-proteins function as modulators of Ca channel activity, contributing to regulation of I(Ca), excitation- contraction coupling, and the cardiac response to pressure-overload. The goal of this research is to generate a deeper understanding of the physiological ramifications and molecular mechanism of RGK/Ca channel modulation, to speed progress toward the therapeutic exploitation of RGKs in cardiovascular disease. PUBLIC HEALTH RELEVANCE: Voltage-activated calcium channels serve two critical functions: the regulation of calcium entry and the control of heart contraction. Changes in the number, or function, of L-type calcium channels are implicated in a variety of cardiovascular disease, including atrial fibrillation, heart failure, and ischemic heart disease. Thus, understanding the basic mechanisms that regulate calcium cannels is important in understanding both fundamental heart muscle performance and for therapeutic intervention. We have identified a family of proteins that regulate calcium channels and the goal of this research is to speed progress toward the day when these proteins can be used to treat heart disease."}
{"text": "Individuals who develop rapid progression of HIV-1 infection have been shown to have a high burden of relatively homogeneous viruses (quasi species) and a low frequency of cytotoxic T lymphocytes that respond to HIV-1, whereas individuals who progress more slowly or not at all have a lower burden of a much more diverse viral quasi species and evidence of vigorous CTL response to HIV-1. We hypothesize that these individuals' antiviral cellular immune responses (CTL and T helper) are effectively exerting selective pressure upon the viral quasi species, leading to amino acid changes, quasi species diversification, and modified interaction with the host immune system due to altered binding of MHC-binding regions. This proposal describes the development of EpiMatrix, a computer-driven T cell epitope prediction algorithm, and the application of the algorithm to the evaluation of quasi species evolution. It is proposed that the replacement of anchor- based motifs with extended matrix-based motifs (as in EpiMatrix) will improve the predictive capacity of T cell epitope prediction algorithms for selected MHC alleles and will permit evaluation of quasi species evolution. The specific aims of the proposed work are: To develop EpiMatrix, a second generation T cell epitope prediction program. To apply EpiMatrix to HIV-1 quasi species; predict, synthesize, & test MHC-binding regions/T epitopes. To evaluate the impact of quasi species evolution on T cell response to epitopes defined in the previous aim. The use of EpiMatrix of predict MHC-binding regions and T cell epitopes within variable regions will allow us, for the first time, to accurately assess the influence of sequence diversity within both conserved and variable regions upon the ability of the host cellular immune response to recognize and respond to HIV-1 quasi species. The successful completion of this project will lead to an enhanced understanding of the interplay between the antiviral pressure of the cellular immune responses, viral burden, and diversity of quasi species within infected individuals. These fundamental interactions that define HIV-1 infection are critical to our understanding of HIV-1 pathogenesis. In addition, the epitope prediction method and epitopes defined during these investigations will aid in the development of a vaccine against AIDS."}
{"text": "Of great contemporary concern in the U.S., as well as in other countries, is the question of what constitutes a healthful dietary intake of the B-vitamin, folate. Although adequate amounts of folate intake protect against colorectal cancer, accumulating evidence indicates that overly abundant quantities among those who harbor pre-cancerous or cancerous lesions may produce a paradoxical acceleration of tumorigenesis. There are many sources of folic acid in the american diet that supplement the quantities naturally present in foods: voluntarily-fortified foods, vitamin supplements and mandatory fortification. The seminal question is whether these sources are collectively sufficient to produce the cancer-promoting effect of folate;a grave concern for which there is already supportive evidence from several pre-clinical and clinical studies. Due to ethical and chronological constraints, this dilemma cannot be resolved by human studies alone. Consistent with this reality are the conclusions of a recent international conference on the topic, which included among the high priority recommendations the need to pursue the issue in appropriate animal models. The overall goal of this proposal, therefore, is to answer these questions within the context of a highly innovative and powerful animal model. This novel animal model of colorectal carcinogenesis, GEM-1, allows one to monitor development of colonic neoplasms in vivo over a period of several months. This will enable us to determine critical parameters such as the threshold of dose at which dietary folate has a promotional effect and to develop a mathematical model that defines the relationship. Our proposed studies will: 1) characterize the GEM-1 model's response to supraphysiological quantities of dietary folic acid in regard to cancer promotion, 2) define the quantitative nature of the dose-response relationship between supraphysiological folic acid intake and acceleration of tumorigenesis, 3) determine whether delivery of supraphysiological quantities of a natural form of the vitamin, 5-methyltetrahydrofolate, lacks the cancer-promoting effects observed with the synthetic form, folic acid, and 4) provide evidence that the molecular pathway by which excess folic acid is driving carcinogenesis is by providing abundant quantities of the rate-limiting substrate for DNA synthesis, thymidine. PUBLIC HEALTH RELEVANCE: There is great debate in the U.S. and in other countries as to what constitutes a healthful dietary intake of the B-vitamin, folate, because many sources of the vitamin have been introduced into the food stream and there is accumulating evidence to suggest that overly abundant quantities of the vitamin may promote the risk of colorectal cancer. The existing data concerning the possible promotion of cancer is rather equivocal, and yet the potential ramifications are very great, making this a particularly pressing public health issue that needs to be addressed with some urgency. The studies proposed in this application will be conducted in a novel and particularly well-suited animal model and will provide important answers as to whether the intake of folate that is helping optimize health for one segment of the population might be presenting potential risks to other segments of the society."}
{"text": "HIV-1 RT continues to be a key target for anti-HIV therapy. The long term goal of our project is to comprehensively define HIV RT function and the mechanism of drug resistance. There is an increasing realization that proper positioning of template-primer is central to several key aspects of RT function. We intend to delineate the functional role of three specific interactions between HIV-1 RT and the template-primer. These contact points lie along the template-primer cleft, in front of the active site (contacting the template 5'-overhang), behind the active site (contacting the template at penultimate basepair), or further away in the minor groove (contacting the primer at 3rd to 6th basepairs) respectively. Three specific aims are listed. First, we will study the fingers subdomain-template contacts near the active site via nested deletions of 33-[M loop of HIV-1 RT created to disrupt the proposed interactions with the template overhang and/or the dNTP. In addition to testing these mutants for dNTP-binding, accuracy of dNTP insertion, processivity and strand transfer, we will also test the role of J33-j34 loop as a determinant of the natural high susceptibility of HIV-1 RT to 2',3'-dideoxyNTPs. The mutation rate of multi-dideoxynucleoside analog resistant RT variants and whether a proofreading-like activity contributes to the overall fidelity will be tested. Second, we will investigate the role of the template grip 135a, which forms a close contact with the penultimate basepair of the template-primer, in RT function. We will both attempt to delineate the mechanism of frameshift mutagenesis by HIV-1 RT, the precise role of template grip 135a in frameshift fidelity and the mechanistic basis for its global influence on fidelity. Altered template grip mutants of HIV-1 RT will be tested for effects on affinity to the template-primer duplex, processivity, RNase H activity and strand transfer. Lastly, we will test whether the thumb helix clamp involving aH and cxl helices indeed serves a critical function in enzyme translocation subsequent to each dNTP addition cycle. We will also test its role in processive polymerization and in frameshift fidelity."}
{"text": "The CITAR of the University of Texas School of Public Health (UTSPH) is a program for advanced training on HIV prevention research. Fellowships are for foreign health professionals leading to a 4 year doctoral degree at the UTSPH in Houston followed by a 5th post-doctoral year doing supervised research in their home country. Support for CITAR Fellows may come from a variety of sources but to date has been almost exclusively from the Vietnam Education Foundation (VEF) which provides 2 year of stipends and institutional support. An AITRP grant would support the remaining 3 years and travel expenses between Houston and our research and training sites in Vietnam. The goals of CITAR are: 1. Education &training of outstanding [foreign health professionals for research careers in public health in their home country on HIV and HIV related issues and to help them obtain funding to carry out those careers;2. Foster research and research capacity in developing countries;3. Promote understanding and cooperation between the United States and other countries. We chose Vietnam because the combination of the early but rapidly expanding AIDS epidemic, commitment of the Vietnamese government but severe limitations of its current public health manpower make it uniquely suited to benefit from our faculty who are highly experienced in AIDS research and international health. .Presently we have 7 Vietnamese Fellows (6 from VEF and 1 on a VN government scholarship) and 3 more VEF Fellows starting Fall 2005. We expect to have 2 to 4 new additional students in each subsequent year. There are 6 elements to the CITAR training experience: 1. Faculty supervised HIV field research leading to a thesis;2. Didactic academic courses;3. Weekly seminar in which faculty and fellows share presentations and discussions about HIV and related issues, 4. Practica;5. Course on practical issues of international research, and;6. Close mentoring of all aspects of the educational experience. A critical cross-cutting element will be ethics, focusing on the ethical conduct of science which will occur in all of the teaching elements. An AITRP would not only fit the philosophy and goals of CITAR for these Vietnam trainees, it would leverage Fogarty resources by supporting fellows already two years into their doctoral programs, with proven academic performance &commitment to research in Vietnam. We will hold student-faculty activities in VN aimed at successful reentry including short courses &VN based."}
{"text": "[unreadable] We have developed a water-based gel that changes color in response to doses of ionizing radiation, producing a stable, non-diffusing image of the radiation dose distribution. We have also developed an optical scanner system to record and manipulate the three-dimensional images. The system has significant advantages over competing methods. In this Phase I project we will demonstrate whether this system is suitable for use as a quality assurance method for radiation therapy treatment planning. The research program will determine the practical limits of radiation dose sensitivity under clinical conditions, and investigate how changes in gel composition and scanning techniques will affect the sensitivity and reproducibility, as well as cost. We will obtain much of the necessary data using clinical irradiation facilities and procedures. In Phase II we plan to establish the accuracy of the system for dose measurement under clinical conditions, develop software to enhance the user-friendliness of the system and perform clinical trials to support a possible application for FDA approval as a 510(k) device. Our long-term commercial objectives are to bring this technology to market by licensing the gel technology to a specialty chemical company, and licensing the scanner technology to a company that currently sells radiation therapy equipment. [unreadable] [unreadable]"}
{"text": "Our goal is to understand the molecular mechanisms through which obesity and excessive fat consumption increase the risk of cancer development. Epidemiological studies have shown that of all cancers, obesity has the most dramatic effect on the common form of liver cancer - hepatocellular carcinoma (HCC). In addition to its important metabolic functions, the liver is an immune organ that is severely impacted by obesity, resulting in non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH), inflammatory conditions that greatly increase HCC risk. To genetically and molecularly investigate how obesity increases HCC risk, we established mouse models in which liver fat accumulation (hepatosteatosis) strongly enhances development of chemically-induced HCC;models that a recent review found to be highly relevant to the etiology and pathogenesis of human HCC. Using these models, we had demonstrated that obesity-induced liver inflammation plays a key role in HCC pathogenesis by activating the oncogenic transcription factor STAT3. However, hepatosteatosis also results in chronic activation of Target of Rapamycin (TOR) complex 1 (TORC1), thereby suppressing autophagy. We postulate that TORC1 activation and suppressed autophagy are additional mechanisms that contribute to the molecular pathogenesis of obesity-promoted HCC. To investigate this central hypothesis of the current proposal, four specific aims will be pursued: 1) Determine whether inhibition of TORC1 prevents obesity-promoted hepatocarcinogenesis;2) Investigate the contribution of chronic TORC1 activation to DEN-induced hepatocarcinogenesis;3) Identify effector pathways that mediate TORC1 effects on obesity-promoted hepatocarcinogenesis;and 4) Examine whether TORC1 feedback regulation by Sestrin has a role in obesity-promoted hepatocarcinogenesis. Whereas the first two aims will ask whether TORC1 activation is required and sufficient for obesity-promoted hepatocarcinogenesis, the third aim will explore the hypothesis that the most critical pathogenic function of chronic TORC1 activation is suppression of basal autophagy. The last aim will investigate the connection between tumor suppressor p53 and the regulation of TORC1 activity and autophagy, which depends on expression of Sestrins, a small family of p53-regulated proteins that activate AMPK. Studies will be performed mainly in vivo using both constitutive and inducible gene targeting as well as a novel approach for isolation and specific manipulation of pre-neoplastic HCC initiating cells. We will also develop new tools for uncoupling TORC1 from inhibition of autophagy. Collectively, the proposed experiments will shed new light on the pathogenic mechanisms that link hypernutrition and obesity to tumor development and progression via TORC1 and its ability to suppress basal autophagy. The proposed studies will also lead to new preventive and therapeutic strategies that will reduce the toll of obesity-promoted liver cancer as well as other cancers, such as pancreatic cancer, that are also strongly impacted by the obesity epidemic. PUBLIC HEALTH RELEVANCE: The obesity epidemic has precipitated a marked increase in the incidence of hepatocellular carcinoma (HCC) or liver cancer. The mechanisms by which obesity increases HCC risk are not fully known but their understanding is required for the design of effective therapeutic and preventive strategies. We will use recently developed mouse models and innovative technology for isolation of pre-neoplastic cells to fully understand how obesity promotes HCC development."}
{"text": "Objectives: Evaluate the risk of arsenic exposure and mitigation effectiveness in an Arizona mining/smelting community through speciated analysis of environmental media and urine samples. Background: The predominantly Hispanic populations of the Hayden- Winkelman community live adjacent to an open pit copper mine/smelter complex. Previous studies in these towns measured arsenic exposures that exceeded the 95th percentile for Arizona, although the sample size was limited. Community concerns have been expressed over arsenic exposure and the potential for adverse health effects. Specific Aims: To 1) Measure a representative sample of households for multi-media, multi-pathway, exposure to arsenic species; 2) Measure individual adsorbed dose using speciated urine arsenic; 3) Model bioavailability of arsenic species by media and pathway; 4) Evaluate the contribution of individual factors (age, socioeconomic status, ethnicity, lifestyle, diet, and disease) to arsenic absorbed dose; 5) Measure the effectiveness of interventions to decrease arsenic exposure; and 6) Educate the community through the establishment of a computer learning center linked with the University. Study design: 130 randomly selected and 20 community designated households will be evaluated for speciated arsenic exposure (HG-AAS) in indoor and outdoor air (PM10, PM25, vapor phase), vacuumed house dust, surface soil, water (drinking and tap), food and beverage collected using trained community members with quality assurance audits. In association with questionnaire data, regression analysis will be used to determine the effect of media, route of exposure, and individual factors on urine arsenic species concentration, and compared with existing statewide data (NHEXAS). Sixty-five intervention and 20 control households will be reevaluated for the effect of substitute or treated water and HEPA vacuuming. A public computer learning center will be developed and staffed by the community. Expected Outcome: Community members will earn the extent of their exposure to arsenic, the effectiveness of low-tech interventions, and how to obtain information on environmental exposures. There is also potential for future research, including evaluation of biological markers of effect of low-level arsenic exposure."}
{"text": "Stroke is the 3rd leading cause of death and the primary cause of severe, long term disability. Each year 795,000 Americans have a stroke and the annual costs to the economy are $57.9 billion. The vast majority of acute ischemic strokes are caused by a thrombus (blood clot) which occludes the blood vessel and stops blood flow to the brain. Tissue plasminogen activator (TPA), an agent that catalyzes the dissolution of blood clots, is the only effective, FDA-approved treatment for ischemic stroke. Unfortunately, TPA is associated with significant risks, delays in treatment, and is unsuccessful in up to 70% of patients at dissolving blood clots in sufficient time to protect the brain. There is a need for a safer, more effective therapy that facilitates early treatment, saves lives, reduces disability and lowers health care costs. In pre-clinical studies, we have shown that these goals might be achieved by a molecule that inactivates the major inhibitor of plasmin and, dissolves clots through a unique mechanism that avoids the risk of hemorrhage and neurotoxicity associated with TPA therapy. Following FDA guidance, we converted this molecule into a biologic drug candidate for stroke (stromab) that potently accelerates the dissolution of human clots. The goal of this Fast Track application is to move stromab further towards human trials by following FDA guidance to: 1) determine the optimal formulation and therapeutic time window for treatment, 2) produce and purify stromab under GLP conditions, 3) investigate the safety, pharmacokinetics and pharmacodynamics of stromab and, 4) submit an IND to the FDA. PUBLIC HEALTH RELEVANCE: Each year 795,000 Americans have a stroke and the annual costs to the economy are $57.9 billion. Tissue plasminogen activator, the only approved treatment for ischemic stroke, is associated with significant risks, delays in treatment, and is unsuccessful in up to 70% of patients at dissolving blood clots in sufficient time to protect the brain. This project seeks to develop a novel therapy for stroke that could markedly reduce death, disability and costs."}
{"text": "ABSTRACT Complex lymphatic anomalies, which include a variety of diagnoses: lymphangiectasia, Central Conducting Lymphatic Anomaly (CCLA), Generalized Lymphatic Anomaly (GLA), Kaposiform Lymphangiomatosis (KLA), and Gorham Stout Disease (GSD), are chronically debilitating and often life-threatening diseases. Unfortunately, for most patients, physicians can offer only palliative care that requires multiple outpatient visits and hospital admissions. The absence of data on the molecular etiology, and lack of understanding of the underlying molecular mechanisms in lymphatic anomalies have greatly hampered further research and precision medicine focused clinical trials. Our long-term goal is to identify efficacious therapies for complex lymphatic anomalies. The objective of this application is to uncover novel disease-causing genes/mutations and use in vitro and in vivo models established in our previous studies to determine optimal treatment strategies. Our preliminary studies have revealed multiple genes converging on the Mitogen-Activated Protein Kinase (MAPK) signaling pathway and modeling mutations in cellular and zebrafish systems have recapitulated the essential morphological features seen in the lymphatic anomaly patients. We have found that a handful of MEK/ERK inhibitors showed the biochemistry and morphological reversal of the effects of mutations in RASopathy genes. This proposal will test the hypothesis that sequencing of highly informative patients referred by an integrated multidisciplinary lymphatic anomalies clinic will unveil novel RASopathy genes and mutations, and these can be rapidly interrogated through our established cellular and zebrafish models to further investigate the mutation phenotype spectrum effect and correlating molecular biomarkers. The specific aims of this proposal are to: 1) Discover additional RASopathy genes and mutations through exome sequencing of patients with complex lymphatic anomalies; 2) Delineate the molecular mechanisms of newly identified genes and mutations in cellular and zebrafish models; and 3) Leverage novel disease models for therapeutic rescue to explore potential future therapeutic targets for human disease. The results from these experiments will have a significant impact on the field because they will answer fundamental questions regarding the genetic etiology, molecular mechanisms, and treatment options, and most importantly, provide validated pre-clinical data for molecularly implemented precision-based therapies for clinical trials. This knowledge will significantly advance our understanding of different types of lymphatic anomalies."}
{"text": "The simian immunodeficiency virus, SIVmne, has a high degree of genetic homology to the human lentivirus HIV-2 and a lower but still quite significant homology to HIV-l, both of which are etiologic agents of human AIDS. SIVmne, like the other SIVs isolated recently causes a severe immunodeficiency in the nonhuman primate, Macaca nemestrina, which selectively involves CD4+ cells and is virtually identical to AIDS in man. We have found a linkage between the expression on a subset of CD4+ lymphocytes of adhesive receptors for high endothelial venules (HEV) and selective depletion and perhaps susceptibility to infection with SIVmne. This arose during our work to develop the macaque as a model system for analysis of the role such receptors have in directing organ- specific migration of lymphocytes through lymph nodes, Peyer's patches, and inflammatory sites. With the monoclonal antibodies (Mabs) Hermes-l and recently with Hutch-l we have defined two distinct types of CD4+ lymphocytes, HEV receptor/hi and lo, which differ markedly in response to mitogens, and capacity to recirculate through lymph nodes. HEV-receptor high cells are lost selectively in simian AIDS before the onset of clinical illness. Our future goals are a) to determine the mechanisms which underly the selective affects of SIVmne on these two cells, b) determine if Mabs to HEV receptors can be used as sensitive prognostic indicators in AIDS, and e) further define the role of HEV-receptors in directing lymphocyte migration and their structural features which confer organ-specific recognition of endothelial cells. We will also explore the capacity of interleukins to regulate expression of a) HEV-receptors on lymphocytes and b) their ligands on endothelium. We propose to examine linkage of these homing receptors to specialized lymphocyte functions and determine the in vivo migratory properties of normal lymphocyte subsets and T cell clones. Various aspects of the project use cellular immunology techniques as well as hybridoma technology and molecular biological methods such as subtractive hybridization cDNA cloning and in situ hybridization with SIV and interleukin specific probes."}
{"text": "SUMMARY Computational Chemical Biology (CCB) Core will provide comprehensive computational support, including chemical virtual screening, physical property prediction, target validation, hits identification, lead optimization, pharmacophore identification, and pharmacokinetic and dynamic modeling. In addition, the CCB core will provide information management infrastructure supporting the creation and management of scientific data analysis workflows, project reporting, and database management for chemicals including their structures and biological properties. These activities will be fully integrated with projects and with Cores B and D. For example, the analysis of high-throughput screening data and chemical structure-activity relationship data generated by Core B will be stored, indexed, computationally analyzed, and visualized at the CCB Core."}
{"text": "The objectives of this project are to make homogeneous preparations of the enzymes undecaprenyl (C55) pyrophosphate synthetase and undecaprenol phosphokinase for Lactobacillus plantarum and Micrococcus lysodeikticus. They will be characterized with respect to their substrate specificity, phospholipid or membrane reqirements and the stereochemical structure of their substrates and products. Application of various alkyl-agarose columns will be made for possible purification of the partially purified C55-PP synthetase. Synthetase specificity will be determined with stereochemically pure C15 and C20 pyrophosphates and kinetic constants will be compared fo the two bacterial systhetases. The stereochemical purity of the enzyme products will be evaluated using 2R-23H and 2S-2 3H-IPP. Analogues of isopentenyl pyrophosphate will be synthesized and tested as substrates for the synthetase. Phospholipid and detergent requirements will also be compared for these two bacterial synthetases. Undecaprenol kinase will be further studied to improve the stability of the solubilized enzyme, greatly increase the degree of purification, and investigate more thoroughly the detergent and phospholipid activation of the enzyme."}
{"text": "This grant has supported studies on various aspects of class switching and lymphokine induction in the spontaneous murine B leukemia, BCL1. Our findings during the last period have prompted a broader investigation of B cell differentiation using this model system and normal B cells. Based on significant supporting data, we have condensed our continuing efforts into three projects. First, we want to determine the precise mechanism and its control for allelically excluded, dual synthesis of mu and gamma1 chains in BCL1 subclones. We know that this occurs by some form of discontinuous transcription, and not by production of a 'long transcript' covering the entire VDJ to C-gamma1 region. Second, we want to determine precisely how IL-5 and IL-6 in conjunction with antigen induce increased rates of u transcription and DNA replication in BCL1 cells rendered antigen-specific by transfection. We have identified the target sequences and the protein-DNA interactions for IL-5+Ag. We want to do the same with IL-6 plus go after the factors induced by both lymphokines. Third, we want to study proteins expressed only at the immature/mature B cell differentiation stage based on the premise that functions relevant to Ig gene expression will be discovered. We have cloned from BCL1 two mRNAs that satisfy the criteria of cell type- and stage-specificity."}
{"text": "Kinetic and thermodynamic studies of various antibody aggregates interacting with mobile Fc receptors on cell surfaces."}
{"text": "For fifteen years the Biomedical Computing Shared Resource (BCSR) has promoted high quality innovate cancer research by providing members of the UWCCC with an advanced biomedical computing environment. As a matter of policy, computing support within the University and Medical School is decentralized and largely the responsibility of individual departments and centers such as the UWCCC. Within this context, the BCSR plays an essential role by providing both technical and intellectual resources that address the unique and specific needs of the UWCCC in an efficient and cost effective manner. The BCSR supports the UWCCC research enterprise in five years. First, it is responsible for much of the network and I/T infrastructure used on a daily basis by every connected UWCCC member. Second, it provides an array of computing services to UWCCC investigators, other shared resources, and to UWCCC administration. Third, it develops and maintains specialized research resources targeted to the particular needs of UWCCC investigators. Fourth, BCSR staff provide critical software engineering, project management and computer science expertise for specific projects. Fifth, the staff of the BCSR fulfill an important leadership role by tracking or anticipating the needs of the UWCCC research community and then developing the resources to meet these needs. The facilities of the BCSR have been used by Drs. Gould, Moser and Newton to build genetic maps of mice and rats. Drs. Newton and Reznikoff have used them to test new methods for assessing gene loss. Drs. Lindstrom and Kinsella have used these facilities to assess models for therapeutic radiation damage to tumors. Drs. Love and Sondel have used these facilities to build and maintain research databases. In addition, the facilities and staff of the BCSR are used by scientific-oriented shared resources such as the Clinical Trials Research Office for patient and protocol tracking, the Analytical Instrumentation Laboratory and Flow Cytometry for computing support and consultation, and the Biostatistics Shared Resource for computational and database resources. The BCSR also provides essential support and services to UWCCC Administration."}
{"text": "This proposal is concerned with mutagenesis and carcinogenesis induced by aromatic polycyclic hydrocarbons which are environmental pollutants. The experiments described will show whether, and how, DNA adducts formed from the proximate carcinogens produce mutations. Our study offers a new approach: we will use the mammalian virus SV40, with its DNA modified in vitro by treatment with the proximate hydrocarbon carcinogen. The modified and characterized SV40 DNA vector will then be studied in infected cells from which we will learn the molecular nature (base change, insertion, deletion) and location of mutations caused by the hydrocarbon adducts. Our studies will also help to elucidate the role of DNA repair in mutagenesis. We will study both pre- and post-replicative repair and will determine whether the repair process itself is error-prone. A close correlation between mutagenesis and carcinogenesis will be made possible by examining the extent of mutagenesis of SV40 in the same cells in which transformation by the hydrocarbons will be studied. This last approach will also provide a basis for the screening of potential carcinogens."}
{"text": "Ascaris lumbricoides var. suum is a parasitic intestinal nematode which possesses an anaerobic energy metabolism. Carbohydrates are dissimilated to a mixture of volatile acids and succinate. Studies are continuing on the pathway of formation of the volatile acids, particularly alpha-methylbutyrate and alpha-methylvalerate. Ascaris muscle differs biochemically from mammalian muscle in many respects. It functions anaerobically. Pyruvate kinase activity is barely detectable. Lactate is not formed by intact worms. In spite of the anaerobic nature of Ascaris muscle, mitochondria are present. Studies are in progress to better define the relationships between cytoplasmic and mitochondrial metabolisms. Malate, formed in the cytoplasm via CO2 fixation into phosphoenolpyruvate, serves as the mitochondrial substrate. In the mitochondrion, malate dismutates to form succinate and pyruvate with the concomitant anaerobic formation of ATP. A number of anti-cestodal agents have been found to inhibit the P32-ATP exchange reaction in both cestode and Ascaris mitochondria. It is proposed to continue these studies on phosphorylating Ascaris mitochondrial systems in an attempt to better localize the possible mode of action of these drugs. Effects of these agents on net ATP forming systems in both Ascaris and Hymenolepis diminuta are being investigated. Studies of the intermediary metabolism of the two filarial worms, Litomosoides carinii and Dipetalonema witei (vitae) and Brugia pahangi are being initiated."}
{"text": "DESCRIPTION: The interactions of molecular tracks and motors result in movements in cells. Fundamental to our understanding of the way in which motility is accomplished in cells is a detailed description not only of the atomic structures of the complexes involved, but also the way in which the activity of these complexes is regulated in response to the needs of the cell. The long term goals of the work described in this application are to understand in structural terms, the way in which myosin motors are regulated. Three types of myosin-based regulation will be investigated: (I) Regulation mediated by light chain phosphorylation will be investigated by examining a number of smooth muscle myosin constructs. (ii) Regulation by light chain dissociation will be examined in brush border myosin l and myosin V. (iii) Regulation by heavy chain phosphorylation will be investigated in Acanthamoeba and Dictyostelium myosin ls. The methods to be used include cryoelectron microscopy, helical image analysis and modeling using the x-ray structures of the myosin head and regulatory domain. The experiments have been designed to visualize actomyosin conformations which are the basis of the regulatory mechanism. Atomic models of the regulated and activated states will be built from the EM and X-ray data. The results should provide detailed insights into the structural basis for regulation in myosin involved in uterine contractions, blood pressure maintenance and intestinal peristalsis (smooth muscle myosin), and in intracellular trafficking and endocytosis (myosin ls and Vs). Some exploratory experiments on dynein are planned."}
{"text": "ZetrOZ has developed a prototype novel, miniature, portable, high-power, ultrasound system for would healing. Wound healing is a major clinical issue, and can present significant clinical challenges both when wounds are acute--occurring due to trauma, burns, surgery, or due to more chronic health problems. Wound healing is an intricate fibroproliferative response to repairs damaged tissue following injury. This process is fragile and susceptible to interruption or failure, leading to non-healing wounds. Historically, wounds have been historically treated in a sub-optimal fashion with basic wound care products designed to cover wounds and absorb exudates. Ultrasound (a safe, commonly used, FDA-approved treatment modality) has been shown to have beneficial effects on factors associated with tissue healing in both in vitro and in vivo animal models, including promotion of histamine release, angiogenesis, and collagen deposition (thereby increasing wound breaking strength), and ultimately results in a reduction in wound size. Although ultrasound therapy has been previously demonstrated to accelerate wound healing, conventional techniques utilize shorter bursts of high-intensity treatments. Such treatment is typically limited to administration by a medical provider. In contrast to current technologies, our device significantly reduces the size, cost, and power requirements of ultrasound therapy when compared to traditional ultrasound devices. The system can deliver therapeutic acoustical energy waves at lower voltages than those in conventional ultrasound systems. During this project we aim to demonstrate that a human wearable low intensity therapeutic ultrasound system enhances wound healing. This will be accomplished through testing the system on a single battery charge in an animal cadaver. We will also demonstrate that low intensity therapeutic ultrasound improves wound healing, as measured by therapy on the rate and extent of epithelialization and granulation tissue formation (the major components in human wound healing) in wild-type and diabetic murine excisional wound models. The proposed experiments will investigate two different power levels to further understand impact of intensity on would healing."}
{"text": "Melanins (eumelanins, pheomelanins) are photoprotective pigments. They remove light energy, dissipating it either as heat or in a chemical reaction in which oxygen is consumed. Recent work has shown that they also scavenge toxic species, e.g. superoxide and singlet oxygen. Protection may arise either from light filtering, removal of oxygen, or such scavenging. The cost of the protection is measured in terms of the chemical or thermal damage that may result. If the damage outweighs the protection afforded by the pigment, phototoxicity results: it has been hypothesized that damage may be more pronounced with pheomelanins than with eumelanins and may promote skin cancer in red-haired individuals. New experiments are proposed to: (i) measure the effectiveness of isolated pigments in scavenging superoxide and singlet oxygen and the resulting damage to the melanin (e.g. bleaching, irreversible free-radical production); (ii) determine whether scavenging reactions are effective in melanosomal and cellular systems; (iii) determine the effect of melanin on cell respiration and survival following exposure either to visible or UV light in the absence or presence of sensitizers. These experiments will include a comparison of eumelanins and pheomelanins, and an assessment of the utility of sensitizers in promoting chemical or thermal damage in pigmented cells will be made. In these studies use will be made of recently-developed electron spin resonance (ESR) procedures for measuring oxygen consumption and superoxide production in photolytic and cellular systems and of a new model pigmented cell system: Chinese hamster ovary (CHO) cells containing phagocytized melanin."}
{"text": "Project Summary and Relevance The aim of this Phase II SBIR proposal from Applikate Technologies, LLC, is to build on the successful completion of our Phase I milestones with the design, construction, and testing of prototype devices and software for implementing the ClearView tissue histology system in clinical settings by laboratory personnel. ClearView is a novel approach to performing complete and non-destructive imaging of tissue specimens for lung and other cancers. Lung cancer biopsy diagnosis error rates may run as high as 15%. Although there are many potential sources of error, important aspects relate to limitations of current methods for tissue processing and analysis. Standard methods limit the amount of tissue that can be visualized by the diagnostic pathologist and that is available for personalized-medicine testing. They also are not amenable to consultation with off-site experts and are dependent on manual processing by adequately trained histotechnologists. Applikate Technologies has developed ClearView, a complete, non-destructive, fast, tissue processing and imaging approach to histology that improves on limits of slide-based histology by providing complete tissue visualization, reducing artifacts of preparation, permitting improved quantitative interpretation, showing additional growth pattern information, and eliminating extensive manual preparation. The ClearView system yields images that are, in many cases, superior even to traditionally processed histology slides with pseudo-coloring that is virtually indistinguishable from conventional staining, all in an entirely digital format ready for remote access and storage. And ClearView is fast enough to provide diagnostic quality images in under two hours for same day diagnosis. We have also demonstrated, during our Phase I funding, that the ClearView process yields orders of magnitude more DNA for molecular analyses and is compatible with immunohistochemical staining. Furthermore, the methodology is applicable not only to lung cancer diagnosis, but to evaluation of practically any solid tissue cancer. The specific aims of the proposal are to: 1) Develop an application-specific, high-speed multiphoton microscope and associated software; 2) Develop the ClearView prototype tissue chamber, and 3) Develop the ClearView prototype tissue processor. These three elements, along with proprietary reagents, form the core of the ClearView system. Following in-house testing, two complete prototype systems will be placed in two hospitals, one a large tertiary care center and the second a mid-sized hospital, for on-site testing and feedback by non-expert users in a clinical setting. Successful completion of these aims will set the stage for a Phase III transition to regulatory approval and provide a proven platform for early implementation through service work for academic research and clinical research groups."}
{"text": "Background: Essential to high-quality palliative care assessment and management is the availability of psychometrically-sound and clinically-relevant screening, diagnosis, and outcome evaluation tools that will enable healthcare providers to attain optimal patient evaluation, prognosis, treatment selection, patient satisfaction, and quality of life improvement. We propose to develop a patient-centered, model-driven, evidence-based system called the System for Interactive Assessment and Management in Palliative Care (SIAM-PC) using innovative methodologies and technologies. Aims: Our four distinct but related specific aims are to: 1) identify and refine the domains of palliative care, using existing theoretical frameworks as a guide; 2) compile the items for multidimensional palliative care item bank, drawing from existing measures and supplementing with newly written items; 3) empirically construct a palliative care item bank using item response theory (IRT); and 4) pilot test a computerized adaptive testing (CAT) platform to dynamically administer palliative care assessment in clinical settings. Methods: First, we will identify and refine the key domains of palliative care, guided by existing frameworks, from in-depth individual interviews (n=50; 20 patients, 20 family members and 10 clinicians) and literature review (Aim 1). Second, we will compile items pertinent to these domains from existing measures, writing new items for un-covered domains (Aim 2). Third, we will test these items by collecting and analyzing data from patients (n=200) receiving palliative (Aim 3). Finally, we will pilot test the CAT system (n=40) in clinical practice (Aim 4). Outcomes: At the end of this R21 project, we expect that (1) the conceptual model of palliative care measurement can be improved; (2) the initial sets of palliative care domains/items can be constructed; (3) items displaying DIF can be identified and revised; and (4) the CAT prototype will demonstrate how the system should be implemented in clinical settings. Palliative care is a major public health concern. There is significant consensus and ample evidence in the literature to call for a comprehensive, interdisciplinary approach to address this concern. This comprehensive assessment and care is demanding in that it depends upon multiple sources of information, and yet the practicalities of real-life care depend upon concise information. Our proposed study is to create valid methods to connect comprehensive assessment with concise information in patient assessment and care. [unreadable] [unreadable] [unreadable]"}
{"text": "Cerebrovascular amyloid beta-protein (ABeta) deposition is a hallmark of Alzheimer's disease (AD) and related disorders including hereditary cerebral hemorrhage with amyloidosis Dutch-type (HCHWA-D). Cerebrovascular ABeta deposition in these disorders is prominently associated with smooth muscle cellular pathology in the cerebral vessel wall and can lead to a loss of vessel integrity and hemorrhagic stroke. The reasons for this remain unclear. The overall hypothesis that forms the basis of this competing continuation grant proposal is that ABeta deposition in the cerebral vessel wall can modulate the function of hemostatic enzymes thus creating a microenvironment that is conducive to hemorrhagic stroke. The objectives of this proposal are to characterize the effects of ABeta on the regulation of certain hemostatic enzymes at the biochemical and cellular levels. The specific aims of this proposal are as follows: First, the effect of soluble ABeta, solution-assembled fibrillar ABeta, and cell-surface-assembled, fibrillar ABeta on the inhibition of factor XIa (FXIa) and certain other coagulation enzymes by PN-2/AbetaPP will be characterized. The inactivation rates of FXIa and other coagulation enzymes by PN-2/AbetaPP in the presence of soluble and the different fibrillar ABeta peptides will be determined. The proteolysis of ABeta peptides by coagulation enzymes will be investigated. Second, studies will be conducted to determine the effect of soluble ABeta, solution-assembled fibrillar Abeta, and cell-surface-assembled fibrillar ABeta on the stimulation of tissue plasminogen activator (tPA) activity. The effect of ABeta peptides on cellular tPA levels and plasminogen activation activity will be explored. In addition, proteolysis of ABeta peptides by tPA and plasmin will be investigated. Third, investigations will compare the pathologic effects of intact ABeta and hemostatic enzyme proteolyzed ABeta on cultured human cerebrovascular smooth muscle (HCSM) cells, a cell type intimately associated with the cerebrovascular pathology of AD and HCHWA-D. The proposed studies will provide a better biochemical understanding of the influence that ABeta has on the inhibition of certain coagulation proteases and the stimulation of plasminogen activation, two processes that together can create a milieu which could result in loss of vessel integrity and hemorrhagic stroke."}
{"text": "This project involves the development and testing of a novel MHC based vaccine designed to provide both humoral and cellular immunity against viral attack. Such vaccines have particular relevance for protection against AIDS. These vaccines are based on linking viral epitopes through a tether to B2-microglobulin. This tripartite construct can replace normal peptides present in MHC-l binding sites, leading to the generation of virus-specific T-cell responses. Initial studies with a model mouse Sendai virus construct have provided proof of principle for this approach. This includes the cloning, refolding and purification of the vaccine and demonstration that it binds/exchanges with B2-microglobulin on cell surfaces, upregulates MHC expression on cells expressing empty class I MHC and stimulates T-cell hybridomas that are reactive to the Sendai virus epitopes present on the vaccine. The proposed studies involve: A. Further studies with the Sendai virus model including 1) additional investigation with the Beta2-linker-Sendai epitope construct to determine the effect of different linker sizes on binding to the MHC and on subsequent CFL responses 2) development and testing of a DNA version of the vaccine construct 3) determining whether vaccination of mice with these constructs will provide protection against challenge with the Sendai virus. As part of these studies the optimization of the route, dosage and duration of effectiveness will be determined. B. Development and testing of AIDS MHC-vaccine constructs, including I) using a p17 (Gag) sequence 2) using a Pol sequence 3) using a Nef sequence. These epitopes were chosen because of their highly conserved nature, their cross-clade reactivity, and their role in the spread of the disease."}
{"text": "The 1980s might be called the decade of awakening private sector interest in containing medical care costs. Employers have begun to take initiatives to control their outlays for health insurance premiums paid as a fringe benefit. Employers are redesigning benefit plans to increase consumer cost sharing, offering alternative health care plans, and changing the methods used to finance premiums. Since insurance benefits provided by employers constitute about one-third of all health care expenditures, these private sector initiatives are viewed as a new potential mechanism to moderate the spiralling rate of inflation in the health care sector. The proposed study has four objectives: (1) to describe what employers are doing to control their health benefit costs, (2) to estimate the savings associated with alternative cost cutting measures, (3) to estimate the demand for particular innovations by medium and large sized firms, and (4) to assess the implications of our findings for policy reforms relating to employer-based health insurance. The project will utilize Bureau of Labor Statistics data, 1981-1984, on the benefit offerings of some 1200-1500 firms nationwide. Three analyses are planned. The first is a cross tabular examination of the extent and kinds of initiatives that employers have taken over the period 1981 through 1984. The second is a regression analysis of premiums that includes benefit provisions and other plan characteristics among the explanatory variables. The third is a regression analysis of the demand for selected benefit characteristics. The implicaitons of our results for policy reforms affecting employers will be examined in detail."}
{"text": "The objective of this study is to develop an in vitro potency assay to replace the guinea pig death test portion of the official release test for the diptheria component. VERO cells will be used as a target for the diptheria toxin. After screening several lot of fetal bovine serum for diptheria antitoxin, a lot has been approved and purchased. After examining several methods for determining viable cells, the Neutral Red Assay has been selected. The U.S. Standard Diptheria Toxin Lot 35119 will be used for the cell death assay. It is currently used in the animal death test and will be used in the rabbit skin test. A reproducible minimum cytopathic dose is being established for the 4 day assay. Inhibition of cell death will be measured using the U.S. Standard Diptheria Antitoxin, Immune Serum Globulin, IGIV and guinea pig immune serum."}
{"text": "Aggressive/disruptive behavior in the elementary school years is a strong indicator of antisocial behavior, drug abuse and low educational and occupational attainment in adolescence and young adulthood. The Good Behavior Game (GBG) and Promoting Alternative Thinking Strategies (PATHS) represent two of a handful of universal, elementary school, preventive interventions which have been shown in large scale, randomized controlled trials to have an immediate and beneficial impact on aggressive/disruptive behavior. PATHS seeks to accomplish reductions in such behavior via teacher led instruction aimed at facilitating emotion regulation and social problem-solving, whereas the GBG provides teachers with an efficient means of reducing aggressive/disruptive behavior using social learning principles within a game-like context. Importantly, however, the effects of the GBG on aggressive/disruptive behavior proved modest in our 1st and 2nd generation Johns Hopkins University Preventive Intervention Research Center (JHU PIRC) field trials. This has been the case for PATHS as well. We recently completed a 27-school, randomized controlled trial examining whether the combination of these interventions, which we refer to as PATHS to PAX, would yield significantly greater impact on aggressive/disruptive behavior than the GBG alone. Our rationale for expecting greater impact was that the use of the GBG should result in reductions in aggressive/disruptive behavior, which should then facilitate the acquisition of the emotion regulation and social problem-solving skills taught in PATHS. PATHS to PAX did result in a modestly greater reduction in aggressive/disruptive behavior than the GBG alone at 1-year post-test. Yet, the most aggressive/disruptive students still failed to sufficiently benefit from te PATHS to PAX intervention. Accordingly, in this application, we propose to examine whether the addition of the Incredible Years (IY), an evidence-based preventive and treatment intervention aimed at reducing aggressive/disruptive behavior, would yield greater impact on these behaviors than the PATHS to PAX intervention alone. We also propose to examine whether the combination of the PATHS to PAX + IY results in increased frequency of implementation of the PATHS to PAX intervention. We hypothesize that relative to teachers in the PATHS to PAX alone condition, teachers in the PATHS to PAX+IY condition will perceive PATHS to PAX as more efficacious and will therefore be more likely to implement it. Four cohorts of 12 schools each will be recruited with schools randomly assigned to 1 of 3 intervention conditions: 1) Control; 2) PATHS to PAX Alone; or 3) PATHS to PAX+IY. Assessments of student outcomes will be carried out at pre-test and post-test in the fall and spring of the initial school year foreach cohort and at a 6-month and one year follow-up."}
{"text": "[unreadable] [unreadable] This application is for competitive renewal of a PHS Institutional National Service Award at the David Geffen School of Medicine at UCLA to prepare fellows for an academic career in Hematology. This award was initiated in July, 2002, and supports 4 fellowship slots in years 2 and 3 of Hematology/Medical Oncology training. Of 11 fellows who have entered the program, 4 are currently in training and 7 have completed their fellowship, of which 4 are on the full-time faculty at UCLA, two are in clinical trial development with industry, and one has entered clinical practice. The faculty consists of 31 academic scientists and physicians, grouped into four areas of mutual research interest, Hematopoiesis and Stem Cell Biology (Kenneth Dorshkind, PhD, Director), Leukemia (Gary Schiller, MD, Director), Lymphocyte Biology and Lymphoma (Alan Lichtenstein, MD, Director) and Hemostasis, Thrombosis and Vascular Biology (Victor Marder, MD, Director). The entire faculty have extramural research grant support, including 24 (77%) with NIH grants. Interaction with other training programs in complementary disciplines emphasizes our goal of interactive basic and translational research careers. A research retreat is; held each spring, during which time each mentor presents an overview of the laboratories' research interest, followed by a report of the respective fellows' progress. Formal review of the mentor's effectiveness and the fellow's progress and goals are prepared by the Steering Committee, made up of the four Focus Directors. A core curriculum is offered to all fellows, including a \"Clinical Research Course\", which covers subjects on research design and analysis, ethics, regulatory, issues, grant preparation and responsible conduct of research. Fellows are encouraged to obtain an advanced degree as part of the STAR program, with formal course work in appropriate departments for one or two additional years. Of the 7 fellows who have completed their program during the interval 2002 - present, 2 have obtained the Master's degree and 1 has received a PhD degree. The training of hematologists with expertise in basic, clinical and translational research is an important priority for the David Geffen School of Medicine at UCLA and this application seeks support for the pivotal period between clinical training and an eventual career in academic hematology. [unreadable] [unreadable] [unreadable]"}
{"text": "A non-hemin regulated translational repressor protein has been purified partially from the postribosomal supernatant of Friend leukemia cells grown in the absence of dimethylsulfoxide. This repressor inhibits globin synthesis in lysates from rabbit reticulocytes and in a highly active fractionated system using brine shrimp ribosomes, rabbit globin mRNA, peptide elongation enzymes from reticulocytes and initiation factors from either reticulocytes or Friend cells. It has no effect on poly(U) directed peptide synthesis, nor on the extension or release of nascent globin peptides that were initiated in intact reticulocytes. In contrast to the hemin controlled repressor from reticulocytes, it does not block mRNA independent synthesis of methionylpuromycin but it does inhibit mRNA dependent synthesis of methionylvaline. A block in binding of mRNA to ribosomes appears to be involved. Current work is directed towards evaluating the physiological role that this repressor protein plays in the virus induced block in differentiation of murine cells of the erythroid line. Such a block in differentiation may account for the accumulation of proerythroblasts and the pathological state defined as Friend virus induced leukemia in infected mice. BIBLIOGRAPHIC REFERENCES: Globin mRNA Translation on Artemia salina Ribosomes with Components from Friend Leukemia Cells. G. Kramer, P. Pinphanichakara, D. Konecki and B. Hardesty. Eur. J. Biochem. 53: 471-480 (1975). Aminoacyl-tRNA Specificity of a 40S Ribosomal Subunit Binding Factor from Rabbit Reticulocytes, J. Cimadevilla and B. Hardesty, Biochem. Biophys. Res. Comm. 63: 16-23 (1975)."}
{"text": "Treatment for head and neck cancer (HNC) is arduous and debilitating. As a result, up to 40% of patients develop depression which is rarely recognized or treated. Untreated depression in cancer patients lengthens hospital stays, and reduces adherence with medical treatment, quality of life, and survival. Patients with HNC typically develop depression within the first 3 months of their diagnosis. Developing depression can lead to interruptions or delays in cancer treatment which in turn may diminish the overall prognosis. Depression can also lead to suicide;patients with cancers of the larynx and tongue account for 2 of the 3 highest suicide rates in cancer patients. We are proposing a groundbreaking approach to the problem of depression in HNC. Patients with HNC have the greatest risk for depression in the several months after diagnosis and during treatment for their cancer. We seek to prevent major depression before it can develop by initiating prophylactic treatment with the antidepressant citalopram soon after the diagnosis of HNC is made, thus potentially avoiding many serious sequelae including treatment interruption or delays. Depression also has an enormous impact on quality of life in patients with HNC. Additionally, an overwhelming majority of HNC patients use alcohol and tobacco, and despite adverse effects on prognosis, it is often difficult for them to decrease use of these substances. We will therefore evaluate whether prophylactic use of citalopram can preserve quality of life during and after treatment for HNC and reduce alcohol and tobacco use by these patients. We are therefore proposing a randomized, double blind, placebo-controlled trial investigating the prophylactic utility of the antidepressant citalopram in nondepressed patients who are about to begin treatment for newly diagnosed or recurrent head and neck cancer. We will evaluate whether such prophylactic treatment can significantly reduce the development of major depression, improve adherence to cancer treatment, preserve quality of life and reduce tobacco and alcohol use. This proposal has considerable relevance to public health in that it not only tests whether depression can be prevented in patients with HNC during treatment but also examines whether this intervention can also improve timely completion of the cancer therapy. Additionally, we will examine whether the quality of life of such patients can be better preserved during treatment and whether alcohol and tobacco use can be diminished. If successful, this approach may be applicable to other medical illnesses that have high rates of comorbid depression."}
{"text": "DESCRIPTION: (Applicant's abstract) Long-term potentiation of synaptic transmission in the hippocampus is a widely used model for studying the synaptic plasticity which underlies learning and memory. Most previous studies have focused on a form of LTP which requires the NMDA type of glutamate receptor. This proposal is to study a form of LTP which does not require NMDA-Rs, but instead requires voltage-dependent calcium channels (VDCCs). In both forms of LTP, calcium entry into postsynaptic neurons, through either NMDA-Rs or VDCCs, is required for induction of the potentiation. While much is known about the subsequent cellular events underlying maintenance of NMDA-R dependent LTP, little is known about the maintenance of NMDA-R independent LTP. It is unclear if cellular processes following induction are shared by the two forms of LTP, or if a distinct set of processes underlies each form of LTP. The major aim of this proposal is to define the signaling pathway leading from the induction of NMDA-R independent LTP, through the maintenance of this LTP. Multiple components of potential signaling pathways will be investigated. These include the ion channels., neurotransmitter receptors, second messengers, and the protein kinase targets of these second messengers. This proposal also aims to determine the roles played by metabotropic glutamate receptors in NMDA-R independent LTP. These roles may include: modulation of LTP induction by regulation of postsynaptic membrane potential, inhibitory interneuronal circuitry, or presynaptic neurotransmitter release, as well as regulation of second messenger systems. These experiments will be done using an in vitro brain slice preparation of rat hippocampus (area CA1). Synaptic transmission and LTP will be monitored by electrophysiological measures (extracellular field potential and whole cell/patch clamp recordings of evoked synaptic responses). Electrophysiological and pharmacological manipulations will be used to test possible components of the signaling pathway for NMDA-R independent LTP."}
{"text": "An important limitation in animal models of cognitive learning has been the difficultly in specifying how animals code (or represent) events. Coding processes are particularly important in the development of stimulus classes. Recent evidence suggests that pigeons (and other animals) can form stimulus classes from originally unrelated stimuli. The characteristic of members of such stimulus classes is that they are able to substitute for one another in much the same way that arbitrarily-designated spoken and written words can be substituted for objects and actions in human language. The proposed research with pigeons will use delayed-matching procedures in which pigeons have been shown to develop single-code-default strategies (one of two samples is coded - the other comparison is chosen by default). Evidence for single-code-default strategies restricts the class of possible codes and allows one to gain access to the underlying codes. The immediate goals of the present research are (a) to examine the coding processes used by pigeons, (b) to determine the conditions under which these coding processes are used, and (c) to identify the nature of the underlying codes. In the first section of this proposal we will examine commonalities in the coding of present/absent samples. In the second section we will apply similar strategies to the analysis of duration sample coding. Finally, we will examine the similar coding of samples that are related only through their association with a common comparison. The long-term goal of this research is to be able to specify the bases of stimulus class formation by animals so one can determine which components might be missing from humans who fail to acquire the stimulus classes needed for language development."}
{"text": "This is a request for support of a Gordon Research Conference on Proteolytic Enzymes and Their Inhibitors. The objective of this conference will be to bring together thirty-two international authorities in this field and to have them discuss current work, new directions and controversial issues before a critical audience of 100 or more working experts from academic, industrial, and governmental institutions throughout the U.S. The subject is of great importance for widely diversified groups of scientists ranging from protein chemists and crystallographers to immunologists, hematologists, rheumatologists and biomedical investigators interested in inflammation, connective tissue disease, emphysema and cancer. Proteases and their endogenous inhibitors play a central role in the control of immune responses (complement pathway), blood clotting and fibrinolysis, and the generation of peptide mediators of inflammation and blood pressure regulation. Proteases are also key regulators of sperm penetration, ovulation and embryo-implantation and perhaps of cancer cell invasiveness and metastasis. They are also instrumental in the post-translational processing of peptide hormones, including neuropeptides, and of collagen and other connective tissue macromolecules. All of these aspects, from molecular structure and catalytic mechanism to physiological and pathological functions of proteases and their inhibitors, will be covered in this five-day conference (31 major speakers plus two poster sessions). The subject matter of the Conference is of considerable importance to human health; PHS support would be consonant with the mission of the DHEW."}
{"text": "Clostridium difficile infections (CDI) have become more difficult to treat due to the rise of hypervirulent strains that have increased morbidity as well as mortality and the likelihood of persistence and relapse in infected patients. To address the dire need for new anti-difficile agents, we explored the premise that nature utilizes good bacteria (i.e. probiotics), including Lactobacillus spp., to suppress gut pathogens such as C. difficile by producing novel antimicrobials, which were optimized by evolution to the microenvironment of the gut. In this regard, we explored the potential for developing reutericyclin from Lactobacillus reuteri as a natural anti- difficile agent. Probiotics are emerging as alternate treatments for CDI. However, there is ongoing debate on whether live probiotics may be used treat severe CDI, especially in immunocompromised patients. We therefore hypothesize that developing the antimicrobial produced by the probiotic specie would harness one of its natural therapeutic properties for localized killing of C. difficile in the gut, providing a more reliable and efficacious treatment strategy. Application of this concept to our studies on reutericyclin revealed that it has impressive antimicrobial and pharmacological properties for treating CDI. These include: rapid killing of nongrowing, toxin- producing stationary phase C. difficile; a novel mechanism of action specific to the bacterial membrane; a narrow spectrum of activity; lack of cytotoxicity against gut epithelia; stability to proteolysis; ability to achieve high non- absorbed concentrations in gut for killing; a low molecular weight and ease of synthesis that will allow advanced chemical optimization. Importantly, the killing of toxin-producing stationary phase cells is not shown by currently prescribed antibiotics vancomycin and metronidazole, which only kill actively growing C. difficile. We believe that this proposal for developing probiotic-derived reutericylcin derivatives to treat CDI is highly innovative and will be achieved through three iterative aims: (i) Synthesis of an expanded sets of reutericyclin analogs to optimize anti-difficile activity and increase affinity for the membrane target site ; (ii) Lead development and characterization involving the stepwise progression of compounds through three stages of tests that include antimicrobial assessment, pharmacokinetic testing, toxicologic and in vivo efficacy experiments. Compounds meeting the selection criteria of the tests will move onto the next stage such that a lead candidate is obtained with potent in vivo efficacy and excellent safety profile; (iii) Mode of action studies to explore the fundamental antibacterial effects of targeting the clostridial membrane. The long-term goal of this project is to develop optimized lead analogs of probiotic derived reutericyclin that exhibit novel modes of action at the membrane target and have characterized antibiotic properties that would allow their progression into advanced preclinical studies as candidates for treating CDI."}
{"text": "Transcription and regulation of gene expression are temporally mediated by modification of the C terminal domain (CTD) of subunit 1 of RNA polymerase II. The combinatorial phosphorylation pattern of the CTD, a 52 consensus repeat of a YSPTSPS heptad, coordinates RNA biogenesis in the transcription cycle. Progress in resolving the regulatory roles of the CTD has been impeded by lack of suitable methods for site-specific and quantitative mapping of CTD phosphorylation, an issue that directly ties structure to function. This proposal addresses this challenge by the development of ultraviolet photodissociation (UVPD) mass spectrometry to map the phosphorylation pattern and occupancy of the CTD in conjunction with label-free quantitation. The three proposed aims include: (1) Analysis of the combinatorial phosphorylation pattern of the CTD using 193 nm UVPD. The ~26 kDa CTD peptide of RNA pol II will be isolated, digested by Proteinase K, and the resulting peptides analyzed by nanoLC-UVPD-MS in the negative mode. Both the phosphorylation sites and occupancies will be characterized. (2) Quantitative analysis of global CTD phosphoryl occupancy and exposure to cell stressors. Changes in the CTD code as a function of particular cell stressors, including heat shock, exposure to salt, dithiothreitol, lipopolysaccharides (LPS), sorbitol or hydrogen peroxide, will be elucidated by label-free quantitation. (3) Quantitative analysis of global CTD phosphoryl occupancy between a control and a yeast strain with a functionally deficient Ssu72 phosphatase. Ssu72 displays specific phosphatase activity toward Ser2 and Ser5 of the CTD heptad repeat, two sites that play a critical role in the temporal regulation of transcription. RNA pol II from Ssu72-deficient and control yeast strains will be harvested, digested with trypsin, and processed to isolate the CTD peptides. The resulting phosphorylated peptides, which will differ in occupancy and abundance for each cell state, will be evaluated by label-free quantitation. This application of innovative UVPD technology will provide critical insight into the role of CTD phosphorylation in transcriptional processing."}
{"text": "Proprotein convertase subtilisin kexin type 9 (PCSK9) regulates plasma cholesterol levels by enhancing the intracellular degradation of the LDL receptor (LDLR), the primary driver of LDL clearance from plasma. Human PCSK9 is composed of 692 amino-acids. After removal of the signal peptide (residues 1-30), the secreted PCSK9 presents three domains: 1. The N-terminal pro-domain (PD, residues 31-152), which is cleaved but remains associated with the rest of the protein, and displays a highly disordered N-terminus (residues 31-60); 2. The catalytic domain (CA, residues 153-449), which contains a proteolytic site masked and neutralized by the associated PD; 3. The C-terminal domain (CD, residues 453-692), which consists of three tightly packed modules, CM1 (residues 457-527), CM2 (residues 534-601) and CM3 (residues 608-679). The consensus view is that PCSK9 binds to the epidermal growth factor type A (EGF-A) repeat of the LDLR via its CA, but does not cause direct proteolysis of the LDLR. Understanding the function of PSCK9 is central to the development of novel therapeutic agents to reduce cholesterol levels and cardiovascular risk. Although PCSK9 primarily targets the hepatic LDLR, it may also function on other lipoprotein receptors, such as apoER2, LRP1, and VLDLR, as well as on LDLR in extra-hepatic tissues. Our published work and preliminary studies have addressed the role of different PCSK9 domains in the secretion and function of the protein. We have found that: 1. Deletion of CM2 produces a protein that is secreted and functional; 2. Deletion of either CM1 or CM3 produces a protein that is neither secreted nor functional; 3. Deletion of the entire CD produces a protein that is secreted but not functional; 4. The PD of a PCSK9 fragment lacking CD facilitates the secretion of a PCSK9 fragment lacking PD; 5. The discrete CD of PCSK9 binds the ectodomain of the LDLR in solution and dose- dependently inhibits the LDLR-degrading effect of full-length PCSK9 cells; 6. PCSK9 undergoes pH-dependent self-association, which is mediated by the CA and increases the LDLR-reducing functionality; 7. Over- expression of PCSK9 reduces LDLR, apoER2, and LRP1 in HEK293T cells, and reduces LDLR levels in macrophages; 8. Transgenic expression of human PCSK9 in macrophages reduces liver LDLR and raises plasma cholesterol levels in mice. The central hypothesis of this proposal is that PCSK9 uses multiple domain interactions for secretion and function, and contributes to atherogenesis by targeting multiple lipoprotein receptors. We plan to: 1. Examine the role of the C-terminal domain in PCSK9 secretion and LDLR binding; 2. Test the hypothesis that domain interaction and self-association modulate PCSK9 secretion and function; 3. Test the hypothesis that PCSK9 secreted by macrophages modifies the biology of the atheroma through multiple effects on lipoprotein receptors. Investigating these novel roles of PCSK9's C-terminal domain and pro-domain may provide new leads in the development of inhibitors and advance our understanding of plasma cholesterol regulation."}
{"text": "Ross Products Division has developed a complete pediatric elemental formula (PEF) suitable for feeding to children on allergen free diets. The product is based on free amino acids and meets the recommended dietary allowances (RDA) for the 1 to 3 year old child in 10000 kcal and for the 4 to 6 year old child in 1500 kcal. The purpose of this study is to assess whether PEF will support adequate growth, tolerance and biochemical response in children requiring an elemental formula as their primary source of nutrition for 4 months while maintaining the appropriate allergen avodance diets. The primary indicators used to test the research hypothesis will be weight, height, intake, stool patterns, and blood biochemistries (serum albumin, ferritin, transthyretin, retinol binding protein [RBP], retinol and urea nitrogen, and plasma amino acids). Secondary variables will include allergic and gastrointestinal symptoms. Subjects will be children with documented AEG currently maintained on NeoCate or NeoCate 1 as their primary feeding."}
{"text": "Dengue virus is a major human pathogen that presents a daunting challenge to treatment design and implementation. No antivirals are currently available and, as for all positive-strand RNA viruses, it is anticipated that multi-drug therapy will be needed due to the high error rate ofthe viral polymerases which can lead to the rapid selection of drug-resistant viruses. However, we have developed a strategy to identify dominant drug targets' in RNA viruses and are testing this strategy with Dengue viruses. Specifically, when the target of a drug is an oligomeric protein or is involved in the generation of an oligomeric species, it is often true that drug-sensitive viruses are genetically dominant over the inevitable drug-resistant ones. Thus, the drug-resistant viruses are not selected from the cells in which they were generated. For Dengue virus, candidate proteins are the core protein C, the envelope protein E, the protease NS2/3, and certain inhibitors ofthe polymerase NSS. We have obtained inhibitors of E, NS2/3 and NS5 and selected and partially sequenced drug-resistant variants of each. For these and other inhibitors of Dengue virus, we will test in coinfections whether drug susceptibility or drug resistance is dominant. Then, we will test the frequency of the emergence of drug resistant viruses in murine infections. We anticipate that, for some drug targets and not others, drug resistance will be dramatically suppressed, paving the way for the use of fewer compounds to treat Dengue virus infection, and possibly even monotherapy. The development of logical strategies to counteract drug resistance is crucial to combatting many pathogens, with Dengue virus as a currently pressing example."}
{"text": "Recent advances in biotechnology and molecular biology have brought about new concerns in protein purification. The proposed protein purification knowledge base addresses some of these issues by providing new computer- readable protein purification databases coupled with an expert system to suggest protocols for purifying new proteins. Experimental data will be scanned, digitized and analyzed for relevant information that the expert system can use to update the working purification strategy. This knowledge base system can decrease the costs of developing new biotechnology products by optimizing the development of large-scale protein purification processes and by using the expertise of key protein purification personnel more efficiently. In addition, a subset of the knowledge base can be used to train students in protein purification techniques. The purification databases will also be distributed independently of the expert system and will be updated on a regular basis."}
{"text": "Many studies suggest that overexpression of DNA repair factors could contribute to resistance to radiochemotherapy. Therefore, studying this pathway has important implications in cancer pathogenesis and cancer therapy. We found that UCHL3, a deubiquitinase, is overexpressed in breast cancer. In addition, increased expression of UCHL3 correlates with poor prognosis for breast cancer patients. However, whether or not UCHL3 casually contributes to clinical phenotypes is unclear, because the cellular function of UCHL3 remains unclear. Here we show for the first time that UCHL3 is involved in DNA repair. We found that UCHL3 interacts Rad51 and deubiquitinates Rad51 without affecting Rad51 levels. UCHL3 itself is phosphorylated following DNA damage and is recruited to double strand breaks. In addition, UCHL3 is important for the recruitment of Rad51 to double strand breaks and homologous recombination. Downregulating UCHL3 sensitizes breast cancer cells to radiation and PARP inhibitors, while overexpressing UCHL3 renders breast cancer cells resistant to these treatments. Based on these Preliminary Data, we hypothesize that UCHL3 is a new factor in DNA repair. UCHL3 deubiquitinates Rad51 and promotes Rad51 function and homologous recombination. Increased UCHL3 contributes to resistance to radiation and chemotherapy by enhancing DNA repair. In this application, we will further explore how UCHL3 regulates Rad51 and how UCHL3 itself is regulated. We will also test the role of UCHL3 in radiochemoresistance using clinically relevant models. Our Specific Aims are: 1. Investigate the regulation of Rad51 by UCHL3; 2. Study the regulation of UCHL3; 3. Investigate the role of UCHL3 in breast cancer therapy. Our studies will reveal a novel function of UCHL3 in DNA repair and response to radiation and chemotherapy. In addition, it will reveal a new therapeutic target in sensitizing breast cancer cells, especially those overexpressing UCHL3, by targeting the UCHL3-Rad51 pathway."}
{"text": "It has recently been established that certain benzodiazepine (BZ) receptors are closely associated with receptors for gamma-aminobutyric acid (GABA) in (neuronal) membranes. The physiological significance of this close association is at present entirely unknown, but may be the first example of a more general phenomenon, and could be related to the co-release of two neurotransmitters from the same neuron. I propose to investigate this close association using two general methods: 1) the ability of type-1 GABA mimetics (e.g., GABA, muscimol) to protect associated BZ receptors from heat inactivation, and 2) the ability of type-2 GABA mimetics (e.g., THIP, isoguvacine, piperidine-4-sulphonic acid, 3-amino-propane sulfonic acid) to prevent binding of 3H-BZ ligands to GABA-associated BZ receptors. Type-1 and type-2 GABA mimetics are mutually antagonistic in a competitive way. Preliminary results indicate that some but not all BZ receptors are associated with GABA receptors, and that these GABA receptors are of two types. First we will do time courses of heat inactivation of BZ receptors in the presence of GABA mimetics of both types. We will then do dose-response experiments with all active GABA mimetics using a single-time (usually 30 min.) at 60 degrees C to determine whether one or more GABA receptors are involved. The ability of type-2 mimetics to antagonize the stabilizing effects of type-1 mimetics will be determined. The inhibitory effects of THIP and isoguvacine as a function of heat inactivation in the presence of GABA will be investigated. Several new potential GABA mimetics will be synthesized and tested in the above systems."}
{"text": "TORSIN A AND THE PATHOPHYSIOLOGY OF DYSTONIA Mutations of the DYT1 gene cause early onset torsion dystonia. This gene encodes torsinA, a member of a novel family of proteins with homology to the HSP 100/CLP family of heat shock proteins. In preliminary studies, we have found that the gene for torsin A is active in nerve cells in several regions of normal adult human brain, including the dopamine neurons of the substantia nigra pars compacta. We have also found that the gene is always active in the brains of patients who are ill for more than a day but not in the brains of those who die suddenly. This finding is consistent with, but does not prove, that torsinA is a stress response protein. In this application we seek funding to obtain detailed information about the localization and regulation of the torsinA mRNA and protein in both human brain and genetically engineered rodent models. We will determine exactly which cells in human and mouse brain contain the mRNA and protein, where in the cells the protein is located, whether mRNA expression is regulated by stress and whether the mutation effects the location or amount of mRNA expression, the subcellular location of the protein or the function of dopamine neurons. These data are essential to the construction of mechanistic models of the neural dysfunction which produces dystonia."}
{"text": "PROJECT SUMMARY Chemo-radiation therapy is a standard treatment regimen for locally advanced head and neck squamous cell carcinoma (HNSCC). The treatment regimen, however, is difficult for patients as they experience high rates of grade 3 or higher toxicities including leukopenia (42%) and the need for feeding tube (52%). Recent studies showed that a subgroup of HNSCC patients with human-papilloma virus (HPV)-positive oropharyngeal (OP) SCC have significantly better prognosis. These clinical data lead to important considerations to de-intensify treatment for this low-risk, younger population in order to reduce acute and chronic toxicity without compromising disease control. It has been suggested that the adaptive de-escalation of treatment can be tailored for individual patients based on the early tumor volume change. However, volumetric assessment is often inadequate because the treatment response of a tumor can be heterogeneous in terms of (i) cell viability, (ii) cellular metabolism, and (iii) perfusion that are relevant to the success of any chemoradiation therapy. These complex changes may not be adequately represented by tumor volume change at the early stage. The proposed study is based on a combination of the quantitative diffusion MRI (dMRI) methods with their own technical innovations that can also be applied to other clinical studies. dMRI is a unique in vivo imaging technique sensitive to cellular microstructures at the scale of water diffusion length on the order of a few microns. However, quantitative dMRI remains challenging as dMRI data represent different biophysical properties of tissue depending on diffusion weighting strength (q) and diffusion time (t) used for the measurement. The scientific premise of the proposal is that this study will establish a quantitative way to utilize both q- and t-dependent dMRI data as a tailored approach to quantify cell viability, cellular metabolism and perfusion from this non-contrast MRI method. We demonstrated that both diffusion coefficient D and diffusional kurtosis coefficient K are promising imaging markers for cell viability. Cellular metabolism can be evaluated in terms of the water exchange ?ex, measured by the diffusion time-dependent K, that is regulated by the ATP- dependent trans-membrane ion channels co-transporting water molecules. Intravoxel incoherent motion MRI metrics (pseudo diffusivity, Dp; perfusion fraction, fp) can provide information about perfusion flow. Ultimately, these dMRI measures will better identify patients who have the potential to benefit from adaptive de-escalation or escalation of therapy. In this proposal, we will further optimize and establish a set of quantitative non-contrast imaging markers of cell viability (D and K), cellular metabolism (?ex), and perfusion (fp?Dp) as a clinical tool for assessment of treatment response and validate it in a clinical trial. The data acquisition and analysis software tools to be developed in this study will enable comprehensive and quantitative assessment of cancer treatment response to tailor chemoradiation therapies for individual patients."}
{"text": "The Genomics Core will facilitate transcriptional profiling of cells and vascular tissues obtained in the studies of the individual projects of the SCOR using high-throughput technologies. Services provided include: 1) nucleic acid (RNA or cDNA) hybridization to commercial arrays purchased by the individual projects and 2) array printing and analyses of subsets of genes pertinent to the specific focus of each project to provide maximum flexibility in customization/high throughput of cell and tissue samples. Core services will include optimization studies, array imaging, and analytical assistance. Procedures for assuring equitable sharing of the instruments and coordinated scheduling will be developed. Activities will be facilitated by a GeneMachines OmniGrid Sample Microarraying Robot, and a Packard Biochip Scanarray 4000 Scanner, together with a dedicated computer. Additional components of the system, underwritten by other resources and at no cost to this grant are a Biomek 2000 Laboratory Automated Workstation for multipurpose fluid handling, and array software licenses. An in-house microarray printer and scanner are central to the Core's function and will permit the SCOR investigators great flexibility in high throughput genomics capability. The arrayer will provide customized printing of selected cDNAs or oligomers prepared by the investigators' own labs (or outsourced synthesis) to facilitate detailed analysis of gene expression relationships based on specific project-focused questions. The instruments are also central to the use of commercial microarrays for whole genome screening. The Associate Director and Director have 3 years experience with microarray analyses. The Genomics Core will coordinate activities with the Proteomics Core (Core B) to seek concordant and discriminant patterns of mRNA vs protein expression in response to pro- and antioxidant interventions by providing genomic profiling complementary to proteomic analyses. This is an important and necessary approach for investigations of frequently divergent gene and protein expressions in the same cells/tissues. A similar pattern of paired analysis will be applied to studies of circulating cells, initially polymorphonuclear leucocytes, as potential markers of gene and protein expression in the vasculature."}
{"text": "Disorganization (Ds), a single gene mouse mutation, has profound effects on mouse development. Unlike most mutations where particular traits with diagnostic features are inherited among generations, the heritable trait in mice with the Ds mutation is the propensity for the body to be formed in an unpredictable manner. No two mice are affected in identical ways. Affected mice may show agenesis, duplication or fusion of structures, and all morphological structures seem prone to anomaly. Remarkably, these mice are not prone to cancer, spotting, behavior, and longevity or fertility problems. Ds is one of the few examples in mammals of a true dominant, gain-of-function mutation. Determining the identity of the Ds gene is important because deep insights into the regulation of pattern formation during development can be gained. In addition, insights into the molecular nature of mutations that show variable expression and low penetrance may be forthcoming. Ds has been located to a 0.2 cM segment of mouse Chr 14, cloned in a 360 kb BAC contig and 80 kb of sequence has been obtained. Cloning of the mouse Ds gene is proposed. The genomic sequence of the Ds locus will be determined in wild-type mice in order to identify genes and markers, evidence for rearrangement in Ds will be sought, cDNA expression and sequence will be assessed in wild type and Ds mice, and the genomic sequence of the Ds locus will be determined. In addition, it is proposed that the identity of Ds will be verified by generation and characterization of transgenic mice with the candidate Ds mutation."}
{"text": "This 3-generation study of families at high and low risk for Major Depressive Disorder (MDD) has documented the strong familial transmission of mood disorders across generations. We have conducted 5 waves of assessments in this cohort over 25 years. In the 5th wave, we added Magnetic Resonance Imaging (MRI) measures and found a remarkably robust association of familial risk for MDD with asymmetries in cortical thickness, with a nearly 30% reduction in thickness observed in the lateral parietal, temporal, and frontal cortices of the right hemisphere of the high risk group. These MRI findings are consistent with EEG findings from the 4th wave of assessments that demonstrated reduced activity over the posterior cortices of the right cerebral hemisphere. Both the MRI and EEG findings were present in high-risk individuals who never had MDD in their lifetimes, suggesting that these abnormalities were not simply a consequence of previously having been depressed or having been treated for depression. Thinning of the cortical mantle and reduced electrophysiological activity in the right hemisphere therefore may constitute related endophenotypes for familial vulnerability to developing MDD. We are proposing a 6th wave of study to gain a deeper understanding of the right hemisphere abnormalities in familial MDD in the 216 individuals imaged thus far. This represents the largest MRI study published for MDD to date, and it is the only sample studying 3 generations of individuals at high or low risk for MDD. We are proposing to collect additional MRI and EEG measures, as well as clinical and cognitive neuroscience data, that will inform us about the neural bases of the right hemisphere thinning and their consequences for brain function and emotional processing. We will also determine whether additional cortical thinning in the left cerebral hemisphere predicts new or recurrent MDD in those people who were imaged in Wave 5. Our cohort has exceptional attributes that will aid our search for detecting MDD endophenotypes, including (1) an elevated clinical risk for developing MDD in the high risk group that has been amply demonstrated; (2) multiple prospective assessments, conducted blind to risk status, provide great confidence in the accuracy of the clinical diagnoses; and (3) the sample contains individuals who have not yet become ill but who are nevertheless at elevated risk of becoming ill, thereby allowing us to disentangle the neurobiological determinants of vulnerability from the compensatory responses that would be present in already-affected individuals. MDD is an early-onset, highly prevalent disorder with significant morbidity, and it afflicts people in their most productive years. Identifying the biological vulnerability for depression before the onset of illness has important implications for prevention and public health."}
{"text": "In the past year, effort in nuclear magnetic resonance imaging has been expanded on several research fronts. A. Following the failure of the magnet manufacturer to install successfully the clinical NMR imaging system in the Department of Radiology, Clinical Center, the research group took over the homogenizing of the magnetic field necessary for clinical imaging, and, using a new technique, achieved the specified homogeneity, thereby enabling the Radiology Department to produce high quality images. B. Considerable controversy exists within the NMR image community over the choice of an optimal field strength for imaging. Bench methods were developed for accurately assessing the signal-to-noise ratio from, and radio-frequency power deposition in, the human body at any frequency used for imaging. It is hoped these results will help resolve the matter. C. A pulse for highly selective spin population inversion has been discovered. The result is of considerable experimental and theoretical importance for it represents only the second known analytical solution of the non-linear differential equations governing the motion of an NMR spin system. Further, above a critical threshold, the inversion is independent of applied power. D. A so-called \"quadrature\" probe system has been invented which reduced radio-frequency power dissipation in the body by almost a factor of 2 while improving signal-to-noise ratio by almost 40%. E. The electronic upper frequency limit for adult head imaging has been pushed from 84 MHz to 130 MHz with the aid of a novel phased-array receiving coil."}
{"text": "The Operations Core (OC) supports OAIC investigations of multifactorial geriatric conditions by recruiting and retaining diverse populations of older persons, seeking input from the local community in research planning and dissemination, monitoring participant safety, ensuring regulatory compliance, developing surveys and instruments, designing Information Technology (IT) systems to implement research, collecting and preparing data for statistical analysis, and providing continuity and shared knowledge across projects. The overall goal of the OC (RC1) is to ensure the successful implementation of research focused on multifactorial geriatric conditions. This goal will be accomplished by leading, managing, and coordinating the effective, efficient and innovative use of facilities, data, staff, resources, and space. There is a consistent demand for experienced personnel with the ability to quickly execute aging-focused research and an increasing need for informatics skills and technology to streamline work. The Specific Aims of the OC are: 1) to provide an infrastructure suitable to support Yale OAIC-funded projects and External Projects. This infrastructure includes experienced personnel, staff training modules, standard operating procedures for conducting field studies, and comprehensive clinical research informatics (CRI) services; 2) to collaborate with the REC, PESC, and Biostatistics Core (RC2) to develop specific CRI and field operations systems for Yale OAIC-funded projects and External Projects; and 3) to conduct innovative OC Development Projects that advance the mission of the Yale OAIC. During the current funding cycle (2013-present), the Operations Core was integrally involved in research support of 80 OAIC-funded projects and External Projects, numerous collaborations within Yale, and engagement with several other Pepper Centers. Altogether, these efforts have resulted in 114 peer-reviewed publications and 81 extramurally funded grants (>$145 million total costs) of which 25 involved 18 junior faculty specializing in aging/geriatrics research. During the next funding cycle, the OC will: (1) collaborate on 4 of the 5 proposed REC and PESC projects, 24 of the 29 funded External Projects, and 7 pending projects from grant submissions involving more than $13 million in total costs; (2) substantially expand and enhance the clinical research informatics infrastructure at Yale School of Medicine, in partnership with leaders from each clinical research support center; and (3) lead an innovative Development Project, the Clinical Decision Support System (CDSS) Toolkit, that will make it possible for any research team having access to REDCap to implement a sophisticated, HIPAA-secure CDSS. The CDSS toolkit will support the growth of projects related to the OAIC subtheme of individualized decision making for older persons with multiple chronic conditions."}
{"text": "Muscles must be able to relax as well as contract. Contraction and relaxation are regulated by molecular switches on the thick (myosin-containing) and thin (actin-containing) filaments, which together make up the contractile machinery. Defects in regulation can lead to muscle disease. Our long term goal is to understand the structural basis of muscle regulation; our focus in this application is the relaxed state. During the current grant period we achieved two key breakthroughs in defining the molecular structure and regulatory mechanism of myosin filaments, resolving basic questions dating back more than 40 years. We also gained new insights into troponin-tropomyosin regulation of actin filaments. Our insights raise key new mechanistic questions, which we propose to investigate in this renewal. To understand how muscles relax, the structures of the actin and myosin filaments in the relaxed state must be determined. To achieve this, state-of-the-art electron microscopic and 3D image reconstruction techniques will be applied to native filaments and their isolated component molecules. Using these approaches: (1) The molecular interactions underlying the relaxed state of striated and smooth muscle myosin filaments will be defined in detail. (2) The specific amino acid residues and protein domains essential to these relaxed-state interactions will be determined by analyzing the effects of targeted mutations on the structure of expressed single myosin molecules. (3) The structural basis of thin filament relaxation will be defined by analyzing the native, relaxed-state organization of the regulatory components troponin and tropomyosin, and the thin filament template protein, nebulin. In each study, reconstructions will be fitted to atomic resolution crystal structures of thick and thin filament subunits, defining molecular contacts at near-atomic resolution. Our studies of myosin molecules and filaments will provide new insights into general mechanisms of thick filament regulation, and especially into the nature of the relaxed state in vertebrate striated muscle. Further advances in our studies of troponin-tropomyosin regulated thin filaments will provide new information on native thin filament structure, the 3D organization of troponin and tropomyosin, and the structural contribution of nebulin to the relaxed state. Preliminary data demonstrate the feasibility of our aims. The new insights arising from these proposals will provide a deeper understanding of the structural basis of muscle relaxation. Defining molecular mechanisms in healthy muscle is essential to understanding structural defects that underlie muscle disorders."}
{"text": "The broad goal of the proposed research is to further understand the role of intrinsic brain networks in cognition. Intrinsic networks (INs) are collections of disparate brain regions that are consistently identified in task-free functional magnetic resonance imaging (fMRI), and are presumed to underlie sensory, motor, and cognitive functions. Further understanding requires detailed knowledge about the dynamics of the networks, both in task-free \"resting state\" as well as during cognitive function. What are the interactions between Ins and the traditional \"task-active\" regions? Is there an integration of information along the networks that may [unreadable] subserve task responses? The current proposal focuses on using fMRI to elucidate dynamic properties of a particular IN known as the default-mode network (DMN). [unreadable] In Specific Aim 1, we model directions of task-evoked information flow among the DMN and task activated regions. The chronometry of initial responses to a brief, attentionally demanding task will be obtained using onset latency analysis, and the evolution of temporal dependences in the (long) poststimulus interval will be quantified using time-varying Granger causality. Specific Aims 2 and 3 pertain to methodological issues in studying INs using fMRI. In Aim 2, we quantify the stationarity of coupling strength and phase differences among regions of the DMN in task-free resting state. Currently, the majority of studies define INs using algorithms that assume that the coupling of IN regions remains constant over time. We employ time-varying functional connectivity and ICA over a long resting-state scan to examine the degree of change overtime, and relate changes in connectivity strength to physiological variables that may signify changes in arousal or awareness level. In Aim 3, we correct for regional differences in hemodynamic latency due to vascular reactivity, so that the relative timing of BOLD signals reflects underlying neural interactions more closely. Our approach is to measure vascular response latencies across the brain using a breath holding task, and develop methods for correcting for these non-neural latency differences. [unreadable] Investigating the temporal behavior of the DMN will provide valuable insight into how the network underlies cognitive dysfunction, as well as function. While the DMN is receiving widespread attention for its purported role in episodic memory and task performance, it is also proving effective as a biomarker for disorders such as Alzheimer's Disease, major depression, ADHD, and schizophrenia. The current proposal provides a framework for studying the temporal behavior of the DMN (and other INs), which will serve as a basis for further investigation of network responses and connectivity in patient populations. [unreadable] [unreadable] [unreadable]"}
{"text": "RNA editing via the ADAR enzymes (adenosine deaminases that act on RNA) has emerged as a novel and significant post- transcriptional mechanism for altering protein products encoded by a single genetic locus. ADAR activity has been detected across phyletically distant organisms and within a variety of tissues within an organism. Paradoxically, the number of mRNA targets of ADARs remains small and limited primarily to ligand- gated ion channels of the mammalian nervous system. A limited number of well characterized mRNA substrates for ADARs have been characterized and a general requirement for a dsRNA intermediate in pre-mRNA has been proven. Still, central questions with no consistent answers remain such as; What determines ADAR specificity? What comprises an RNA editing site and can we predict their existence rather than relying on serendipity? What are the phenotypic consequences for the organism of editing of a particular RNA editing site? What is the global role of RNA editing in gene regulation? In this proposal, these major areas are addressed using Drosophila as a model genetic system. First, known RNA editing sites in several Drosophila voltage- and ligand-gated ion channels as well as one in an enzyme will be utilized to elucidate the cis-determinants of pre-mRNA editing sites via in vivo transgenic and in vitro methods. Second, a genetic and molecular approach will determine the effects of knocking out or reducing RNA editing of a specific RNA editing site in a ion-channel gene known to have effects on nervous system function and behavior. Lastly, a recently isolated knock- out mutation of the Drosophila ADAR, dADAR, will be characterized at the molecular, genetic and behavioral level to assess the global significance of specific RNA editing."}
{"text": "The long-term objective of this proposal is to understand in molecular terms the mechanism by which Agrobacterium tumefaciens interacts with its host resulting in Crown Gall tumor formation in a wide variety of plants. This proposal follows the same general approach that we have used in previous years to gain insight into these mechanisms, namely, to analyze mutants altered in tumor formation. However, the experiments described are much more focused because we now know the sequence of the Agrobacterium genome and we have access to microarray chip technology. With this new information and technology we can carry out directed mutant studies and types of analyses that were not possible previously. The information revealed by the sequence of the genome has raised questions but also the means to answer these questions which we could only speculate on previously. Thus, we now know that far more genes are activated in Agrobacterium by interaction with host plants than was recognized previously. Using microarray chip technology we will identify and characterize global regulatory circuits that are directly activated or suppressed by plant signal molecules. Using microarray technology, we will expand on previous observations by determining the alterations in the global expression of genes in Agrobacterium grown with plant cells at a temperature optimum for the growth of Agrobacterium but inhibitory for the transfer of T-DNA (28[unreadable]). We will also continue our studies on the mechanism by which T-DNA is transferred from Agrobacterium into host cells, focusing on the role of VirJ in this process. Finally, the genome sequence of Agrobacterium has revealed many genes that serve pathogenicity functions in related bacteria. We will mutate these genes to determine if they are also required for pathogenicity of Agrobacterium. The studies proposed in this application should open up new vistas and provide an increased understanding of the many levels of interaction of Agrobacterium with its variety of eukaryotic hosts."}
{"text": "The impact of recent legislative changes focused on improving our knowledge of drugs used in the treatment !of children is slowly being realized, but current studies have not investigated the differences in drug disposition afforded by disease state. This is most notable for critically ill children, in who changes in organ function, total body water, tissue and plasma proteins, can all affect the disposition and action of therapeutic drugs. The long-term research goal of this proposal is to optimize the safety and efficacy of drugs used in the treatment of critically ill children by bridging our gap in knowledge of the clinical pharmacology of drugs frequently used in these patients. We will use sophisticated pharmacokinetic-pharmacodynamic (PK-PD) modeling techniques and clinical trial simulation to streamline the development and performance of clinical studies. Our central hypothesis is that informative clinical trials can be designed more efficiently when well characterized models that account for sources of variation in pharmacokinetics and pharmacodynamics are utilized. We plan to initially construct select models based on published clinical pharmacologic data, and then perform clinical trials simulations for the design of prospective studies of drugs frequently used in the care of critically ill children. Based on the results of our models and trial simulations, we will then carry forward and perform clinical studies of two frequently used drugs (pentobarbital and dopamine) in critically ill children. lastly, we will integrate results from these clinical studies back into our models to allow for pharmacologically more rational dosing in this difficult to study pediatric population. For this mentored training, two complementary approaches will be pursued. The first will be formal coursework covering a range of topics iintegral to the field of clinical pharmacology. The second approach will be through a mentored clinical and laboratory research program. This intensive didactic and mentored training will allow for the development of a future independent pediatric clinical investigator."}
{"text": "Actinobacillus actinomycetemcomitans(Aa), a major pathogen in certain human periodontal diseases, elaborates a potent leukotoxin (Ltx). This polypeptide kills human neutrophils, monocytes and T lymphocytes and may impair host antibacterial resistance by destroying or perturbing these cells in infected tissues. Results of current studies indicate that the toxin disrupts the permeability barrier of the plasma membrane of susceptible cells. This results in a 2+rapid rise in Ca (and probably other ions) levels within the cell which ultimately leads to cell death. This proposal will determine the molecular mechanism(s) for this observation. Two separate lines of experimentation form the basis for this proposal: l) Ltx-treated lymphocytes and NK cells exhibit many of the morphological characteristics of apoptosis; and 2) the Ltx receptor is a beta2 integrin, a molecule which when stimulated is capable of elevating cytosolic 2+Ca levels. In Specific Aim #1, Ltx-treated cells will be evaluated by DNA degradation, macromolecular synthesis, and FACS-associated morphologic alterations to determine if programmed cell death is occurring. Specific Aim 2 will focus on the expression of genes known to be involved in programmed cell death such as Bc1-2, Bc1-x, ICE, Bax, CPP32 and p53 to determine a molecular mechanism for cell death. Specific Aim 3 proposes experiments to define the nature of the toxin/receptor interaction. We envision that the first contact that the Ltx contact on a cell is a (32 integrin cell surface molecule. It is our goal to define these earliest consequences of this interaction such as determine the signal 2+pathway that is activated and the relationship to elevated cytosolic Ca +2 levels. Finally Specific Aim 4 will identify genes which are unique to Ltx- mediated apoptosis. It now emerging that the process of apoptosis involves different pathways when different agents are used to initiate the process. From these experiments we will have a better understanding of the pathway that is utilized by the toxin to kill cells. These studies provide fundamental insights into the pathobiology of the Aa Ltx and will bring us closer to understanding of its role in human periodontal infections on a molecular level. In addition, detailed studies on the action of the Aa Ltx will contribute to a better understanding of the biology of other membrane-active bacterial cytolynsins."}
{"text": "We are trying to understand how mRNA polyadenylation occurs in male germ cells in the absence of the canonical AAUAAA polyadenylation signal. We discovered and characterized a variant form of the CstF-64 polyadenylation protein that is expressed at high levels in meiotic and postmeiotic germ cells, and in no other tissue except the brain. This new protein, called \"(CstF-64,\" is the leading candidate to account for AAUAAA-independent polyadenylation in germ cells. Our overall laboratory hypothesis is that (1) the somatic CstF-64 gene (Cstf2) is on the X chromosome in mice and humans, (2) the X-chromosomal CstF-64 is inactivated during male meiosis, (3) since CstF-64 function is essential, an auxiliary CstF-64 gene must be expressed from an autosome, and (4) the auxiliary CstF-64 protein is responsible for AAUAAA-independent polyadenylation of mRNAs in male germ cells and is essential for spermatogenesis. In the previous grant period, we addressed items 1-3 of our hypothesis. The goals of this grant proposal are to address item 4. This Independent Scientist Award (K02) will assist me in accomplishing those goals. Specifically, I am seeking to receive salary support to release me from some or all of my teaching and administrative duties so that I can devote a greater portion of my time to research and career development. For the research portion, I will be able to spend more time in the laboratory myself, and to direct the research of my workers more effectively. For the career development portion, I will be able to attend national and international meetings, and spend short periods visiting laboratories of collaborators to learn new techniques relevant to our work. These activities will be especially helpful, because my background is in biochemistry and not in reproductive biology. The Department of Cell Biology & Biochemistry is an excellent place from which to achieve these goals: the major strength of the Department is in many aspects of reproductive biology, and my colleagues have been exceedingly generous in sharing their ideas and expertise. This environment, supplemented with trips to meetings and visits to collaborators, will provide excellent support for my endeavors."}
{"text": "More than 20 million people in the US have chronic kidney disease, and the cost of end stage renal disease management is over $40 billion annually in public and private funding. For renal failure patients on hemodialysis, broad consensus is that native arteriovenous fistulas (AVF) are the preferred access type, with better survival, fewer complications, longer patency, and reduced costs relative to other options. However, because up to 60% of attempted AVF never mature to the point of usability for dialysis, early AVF failure is a key barrier to maximizing health outcomes in these patients. The high AVF failure rate contributes to initiation of catheter-based dialysis in a large percentage of patients, with increased risks of infection and premature death. Maturation failure is thought to be related to peri-anastomotic stenoses resulting from intimal hyperplasia, inflammation, and negative wall remodeling. These factors occur in the presence of metabolically active perivascular adipose tissue which we theorize impacts overall remodeling. Chronic systemic inflammation is linked to the uremic milieu of patients with advanced kidney disease, and emerging evidence directly links elevation of pro-inflammatory mediators to adipose tissue. Furthermore, our pilot studies on AVF patients suggest clear links between base adipose phenotype and eventual fistula maturation. In addition, a genetic contributor to the process of atherogenesis may also lead to high rates of AVF failure. Polymorphisms associated with the ApoL1 gene on chromosome 22 have been shown to cause kidney failure in African Americans. The gene codes for a protein whose function incompletely understood, but is a component of HDL in circulation and is expressed in higher levels in patients with known comorbid predictors of atherogenesis. This indicates that the protein itself may be either associated with or a marker for development of atherogenesis and therefore may also contribute to increased rates of AVF failure. Thus for the current project we hypothesize that peri-anastomotic AVF adipose inflammation and APOL1 SNP status/gene expression patterns drive early AVF negative wall remodeling, contributing to failure to mature. These hypotheses will be tested via the following Specific Aims: 1) Identify adipose tissue expression profiles and clinical determinants of AVF failure. 2) Determine relationships between circulating and tissue levels of adipocyte-related mediators. 3) Understand the impact of ApoL1 gene status and protein expression on AVF remodeling and maturation. Completion of these aims will provide insights into the mechanisms of fistula maturation which may ultimately translate into novel clinical and therapeutic approaches in this extremely ill patient population. Furthermore, this work will be executed in a setting where I will gain enormous personal training experience by combining didactic coursework with clinical and basic research in a highly structured, actively mentored environment."}
{"text": "Two population genetic features of the human histocompatibility (HLA) system make it ideally suited for the study of evolutionary forces acting on a population. These are the high level of polymorphism exhibited by many of the loci of the HLA system, and the existence of significant linkage disequilibrium (non-random association) between certain pairs of antigens at different loci. The distribution of allele frequencies at the HLA loci, and the patterns of linkage disequilibria, are consistent with selective events acting in the region. This proposal involves: 1) population data analysis of a number of very large data sets, including the Caucasian and Japanese data from the 8th (1980) and 9th (1984) International Histocompatibility Workshops, Japanese data from the All Japanese HLA Workshops of 1983 and 1985, Chinese data from Singapore, Indian data from New Delhi, extension of our previous analysis of a large Danish study to include additional HLA loci, and the data that will be available from the Third Asia Oceania Histocompatibility Workshop (Sapporo, Japan, 1986); 2) simulation of two and three locus neutrality models, including migration, bottleneck and founder effects; 3) statistical analysis of the distributional properties of population wide measures of disequilibrium and application of bootstrap and jackknife techniques to determine confidence limits of the measures; 4) deterministic analysis of selection models; 5) comparison of generated population genetic parameters under neutrality and selection models with the HLA data; 6) correlation of HLA antigen disease association patterns with linkage disequilibrium patterns, including comparison of patterns in different racial groups, to study the evolution of disease predisposing genes. The nature of selective events acting in the HLA region will be delineated, and the mechanism by which disease predisposing genes become common in a population investigated. The methods to be developed have applicability to other multigene families, marker trait associations and restriction fragment length polymorphism data, in all organisms."}
{"text": "This research uses mathematical and computational techniques to determine the functional properties of local-circuit neurons, based on the well-founded results of cable theory, compartmental analysis, and biophysis of ionic fluxes across nerve-cell membranes. Five specific problem areas are addressed: (1) interaction of chemical and electrical synapses in neuronal circuits, (2) integrative properties of nonspiking neurons, (3) dynamics of reciprocal synapses, (4) functional roles of dendritic spines, and (5) properties of neurons exhibiting dendritic spikes. The aim of the studies is to provide realistic, quantitative, and consistent data for the behavior of these systems, so that both experimental and theoretical investigations can be rigorously based on solid theory, rather than on qualitative estimates."}
{"text": "The Epidemiology Branch is conducting a number of birth defect studies in collaboration with the Health Research Board and Trinity College, Dublin, Ireland. The main objective of these studies is to determine the relationship between folate and birth defects. The birth defects studied to date are neural tube defects (NTDs), oral clefts, congenital heart defects, Down syndrome and omphalocele. These studies focus on biochemical factors in the area of folate metabolism, and on genetic mutations in folate related genes associated with birth defects. In the past we have shown that elevated homocysteine is a risk factor for NTDs, that a mutation in the methylenetetrahydrofolate reductase (MTHFR) gene 677C->T is a risk factor for NTDs, and that a small dose of folic acid (100-200 micrograms) can raise red cell folate to levels that can prevent a fifth to almost a half of NTDs. We have shown that methylenetetrahydrofolate reductase (MTHFD), an important gene in the production of purine and pyrimidine for DNA synthesis is a risk factor for NTDs. Mothers who have the R653Q variant of this gene are at increased risk of having a child with an NTD. [unreadable] [unreadable] We have expanded our work on MTHFD, confirming in a second population that the MTHFD R653Q variant is a maternal risk factor for NTDs. We have examined other polymorphisms and variants related to folate metabolism as potential NTD risks. The reduced folate carrier is essential for transfer of folate into cells. A variant of the enzyme gene, H27R, has been reported to be a risk factor for NTDs, but this is uncertain. We stuied this variant in a large Irish population and showed that it was unrelated to NTDs in cases, their mothers and their fathers. [unreadable] [unreadable] Both the MTHFR C677T and A1298C variants have been reported to reduce the risk for some cancers. The biochemical effect of the MTHFR C677T variant is clear--it reduces folate stores; however, the biochemical effect of the MTHFR A1298C variant is unclear, perhaps because it is in linkage dysequilibrium with the MTFHR 677C wild type SNP. We were able to measure the effect of MTHFR A1298C independently of the MTHFR 677 effect, showing the the A1298C variant produces increased cellular (red cell) folate levels. This is a rare example of SNPs on the same exon producing opposite effects and it suggests that MTHFR 1298C influences cancer risk by a different mechanism than MTHFR 677T. We continue to investigate the role of other genes as NTD risk factors.[unreadable] [unreadable] We recently explored the role of another critical folate enzyme gene, dihydrofolate reductase as a risk factor for NTDs. Previous work did not show a protective effect of a 19bp intron deletion in mothers of NTD cases. Our study demonstrated that in the Irish population either one or two copies of the allele was significantly protective (p=.01) in mothers. There was a small increase in expression, a 1.5 fold increase in mRNA, as well. The difference did not reach statistical significance. Thus, the presence of the 19bp intron deletion may be protective against NTDs when present in mothers."}
{"text": "Amphotericin B plays a critical role in the clinical treatment of life threatening systemic fungal infections. It is often considered the gold standard drug of choice in these instances due to its broad spectrum efficacy and the lack of alternatives. However, amphotericin B is also well known for its severe and potentially lethal side effects, including severe nephrotoxicity. Doctors are often left with the difficult choice of balancing the risk of infection with the risk of treatment. A number of strategies have been employed to decrease nephrotoxicity of amphotericin B including, but not limited to, generating glycosylated derivatives at a fixed position to improve solubility, purifying the analogs more stringently, and creating liposomal formulations. These efforts have met with limited success, decreasing toxicity to allow for longer treatment regimens, but also showing considerable different pharmokinetic characteristics compared to standard amphotericin B. Strikingly, medicinal chemistry has not had much success altering the characteristics of amphtotericin B. This is largely true because there are few positions in polyene natural products that are accessible to traditional medicinal chemistry. Still, the few published studies that generate even minor chemical modifications of the core scaffold demonstrate that it is possible to dramatically alter the activity and binding specificity of the compound, supporting the possibility that the antifungal activity of amphoterici B and its toxicity to humans can be decoupled. This would be of enormous value to the infectious disease field. Radiant Genomics believes that the enormous diversity of natural product gene clusters that can be identified through a combination of bioinformatics and next generation sequencing of metagenomes and individual organisms can now be mined to provide candidate natural amphotericin analogs that may have decreased toxicity while maintaining its antifungal properties. These natural analogs will have modifications and structural changes in the core amphotericin B scaffold that are impossible to duplicate through synthetic medical chemistry. Successful production and characterization of a range of natural amphotericin analogs will provide strong proof of concept that the Radiant Genomics natural product discovery platform can allow scientists to quickly interrogate the natural diversity surrounding a targeted natural product, an impossibility using traditional natural product discovery methods, and would revolutionize the field of natural product discovery. A less toxic version of amphotericin B would be primed to become the standard treatment for all systemic fungal infections."}
{"text": "The Illinois Center for Health Statistics, Illinois Department of Public Health, has submitted a grant application to CDC to continue its longstanding PRAMS survey as part of Illinois' effort to reduce infant mortality and morbidity, and improve maternal outcomes of its citizens. These survey data will guide maternal and child health policy and resource allocation, direct immunization strategies for reaching pregnant women, help determine the effectiveness of Medicaid and other health insurance funding related to deliveries, be used to promote breast- feeding, broaden our understanding of postpartum depression and strategies to address it, and be made available for a myriad of related research. Illinois has a proven record of achieving high response rates, especially for a larger state with a diverse population, and thus ensuring the usefulness of PRAMS data for an array of health programs, their policies and related research."}
{"text": "The epithelial barrier presents a significant obstacle to the delivery of macromolecules in the size range of 20 - 150 kDa. In particular, the tight junctional complex, which links adjacent cells and occludes the paracellular space, presents a significant obstacle to delivery of macromolecules. To improve the transport of macromolecular biologics across epithelia, new approaches need to be developed that enhance paracellular drug transport by specifically and reversibly modulating tight junctions. In this proposal, we investigate the effect of nanostructured surfaces on the modulation of tight junction permeability and transport of key therapeutic molecules in vitro. We seek to determine the mechanisms through which epithelial permeability is enhanced by nanotopography and optimize nanostructured materials to broaden the types of drugs that can be delivered paracellularly. It is expected that the fundamental knowledge gained in these studies will enhance the development of new epithelial drug delivery systems."}
{"text": "Perturbation of the metabolism of the brain has serious negative effects on the well-being and survival of the organism. The contribution of the various cell types in the brain to overall metabolism cannot be deter- mined by in vivo experiments. Growth of these sub-populations separately in tissue culture permits examination of their metabolism independently. Furthermore, the effect of varying the extracellular concentration of glucose to mimic conditions seen in vivo in pathologic conditions can be assessed on the separate cell populations. Results from such studies demonstrate that neurons and astroglia handle 2-[14C]DG in a similar fashion under normoglycemic (1.0 - 2.0 mM glucose), slightly hyperglyce- mic (5.0 mM glucose) and hypoglycemic (0.1 - 0.5 mM glucose) conditions; the intracellular:extracellular distribution of 2-[14C]DG is similar in both cell types. Results from studies on glucose were the same as that for 2-[14C]DG at normoglycemic and hyperglycemic levels of glucose. However, results with glucose in the hypoglycemic range were impossible to interpret due to the presence of an intracellular pool of glucose apparently separate from the metabolically active pool. Since the 2- [14C]DG appears to be in equilibrium with the metabolically active glucose pool , movement of glucose at the hypoglycemic levels will have to be inferred from the 2-[14C]DG data. Thus, the ability of neurons and astroglia to transport glucose into the cell's metabolically active pool does not appear to differ."}
{"text": "Pain is one of the most devastating, persistent and incapacitating symptoms in patients with cancer. More than eighty percent of patients with advanced cancer develop pain before death. The great majority of these patients will require opioid analgesics for appropriate pain control. Morphine is recommended as a first line opioid by the World Health Organization Cancer Pain Relief Guidelines. However, the active metabolites of morphine can accumulate and result in opioid induced neurotoxicity, and repeated administration will lead to the need for higher dose or opioid rotation. In addition, the price of morphine can be prohibitively expensive for developing countries, and therefore, most patients with cancer elsewhere in the world die without having ever received a single dose of strong opioid analgesic. Methadone has demonstrated its excellent oral bioavailability, rapid onset of analgesic action, a long life associated with prolonged analgesic effect requiring infrequent administration, lack of known active metabolites. Further, methadone is not opioid derivative, and is much less expensive. Our pilot randomized controlled trial demonstrates similar levels of pain relief in patients receiving methadone or morphine in a 4-week long trial. This leads us to hypothesize that over time the maintenance of pain control in patients receiving methadone due to its absence of NMDA production and opioid metabolite accumulation will result in less frequent opioid induced toxicity, dose escalation and treatment failure, and therefore, improved quality of life. Our long term goal of this study is to provide superior analgesic control for patients with advanced cancer. In this randomized, double blind, parallel study, we aim to determine the clinical efficacy (measuring pain intensity, toxicity, dose escalation, treatment failure and quality of life), to perform economic evaluation (comparing the costs and clinical benefits). 250 patients will be enrolled in the study, half of the patients will receive slow release morphine every 12 hours plus rescue opioid dose, and half will receive methadone every 12 hours plus rescue opioid dose for a total of 12 weeks. All patients will be titrated up to their optimal opioid dose level. Validated instruments will be used to assess pain, symptoms, toxicities and quality of life. Cost-effectiveness analysis will be performed using resource utilization, and medical and non-medical costs. Relevance to Public Health: In this study, we will test if methadone is better than morphine in cancer pain control, quality of life, and is more cost-effective."}
{"text": "The purpose of this study is to investigate by sonography the impact of intracatheter nitroglycerin infusion upon the incidence of vascular thrombosis following percutaneous placement of femoral venous catheters in critically ill children. We hypothesize that intracatheter nitroglycerin infusion will be associated with a lower incidence of thrombosis when compared to control catheters. The study will include a total of 62 children ages birth to 6 years of age. Two groups will be studied. Group 1 (nitroglycerin; n=31) will include critically ill children admitted to the intensive care unit in whom the primary care team has elected to place a double or triple lumen femoral venous catheter. Group 2 (D5W; n=31) will also include critically ill children admitted to the intensive care unit in whom the primary care team has elected to place a double or triple lumen femoral venous catheter. Ultrasound examinations of the catheterized femoral vein will be performed and analyzed by the Radiology investigator within 2 days of catheter insertion and a final study will be performed prior to discharge from the hospital. The primary outcome variable is the difference in the incidence of ultrasound-diagnosed thrombosis between the nitroglycerin and D5W groups. Secondary outcome variables include difference in clinical evidence of thrombosis between the two groups, difference in the incidence of catheter-related infection between the two groups, impact of TPN, intralipid, and heparin infusions on thrombosis in each group, and impact of catheter duration on thrombosis in each group."}
{"text": "This project involves the development of new methods for estimating dose-response relationships between environmental carcinogens and cancer morbidity, and the application of these methods to new and existing datasets. The high resolving power of the new nonparametric procedures will be utilized to better visualize the characteristcs of environmental exposure and cancer morbidity data. For purposes of validation, results obtained through use of these new procedures will be compared to previously completed analyses of existing data sets. New data from several morbidity studies will also be analyzed in order to further fieldtest, and ascertain the properties of, the new techniques."}
{"text": "Socioeconomic and other factors such as race, ethnicity, income, education, and place of residence are associated with reduced access to and poorer quality healthcare. Clinicians serving disadvantaged populations are often overloaded, and have little chance to gather a comprehensive biomedical and psychosocial (biopsychosocial, BPS) evaluation of patients. This situation is unfortunate because many physical symptoms are medically unexplained, reflecting a pattern of high symptom reporting or underlying psychosocial issues, such as depression, trauma, or poorly-tolerated stress. These BPS factors are particularly problematic for patients from disadvantaged populations. Lastly, severe, medically unexplained symptoms, psychosocial issues, and poor quality of life (QOL) occur together in a subset of at-risk individuals (ARI), which comprises about 20% of typical clinic populations. ARI are at risk for poor outcomes and high costs from inappropriate care, a problem that is compounded in disadvantaged and minority populations where access to primary care is limited and urgent care is more prevalent than preventive care. SOLUTION: Our objective is to deploy a clinical methodology and infrastructure to ensure delivery of patient-centered care to minority and disadvantaged patients. Our work builds on an online, patient self-assessment system called CarePrep, now operational in clinic. CarePrep allows patients to easily enter and track BPS and QOL data over the Internet from home or clinic. However, just handing a diagnosis of depression, for example, to a clinician does not change outcomes. Therefore, we will also deploy an integrated intervention to support BPS care. APPROACH: 1) We will adapt, enhance cultural sensitivity, deploy, refine, and initially validate CarePrep in a primary care setting. Our goals are to meet the needs of patients and clinicians for enhancing communication and patient- centered care, ensuring that BPS issues are uncovered. Patients will be asked to do CarePrep before clinic. We will assess validity, feasibility, and CarePrep's ability to accurately identify patients who warrant extra attention (ARI) by findings such as severe symptoms, psychosocial issues, impaired QOL, or substance abuse. 2) We will analyze and develop a plan for supporting culturally competent, BPS care using some combination of automated CarePrep functionality, training and supporting care managers or existing clinic staff in delivering simple, brief BPS interventions, and telehealth support. IMPACT stems from 1) creating a system that generates sufficient value to patients, clinicians, and administrators to warrant routine use; 2) creating a streamlined, branching assessment that minimizes redundancy and maximizes relevance, thereby supporting collection of the full spectrum of relevant data and delivery of context-relevant education and guidance; 3) using this technology and clinical methodology to transcend barriers to care for minorities and disadvantaged populations, and 4) identifying, characterizing, and targeting care to the patients with the greatest need and highest utilization within such populations, thereby facilitating cost-effective use of limited healthcare dollars."}
{"text": "In the United States there are approximately 10 million diabetics; over 75,000 diabetic cataracts are surgically removed annually. Lens aldose reductase has been implicated in the pathogenesis of sugar cataracts; therefore, inhibition of this enzyme may represent a therapeutic approach for the prevention or possible delay of cataractogenesis. Considerable work sponsored by NEI has established the in vitro activity of potential aldose reductase inhibitors. However, little is known about their effect in vivo. In our proposed investigation, we will employ an in vivo system to establish the concomitant protective effect of 3 types of aldose reductase inhibitors on both morphological and biochemical changes during sugar cataractogenesis. The 4 lenticular parameters are: lens growth, fiber structure, protein components (alpha, beta and gamma crystallins) and sugar levels. These parameters can be quantitated; they provide a comprehensive index of lens integrity. These parameters will be correlated with clinical findings (slit lamp and blood sugar levels). Then the effect of aldose reductase inhibitors on normal lens physiology will be analyzed in a similar manner. In order to determine which structure provides maximal therapeutic activity in vivo, dose response studies will be performed on a flavonoid, quercetin, a sphirohydantoin, CP, 45, 634, and a third structure ALO-1349. Our in vivo system is the rat neonate. The fused eyelids provide an orbital pouch for the topical administration of aldose reductase inhibitors. The reasons for selecting this model are explained on page 18. In addition to the clinical relevance, this work will test the hypothesis of lenticular aldose reductase in vivo and provide a model for further investigation of lens therapy."}
{"text": "The Department of Obstetrics and Gynecology at the University of Tennessee, Memphis has been part of the Maternal-Fetal Medicine Network since April 1986. With this application, we hope to participate in a large multicenter network designed to develop and conduct clinical trials in the field of Maternal-Fetal Medicine, which could not be undertaken in a single center. In comparison to our previous application, we have recently expanded our resources and facilities to include patients at all major hospitals in the city. The obstetric population will now total approximately 13,000 women of various ethnic and economic groups. We are particularly interested in pursuing trials which require large sample size in order to adequately address specific questions and those regarding rare events of obstetric interest. The Principal Investigator, Dr. Baha Sibai, and the Alternate Principal Investigator, Dr. Brian Mercer, as well as the faculty in the Division of Maternal-Fetal Medicine continue to be active in Network administrative activities, and the design and conduct of protocols. The Principal Investigator currently participates on the Concurrent Research Committee, the Ad Hoc Committee on Preterm Studies, and the \"High-Risk\" Aspirin Protocol, the Interim Progesterone, and the Preliminary Terbutaline subcommittees. The Alternate Principal Investigator chairs the \"Premature Rupture of Membranes\" protocol and the Chart Review Subcommittee. He serves on the Capitation Subcommittee, Preterm PROM Pathology Subcommittee, Preterm Prediction Protocol Subcommittee, Obstetrical Determinants of Neonatal Survival Protocol Subcommittee, Neural Tube Defect Protocol Subcommittee and the Preliminary MgSO4 Subcommittee. He has recently submitted for consideration a clinical trial regarding tocolytic, corticosteroid and antimicrobial therapy after PROM. We are applying to continue as a clinical research center within the Network and agree to join protocols in existence and participate in the design of protocols in cooperation with other centers selected by the NICHD. The University of Tennessee, Memphis, and the Department of Obstetrics and Gynecology are committed to collaborative Maternal-Fetal research as documented by listed publications and the enclosed letters of commitment. The Division of Maternal-Fetal Medicine agrees to cooperate with the policy of capitation of research costs for each individual protocol, in addition to a base budget."}
{"text": "This proposal seeks to determine the role of the secondary heart field in the morphogenesis of the outflow tract during cardiogenesis in the mouse. The specific aims are 1) To establish the basal proliferation and apoptosis rates in different areas of the outflow tract and secondary heart field during normal mouse development. 2) To establish the fate of labeled cells that originate in the secondary heart field in a mouse model in which there is loss of Fgf8 activity in the secondary heart field. 3) To determine the effect that loss of FGF8 signal in the secondary heart field derived outflow tract cells has on apoptosis and proliferation rates in the outflow tract. We hypothesize that the secondary heart field cells have a unique and previously unrecognized role in the morphogenesis of the outflow tract. The fates of cells derived from the secondary heart field during normal heart development will be determined and compared with those in a model of conotruncal defects. This will help elucidate the role of the secondary heart field in the remodeling and septation of the outflow tract. [unreadable] [unreadable]"}
{"text": "Project Summary: The goals of this application are to understand to what extent replication fitness influences the emergence of HIV variants resistant to non-nucleoside reverse transcriptase inhibitors (NNRTIs), and to delineate the underlying biochemical mechanisms for how drug resistance mutations interact to influence replication fitness. NNRTI-resistant variants of HIV, usually cross-resistant to other NNRTIs, develop rapidly if viral replication is not suppressed. We have shown that HIV replication fitness, as measured in cell culture, is reduced in NNRTI-resistant mutants that are infrequent clinically, even though they confer higher levels of resistance than the most common mutant, K103N. These poorly replicating mutants also reduce RNase H cleavage by reverse transcriptase. Two mutants, G190S and A, also reduce priming from tRNA(Lys,3), raising the question of whether defects in RNA priming also contribute to the reduced fitness of NNRTI- resistant mutants. We also have shown that the nucleoside (nRTI) resistance mutation L74V compensates for the reduced fitness of K103N+L100I, but does not improve its reduced RNase H cleavage rates. Published studies by others have shown that L74V reduces RNA priming. We therefore propose that L74V can compensate for the reduced RNase H activity of poorly replicating, highly NNRTI-resistant variants by improving steps in reverse transcription, such as strand transfer and strand displacement synthesis, that compensate for RNase H cleavage defects. We postulate that selection for highly NNRTI-resistant variants that have improved fitness from compensatory nRTI resistance mutations can lead to early virologic failure. These studies have important implications for the more rational design of nRTI-NNRTI combination regimens. During the next funding period, we plan to: 1. Evaluate the association of nRTI and NNRTI resistance mutations in clinical samples. 2. Determine the effects of nRTI resistance mutations on the replication fitness and drug resistance of NNRTI-resistant mutants. 3. Characterize the biochemical basis for how L74V improves the fitness of NNRTI-resistant variants. Relevance: The proposed studies will evaluate what factors lead to the development of drug resistant strains of HIV, how changes in reverse transcriptase function affect replication of HIV, and whether combinations of drugs can be chosen to influence which drug resistant strains develop."}
{"text": "Literacy requires a prior set of emergent literacy skills that, in turn, require both print-related experiences and a foundation of early oral language competence. For children from Spanish-speaking homes, limited exposure to English and/or limited oral language competence in either Spanish or English may limit the development of their emergent English literacy both directly and indirectly. The eventual results of these limitations contribute to academic underachievement (Goldenberg, 1987), a persistent problem in the US Hispamc population in which only 63% of students graduate from high school (Healthy People 2000). These limitations may be ameliorated by early exposure to English language in addition to an enriched home language environment. The Bilingual Early Language and Literacy Support (BELLS) project was funded by NIH and OERI (Contract# 5-R01- HD39501) to test the language and emergent literacy outcomes of Spanish-speaking/bilingual children who either were enrolled in an early childhood program that included English exposure/immersion and home language and literacy support or were in a community, where limited early childhood experiences were available. BELLS was funded to assess children up to Kindergarten entry. However, many of the children enrolled in BELLS will be entering kindergarten prior to the end of the BELLS project and many will be in first grade when the project ends. The literature supports longitudinal research that follows children from early childhood into the school setting (Dickinson & McCabe, 2001). The purpose of this supplemental study will be to follow BELLS children into Kindergarten and first grade. This will allow us to examine school and home pathways to reading achievement."}
{"text": "The objectives of the project remain to study the concept of collagenolytic activity as a proximal event in the acquisition of an invasive potential by tumor cells and to define inter-relationships between tumor protease (collagenase) activities, host protease (collagenase) inhibitor(s) and tumor growth characteristics (non-invasive vs. invasive growth) in cell lines derived from chemical carcinogen (FANFT)-induced transitional cell carcinomas arising in the urinary bladder of Fischer rats. Normal epithelial cells, microvascular endothelial cells, and fibroblasts will be studied for their involvement in the regulation of the connective tissue turnover at the epithelial-stromal interphase (protease (collagenase) activities; protease (collagenase) inhibitors) and invasive carcinoma cells analyzed for mechanisms involved in the degradation of connective tissue barriers."}
{"text": "The objective of this proposal is to develop ultra-sensitive ELISA (Enzyme-Linked ImmunoSorbent Assay) methods for quantification of HIV p24 antigen. At present, the p24 antigen test is relatively insensitive, being able to detect only 5- 10 pg/ml. This quantity of antigen may not be present in the serum of infected individuals, even when the virus is actively replicating. In fact, only about 50-60% of AIDS patients, 30-40% of ARC patients, and 10% of asymptomatic patients will have p24 antigenemia that is detectable. Therefore, the development of more sensitive p24 antigen tests is of great importance. It is in this context that we are proposing to develop a highly sensitive p24 antigen assays that will be less expensive than RNA viral load tests and appropriate for use both in developed and developing countries in which the limitations of infrastructure and laboratory capability prohibit nucleic acid testing. The specific aims of the Phase I and Phase II, in part, include 1) analyzing and optimizing of all variables affecting the accuracy and functionality of the p24 tests;and 2) validation of the HIV-1 p24 detection assay. Advantages of the proposed assays such as the increased sensitivity, signal-to- noise ratio, and dynamic range, reduced time and low cost of the analysis substantiates our belief that the proposed amplification method has a prominent commercial and scientific potential. PUBLIC HEALTH RELEVANCE: The development of the proposed method will allow using HIV p24 antigen tests for monitoring HIV infection, blood screening, identification of acute infection, to assist in the diagnosis of infection in the newborn, and in detecting antigen in supernatants from cultures. The performance of the developed tests will be improved significantly as compared with that for conventional assays. Successful completion of these studies will be beneficial for public health, and make available all of the technology needed for a substantial business opportunity to license the technology and manufacture commercial products."}
{"text": "Recent work from several laboratories has shown that murine gamma interferon induces the biosynthesis and cell surface expression of immune-associated (Ia) glycoprotein determinants on both normal and neoplastic mouse mononuclear phagocyte lineage cells. Experiments carried out by the principal investigator demonstrate that gamma interferon activates a murine macrophage tumor cell line, (P-388.D1), to release a second, noninterferon factor activity which, in turn, stimulates Ia expression on mouse macrophage tumor cells. The major goal of this research proposal is to test the hypothesis that gamma interferon regulates the positive expression of macrophage Ia determinants by stimulating the induction of a second group of Ia-inducing factors (IaIF) by the macrophage which subsequently control the cell surface expression of individual Ia subregion determinants. Thus, a variety of murine macrophage tumor cell lines and normal mononuclear phagocyte subpopulations will be cultured in vitro using different Ia induction procedures, and conditioned medium from these cells will be tested against neoplastic and normal Ia negative macrophage target cells for the ability to induce Ia expression. Quantitation of Ia induction will be carried out using direct and indirect immunofluorescence with anti-Ia monoclonal antibodies and flow cytometry analysis (FcM). Where active IaIF supernatants are found, they will be examined using standard biochemical analysis techniques in an effort to characterize the material and show if there are multiple IaIF activities which control the expression of discrete Ia subregion glycoproteins. Normal or neoplastic macrophages which are induced by IaIF activity for one or more Ia subregion antigens (i.e., I-A, I-E/C, or I-J), will be studied for functional activity as antigen-presenting cells in T-cell proliferation and activation assays using MHC-restricted, antigen-specific, continuous helper T-cell lines, or T hybridomas as effector cells. I-J-inducible macrophages will be tested as accessory cells in the induction of a primary in vitro burro red blood cell IgM response, and as antigen-presenting cells in the activation of third-order suppressor T cells (Ts 3) in the azobenzenearsonate (ABA) system. This study will provide basic information describing the mechanism by which gamma interferon may regulate the cell surface expression of individual Ia subregion antigens on phenotypically and functionally unique subsets of mononuclear phagocytes. (SR)"}
{"text": "This is an application requesting support for the 6th Gordon Research Conference (GRC) on: In Vivo Magnetic Resonance (MR). This meeting will cover an array of topics spanning the use of MR imaging and spectroscopy in cells, animals, and humans;addressing both basic science and clinical research issues. Particular emphases will be placed on emerging technological, biological, and health related matters that will significantly impact the growth of this remarkable imaging field. The 6th Conference will be held July 25 - 30, 2010, at Proctor Academy, Andover, NH. The Chair of the 6th Conference is Risto A Kauppinen, MD PhD, Director of the Biomedical NMR Research Center and Research Professor of Radiology, Dartmouth College, Hanover, NH;the vice-Chair is Craig Stanisz, PhD, Associate Professor, Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada. The 5th In Vivo MR GRC was held in 2008 in the same location. The previous Conference was extremely successful. Significant advances in instrumentation, imaging speed, and in a wide array of advancements in coil and gradient technologies continue to create a rich environment for rapid growth of MR and its in vivo applications. It is both timely and necessary to gather together the leading individuals and future investigators to address, in depth, as is done at no other scientific venue, the next ventures of this important imaging science field. The In Vivo MR GRC will include scientific sessions, each with two to four speakers and a discussion leader. There will also be four poster sessions (from experience gathered in previous GRCs most participants will present a poster) and discussions designed to facilitate close interaction between the participants. By design, GRC meetings are small by conventional standards (approximately 140-150 participants) but participants will be invited/accepted in a manner to ensure attendance by a diverse group of junior and senior investigators representing academic and government institutions and individuals from corporate research programs. Students and postdoctoral fellows will be encouraged to attend. Junior investigators will be given opportunity for oral presentations. PUBLIC HEALTH RELEVANCE: Support is requested for the 6th Gordon Research Conference (GRC) on :In Vivo Magnetic Resonance: (July 25-30, 2010). Magnetic resonance (MR) techniques play a critical role in the delivery of high quality health care and MR is an essential player in the modern molecular medicine. The GRC meeting will emphasize emerging and cutting-edge technological, biological, and health related issues that will significantly impact the growth and application of this remarkable imaging field."}
{"text": "Type 1 pili of Escherichia coli and other gram negative enteric bacteria are filamentous, proteinaceous appendages composed primarily of a subunit called pilin. Type 1 pili mediate the mannose-sensitive adhesion of E. coli to a variety of eucaryotic cells and are a factor in promoting colonization in the lower urinary tract. The long term goal of this project is to establish the molecular nature of the genetic control, assembly and receptor binding activity of type 1 pili and in doing so, contribute to the understanding of the control and assembly of supramolecular structures and receptor-ligand interactions in general. Also, it is hoped that this research will lead to a better understanding of the role of bacterial adhesion in the pathogenesis of infectious disease. The research herein proposed uses a variety of genetic and biochemical techniques to: 1) Discern the factors effecting pilA expression. The pilA gene encodes the major subunit of pili and is subject to at least two types of transcriptional control. One type is a repression effected by the product of an adjacent gene, the other type is a metastable (ON or OFF) control effected by the inversion of the pilA promotor. Ways are proposed to create and analyze lesions affecting these two types of transcriptional control. 2) Discern the molecular interactions required for pilus assembly. Methods are suggested for the analysis of mutants having point mutations in pilE, a gene encoding a minor component of pili that is required for receptor binding. Projects include the isolation of extrageneic compensatory mutations, analysis of conditional pilE mutations and localization of the pilE protein. In addition, methods for isolating and examining mutants having lesions affecting two other assembly related genes, one of them affecting pilus length, are proposed. 3) Discern the molecular interactions required for receptor binding. Methods are outlined for isolating and analyzing mutants with an altered receptor specificity. Also, ways for isolating the pilE gene product and assessing the role of the pilE protein in receptor binding are outlined. In addition, the role of receptor binding and pilus length on the interaction of piliated E. coli with macrophages is examined."}
{"text": "Investigators who need to use morphological techniques frequently do not have the facilities in their own laboratories. In addition, project personnel, especially young investigators, often come to the Council without training in these techniques. The Morphology Core provides a number of avenues which enable investigators to produce high quality work involving light and electron microscopy, immunocytochemistry, in situ hybridization, and photography."}
{"text": "The long-term objective of the proposed research is to test the hypothesis that in systemic lupus erythematosus (SLE) there is an increase in the antigen driven autoimmune response through delayed clearance of DNA and nuclear-associated proteins due to an abnormally low C-reactive protein (CRP) response. This hypothesis is based on observations that in the SLE the CRP response during inflammatory episodes related to disease is markedly diminished compared to the degree of inflammation and tissue damage present. The mechanisms responsible for this reduced CRP response are not known. In addition, CRP has been shown to localize in the nuclei of damaged cells and to bind to chromatin. Given the inappropriate CRP-response and the characteristic antibody response to DNA and nuclear proteins in SLE, we, and others, hypothesize that CRP reacts with DNA associated components in damaged tissue and aids in their removal through interactions with the complement system and cells of the phagocytic system. We propose to use human CRP-transgenic (NZB X NZW)F1 mice to test our hypothesis. (NZB X NZW) F1 mice have been utilized as a model for SLE and display a well characterized anti-DNA and anti-nuclear antigen response. Therefore, transgenic (NZB X NZW)F1 mice which have the human CRP gene should be an exceptional tool for studying the effects of CRP on the development of anti-DNA and anti-nuclear antibodies in SLE. Specifically, we plan to determine the effects of human CRP on the development of the lupus like disease of (NZB X NZW)F1 autoimmune mice. We will construct transgenic CB6 mice which express human CRP in an acute phase manner, and then transmit the human CRP on the development of the lupus like disease of (NZB X NZW)F1 autoimmune mice. We will construct transgenic CB6 mice which express human CRP in an acute phase manner, and then transmit the human CRP gene to autoimmune mice through selective breeding. If there is a disease associated CRP response, the development of disease in the CRP transgenic (NZB X NZW)F1 mice will be compared to (NZB X NZW)F1 mice. If there is no significant, disease associated-CRP response in the (NZB X NZW)f1 transgenic mice, we will determine whether these mice have normal CRP expression in response to other stimuli and if the low response is due to some disease related phenomenon. In addition, we will examine the effect of constant expression of CRP on the development of disease in (NZW X NZW)F1 transgenic mice by introducing into the mice DNA constructs of CRP genomic DNA and the mouse metallothionein I enhancer/promoter sequences. The data from the proposed experiments will be an important addition to our knowledge of CRP's role in the development of the autoimmune response, as well as the regulation of CRP's expression in SLE. Finally, these studies will also provide valuable information on regulation of CRP expression within a normal background."}
{"text": "The development of ventricular fibrillation (VF) and sudden cardiac death include several key steps, such as initiation of reentry, degeneration to multiple reentrant waves, and maintenance of VF. issue heterogeneity has been traditionally considered as the major cause for these key steps leading to VF. However, recent studies indicate that dynamic wave instability operates synergistically with pre-existing tissue heterogeneity to promote wavebreak. Dynamic wave stability is regulated by multiple factors, including electrical restitution, intracellular Ca (Cai) cycling, cardiac memory, and electrotonic currents. The goal of this project is to use computer simulations with simplified models to developed theories which identify the critical parameters controlling the development of VF, as the theoretical basis for novel therapeutic strategies. The central task of this project is to investigate how voltage, Cai, and tissue heterogeneity interact to regulate vulnerability to rentry and maintenace of VF. The first aim is to develop simplified models to investigate the nonlinear dynamics of Cai cycling coupled to membrane voltage, in order to understand dynamical mechanisms of cardiac alternans. The second aim is to use physiologically-detailed AP models in homogeneous tissues to determine the physiological mechanisms by which voltage-Cai dynamics promotes spatially discordant alternans, dispersion of refractoriness, wave instability and arrhythmogenesis. The third aim is to determine how voltage-Cai dynamics interacts with tissue heterogeneities to regulate spatially discordant alternans, dispersion of refractoriness, wave instability and arrhythmogenesis. This project will develop general theories which will then be validated in more realistic settings in Projects 1, 3, and 4. The approach in this project will allow us to extensively explore the parameter space and systematically investigate the mechanisms of VF initiation and maintenance to identify the critical parameters essential for developing novel therapeutic strategies."}
{"text": "Mitochondrial diseases are devastating disorders for which there is no cure and no proven treatment. About 1 in 2000 individuals are at risk of developing a mitochondrial disease sometime in their lifetime. Half of those affected are children who show symptoms before age 5 and approximately 80% of these will die before age 20. The human suffering imposed by mitochondrial and metabolic diseases is enormous, yet much work is needed to understand the genetic and environmental causes of these diseases. Mitochondrial genetic diseases are characterized by alterations in the mitochondrial genome, as point mutations, deletions, rearrangements, or depletion of the mitochondrial DNA (mtDNA). The mutation rate of the mitochondrial genome is 10-20 times greater than of nuclear DNA, and mtDNA is more prone to oxidative damage than is nuclear DNA. Mutations in human mtDNA cause premature aging, severe neuromuscular pathologies and maternally inherited metabolic diseases, and influence apoptosis. The primary goal of this project is to understand the contribution of the replication apparatus in the production and prevention of mutations in mtDNA. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we have focused this project on understanding the role of the human pol gamma in mtDNA mutagenesis. Human mitochondrial DNA is replicated by the two-subunit gamma, composed of a 140 kDa subunit containing catalytic activity and a 55 kDa accessory subunit. The catalytic subunit contains DNA polymerase activity, 3'-5'exonuclease proofreading activity, and 5'dRP lyase activity required for base excision repair. As the only DNA polymerase in animal cell mitochondria, pol gamma participates in DNA replication and DNA repair. The 140 kDa catalytic subunit for pol gamma is encoded by the nuclear POLG gene. To date there are over 150 pathogenic mutations in POLG that cause a wide spectrum of disease including Progressive external ophthalmoplegia (PEO), parkinsonism, premature menopause, Alpers syndrome, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) or sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO). Presently, there are over 150 pathogenic disease mutations in the POLG gene that cause PEO, ataxia-neuropathy and Alpers syndrome. We described a novel POLG missense mutation (c.3218C>T;p.P1073L) that, in association with 2 previously described mutations, caused an Alpers-like hepatocerebral syndrome in 4 children. Four children, 2 related and 2 unrelated, with the novel p.P1073L mutation (all patients) and either the p.A467T (2 patients), p.G848S (1 patient), or p.W748S (1 patient) mutation presented with psychomotor delay, encephalopathy, and liver failure. Detailed clinical and laboratory examinations including brain magnetic resonance imaging, muscle biopsy, measurement of mitochondrial DNA, and sequencing of the POLG gene. All 4 patients had psychomotor delay, seizures, and liver disease. Three patients had severe gastrointestinal dysmotility, which may be associated with the new p.P1073L mutation. The heterozygous presence of the novel p.P1073L mutation in trans with another recessive POLG mutation causes a hepatocerebral disorder identical or very similar to Alpers syndrome. This adds to the already striking clinical heterogeneity of POLG mutations. In the Belgian patients, the familial occurrence without consanguinity is related to the high frequency of the recessive p.A467T and p.W748S mutations in northwestern Europe and reveals a pitfall for diagnosis and genetic counseling. The severity and dominance of many POLG disease-associated mutations are unclear, because they have been reported in sporadic cases. To understand the consequences of pol gamma disease-associated mutations in vivo, we identified dominant and recessive changes in mtDNA mutagenesis, depletion and mitochondrial dysfunction caused by 31 mutations in the conserved regions of the gene, MIP1, which encodes the Saccharomyces cerevisiae ortholog of human pol gamma. Twenty mip1 mutant enzymes were shown to disrupt mtDNA replication and may be sufficient to cause disease. Previously uncharacterized sporadic mutations, Q308H, R807C, G1076V, R1096H and S1104C, caused decreased polymerase activity leading to mtDNA depletion and mitochondrial dysfunction. We present evidence showing a limited role of point mutagenesis by these POLG mutations in mitochondrial dysfunction and disease progression. Instead, most mitochondrial defective mip1 mutants displayed reduced or depleted mtDNA. We also determined that the severity of the phenotype of the mip1 mutant strain correlates with the age of onset of disease associated with the human ortholog. Finally, we demonstrated that increasing nucleotide pools by overexpression of ribonucleotide reductase (RNR1) suppressed mtDNA replication defects caused by several dominant mip1 mutations, and the orthologous human mutations revealed severe nucleotide binding defects. Defects in mtDNA replication are the principle cause of severe, heritable metabolic disorders classified as mitochondrial diseases. In vitro analysis of the biochemical mechanisms of mtDNA replication has proven to be a powerful tool for understanding the origins of mitochondrial disease. Mitochondrial single-stranded DNA-binding protein (mtSSB) is an essential component of the mtDNA replication machinery. To facilitate ongoing biochemical studies, a recombinant source of mtSSB is needed to avoid the time and expense of human tissue culture. This chapter focuses on the subcloning, purification, and initial functional validation of the recombinant human mitochondrial single-stranded DNA-binding protein. The cDNA encoding the mature form of the human mtSSB protein was amplified from a HeLa cDNA library, and recombinant human mtSSB was overproduced in Escherichia coli. A procedure was developed to rapidly purify milligram quantities of homogenous, nuclease-free mtSSB that avoids DNA-cellulose chromatography. We show that, similar to E. coli SSB, human mtSSB assembles into a tetramer and binds single-stranded oligonucleotides in a 4-to-1 protein:oligonucleotide molar ratio. Missense mutations in the human C10orf2 gene, encoding the mitochondrial DNA (mtDNA) helicase, co-segregate with mitochondrial diseases such as adult-onset progressive external ophthalmoplegia (PEO), hepatocerebral syndrome with mtDNA depletion syndrome (MDS), and infantile-onset spinocerebellar ataxia (IOSCA). To understand the biochemical consequences of C10orf2 mutations, we overproduced wild type and 20 mutant forms of human mtDNA helicase in Escherichia coli and developed novel schemes to purify the recombinant enzymes to near homogeneity. A combination of molecular crowding, non-ionic detergents, Mg(2+) ions, and elevated ionic strength were required to combat insolubility and intrinsic instability of certain mutant variants. A systematic biochemical assessment of the enzymes included analysis of DNA binding affinity, DNA helicase activity, the kinetics of nucleotide hydrolysis, and estimates of thermal stability. In contrast to other studies, we found that all 20 mutant variants retain helicase function under optimized in vitro conditions despite partial reductions in DNA binding affinity, nucleotide hydrolysis or thermal stability for some mutants. Such partial defects are consistent with the delayed presentation of mitochondrial diseases associated with mutation of C10orf2."}
{"text": "X-ray crystallography and cryo-electron microscopy are to be used for structural studies of enveloped viruses and their self-assembled cores. The work will concentrate on alpha- (Sindbis, Semliki Forest, Ross River, Aura), rubella and flavi- (hepatitis C and yellow fever) viruses. Rubella virus is a major problem to the human fetus in pregnant mothers. Hepatitis C virus infections are a worldwide problem, affecting as much as 20% of the population in some countries. Alphaviruses can sometimes cause fatal diseases such as encephalitis fever and arthritis. Currently, little structural information is available about enveloped viruses. Structural studies are likely to provide understanding of the mechanisms by which these positive-stranded RNA viruses disassemble on cell entry and assemble prior to cell exit. Many laboratories have attempted to crystallize alphaviruses, but only very poorly diffracting crystals have been produced. We suspect that the probable cause for the crystalline disorder may be the presence of heterogeneous carbohydrate moieties. Hence, site-directed mutations are being made (in collaboration with Richard Kuhn) to eliminate or reduce the number of potential glycosylation sites on the surface glycoproteins of Sindbis virus. The most difficult and time-consuming part of the work will be in sample preparation involving site-specific mutagenesis to deglycosylate the surface glycoproteins to obtain homogeneous samples of in vitro assembled cores. Although the electron microscopic, image reconstruction and crystallographic studies will take time and effort, the technologies are well established. We have attained good expression of Sindbis virus, Ross River virus, rubella virus and hepatitis C virus capsid protein in E. coli, although crystallization of the capsid protein has so far been achieved for only Sindbis virus. Mutational and structural studies of these proteins will be pursued similar to those previously used in the analysis of Sindbis virus. Self-assembled core-like particles using various nucleic acids, including genomic RNA and a DNA 48-mer, have been obtained for all the above mentioned capsid proteins. Cryo-electron microscopy studies and crystallization trials are underway. Reproducible small crystals (less than or equal to 0.05 mm diameter) of the in vitro assembled Sindibis cores can be produced routinely."}
{"text": "The human gut microbiota provide physiologic attributes that we have not had to evolve on our own, including the ability to process otherwise indigestible dietary polysaccharides. Bacteroides thetaiotaomicron (B. theta), a prominent member of the normal human distal gut microbiota, has an expanded capacity to process both dietary and host-derived polysaccharides, a feature that likely enhances its fitness in the crowded gut ecosystem. A fundamental question is how this prototypic gut symbiont recognizes and adapts to changing carbohydrate availability. The B. theta genome encodes 50 extra-cytoplasmic function sigma (ECF-?) factors, 26 are located adjacent to genes encoding anti-sigma (anti-?) factors, which are predicted transmembrane proteins with periplasmic sensor domains. Most ECF-? /anti-? pairs (25/26) co-localize with a third gene, encoding a SusC-like outer membrane porin. SusC paralogs are implicated in polysaccharide binding and are predicted to interact directly with anti-? factors, comprising a series of cell envelope- spanning switches that transduce external cues (SusC and anti-?) to effect transcriptional changes (ECF-?). My results from in vivo and in vitro experiments indicate that B. theta adapts its physiology to utilize host-derived mucopolysaccharides via an ECF-? /anti-? dependent mechanism. I have identified four prototypic loci (44 genes), each containing an ECF-?/anti-? switch and polysaccharide catabolic functions. I propose to probe the function of these 4 systems by investigating their mechanism(s) and specificity of signal transduction as well as their contribution B. theta fitness in the mouse intestine. Aim 1 will test a working model of ECF-?/anti-? signal transduction through genetic disruption of its predicted components and subsequent assay of function. Yeast 2-hybrid analysis will be used to probe predicted protein-protein interactions between signaling components and, because these signaling components are expanded in B. theta, the potential for cross-talk between paralogs. Aim 2 seeks to chemically define mucopolysaccharide components that trigger ECF-?/anti-? switches and, in conjunction with a bioassay for locus induction, will provide valuable insight into the chemical language through which B. theta perceives its environment. Aim 3 addresses the hypothesis that mucopolysaccharide utilization by B. theta enhances in vivo fitness. The ability of B. theta to turn on these systems will be eliminated through genetic manipulations, and the colonization behavior of mutants evaluated in competition with wild-type B. theta in gnotobiotic mice. These studies will expand our understanding of how bacteria inhabiting the gut can shift their metabolism to coincide with changing availability of carbohydrate resources in the intestine. [unreadable] [unreadable] [unreadable]"}
{"text": "Dithiobiuret (DTB), H2N-C-NH-C-NH2, is used in the synthesis of insecticides, rodenticides and resins and as a plasticizer and rubber accelerator. There is a paucity of studies but Astwood et al. (Science 102:196, 1945) reported skeletal muscle paralysis in rats after several days of treatment. The paralysis started in the hindlimbs and then rapidly ascended to involve forelimbs and head and neck muscles. If treatment was continued the animals died apparently of respiratory paralysis. The gross toxicologic effects appear similar to environmental agents that produce neuropathy and myopathy. Acrylamide produces muscle weakness by peripheral nerve damage and triorthocresyl phosphate through damage to motoneurons. Leptophos causes CNS-PNS distal axonopathy and paraboxon treatment myopathy. Thus, it is of interest to see if weakness produced by DTB is due to nerve and/or muscle damage. The objectives are to describe experimental conditions under which DTB treatment produces paralysis in rats and cats, see what tissues or organs are involved, attempt to determine its mechanism of action and develop antidotes or antagonists which in turn may give insight into its mechanism of action. Muscle weakness in the whole animal will be evaluated by a treadmill test, brain function by direct and indirect methods, spinal cord function by assessing spinal reflexes and neuromuscular function by nerve-muscle studies. Histological assessment of nerve and muscle tissue will be included. Metabolic activation will be assessed by seeing if a relationship exists between activity of the hepatic MFO system and delayed toxic effects. An indirect benefit may be establishment of DTB-treated animals as a model for the muscle weakness seen in either a myasthenic disease, myotonic muscular dystrophy or acute intermittent porphyria neuropathy in man."}
{"text": "Human studies show that cocaine dependence affects the microstructure of white matter, probably as a result of vasoconstrictive effects of the drug. Little is known, however, about the temporal development of the white matter injury and the changes in cerebral vasculature during the dependence period, or about the recovery of both with cessation of the drug use. Magnetic resonance imaging (MRI) provides tools that are highly suitable for investigating in a non-invasive manner the anatomical, structural and functional and chemical characteristics of the brain. In this application we propose to implement a set of MRI techniques for investigating the effects of the use of cocaine on monkey brain, in particular its effects on white matter microstructure on gray/white matter volumes, on the vascularity and perfusion of gray and white matter and on the neurochemical profile of the monkey brain. These techniques will be used to perform a preliminary study on a small and well-controlled population of monkeys. Aim 1. To develop a robust methodology for investigating structural, anatomical and functional measures in monkey brains. Aim 2. To collect preliminary data on the effects of drug use and its cessation on monkey brain. We hypothesize that: 1. Long term cocaine self-administration will result in brain changes in the monkeys similar to those seen in human studies. 2. Cessation of drug use will result in a normalization of brain measures. This will address the important issue of whether or not there is significant brain recovery following cessation of drug use."}
{"text": "The purpose of this study is to treat patients with metastatic brain tumors using Gadolinium Texaphyrin (PCI-0120) injection two to five hours before radiation treatments for ten consecutive days (excluding weekends and holidays); to establish an appropriate intravenous dose of Gadolinium Texaphyrin (PCI-0120) Injection; to determine the maximum tolerated total dose (MTD) of Gadolinium Texaphyrin (PCI-0120) Injection for palliative radiation therapy of metastases to the brain; to evaluate an additional 20 patients at the MTD to establish the safety profile of Gadolinium Texaphyrin (PCI-0120) Injection; to evaluate accumulation of Gadolinium Texaphyrin (PCI-0120) Injection in metastases and changes in tumor volume (i.e., response to treatment); to determine the dose limiting toxicity of repetitive doses of Gadolinium Texaphyrin (PCI-0120) Injection, where each dose is followed by a radiation treatment, in patients with metastatic cancer to the brain; to determine the biolocalization of Gadolinium Texaphyrin (PCI-0120) Injection in cancer (e.g., brain metastases) and normal tissue(s) (e.g., brain tissue), and the change in cancer volume, using MRI and the high (white) signal intensity Gadolinium Texaphyrin (PCI-0120) Injection produces on MRI scans; and to determine the in vivo pharmacokinetics of Gadolinium Texaphyrin (PCI-0120) injection in patients treated at the MTD."}
{"text": "Cellular senescence limits the proliferation of human cells and can be induced by a variety of cellular alterations, both intrinsic and extrinsic. Senescent cells accumulate in human tissues, including the prostate, with increasing age. These senescent cells have altered function, including increased expression of proinflammatory cytokines that can alter the function of adjacent cells. Benign prostatic hyperplasia (BPH) is the single most common pathology of aging men. Based on our published studies we hypothesize that senescence of a subset of epithelial cells in BPH tissue leads to release of cytokines and growth factors that, through direct and indirect actions, drives increased proliferation of adjacent non-senescent epithelial cells and stromal cells and ultimately prostatic tissue growth in aging men. We propose to characterize the mechanisms by which cellular senescence can promote the development of benign prostatic hyperplasia. Two Specific Aims are proposed. In Specific Aim 1, we will examine the underlying cellular alterations leading to prostatic epithelial senescence in vitro and in vivo;determine the types of cytokines and growth factors expressed by senescent epithelial cells in vitro;evaluate whether these same proteins are expressed at increased levels in BPH tissue in vivo and quantitatively evaluate the extent to which there is coexpression of these cytokines and growth factors at the cellular level in vivo with markers of senescence, including key cell cycle regulator proteins such as p21 and p16. In Specific Aim 2 we will use primary cultures of prostatic epithelial and stromal cells, the reactive stroma model system and transgenic models to examine the biological activities of the identified cytokines/growth factors and model potential autocrine and paracrine activities of those factors that are increased in BPH in vivo. In addition, we will establish a transgenic mouse model of epithelial senescence and examine the biological impact of epithelial senescence in this mouse model. Benign prostatic hyperplasia causes considerable morbidity in older men, with up to 30% of men requiring treatment for this condition, and with more than one billion dollars spent on the medical and surgical treatment of this disease annually. These studies will make a fundamental contribution to our understanding of the role of cellular senescence in the pathogenesis of this common disease and lead to more effective preventive treatments and medical therapies. PUBLIC HEALTH RELEVANCE: Benign prostatic hyperplasia causes considerable morbidity in older men by blocking the urinary tract and up to 30% of men will require treatment for this condition at some time in their lives, with more than one billion dollars spent on the medical and surgical treatment of this disease annually. We believe these studies will make a fundamental contribution to our understanding the causes of this common disease and by doing so lead to more effective preventive treatments and medical therapies."}
{"text": "Spinal muscular atrophy (SMA) is characterized by loss of motor neurons and atrophy of muscle. Proximal SMA is the second most common genetic cause of infant death. As in many neurogenetic disorders, the mutated protein SMN is ubiquitously expressed, yet only a particular type of neuron is affected. SMA is caused by loss of the SMN1 gene and retention of the SMN2 gene, leading to low levels of wild-type SMN, which is insufficient for motor neuron survival. Increasing SMN levels by increasing SMN2 copy number modulates the severity of the phenotype. In recent years the investigators, and others, have identified drug compounds that can induce SMN2 to produce more SMN protein. In addition, when the drug is given prior to motor neuron loss certain compounds can modify the SMA mouse phenotype. It has now become possible to perform newborn screening for SMA. However key questions remain concerning optimal deployment of therapeutics in SMA, including compounds that activate SMN2, oligonucleotides that stimulate incorporation of SMN exon7 by SMN2, or gene therapy. In this application the investigators will define the spatial and temporal requirement for high levels of SMN to correct SMA. Additionally, they will study modifiers of SMA and the effectiveness of combination drug treatments. These are essential components in order to optimize treatment regimes for SMA. In this application four aims are proposed using SMA mice. 1) Determine the importance of high levels of SMN in tissues other than neurons or motor neurons by using mouse lines that selectively create or correct the SMA condition in neurons or motor neurons. This will address if there is any benefit in SMA animals of increasing SMN levels outside the nervous system. 2) The temporal requirement for high levels of SMN to correct SMA will be determined using a SMN inducible system. This will allow determination of when SMN must be increased in SMA and whether induction of SMN must be continuous or just during a specific window of time. 3) Study the role of phosphorylation in SMN's ability to correct SMA. Additionally, they will determine if combinations of drug increase survival in SMA mice. The activity of current drugs must be increased in order to have a major impact in the SMA mouse. Combinations of drugs that activate SMN, as well as alteration of the phosphorylation state of SMN, may significantly impact the SMA phenotype. 4) Lastly, a modifier of the SMA phenotype, plastin3, has been reported. The investigators will determine if plastin3 is truly a modifier of SMA by asking whether increased plastin3 expression can correct SMA mice. The aims proposed here will indicate the temporal spatial requirement for SMN induction, identify the optimal compound combination for induction of SMN, and indicate the importance of a reported modifier of SMA in mice. This will result in optimization of therapeutics for SMA."}
{"text": "In this project, we will investigate the biomechanics that enable high-frequency (JHF) sound, greater than 10 kHz, to be transduced from middle-ear to cochlea in a group of terrestrial mammals. Anatomical studies indicate that in larger mammals, the malleus may-assume a twisting mode of vibration, rather than the classical hinging mode. Twisting would transfer sound energy more efficiently at HF because relative mass inertia is greatly decreased. At lower frequencies, the hinging mode is utilized due to the stiffness of the connecting ligaments and tendons. For smaller mammals, the impedance due to mass is not prohibitive at HF, so a twisting mode is not necessary. Accordingly, features of their anatomy seem to preclude twisting. ~ Here we propose an interspecies (human, cat, chincilla, and gerbil) investigation of the hypothetical twisting mode using physiologic measurement, morphologic observation and mathematical analysis. ~ SA1: Empirically determine impedance as a function of frequency for the hinging and twisting vibrational modes by inducing these motions through the malleus and measuring the cochlear output. Two magnetic voice coil motors will drive the anterior and posterior quadrants of the eardrum with independent phase. This causes hinging and twisting with equal and opposite phase respectively. A novel cochlea preparation that introduces a 'third window\" will be created to measure the spiral ligament, which moves with stapes pressure, by laser Doppler. This gives a relative indication of cochlear output. SA2: Morphometrically quantify the primary anatomical structures of the eardrum and ossicles that could contribute to a twisting vibrational mode by multimodal imaging. Middle-ear morphometry will be Imaged by micro-CT. Collagen fiber structure at select areas in the eardrum will be imaged by multiphoton and transmission electron microscopy. Image analysis software will be used to obtain quantitative data from these Images. SA3: Analytically deduce the conditions required for a twisting vibrational mode by developing simple and finite element models of the eardrum and ossicles that can incorporate the methods and data acquired from SA1 and SA2. A simple model for the hinging and twisting modes will be formulated based on micro-CT data. This will guide the construction of a finite element model that can incorporate data from the other specific aims. Assessment of the parameters derived from the four species studied will allow us to formulate the fundamental physical and physiological basis for a twisting mode. ~ This research is relevant to current medicine because an improved understanding of eardrum and ossicle biomechanics is required to develop more effective technologies related to myringoplasty. This may be particularly important for post-surgical HF hearing preservation."}
{"text": "The Sickle Cell Scholar training program is an integral part of the education component of the Comprehensive Sickle Cell Center grant. The Sickle Cell Scholar will be chosen according to the guidelines in the RFA. Under the auspices of the Los Angeles Sickle Cell Center, the scholar will have the opportunity to do clinical and translational research related to the efficacy of L-glutamine in ameliorating the symptoms of sickle cell disease, the genesis of cardiac dysfunction in transfused sickle cell patients, the health services research related to provision of adult care, or translational research related to the role of viscosity in alteration of blood flow and oxygen delivery in humans and the use of engineering techniques to detect evidence of sickling. Unique to this program, the scholar will be supervised by the Center education director who is a cell biologist and doctoral level educator. This individual will be responsible for setting educational milestones and systematically monitoring the progress of the scholar to the training program. Also described here is a plan for the management of the summer high school student program that will also be run by the Center education director."}
{"text": "The search for the origins of diversity in primary vestibular afferent response dynamics has been a dominant theme of vestibular research and is the major subject of the present proposal. The long-term goal of the work is to study the role of transmitter systems in shaping afferent responses, and the immediate objective is to identify the impact of GABA and glutamate as hair cell transmitters. The central hypothesis of the research is that the crista performs a mathematical differentiation (differential calculus) of some velocity-sensitive input signals in processing convergent excitatory (glutamatergic) and inhibitory (GABAergic) hair cell synapses onto dendrites of single afferent neurons and that this push-pull input accounts, at least in part, for the wide range of observed afferent responses. Three specific aims will be pursued. These aims embrace a multidisciplinary approach utilizing techniques and experimental models selected for their particular suitability for the experiments, the research experience of the investigators, and the applicability of the results to understanding human vestibular disorders. Anatomical, physiological, pharmacological and mathematical tools are employed concomitantly to address the aims and test hypotheses from multiple perspectives. The first aim is to relate the chemical anatomy of the hair cells in the horizontal canal crista ampullaris to primary afferent response dynamics. The second aim is to identify the relationship between hair cell transmitter phenotype, hair cell morphology, and hair cell activation and response kinetics. The third specific aim is to evaluate the effects of pharmacological manipulation of GABA transmission on the horizontal canal nerve and on individual afferent fibers. The role of transmitter phenotype in shaping primary afferent activity is a fundamentally and clinically important aspect of vestibular function that is poorly understood. Most studies will be conducted in toadfish, which are experimentally advantageous because of their availability, the ease of exposing and extracting their labyrinths, and the similarity of vestibular endorgans throughout the vertebrate phyla. Additional studies in mouse are proposed in order to relate findings in toadfish to a mammalian vestibular system, thus establishing the generality and human health relevance of the results. The proposed research is unique, and will have direct bearing on the development of more effective therapeutic interventions, both medicinal and device-oriented, for peripheral vestibular disorders. [unreadable] [unreadable] [unreadable]"}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. During the reporting period, we have completed the characterization of the prairie vole BAC library and published a paper describing the library. Using end sequencing of random BAC's we have identified more than 2000 SNPs in more than 300 loci. In addition, we have isolated BAC clones containing the genes for 20 behaviorally relevant genes (e.g. oxytocin, vasopress, CRF and their receptors, steroid receptor, etc), and a manuscript is in preparation describing these loci. Using the SNPs and a pedigree with more than 300 individual prairie voles we have created a first generation linkage map of the prairie vole genome. This map will be useful in assembling the prairie vole genome. The Broad Institute is currently sequencing the prairie vole genome."}
{"text": "Quantifying Environmental Variables Affecting Airborne Influenza Transmission In most temperate climates, influenza prevails in cold, dry winter months. However, in some temperate and tropical regions, influenza epidemicity is correlated with extremes of precipitation, not dryness, and can circulate at low levels essentially year-round or appear in uni- or bi-modal annual outbreaks. How environmental variables affect influenza circulation in the human population remains poorly understood, in part because the science behind airborne respiratory virus transmission crosses disciplinary boundaries between the biomedical and physical sciences, encompassing fields as diverse as virology, physiology, epidemiology, fluid mechanics, aerosol science, and climatology. Here we seek to understand how individual environmental variables - such as temperature, humidity, and airflow - cumulatively affect the transmission probability of influenza viruses in a representative mammalian experimental system. The theoretical framework behind these studies is a novel quantitative model, based upon data gathered in experimental guinea pigs, which attempts to characterize the impact of the environment on influenza virus transmission between infected and susceptible hosts. This project bridges the gap between virology and engineering in bringing together three co- investigators with relevant and complementary skill sets: Dr. Nicole Bouvier, a physician-scientist with extensive experience in the transmission of influenza viruses among guinea pigs; Dr. William Ristenpart, an engineer with expertise in the application of high-speed imaging technologies to investigations of complex fluid dynamics; and Dr. Anthony Wexler, an authority on aerosol transport who has developed novel techniques for high-resolution imaging of aerosol deposition in the rodent respiratory tract. Our preliminary theoretical modeling has generated innovative interpretations of the experimental data, yielding three testable hypotheses, which form the basis of this proposal: (1) Influenza virus transmission probability will decrease with increased airflow speed, (2) transmission probability will decrease with the degree of turbulence, and (3) transmission probability will increase with the time integral of the viral concentration within the inoculated animal. Rigorously controlled laboratory studies, designed to isolate a single variable for analysis while others are held constant, will provide a quantitative framework for understanding the cumulative effects of temperature, humidity, airflow velocity, turbulence, and position on the transmission of human influenza viruses in a relevant animal model. Quantifying these environmental variables, individually and cumulatively, will enable their extrapolation to larger environments and time scales, with the potential to transform our understanding of the epidemiology of seasonal and pandemic influenza."}
{"text": "Enabling diagnostic technologies that merge the use of nanoscale components and miniaturized detector systems could have a great impact in point-of -care diagnostics. In particular, diagnostic systems that are sensitive, robust, portable, low-cost, and can accurately quantify multiple molecular targets in parallel, would have a tremendous impact in biomedical research and molecular diagnostics. In this application, we propose to use a magnetic nanosensor technology, in conjunction with a portable magnetic relaxometer, to develop a sensitive diagnostic test for intracellular pathogens and toxins in clinical samples. Bacterial infections are on the rise in the United States and their economic impact on the healthcare sector is significant. The presence of enterohemorrhagic E. coli O157:H7 in tainted produce, the B. anthracis attacks in 2001, and the recent identification of drug resistant Mycobacterium tuberculosis have sparked public concern and underscore the need to develop sensitive and portable diagnostic technologies for fast and accurate detection of bacteria and toxins in food and clinical samples. Our approach utilizes \"magnetic relaxation nanoswitches\" (MRnS), i.e. magnetic nanoparticles that selectively change the spin-spin relaxation times (T2) of surrounding water molecules (NMR signal) upon specific molecular target interaction. The principal investigator of this grant application has previously shown that this technology can detect oligonucleotides in the femtomolemole range with extremely high molecular specificity [Nature Biotech. 2002;20:816-820], as well as other molecular targets such as proteins and viruses, without the need of target amplification. Most recently, we have developed magnetic nanosensors for the detection of a particular bacterium in blood and milk [Nanoletters 2007; 7, 380]. Also, with collaborators at the Army's Edgewood Chemical and Biological Center, we have gathered preliminary data to detect ricin toxin, using magnetic nanosensors. As a detector, we have used a newly developed minituarized and portable NMR relaxometer that allows substantial improvement in detection threshold and speed. The overall goal of this application is to extend research on magnetic nanosensors in an effort to develop highly sensitive, minituarized and portable detection systems to screen for the presence of a particular bacterium and toxin in clinical samples. We hypothesize that our magnetic nanoparticle system can provide a single homogeneous assay that can be used to detect the presence of intracellular pathogens and toxins in blood, without the need for target amplification. As an intracellular bacterial pathogen model, we will use Mycobacterium avium spp. paratuberculosis (MAP). As a toxin model, we will use anthrax toxin (AT), which is produced by another intracellular pathogen; B. anthracis. PUBLIC HEALTH RELEVANCE In this project, we will develop a diagnostic technology that merges the use of magnetic nanoparticles and a miniaturized detector to monitor the presence of an intracellular pathogen and toxin in clinical samples. This technology could facilitate quick, selective and sensitive bacterial detection to support clinical decision making. [unreadable] [unreadable] [unreadable]"}
{"text": "Microarray-based information, now routine in most medical research communities, pervades most aspects of diagnostics, sensing, biotechnology, oncology and pathology. Detection limits and selectivities for DNA targets are far below theoretical performance limits, but very little information is reported or known about the chemical, physical or biological fate of full length DNA, cDNA or oligo-DNA probes immobilized on any of the diverse set of microarray surfaces. How immobilized DNA surface disposition influences subsequent hybridization efficiency, and reliability of array data interpretation and assay quantification is unknown. Quantitative interpretation of DNA microarray signal intensity is currently very difficult since factors influencing DNA probe-target interactions at microarray surfaces have not been analyzed with high-resolution surface analytical methods often applied to other biomedical surface problems. Our hypothesis is that DNA microarray target hybridization efficiency and diagnostic target detection limits in biomedical samples are correlated directly with the orientation, density, and immobilization efficiency of probe DNA on microarray surfaces. To investigate this hypothesis, we propose the following Specific Aims: 1. Establish a quantitative understanding of the correlation between immobilized probe DNA density on microarray surfaces and target hybridization efficiency in biological samples using radiometric 32P-DNA assay and optical imaging on several surface chemistry platforms and assess reliability and reproducibility issues in these strategies; 2. Develop reliable, quantitative methods for high-resolution surface analysis of DNA density and orientational populations on ss-cDNA and hybridized ds-DNA on arraying surfaces using modem biomedically relevant methods (XPS, ToF-SIMS and optical anisotropy of immobilized DNA). The overall objective is to correlate high-resolution surface analytical data on DNA arrays with radiometric measurements to establish a non-radiometric 'standard curve' to assess DNA immobilization on microarray surfaces more conveniently and accurately. Moreover, orientational information on immobilized DNA using a combination of innovative spectroscopy methods will be correlated to immobilized DNA density and hybridization efficiency in array formats. All methods will converge to produce a fundamental understanding of microarray surface & hybridization performance limitations currently not available."}
{"text": "Cocaine addiction continues to be a major health and social problem in the United States and other countries. Currently employed pharmacological agents for treating cocaine abuse have proved inadequate, leaving few treatment options. One alternative is to use protein-based therapeutics that bind cocaine, thereby blocking its effects, and/or degrade cocaine via hydrolysis of the benzoyl ester. This approach is especially attractive because the therapeutic agents exert no pharmacodynamic action of their own and therefore have little potential for side effects. The effectiveness of these agents, however, is limited by their inability to act directly within the central nervous system (CNS). We seek to address this deficiency by engineering filamentous bacteriophage, which have been shown to penetrate the CNS when administered intranasally, to display cocaine-sequestering proteins on its surface. Specifically, the aims of this proposal are: (1) construction of phagemid vectors that contain the genes of different cocaine-inactivating proteins as fusions with genes of phage coat proteins; (2) preparation and evaluation of phage displaying the various proteins of interest; (3) use of a locomotor activity rodent model to evaluate the ability of phage-displayed proteins to block the psychoactive effects of cocaine; (4) evaluation of phage distribution in the animals using immunohistochemical and molecular biological methods; (5) evaluation of a potential humoral immune response to the phage in the animals."}
{"text": "The overarching goal of this competitive revision application is to extend the mission of the University of Alabama at Birmingham (UAB)-University of California, San Diego (UCSD) O?Brien Center for Acute Kidney Injury (AKI) Research by defining the impact of AKI on the excess burden of chronic kidney disease (CKD) in the Southeastern US. The clustering of major CKD risk factors such as black race, obesity and diabetes in our region is a key driver of the high prevalence of CKD in the Southeastern US. As such, identifying potential interventions to reduce the incidence and progression of CKD in these vulnerable populations is critical for reducing the substantial social and financial costs associated with CKD and related health disparities (stroke, heart disease, death) in our region. AKI affects as many as 20% of hospitalized patients and is a recognized risk factor for the development of CKD, cardiovascular disease and death. These outcomes may be modifiable by care provided in the post-AKI period, yet optimum care for care of AKI survivors is not well defined and is often fragmented after discharge. Disadvantaged patients are at especially high risk for inappropriate follow-up and low quality of care, suggesting that disparities in AKI outcomes contribute to the excess risk of incident and progressive CKD in high-risk populations in our region. However, progress in this area is hampered by a lack of data on the degree to which differences in AKI outcomes contribute to CKD disparities in the Southeastern US, and potential interventions that may target these disparities. Accordingly, the primary focus of this application is to define the impact of AKI during acute hospitalization on incident and progressive CKD; how these associations differ by race, obesity and diabetes status; and pilot a dedicated AKI follow-up clinic as a potential intervention to address disparities in these outcomes. Specifically, Aim 1 will leverage an established, ongoing regional CTSA collaboration?the Southeastern Shared Health Research Information Network (SE-SHRINE)?to examine all hospitalized AKI patients within four centers (UAB, MUSC, UAMS, U Kentucky) and link them to the US Renal Data Systems to define disparities in AKI outcomes ([i] severity and duration of AKI; [ii] recovery of dialysis-dependent AKI or development of ESRD after discharge; and [iii] associated health disparities such as death and cardiovascular disease) by race, obesity and diabetes. Aim 2 will assess the feasibility and collect key efficacy measures of a dedicated AKI follow-up clinic for patients with AKI discharged to the community at three Southeastern CTSA hubs (UAB, Vanderbilt, U Kentucky). We will leverage the resources available in O?Brien Cores to integrate existing intellectual and technological resources of UAB and facilitate the success of this project. This project will help build a collaborative research network for AKI research that could serve as a platform for multi-center, transdisciplinary science and clinical trials focused on reducing the burden of CKD and related health disparities in the Southeastern US."}
{"text": "Institutional Racism is a process or set of policies which directly or indirectly produces differentially negative outcomes for minorities (Feagin, 1975). The result is an inequitable distribution of resources. Since the availability of resources and alternatives for problem solving is critical for coping with problems of living, Institutional Racism remains an important mental health concern with implications for individual, organizational, and community effectiveness. A workshop or convocation of scholars is planned to discuss and elaborate the unique perspectives and needs of Asian Americans, Afro-Americans, Latinos, and native Americans. In addition it will seek to explore current interventions for Institutional Racism and to exchange strategies of altering social systems in ways that make them more responsive to needs. Finally, following the workshop, a book will be published integrating the notions of Institutional Racism and Community Competence."}
{"text": "We intend to investigate and develop new, efficient and general synthetic methods for stereospecific and regiospecific carbon-carbon bond formation. The photochemistry and alkylation of vinyl sulfides, a potentially interesting, but generally overlooked, organic functionality, will be studied. The proposed photochemical ring annelation method, involving formation of a transient thiocarbonyl ylide followed by reaction with a dipolarophile could be a flexible and highly stereospecific technique for assemblage of a complex ring network. We hope to use this method for the construction of steroid and steroid-like molecules of potential biological interest. Oxidation of photogenerated dihydrothiophenes affords naphtho(2,1- b) thiophenes, derivatives of which have recently exhibited significant antimalarial activity. Regioselective alkylation of enolates derived from Beta- and gamma- keto-vinyl sulfides could be developed into a valuable synthetic method. Sulfur has been chosen as a temporary directing group because of its flexibility in recently reported synthetic operations. We hope to utilize these keto-vinyl sulfides as precursors to prostaglandins and prostaglandin-like intermediates. The undersigned agrees to accept responsibility for the scientific and technical conduct of the project and for provision of required progress reports if a grant is awarded as a result of this application."}
{"text": "DESCRIPTION: (Adapted from the application) Why do some people die at 60, more (in most developed countries) at 80, and a few at 100? Why is the chance of dying at 80 rather than 60 increasing and the chance of dying at 100 rapidly increasing (albeit from a very low level)? How important are genetic vs. environmental, behavioral, and medical factors in determining how long an individual will live? It might be expected that the answers to these questions--and the determinants of longevity more generally--are well understood. Surprisingly little, however, is known. The continuation of this program of demographic research on the oldest-old focuses on mechanisms and determinants of survival and longevity. The theoretical foundation that underlies the research and the conceptual framework that ties the various projects together are derived from the perspectives and methods of demography. The research program emphasizes demographic research on the genetic and non-genetic determinants of longevity, including research on the interaction between fertility and mortality and research on why age-specific mortality decelerates with age. Data from longitudinal surveys of thousands of elderly Danish twins and more than 10,000 Chinese oetogenanans, nonagenanans, and centenarians will be gaffiered and analyzed. Biodemographic experiments on the mechanisms and determinants of longevity will be conducted on several model species, inncluding Ceratifis capitam, Drosophila melanogaster, Melonoplus sanguinpes, Saccharomyces cerevisiae, and Plantago lanceolata. New demographic and statistical methods of survival analysis will be developed and applied to analyze these data. The data will be released as public-use files."}
{"text": "This proposal is designed to define the molecular basis of sickle cell adhesion to the endothelium. Vasoocclusion is believed cause the painful, reoccurring crisis suffered by sickle cell patients. Recent evidence suggests that this vasoocclusion may be precipitated by sickle cell adhesion to the endothelium. The molecules involved on the sickle cell surface, the endothelial cell surface, and any intermediary ligands have not been clearly identified. Our working hypothesis is that sickle cells and possible normal reticulocytes contain a receptor or receptors that bind to a ligand such as unusually large von Willebrand factor (ULvWF), which in turn binds to an endothelial cell receptor(s), precipitating the vasoocclusive crises in sickle cell disease. The specific aims of this grant are fourfold. First, we propose to identify and characterize the receptor(s) on the sickle cell surface that mediate adhesion to endothelial cells. This will involve a variety of approaches such as flow cytometry and sorting, immunoelectron microscopy, monoclonal antibody production, and adhesion assays. Second, we will identify and characterize the receptor(s) on the endothelial cell surface that mediate adhesion to sickle cells, using techniques similar to those listed above, and by testing specific receptor-deficient mutants in an endothelial-like cell line. Third, we will evaluate the role that ULvWF and other potential ligands play in the adhesion of sickle cells to endothelial cells. Finally, we will examine the contribution of activated platelets and platelet secretory products to the adhesion of sickle cells to endothelial cells. Completion of these studies should result in a greater understanding of the molecular events that contribute to the damaging, painful events in sickle cell disease and should provide a rationale for future treatment of this disease."}
{"text": "The overall goal of the proposed studies is to define the mechanisms by which MD-2, a co-receptor to TLR4 participates in LPS-induced signaling, to investigate the regulation of MD2 promoter and to characterize the expression, function and regulation of an alternatively spliced version of MD2 (MD2-S) that occurs in human but not mouse tissues. MD-2 is a secreted surface protein, which is required for LPS/TLR4 signaling. A fundamental gap in our understanding of LPS/TLR4/MD-2 signaling is the exact function of MD-2 and the molecular mechanisms, which make MD-2 a critical component of the LPS/TLR4 signaling complex. The regulatory mechanisms of the LPS/TLR4/MD-2-mediated inflammatory signaling at the level of MD-2 and the role for alternative splicing of human MD-2 in this regulation are also unknown. We have observed that an alternatively spliced version of MD-2 exists in human tissues. The studies proposed in this application will seek to characterize and define the functional role and the regulation of the alternatively spliced form of human MD-2 and will investigate the role of MD-2 as signaling molecule and the regulation of the human MD-2 promoter. The hypothesis: Based on our preliminary data, we hypothesize that altematively spliced isoform of MD-2 (short MD-2 or MD-2 S) is unable to transduce LPS/TLR4 signaling and that it may play a negative regulatory role in LSP/TLR4/MD2-induced inflammatory signaling at the level of MD-2. Based on preliminary data, we further hypothesize that MD-2 participates in LPS signaling, and is tyrosine phosphorylated upon LPS stimulation and internalization and that mutations of the tyrosine sites in MD-2 diminishes it's function to transduce LPS/TLR4 signaling. We propose that MD-2 may provide a potential regulatory step in TLR4/MD-2-mediated inflammatory signaling. We have recently cloned the promoter site of MD-2 and we propose to investigate the regulation of MD-2 expression by investigating the regulation of MD-2 promoter. Thus, MD-2 may provide an additional potential regulatory step in LPS/TLR4/MD2-induced inflammatory signaling. The Specific Aims are: 1- To characterize and determine the tissue expression and function of alternatively-spliced MD-2 isoform (MD-2 S) and expression of recombinant MD-2 S to investigate its function in LPS/TLR4 signaling in in-vitro and in-vivo experiments. 2-To define the molecular signaling mechanisms for the involvement of MD-2 as a signaling molecule in LPS/TLR4-mediated NF-kB activation. Investigate the role of Tyrosine phosphorylation of MD-2 upon LPS signaling. 3-To investigate the regulation of MD-2 promoter, and to determine cytokine responsive elements in the promoter. Significance: Improved understanding of the molecular mechanisms by which MD-2 participates in LPS/TLR4 signaling and the understanding of the functional role of alternative splicing of MD-2 and of the regulation of MD-2 promoter may provide new targets for intervention and prevention of inflammatory diseases where TLR4/MD-2 signaling plays a major role, including gram-negative sepsis and endotoxic shock."}
{"text": "[unreadable] Our goal is to improve the versatility, safety and effectiveness of E. coli as a platform for commercial production of DNA, proteins and metabolites. The natural E. coli genome will be reduced to its essentials by removing unnecessary, detrimental and potentially harmful genes through our Scarless Genome Reduction (SGR) technology. The result will be a robust, benign, environmentally friendly organism which can be modified for production as needed. DNA applications include vaccines, gene therapy and RNAi based therapeutics. Protein applications include drugs, hormones, industrial enzymes and some antibiotics. Metabolites include many antibiotics, vitamins, amino acids as well as industrial chemicals and fuels such as ethanol and hydrogen. As the technical ability to manipulate genomes grows, it will be possible for reduced genome [unreadable]. coli to take on many applications previously reserved for other organisms or chemical processes. In the Phase I work, the E. coli genome was reduced to the point that there were no more IS transposable elements. These elements have the capacity to hop around the genome and inactivate genes including the genes for products. The new IS-free strains have remarkably improved properties, including a greater tolerance for high level product expression, greater cloning ability, and better stability. IS hopping is one of the ways E coli defends itself from the stress of production which, from the bacterial point of view, resembles an infection. For production of medicines, the cleanness of the genome is a benefit because purification of product is less difficult and good quality assurance and quality control are more readily attainable. For injectable DNA preparations, the elimination of IS hopping is a tangible safety benefit, which will avoid the risk of transposition into a chromosome of a patient. Finally, the cells grow robustly to high density in industrial fermentation conditions using inexpensive minimal salts media, alleviating concern about prion contamination from bovine-derived media additives. This Phase II project will explore a variety of commercial applications which have been brought to our attention by companies interested in our product. [unreadable] [unreadable] [unreadable]"}
{"text": "We propose to study the mechanism by which several restriction enzymes recognize, interact with and hydrolyze specific nucleotide sequences. This study will involve a) the physical-chemical characterization of restriction enzymes as proteins; b) the kinetic characterization of DNA hydrolysis; and c) the characterization of the enzyme-DNA binding reaction in the absence of hydrolysis. In addition, we will attempt to grow crystals of at least one restriction enzyme bound to its specific recognition sequence for analysis by x-ray diffraction techniques. One of the major goals of the proposed research will be to develop a method for the physical mapping for restriction enzyme binding sites on a DNA molecule by the electron microscopic visualization of the position of the bound enzyme. This technique would provide a simple and rapid analysis of the positions of specific nucleotide sequences, and would have a wide variety of applications in studying nucleic acid structure and function. We will ultimately attempt to extend this method to an analysis of DNA-RNA hybrid molecules of biological significance, such as reverse transcriptase products of RNA tumor virus genomes. Our proposed study of restriction enzyme mechanisms requires an analytical method capable of resolving and quantitating all reaction intermediates and products. We have developed agarose gel electrophoresis to a degree of resolution which allows us to start with a closed circular DNA in the molecular weight range 3 to 10 million, and to resolve from each other all conformational isomers of a given size, the full-length linear and circular single strands, and all linear intermediate and final cleavage products generated by a given restriction nuclease. We propose to further develop this method for studying DNA-protein interactions, for restriction enzyme analysis of intermediates in DNA replication and recombination, and for other applications requiring ultra-high resolution."}
{"text": "The Jackson Laboratory Cancer Center (JAXCC) is an organizational unit with dedicated administrative oversight that is strategically integrated with the parent organization's administration. Dr. Edison Liu, JAXCC Director, heads the Cancer Center administration, working with other members ofthe JAX executive committee, the Senior Management Team; Drs. Braun, (Center Deputy Director), Donahue (Director of Genetic Resource Science and co-Project Leader Cancer Models Development Resource) and Hewett (JAX Executive Vice President and Chief Operating Officer). Dr. Barbara Tennent, Associate Director for Research Administration provides dedicated administrative support to the JAXCC and reports to Dr. Liu. Two committees comprise the formal administrative structure ofthe JAXCC. The Operations Committee, chaired by Dr. Liu comprises Drs. Braun, Hewett, Nair and Tennent, and works to ensure that the overall direction ofthe JAXCC operations anticipates and supports the needs of JAXCC members. The Shared Resources Management Committee, which comprises Ms. Valerie Scott, Senior Director of Scientific Services, Ms. Yu-Hui Rogers, Site Director, JAX Genomic Medicine, and Drs. Donahue and Tennent coordinate the operations and development of CCSG-supported shared resources across campuses. Funds are requested for partial support of Dr. Tennent's administrative responsibilities. She oversees the necessary planning, policy development, budget development and research developmental activities for the ongoing growth ofthe JAXCC cancer research focus. Dr. Tennent is the primary coordinator for scientific and administrative management of the JAXCC. She is an ex officio member of the Scientific Executive Committee and serves on the Operations Executive Committee and the Shared Resources Committee. She works closely with all members ofthe administrative team to coordinate the activities ofthe JAXCC, including organization of the External Advisory Board meetings, management ofthe pilot project process, and membership records. Dr. Tennent is the primary administrative contact for NCI Cancer Center program officials and is responsible for management ofthe renewal, progress reports and requests from NCI as required. Institutional funds support dedicated effort for Ms. Scoff, who oversees the four institutional scientific services for which CCSG support is requested. She is responsible for advocacy, policy development and application, budget management, operations, quality assurance, technical staffing and development, planned technology acquisition and implementation as well as user communications. Institutional funds also support Ms. Roger's together in implementing processes, procedures and management structure that facilitate efficient resource sharing and access to CCSG-supported resources. The JAXCC is strongly supported by institutional administrative offices including the Office of Sponsored Programs for pre and post award administration, compliance, and financial management of the CCSG; Financial Services for budget development and account management; and Scientific Program Development, for securing external funding from foundations and federal agencies for research, education, and resource programs."}
{"text": "CORE COMPONENT G: ADMINISTRATION The administrative core supports all administrative and budgetary aspects of the Program Project. These support services include: 1. Clinical Coordinator Team - a group of physicians and a clinical specialist who are responsible for the initial contact with referring physicians and patients, patient financial counseling, scheduling and protocol assignments. 2. Protocol Management Team - a group of individuals responsible for coordinating protocol development, review, renewal and monitoring. 3. Grant Coordinator - the individual responsible for financial management of this Program Project grant. 4. Research Nurse Facilitator- a research nurse specialist responsible for facilitating the initiation and conduct of clinical trials. 5. Word Processing andAdministrative Services - a group of individuals responsible for word-processing manuscripts and submitting manuscripts for publication;updating citation and grant databases;creating presentation materials for internal investigator meetings and external scientific conferences;scheduling lectures, visitors and conferences. Relevance to Public Health: By providing essential administrative support, this core assists all projects on this grant in achieving the goal of making allogeneic hematopoietic cell transplantation safer, more effective, increasingly available, and applied more appropriately in the course of disease so as to maximize benefit and minimize risk."}
{"text": "The menisci are C-shaped fibrocartilage disks that occupy the periphery of the knee joint and serve a number of significant mechanical functions including load distribution across articular cartilage, and joint stabilization. In the face of such mechanical demands, it is not surprising that the meniscus can become damaged. Unfortunately, surgical options for the treatment of damaged menisci are limited, primarily due to the poor vascularity and healing capacity of the central two thirds of the meniscus. Allograft implantation is complicated by the threat of transmission of infectious diseases, the difficulty of matching meniscal shape and material properties, and problems with maintaining cell viability. While meniscectomy (the complete or partial removal of the affected tissue) which is the most common orthopedic procedure performed in the United States can lead to the early onset of osteoarthritis. Providing a meniscal substitute engineered to function much in the way of the native tissue could delay or even avoid the onset of osteoarthritis. Meniscal substitutes, whether cell-seeded degradable scaffolds or synthetic implants, should ideally help to distribute loads across adjacent articular cartilage much in the way of the native tissue, thereby protecting the underlying articular cartilage. However, the properties of a substitute that are required to ensure that this functional capability is met are unclear. This is caused in part by the absence of a robust, comprehensive and physiologically relevant model within which the effect of substitute design variables can be parametrically studied. This lack of such a test, or series of tests, makes the design and evaluation of candidate scaffolds impossible, retards the regulatory pathway to commercialization, and leads to a situation where scaffolds are implanted in humans with little information about their ability to perform mechanically in the highly-loaded environment of the knee joint. Our goal is to define the relationship between meniscal substitute material and structural properties and joint contact mechanics under multiple physiological activities across a range of patient populations. The main element of the framework required to achieve this goal is a statistically-based, rapidly computable interpolator that will be built using results from experimentally-validated knee specific computational models. We will gather information about factors that most markedly affect the functional performance of the native meniscus and use this information as a template to identify the design space into which substitutes should fall if they are to function appropriately. Our goal is not to design a meniscal substitute; rather, we will demonstrate that our approach can establish a workable design space for use by those developing solutions for meniscal repair. Our efforts will culminate in the development of a new paradigm for the development and screening of any complicated tissue substitute, whether a non-degradable implant or a cell-seeded degradable scaffold prior to initiating more time consuming and costly animal and clinical trials. PUBLIC HEALTH RELEVANCE: Our goal is to define the relationship between meniscal material and structural properties and joint contact mechanics under multiple physiological activities across a range of patient populations. The main element of the framework required to achieve this goal is a statistically-based, rapidly computable interpolator that will be built using results from experimentally-validated computational models of the intact and meniscal substituted knee. Our approach will serve as a tool for rapid evaluation and design of functional substitutes and provide a new paradigm for the development and screening of any complicated tissue substitute, whether a non- degradable implant or a cell-seeded degradable scaffold."}
{"text": "Success in science requires a variety of professional \"survival\" skills as well as the background and research experience normally provided by pre- and post-doctoral training programs. Thus, ultimately most researchers must learn to communicate effectively (orally or in writing), teach, supervise and mentor, obtain a job and funding, and behave responsibly. Over the past 15 years the PIs have developed a course to provide students with explicit training in these subjects. In addition, they now offer annual trainer-of-trainers workshops in which faculty obtain the instruction and materials necessary to establish such courses at their own institutions. In this proposal, funding is requested to extend their efforts at disseminating their model for providing training in survival skills and ethics. There are three specific aims: (l) To increase the implementation of courses in survival skills and ethics at other institutions. This would be accomplished by providing 36-40 scholarships that will cover the cost of participating in the trainer-of- trainers workshop and provide faculty with small start-up grants for establishing courses in survival skills and ethics at their institutions. (2) To promote the sustainabilitv of courses implemented. This would be done by continuing to assist faculty who attend future (or have attended former) trainer-of-trainers workshop in sustaining courses and workshops in survival skills and ethics by providing them with on-going consultation and support services including updates of materials. (3) To strengthen and expand the evaluation of project effectiveness. This would be done through surveys of participants efforts to implement courses. In these ways the PIs seek to improve graduate and postdoctoral education, promoting explicit instruction in many of the skills that are necessary for success as a professional, yet are often not formally taught in graduate training programs in the sciences."}
{"text": "The research described in this project is designed to ask and provide answers to basic questions about the structure and functions of the outer membrane of Gram-negative bacteria. Specifically, it will yield knowledge about membrane protein tertiary and quaternary structure, provide specific information about how an important class of transmembrane proteins (the porins) form hydrophilic channels through the membrane, and give insights into the relationships between the various components of the outer membrane. Gram-negative bacteria are important enteric and systemic pathogens. The outer membrane and its related structure play key roles in colonization, immunogenicity, viability and the antibiotic response of these organisms. A better knowledge of the structure and function of the outer membrane will aid in the development of antibiotics and treatments for pathological conditions caused by these organisms. The main thrust of the proposed research is the in-depth use of genetic and biochemical approaches to isolate and characterize Escherichia coli mutants which have specific altered properties of the outer membrane. One approach that will be used is to select for the uptake of substrates which normally do not traverse the outer membrane barrier. This selection yields mutants which have altered porin proteins. These alterations impart a hypersensitivity to detergents and antibiotics. The hyperpermeable mutants will be analyzed phenotypically and the exact nature of the lesions in the gene for the porin proteins will be determined at the DNA level. The hyperpermeability phenotype exhibited by these mutants will be used to develop additional selections for the isolation of novel mutants which have acquired regulatory or compensatory mutations which reduce or eliminate the hyperpermeability phenotype. The genetically and biochemically analysis of the many mutant types isolated in this project will help to achieve the goals of the project."}
{"text": "Principal Investigator/Program Director (Last, first, middle): Cole, Ronald, A. RESEARCH & RELATED Other Project Information 1. * Are Human Subjects Involved? l Yes m No 1.a. If YES to Human Subjects Is the IRB review Pending? l Yes m No IRB Approval Date: Exemption Number: 1 2 3 4 5 6 Human Subject Assurance Number 00007232 2. * Are Vertebrate Animals Used? m Yes l No 2.a. If YES to Vertebrate Animals Is the IACUC review Pending? m Yes m No IACUC Approval Date: Animal Welfare Assurance Number 3. * Is proprietary/privileged information m Yes l No included in the application? 4.a.* Does this project have an actual or potential impact on m Yes l No the environment? 4.b. If yes, please explain: 4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an environmental assessment (EA) or environmental impact statement (EIS) been performed? m Yes m No 4.d. If yes, please explain: 5.a.* Does this project involve activities outside the U.S. or m Yes l No partnership with International Collaborators? 5.b. If yes, identify countries: 5.c. Optional Explanation: 6. * Project Summary/Abstract 1582-MENTORAbstract_SUMMARYPhaMseim_1e_Tanypde_:2a[1p]p.plidcfation/pdf 7. * Project Narrative 3538-MENTOR project_narrative[1].pdf Mime Type: application/pdf 8. Bibliography & References Cited 3318-MENTORNIHApr06References_r3[M1]i.mpdefType: application/pdf 9. Facilities & Other Resources 5600-MENTOR facilities.pdf Mime Type: application/pdf 10. Equipment 7794-MENTOR EQUIP.pdf Mime Type: application/pdf Tracking Number: Other Information Page 5 OMB Number: 4040-0001 Expiration Date: 04/30/2008 Principal Investigator/Program Director (Last, first, middle): Cole, Ronald, A. Project Summary Millions of children in the U.S. are at risk for learning to read well despite major advances in our understanding of the causes and cures for poor reading. In this Fast- Track proposal we propose to address this significant need and opportunity with a computer-based reading program that combines the power of one-on-one tutoring with the benefits of self-assessing, individualized, scientifically-based reading instruction. We plan to position this product, Foundations to Literacy\", as a service over the Web, so that it is accessible in classrooms and resource rooms, community learning centers and homes. The goal of the Phase I work is to demonstrate the technical feasibility of delivering an immersive, individualized one-on-one tutoring experience to children in organizations and homes using a scalable, cost-effective Application Service Provider (ASP) delivery model. Success in the Phase I project will be measured by satisfactory learning experiences by 16 children connected to a single server who are using the program simultaneously. The goal of the Phase II research and development effort is to enhance the functionality of the Foundations to Literacy program by incorporating a new set of learning activities into the program that teach children to read text fluently with good comprehension. Improving reading comprehension is a national priority, and research has shown that good comprehension requires sufficient vocabulary and the ability to read words in context fluently and automatically. We propose to develop a novel set of learning activities that facilitate acquisition of basic oral vocabulary-the meanings of spoken words-and also teaches children to recognize printed versions of these words accurately and rapidly. In addition, using speech recognition technology to measure the"}
{"text": "Diabetic patients are at high risk of a range of comorbid complications, including cardiovascular disease (CVD). The increased risk of cardiovascular events seen in patients with type 2 diabetes is associated with a cluster of risk factors for cardiovascular and metabolic disorders that tend to coexists in these patients. Despite successful implementation of evidence based strategies, many individuals are not identified as high risk before their first event and others continue to experience cardiovascular events despite optimal management. While much of this risk is attributable to the presence of conventional risk factors, such as hyperglycemia, hyperlipidemia, hypertension, a substantial burden of this risk remains unexplained. Inflammatory mediators and growth factors are increasingly recognized as playing important roles in the development of atherosclerosis. Therefore the overall objective of this proposal focuses on performing longitudinal assessment to define the role and contribution of a novel biomarker, connective tissue growth factor (CTGF), in the initiation/ progression of macrovascular and microvascular disease in subjects with type 2 diabetes and to determine whether increases in CTGF in the presence of microvascular disease will predict development of macrovascular disease. Our preliminary findings, based on cross-sectional data generated from a cohort of type 2 diabetic patients, suggest that the occurrence of cardiovascular events is independently linked to elevations in CTGF level. Our results demonstrate that diabetic patients with a prior history of myocardial infarction (MI) or coronary artery disease (CAD) have significantly higher levels of plasma CTGF than patients who have not had a prior cardiovascular event. We also uncovered a strong association between circulating levels of plasma CTGF and retinopathy ETDRS scores as well as albumin excretion rate (AER) in these type 2 diabetic subjects. In addition, our findings demonstrate that CTGF expression is induced in aorta of ApoE-/- knockout mice with atherosclerosis compared to control mice. Therefore, based on the preliminary data we generated, we hypothesize that higher levels of circulating CTGF reflect ongoing vascular damage from hypertension, endothelial dysfunction and inflammation, and that elevated CTGF levels will predict greater risk for future cardiovascular events and progressive retinopathy and nephropathy. To test our hypothesis we propose the following specific aims: 1) Determine whether plasma CTGF levels predict future cardiovascular events and progressive retinopathy and nephropathy in individuals with type 2 diabetes. 2) Determine the role and contribution of CTGF to the initiation and progression of diabetic vascular disease. The proposed studies should establish CTGF as a pathologically-important risk factor for diabetic vascular disease that will form the basis for defining new targets for interventional therapy."}
{"text": "Several lines of evidence have suggested that certain strains of Escherichia coli may produce an enterotoxin with physiologic properties similar to cholera-toxin. In this grant we propose to develop an in vitro assay system to measure E. coli enterotoxin. Purification of the toxin is continuing with the goal of producing an immunogenic product suitable for vaccine usage. The toxin assay system will also be employed in further clinical studies. E. coli strains from children with acute diarrhea will be tested for enterotoxin activity. Their serum will be examined for antitoxin activity based on neutralization of the toxin assay system."}
{"text": "The MBRS-IMSD Program at the University of California, Irvine (UCI) started in 1996 as a comprehensive effort to increase the number of underrepresented minority (URM) undergraduate and graduate students pursuing biomedical research careers. During the last funding cycle (2004-07), the program has increased the rate at which undergraduate participants enter PhD programs following graduation and the number of URM students completing PhD programs at UCI. Independent paid research conducted under the direction of faculty mentors serves as a core element to induce MBRS students to pursue graduate school and research-focused careers. Over 100 faculty with funded research programs serve as preceptors of MBRS trainees. The program offers a series of components to increase the interest, motivation and academic preparedness of undergraduates (freshmen to seniors) to enter PhD programs in biomedical sciences, including a peer tutoring/mentoring program of science classes, a seminar series, workshops on laboratory methods, scientific communications, GRE preparation and application to graduate school. The graduate component of the program is designed to provide a comprehensive training for URM Ph.D. students to excel in graduate school. The program provides summer research training for incoming URM PhD students to prepare them for the core graduate classes, a workshop on extramural funding, counseling and orientation about the graduate studies in a non-departmental setting, a workshop to prepare oral exams after the first year in the PhD program and preparation for advancement to candidacy and dissertation research. Public Health Relevance: The inclusion of underrepresented minorities in the biomedical scientific workforce is critical to address the need of improving the health of the people of the United States and eliminating health disparities in the nation. This project will improve the quality and quantity of undergraduate and graduate students from underrepresented groups being trained as the next generation of biomedical research scientists."}
{"text": "The present proposal aims to analyze various physiological and pharmacological properties of adult and developing mammalian extraocular muscles (EOM) and to study some aspects of the ultrastructure of rate EOM using serial sectioning. The study of the electrical properties of rate EOM fibers with intracellular microelectrodes will be continued with emphasis on the slow multiply-innervated fibers and the synaptic transmission. In rabbit EOM, the possibility that the morphological variations described along the multiply innervated fibers are accompanied by functional variations will be explored. The analysis of the different types of contractile responses of rat EOM subjected to various treatments, will be continued to establish the contribution of the different types of fibers to the total tension and to determine the mechanism of action of cholinergic drugs. The changes of the contractile and electrical properties of rate EOM during the first three weeks after birth will be studied to define the differentiation process of these muscles."}
{"text": "Project Summary The expansion of access to ART has the potential to greatly benefit the physical, social and economic well being of individuals and households, as well as produce unintended negative consequences with regard to increased risk behavior and social disruption which can compromise its effectiveness. Drawing on Social Cognitive Theory, the proposed 4-year study will examine (1) how depression influences multiple health outcomes of patients and their households including economic output, household constellation, children's school attendance, and sexual risk behaviors, both directly and as a moderator of impact of ART; and (2) the impact of antidepressant treatment in mitigating the negative consequences of depression on these health outcomes in the context of ART scale-up in Uganda. The study will consist of two phases. In Phase 1, semi- structured qualitative interviews with ART patients, clinic providers, administrators, and support service organizations will be used to explore (1) the impact of HIV and ART on patients' physical, mental, social, economic and sexual health; (2) how mental health and depression influence these ART outcomes; and (3) attitudes towards and acceptability of depression treatment and antidepressants. Findings will help develop and refine hypotheses and measures for use in Phase 2. In Phase 2, patients entering HIV care and starting ART will be followed for 12 months in a longitudinal prospective cohort study. Those diagnosed with depression will be offered antidepressant treatment. Data from our two other just-funded observational cohort studies of ART and non-ART patients in Uganda, which include a subgroup of patients who are depressed but not receiving depression treatment, will provide important additional comparative data for evaluating the effects of depression and antidepressant therapy. These findings could provide critical information for the development of interventions and public health policy aimed at maximizing the benefits of ART in limiting HIV transmission and the social and economic consequences of the epidemic in the developing world, including the prioritization of improved diagnosis and treatment of depression."}
{"text": "We have discovered the first experimental system for in vitro assembly of gap junctions. Assembly of gap junctions is pre-conditioned by disruption of cytoskeletal elements and proceeds even in the presence of inhibitors of protein synthesis. We now want to investigate the role of temperature in the assembly process, the ontogenetic and structural relationship between tight and gap junctions and the role of lipid molecules in the structure of the connexon (its building unit). This project has been interrupted for lack of personnel and aacess to freeze-fracture equipment."}
{"text": "Modern biomedical research relies on interdisciplinary approaches such as bioinformatics that synthesize knowledge and methods from other disciplines to provide an integrated framework for solving biomedical problems. The rapid advancement of high-throughput technologies for measuring biological systems has generated a significant demand at Dartmouth College and other research institutions across Northern New England for interdisciplinary approaches in the quantitative sciences (e.g. bioinformatics, biostatistics, genomics, mathematical biology, proteomics, and systems biology). Integrating high-dimensional research databases with clinical databases from medical schools and hospitals across the region will be needed for translational medicine to become a reality. Unfortunately, the research institutions in Maine, New Hampshire, and Vermont are in a largely rural setting have not kept pace those in larger metropolitan areas such as nearby Boston or New York. The goal of this COBRE program is to establish a Quantitative Biology Research Institute (QBRI) that will support and enhance quantitative biology research across the region and facilitate its integration and synergy with experimental and observational biology. This will be accomplished by 1) establishing a Quantitative Biology Research Institute (QBRI) focused on developing, supporting, and enhancing quantitative biology research in Maine, New Hampshire, and Vermont that will become nationally and internationally recognized, free standing, and will foster meaningful collaborations with experimental biologists thus improving the ability of investigators in the region to compete for NIH funding, 2) recruiting talented tenure track quantitative biologists to Maine, New Hampshire, and Vermont, 3) mentoring the development of four junior quantitative biologists across the region and 4) promoting synergistic collaborations between quantitative biologists and experimental biologists through four research projects, an Administrative Core and an Integrative Biology Core. The scientific focus of the four research projects is gene-environment interaction within the context of environmental health and toxicology. This provides an important unifying and synergistic theme for the COBRE."}
{"text": "Parathyroid hormone (PTH) is a key regulator of bone metabolism and is used as anabolic pharmacotherapy for treatment of osteoporosis. Recent evidence suggests that lipids may affect bone metabolism and that intermittent PTH therapy may fail in the presence of hyperlipidemia. This is clinically important because hyperlipidemia is prevalent in patients with osteoporosis even accounting for age. In the hyperlipidemic condition, bioactive derivatives of low-density lipoproteins (LDL) are generated in the subendothelial space of tissues, triggering chronic inflammatory responses including oxidant stress and expression of cytokines and chemokines. We have found that these inflammatory lipoproteins/lipids are also present in bone and that they inhibit osteoblastic differentiation. In additional studies, we and other investigators demonstrated that hyperlipidemic mice have reduced bone density compared to WT mice. Our preliminary studies, both in vitro and in vivo, now show that lipids inhibit PTH-induced immediate early genes, including Nurr1, a transcriptional regulator of osteoblastic genes, by attenuating cyclic AMP production and that hyperlipidemia blunts PTH-induced osteoanabolism in vivo, primarily in cortical bone. These findings strongly suggest that hyperlipidemia induces PTH resistance. Whether PTH resistance is at the molecular and/or tissue level remains to be determined. Since osteoporosis and hyperlipidemia remain widespread despite treatment, understanding effects of lipids on basal (endogenous) and intermittent (exogenous) PTH may provide new approaches to osteoporosis. We hypothesize that inflammatory lipoproteins, which are increased in hyperlipidemia, reduce PTH anabolic effects. Based on our preliminary studies, in Specific Aim 1, we will test in vitro whether the inhibitory mechanism of lipids on PTH-induced cyclic AMP production is at the level of PTH receptor expression, receptor trafficking, downstream at the level of G-protein activation, or further downstream at the level of adenylate cyclase activation. In Specific Aim 2, we will identify the level at which lipid-induced PTH resistance occurs in vivo: at the level of differentiation of marrow progenitors toward osteogenic vs. adipogenic lineages; anabolic responses of mature osteoblasts/osteocytes; and/or transient expression of osteoclastogenic factors by osteoblasts. We will generate Ldlr-/- mice that express green fluorescent protein targeted to osteoblasts and osteocytes. In Specific Aim 3, we will test whether reducing hyperlipidemia or inhibiting lipid oxidation will reverse PTH resistance in vivo by measuring bone density, histomorphometric parameters and bone turnover markers in the hyperlipidemic (Ldlr-/-) mice that are treated with liver X receptor agonists or that overexpress the anti-oxidant enzyme, paraoxonase-1. These proposed studies will provide insights into how inflammatory lipids inhibit PTH-induced osteoanabolism, pinpoint the site of inhibitory action within the PTH signaling pathway, and demonstrate approaches to reverse lipid-induced PTH dysregulation."}
{"text": "Summary/Abstract Despite ~10% of the human genome being comprised of secretory proteins, little is known about the physical states of granule lumenal and membrane proteins before and during fusion. This proposal is based upon the hypothesis that the mobility characteristics of granule lumenal contents and of granule membrane proteins shape the secretory response. The proposal will provide fundamental new insights concerning secretory granule structure and function in exocytosis and will provide the first quantitative measures of the rotational and translational mobility of granule lumenal and membrane proteins of individual granules. Our research focuses on events in the exocytotic pathway that occur in the highly specialized domain of the plasma membrane-cytoplasm interface. This region is superbly imaged by total internal reflection fluorescence microscopy (TIRFM), a core technique that we use extensively in our studies. The proposal is supported by strong preliminary results demonstrating distinct mobilities of different lumenal proteins, NPY-Cerulean and tPA-Cerulean. tPA-Cerulean, which has a much lower mobility, is released much more slowly upon fusion of individual granules and the fusion is associated with much slower fusion pore expansion. The rotational and translational mobility of lumenal and granule membrane proteins and of individual granules will be measured by novel combinations of TIRFM, polarization, fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). There are several related goals in the proposal: 1) To understand the MOBILITY CHARACTERISTICS OF PROTEINS IN THE GRANULE LUMEN and reveal their influence in determining the rates of protein and catecholamine release, 2) To determine the translational MOBILITY OF GRANULE MEMBRANE PROTEINS and whether the mobility permits their recruitment by diffusion to the fusion site on the granule membrane, 3) To determine the ROTATIONAL MOBILITY OF ENTIRE GRANULES in order to better define the tethered and/or caged state of the granules before fusion, 4) To determine whether the increase in granule travel immediately before fusion reflects a combination of translational and rotational motion that permits the granule to `ROLL' into a fusion competent interaction with the plasma membrane, and 5) To measure for the first time the ABSOLUTE DISTANCE BETWEEN THE GRANULE AND THE PLASMA MEMBRANE using supercritical angle emission, thereby determining the timing of engagement of the granule with the plasma membrane before fusion. In addition, it is anticipated that the new techniques will not only be applicable to secretory cell biology, but more generally to the multitude of cell biological issues at the plasma membrane- cytosol interface."}
{"text": "DESCRIPTION: Colorectal cancer (CRC) is a leading cause of cancer-related death in the U.S. Screening is a cost-effective strategy for reducing both CRC incidence and mortality but has yet to have a major impact on disease burden partly because of poor patient adherence. Shared decision-making is a potentially effective yet unproven strategy for increasing CRC screening rates. The overall objective of this proposal is to develop an interactive, web-based decision aid and evaluate its impact on shared decision-making and patient adherence to CRC screening recommendations. During Phase 1 (Year 1), an interactive, web-based decision aid will be developed that employs state-of-the-art technology to educate patients about the five recommended CRC screening strategies, heighten risk perception, and empower patients to participate in the decision-making process. Content will be developed using a critical review of relevant literature and existing decision aids, expert opinion, and decision-making theory. A personalized risk assessment tool plus pre-/post-test modules to assess demographics, knowledge, beliefs, desire, barriers, and patient preferences will be incorporated. Tailored feedback will also be provided. Once developed, the aid will be subjected to external review by an outside advisory committee for content validity and pre-tested for usability. During Phase 2 (Year 1), an optimal strategy will be defined for implementation of the decision aid in two ambulatory care settings. The research team will interact with physicians and office staff to identify potential logistical barriers and to conjointly develop a tailored strategy for implementation at the targeted ambulatory care sites. Issues to be addressed include resource needs, space allocation, patient identification and scheduling, and tracking. Physician and staff educational seminars will be conducted before implementation. During Phase 3 (Years 2 to 5), a randomized controlled clinical trial will be conducted to evaluate the impact of the decision aid on shared decision-making and adherence to CRC screening recommendations. Eligible patients will be randomized to review the decision aid, the personalized risk assessment tool alone or a control condition. The primary outcome will be patient adherence to CRC screening recommendations using computerized tracking systems; secondary outcomes will include patient knowledge and patient satisfaction using evaluation components of the decision aid or validated instruments."}
{"text": "This application proposes to continue the participation of investigators at Thomas Jefferson University in an interactive network of six centers (the Asthma Clinical Research Network or ACRN ) in conducting studies of novel therapeutic approaches to asthma and in disseminating findings to the practicing community. The need for such a network was suggested by epidemiological data showing increases in mortality, morbidity, prevalence, and costs of asthma, by clinical and basic research studies showing that asthma is linked to inflammation in the airways, and by the accelerating rate of development of potentially effective, but also potentially costly treatments for asthma. Defining the place of these therapies was seen as requiring collaborative, multi-center studies examining large numbers of subjects reflecting the diversity of the U.S. population. In its first 5 years, the ACRN established an infrastructure to meet this need and has added another center at Harlem Hospital in New York, which serves a predominantly minority population. The ACRN has completed studies of the effects of regular beta-agonist use in mild asthma ( BAGS ) and of the efficacy of the anti-inflammatory agent, colchicine, as an alternative to an inhaled corticosteroid in moderate asthma. Now underway are two additional trials comparing the effects of a long-acting beta-agonist, an inhaled corticosteriod, and the combination of the two in altering clinical and physiologic outcomes, and airway inflammation in moderate or severe asthma. A fifth study, establishing doses of different inhaled corticosteroids with equivalent effects on cortisol secretion, is imminent. Completed trials as well as 10-12 ancillary studies designed to improve the performance of clinical research have been presented at meetings of the ATS, ACCP, and AAAAI. Three of these studies have thus far been published in peer- reviewed journals. The ACRN has also reported the results of a subgroup analysis of subjects in the BAGS study showing that subjects with different genotypes for the beta-adrenergic receptor are differently affected by regular use of albuterol. This application proposes continued participation of the Jefferson Center in the multicentered, collaborative trials of the ACRN. Proposed studies include a comparison of the efficacy of doses of different inhaled corticosteriods with equal systemic effects (as estimated from the study described above), a prospective study of the effects of regular use of an inhaled beta-agonist in subjects stratified by genotype for the beta-adrenergic receptor, and a study of the efficacy of a leukotriene pathway antagonist in enabling reduction or elimination of inhaled corticosteriods in subjects with mild or moderate persistent asthma. Other planned studies are also briefly discussed, with the understanding that they could be modified or replaced in response to new information, new therapies, or changing clinical research priorities."}
{"text": "In Orientals, ALDH2 deficiency due to a common polymorphism frequently causes a flushing reaction after alcohol consumption and this aversive reaction is responsible for lower rates of alcoholism in individuals with the inactive ALDH2-2 allele. ALDH2 deficiency was detected in South American Indian populations; however, these findings have never been confirmed and a previous search for the Oriental ALDH2-2 allele in South American Indians was negative. Recently R. Peterson has identified a series of additional markers at ALDH2. By restriction enzyme analysis, SSCP and sequencing enabled him to identify haplotypes characteristic for the ALDH2-2 and ALDH2-1 alleles in different populations. This analysis is shedding light on the origins and functional role of the variant ALDH2-2 allele, which appears to have appeared on a single haplotype lineage and spread among East Asian populations. Although ALDH22R [Glu487Lys] probably originated on a single genetic background, haplotype analysis reveals that it is sufficiently ancient for additional mutations to have occurred subsequently. This result is most compatible with an effect of selection to maintain the Oriental ALDH2 variant, Glu487Lys."}
{"text": "Converging lines of research indicate significant sex differences in schizophrenia. Females have a more benign course, typified by later age of onset, better premorbid adjustment, more affective symptoms, fewer cognitive deficits, better neuroleptic response, shorter hospital stay, fewer relapses, lower risk for suicide, lower family Expressed Emotion (EE) and better social and work adjustment. Nevertheless, evidence of sex differences tends to be underemphasized and hypotheses concerning sex differences have rarely been investigated in a systematic, prospective study. AIMS: This study will assess: (1) sex-related variation in the clinical symptom pattern of schizophrenia; (2) sex differences in premorbid factors and precipitants of onset; (3) sex-related parameters of posthospital recovery and relationship to family environment factors; (4) prominence of sex-related differences at early and later phases of the illness; and (5) the relative influence of neurocognitive, neuroanatomic and family environment factors in prediction of two-year recovery/relapse rates for first- admission, and more chronic, males and females. METHODS: Study 1. From a pool of approximately 1100 annual inpatient admissions, a sample of approximately 700 consecutive admission psychotic patients will be identified over a 30-month period and given a standard (SCID) interview. Patients who meet broad-band (New Haven) criteria for schizophrenia will be cross-classified on more narrow-band (RDC and DSM-III) criteria and the sex ratio will be compared across diagnostic systems to determine the extent of differential exclusion by sex. Study 2. 300 (150 early- and 150 later-phase) patients who meet DSM-III criteria for schizophrenia spectrum disorders will be assessed on premorbid, neurocognitive, neuroanatomic (VBR), and family environment measures to identify sex-related parameters of psychopathology. Study 3. The same 300 patients will be studied longitudinally in order to determine sex differences in neurocognitive, neuroanatomic and family environment factors and their relative importance in predicting recover/relapse and functioning for males and females over the two-year post-admission phase. SIGNIFICANCE: This study will contribute to (1) general knowledge and practice in the diagnosis and classification of schizophrenia; (2) understanding of the pathogenesis and course of illness; (3) increased accuracy of prognosis and selection of treatment strategies for males and females."}
{"text": "CORE 2: DRIVING BIOLOGICAL PROBLEMS Three driving biological problems will focus our biocomputation research. A macroscale DBP focused on neuroprosthetic dynamics, a mesoscale DBP focused on viral and cellular dynamics, and a molecular scale DBP focused on drug target dynamics. We use seven criteria to select our Driving Biological Problems (DBPs): Canonical: The problem should be an archetype of problems in an entire field of inquiry to guarantee broad applicability of the tools we develop. [unreadable] Collectively cover a range of scales: It is critical that activities of the center cover scales from molecular through cellular to organismal levels. [unreadable] Physics-based: The DBP should present a problem that can be addressed by representing and analyzing the geometry and physics of the biological system. [unreadable] Data rich: Biology is dominated by experimental data. These data provide the real-world constraints that drive and validate models and simulations. [unreadable] World-class, engaged experimentalists: We seek close integration and deep interactions among the biological and computational participants. [unreadable] Have important implications for disease: We ensure that our DBPs are related to disease processes or treatments to ensure that they contribute to the advancement of human health. Our past DBPs met these criteria and enabled us to develop software that is now used by a very broad community of researchers. Our next three DBPs also satisfy these criteria and are briefly reviewed below."}
{"text": "This project is aimed at studying renal lymph in experimental hypertension and in experimental renal failure. Essential hypertension in man frequently leads to a gradual deterioration of renal function which is a consequence of both functional and structural vascular damage in the kidney. Similarly, circulatory factors have been shown to play an important role in the pathomechanism of acute and chronic renal failure. In this research alterations of the renal microcirculation in experimental hypertension and renal failure will be investigated. This will be accomplished by studying the renal lymph and obtaining information on: 1. capillary permeability to macromolecules, primarily albumin, 2. total lymph drainage of the kidney, and 3. abnormal constituents in lymph that may reveal the extent of cellular injury. In addition, structural alterations of the renal microcirculation as seen by light and electron microscopy will be correlated with the functional studies. The morphological investigation will be carried out by the Department of Pathology of this school."}
{"text": "The objective of this laboratory is to develop techniques for quantitative 3D light and electron microscopy and to make these available to the scientific community for research and education. A 400 kV transmission electron microscope allows examination of specimens 10-20 times thicker than usual and has been modified for an unlimited range of specimen tilt angles for 3D imaging. A laser scanning confocal light microscope provides optical sectioning in thick specimens, including wet and living specimens, superior to that obtainable using ordinary light microscopes. The foci in the core research are quantitation, comparison of images using different imaging modalities, and the evaluation of the effects of preparation methods and other potential sources of artifacts. Images are processed using image analysis programs imported from other sources or developed here. Programs are available for reconstruction from serial section images and from images taken at various tilts (including stereo images). These programs provide 3D localization of particulate markers (gold particles), reconstruction of membranes and other surfaces, and quantitative analysis of networks. Initially, manual processes are implemented. We plan in the longer term to automate as many processes as possible. Current collaborative research projects are focussed on the localization of surfaces and surface receptor molecules in motile cells, the organization of the cytoplasmic matrix, the structure of protein networks, the structure of the transverse tubular system in skeletal muscle, and the development of the fibrillar system in differentiating cardiac and skeletal muscle cells. Service projects cover a broad range of topics. Users have come from several surrounding states and from Japan. Training is done on-site at the facility, and through workshops presented at the facility and elsewhere. Dissemination of information on the facility is done through publications, lectures and, posters presented at national and international meetings, and direct mailing to biomedical scientists."}
{"text": "This Project will characterize the role of nitric oxide (NO) and NO-derived species in mouse models of inflammatory bowel disease (IBD) and cancer. These studies will use 129/SvEv Recombinase-activating gene 2 (Rag-2) knockout (KO) mice and C57BL/6 T cell receptor (TCR) alphabeta KO mice. Rag-2 KO mice on a 129/SvEv background develop severe IBD and cancer when infected with H. hepaticus. Histopathologic lesions and the pattern of macrophage and neutrophil infiltration will be correlated with biomarkers for nitration, oxidation, and halogenation of DNA and proteins in Rag-2 KO mice. These biomarkers will be further validated by comparing disease in naive and effector CD45RB high T cell-bearing Rag-2 KO mice, and by modulating NO production with N-methylarginine (NMA). TCR alphabeta KO mice develop severe IBD when infected with H. hepaticus. IBD in TCRbeta KO mice, like in Rag-2 KO mice, is a model for Crohn's disease (CD) and features activated macrophages and abundant interferon-gamma (IFN) and tumor necrosis factor-alpha (TNF). TCR alpha KO mice are a model for ulcerative colitis (UC), and are dependent on interleukin-4 (K,-4) but not IFN or TNF for the development of disease. Histopathologic lesions and cytokine levels will be correlated with biomarkers in TCR alpha and TCR beta KO mice. To further characterize the role of NO and NO-derived species in IBD, we will compare pharmacologic inhibition of inducible nitric oxide synthase (iNOS) with genetic inactivation of the enzyme by generating iNOS-deficient TCR alphabeta KO mice. In vitro analysis of somatic mutations arising in vivo in the presence and in the absence of NO and NO-derived species will also be performed. By using a novel IBD associated cancer model and models for UC and CD, we will be able to validate new biomarkers for nitration, oxidation, and halogenation of DNA and proteins. We will also gain a better understanding of the role of NO and NO-derived species across a broad range of carcinogenic events, including genotoxicity, cellular proliferation, cytotoxicity, and angiogenesis. The Specific Aims are: Specific Aim #1. Characterize the role of NO an oxidative stress in IBD and cancer in Rag-2 KO mice Specific Aim #2. Characterize the role of NO and oxidative stress in IBD in TCR alphabeta KO mice"}
{"text": "Transcription will be studied in intact nuclei isolated from tissue culture cells. Products are characterized by size, rapidly hybridizing content, and sensitivity to selective inhibitors. Polymerase systems will be further characterized by their response to prior treatment of cells with specific inhibitors. Nuclei will be sub-fractionated to identify the location of the different polymerase systems in specific structures. The organization of transcription in adenovirus infected cells will be compared to normal cells. The location of adenovirus genes in different polymerase systems in rat embryo transformed cells will be measured. Additional specific inhibitors of heterogeneous RNA synthesizing systems in vivo and in vitro will be sought. The regulation of translation of mRNA at the level of initiation will be studied further. Initiation of adenovirus mRNA will be studied, both in vivo and in vitro. The nature of histone synthesis regulation of DNA synthesis will be studied in synchronized cultures. The mechanism of inhibitors of initiation, such as poliovirus and 5- azacytidine will be studied in adenovirus infected cells, where specific mRNA is identifiable and in vitro studies of initiation may be possible, and in immunoglobulin and growth hormone producing cells. The effect of the inhibitors and such clinically significant compounds as BCNU on the initiation factors will be studied in the adenovirus in vitro system. The messenger RNA lifetime for general protein synthesis and for specialized proteins will be studied using cordycepin, which gives significantly different results than actinomycin. The effects on RNA, protein and DNA metabolism of nucleoside analogues and compounds used in clinical cancer chemotherapy will be examined in detail. Both base and sugar substituted analogues of adenosine and uridine will be studied for their selectivity amongst different polymerase systems and for their effect on RNA metabolism. The inhibition of DNA synthesis and of the initiation of translation of the alkylating agent, BCNU will be studied further."}
{"text": "Research on cannabinoids has progressed tremendously since the discovery of specific recognition sites (receptors) for chemicals like 9-tetrahydrocannabinol (THC), the main active ingredient in marijuana. The endocannabinoid signaling system (ECS) thus likely serves as the biological substrate for the marijuana high. This subjective state presumably underlies human marijuana consumption that may lead to compulsive cannabis intake and dependence disorders. Drug discrimination is a powerful, pharmacologically selective model for assessing subjectively experienced drug effects in animals (and man) and is the major in vivo behavioral technique in this application. Cannabinoid receptor CB1 (CB1R) agonists and antagonists will be trained in drug discrimination using different doses, providing for in vivo assays with different sensitivity levels. This is complemented by observational studies and schedule controlled responding allowing for an in depth characterization of ligands. A major aim of this research is to identify new medications that will translate into better pharmacotherapies for combating marijuana addiction. Alleviation of withdrawal-related effects in physically dependent individuals likely is an important motivational factor in addiction processes. New molecules are designed and synthesized by on site expertise. One focus is to identify and refine in vivo neutral CB1R antagonists. Most current CB1R antagonists also display intrinsic activity (inverse agonism) that may hamper patient compliance in treatment settings. Thus, the studies will expand our understanding of the ECS, comprised of two major endogenous signaling molecules, anandamide (AEA) and 2-arachidonoylglycerol (2-AG) acting on two known receptors, CB1 and CB2, in normal body function and pathophysiology. Additional therapeutic targets for the application concern ligands selectively affecting the enzymes involved in the deactivation of the endocannabinoids, thus potentially avoiding direct receptor activation. The behavioral studies are aided by neuro/biochemical procedures provided by collaborating faculty in pursuing these goals. In addition to AEA, recent developments regarding 2-AG allow for a much more comprehensive understanding of this major endocannabinoid in ECS signaling. By obtaining information on the functions of the endogenous substances (AEA and 2-AG) and the exogenous THC, as well as in vivo neutral blocking agents, these studies will not only further our understanding of ECS signaling in the behavioral neurobiology of cannabis abuse / dependence, but may also lead to the development of effective medications for treating disorders involving cannabis / marijuana, and designer THC-like cannabimimetics."}
{"text": "Activation of cellular tumor suppressor pathways is the cell's major defense against cancer induced by activated oncogenes. The ARF-p53 tumor suppressor pathway, which is one of the most important in mammalian cells, can be activated by a number of viral and cellular oncogenes. Activated ARF induces a p53-mediated block to cell division via a cell cycle arrest or apoptotic cell death. How oncogenic stress activates ARF remains to be elucidated. Polyoma virus middle T-antigen (PyMT) is a potent oncogene able to bind a number of key regulatory cellular proteins and activate a number of important cellular signaling pathways including the ARF-p53 tumor suppressor pathway. We will use PyMT as a model oncogene in order to better understand how ARF is being activated. We hypothesize that PyMT induces ARF by the inappropriate activation of one or more cellular signaling pathways that also mediate the ability of PyMT to transform cells. Our plan is to identify the cellular signaling pathways induced by PyMT that results in activation of ARF. REF52 cells differ from most other established cell lines in containing an intact ARF-p53 tumor suppressor pathway and are distinct in resembling primary cells in their requirement for oncogene cooperation for their transformation. PyMT activates the ARF-p53 pathway, blocking REF52 cell division and will not transform REF52 cells in the absence of a co-operating oncogene. We plan to take advantage of these unique properties of REF52 cells to isolate PyMT and cell mutants that are involved in the activation of ARF. Three interrelated aims will be pursued. Aim-1 will be to use previously isolated PyMT mutants to identify which domains of PyMT are required to activate ARF. Aim-2 will be to use mutagenized PyMT to identify sequences in both defined and undefined regions of PyMT that are required for the activation of the ARF-p53 pathway in REF52 cells. Aim-3 will be to isolate and define REF52 cellular mutants in which PyMT signaling fails to activate the ARF-p53 tumor suppressor surveillance pathway. We believe that such cell mutants will help to differentiate between normal and oncogene activation of important cellular signal transduction pathways. Understanding the mechanism(s) of oncogenic activation of the ARF-p53 tumor pathway will help in designing better drugs and therapies for the treatment of cancer."}
{"text": "People living with HIV/AIDS (PLWH/A) disproportionately smoke cigarettes, putting them at risk for many adverse health outcomes, yet little is known about how to tailor cessation interventions for PLWH/A. Oregon's AIDS Drug Assistance Program (ADAP), known as CAREAssist, serves more than 2,100 low-income PLWH/A by assisting them with health insurance coverage and providing co-pays and prescription drugs. Survey data from 2006 indicated that the smoking prevalence among CAREAssist clients was high (42%), but that most smokers wanted to quit (72%) and were interested in using medicines for stopping smoking if available at no cost (83%). Recognizing an opportunity, CAREAssist in 2008 integrated cessation pharmacotherapy into its drug formulary, making it available at no cost to clients whose insurance plans would not pay for them, and the program started paying for counseling through the Oregon Tobacco Quit Line. Despite the removal of these financial barriers, preliminary data from November 2009 suggests the smoking prevalence among CAREAssist clients is virtually unchanged from 2006. The overall goal of the proposed project is to improve understanding of barriers to CAREAssist clients using free cessation pharmacotherapy and counseling services in order to inform the development of an intervention that would integrate additional cessation support into the integrated HIV system of care in Oregon. Existing survey data on over 1200 CAREAssist clients will be analyzed and in-depth interviews will be conducted with 15 HIV case managers, 15 medical providers, and 45 CAREAssist clients to describe:1) current smoking, quit behaviors, and utilization of prescribed pharmacotherapies and the Quit Line among CAREAssist clients by demographics, housing instability, substance abuse, overall physical health, overall mental health, co-morbidities, adherence to HIV treatment, visits to medical providers, practical and social support, and smoking behaviors among their social networks; 2) client-level barriers to utilizing cessation services, taking pharmacotherapies, and successfully quitting smoking; 3) current HIV medical provider and HIV case manager knowledge, attitude, and practices regarding tobacco cessation; 4) HIV medical provider, HIV case manager, and system-level barriers to screening for smoking behaviors among PLWH/A, providing cessation counseling and referrals, prescribing cessation pharmacotherapies, and integrating these pharmacotherapies into HIV treatment regimens. Project relevance: PLWH/A disproportionately smoke cigarettes, putting them at risk for many adverse health outcomes, yet little is known about how to tailor cessation interventions for PLWH/A. The goal of the proposed project is to improve understanding of the barriers to Oregon AIDS Drug Assistance Program (ADAP) clients using free cessation pharmacotherapy and counseling services. This information will be used to inform the development of an intervention that would integrate additional cessation support into Oregon's HIV system of care and could be implemented with ADAP populations across the nation."}
{"text": "The aim of this research training program is to contribute to a knowledge base about health behavior that: (a) promotes maximal well-being; (b) facilitates early detection of disorder to minimize its effect; and (c) enhances quality of life in illness situations. Research training will be provided annually to three predoctoral and three postdoctoral trainees that develops the following research skills: (a) analysis of concepts and theories from the behavioral and biological sciences that are relevant to health behavior, health behavior change, and quality of life in chronic illness; (b) synthesis of concepts, theories, and research findings from the behavioral and biological sciences to frame research concerned with enhancing quality of life in persons with chronic illness; (c) developing and testing instruments that measure concepts related to health behavior and quality of life in chronic illness; (d) design and conduct of research to advance knowledge related to the development and testing of tailored health behavior interventions; (e) interdisciplinary research collaboration; and (f) scientific writing, research application preparation, scientific presentations, and publications. To provide trainees with such an experience, Indiana University School of Nursing will: (a) recruit diverse and qualified trainees; (b) provide a high quality research training program, with student access to accomplished interdisciplinary mentors; and (c) provide a research-intensive environment, with an expanding portfolio of faculty health behavior research. Important advancements of this research training program for the next five years are: a greater interdisciplinary focus with a larger number of interdisciplinary faculty available as research mentors; a sharpened focus on chronic illness across the lifespan; and an increased emphasis on developing and testing of tailored behavioral interventions, particularly those informed by information technology. Research training includes bi-weekly Health Behavior Research Seminars and Research In Progress meetings, Behavioral Intervention Workshops, and experience on interdisciplinary research teams and similar intensive mentoring experiences, in addition to coursework as appropriate. Predoctoral trainees are supported for 2-3 years and expected to apply for individual NRSA funding; postdoctoral trainees are supported for 2-3 years and expected to submit at least one grant application for external funding. Trainees have access to resources within three research programs embedded within the School of Nursing: the Center for Enhancing Quality of Life in Chronic Illness, the Mary Margaret Walther Program for Cancer Care Research, and the Behavioral Cooperative Oncology Group. Trainees also have access to Clarian Health Partners, the third largest hospital network in the United States [unreadable] [unreadable] [unreadable]"}
{"text": "Project Summary: Prepulse inhibition (PPI) represents a powerful method for understanding a fundamental component of information-processing, sensorimotor gating. PPI refers to the diminution of the startle response when a sudden startling stimulus is preceded 30-500 ms by a barely detectable prestimulus. This basic form of startle plasticity, seen across species from rodents to primates, has been studied extensively as an exemplar of preattentional sensory 'filters'that defend cognitive processes against the information inundation of our sensory world. A prominent theory holds that dysfunction in these filtering systems represents a core endophenotype manifested in schizophrenia, obsessive compulsive disorder, tic disorders, and possibly attention deficit hyperactivity disorder and post traumatic stress disorder. Patients suffering from this diverse range of illnesses share the common feature of an inability to screen out sensory, motor, or cognitive information, and display markedly deficient PPI. Because PPI deficits can be studied across species, PPI has become well-established as a crucial paradigm with which to study deficient sensorimotor gating in animal models of these psychiatric disorders. Much information has accrued regarding the neurochemical systems and circuits underlying PPI, particularly regarding the role of the dopamine and serotonin systems. Nevertheless, there is a striking gap in knowledge about the role of the norepinephrine (NE) system in regulating PPI. Our preliminary evidence indicates that this catecholamine plays a fundamental role in the ability of phencyclidine-like drugs (potent psychotomimetics in humans) to disrupt PPI in rodent models and the unique ability of atypical antipsychotic medications to reverse these PPI deficits. In addition, we have recently discovered that pharmacologically activating the locus coeruleus (LC), the major source of forebrain norepinephrine, markedly disrupts PPI. These data indicate that the LC-NE system represents a critical, yet almost completely overlooked central modulator of PPI. The experiments outlined in this proposal are designed to systematically characterize the role of the LC and its NE-innervated forebrain targets in regulating PPI, and in mediating the actions of psychotomimetic and antipsychotic drugs on PPI. These data may provide insights into novel circuitries underlying the regulation of PPI, and be of great relevance to understanding the neurobiology of the large set of psychiatric disorders of which dysfunctional sensorimotor gating is a central feature. Relevance to Public Health: Information-processing deficits are a major part of several psychiatric illnesses such as schizophrenia. When totaled, these illnesses represent a staggering 9-10% of the United States population. This proposal will study the neurobiology of the information-processing deficits seen in illnesses like schizophrenia in order to ultimately develop new treatments for these diseases."}
{"text": "PROJECT SUMMARY The objective of this SBIR Commercialization Readiness Program (CRP) project is to evaluate chronic toxicity of a new therapeutic for diabetic neuropathy in support of an IND submission. Of the 25 million Americans who suffer from diabetes, approximately 50% will be diagnosed with neuropathy, which is characterized by nerve degeneration. Despite the high prevalence of the disease, there is currently no FDA-approved treatment to either prevent diabetes-induced nerve degeneration or promote nerve regeneration. Thus, there is a substantial unmet need to develop more effective treatments for diabetic neuropathy. The founders of WinSanTor have identified a promising candidate which both prevents and reverses neuropathy in rodent models of the disease. The candidate molecule, pirenzepine, was identified using a novel screening methodology developed in the labs of the company?s founders. Pirenzepine has subsequently been evaluated in over a dozen in vivo tests, and has demonstrated the unique ability to ameliorate both epidermal fiber loss and thermal hypoalgesia. Pirenzepine is an approved drug for other indications in non-US countries, and so it is substantially de-risked as a drug development candidate. In a SBIR Fast-track program, WinSanTor successfully executed an expedited pre-clinical program that included: 1) development and validation of bioanalytical methods; 2) pharmacokinetic analyses; 3) optimization of formulation to enhance delivery; 4) generation of a safety profile; and 5) GMP manufacturing of pirenzepine. These efforts fully support the continued execution of the pre-clinical program through the evaluation of chronic toxicity assessments. The focus of this CRP program will be to evaluate the toxicity of pirenzepine when applied topically for 9 months. A 9-month study was chosen to fulfill the 9-month chronic toxicity study requirement for an NDA submission and to support the duration of the anticipated Phase 2 clinical trial protocol that will involve administration for at least 5 months. This study will be executed in mini-pigs and will use a number of toxicity end points, such as mortality observations, clinical observations, Draize scoring, clinical pathology, and histopathology, to fully define a toxicological profile of pirenzepine. The metric of success for this Aim is to achieve to achieve a NOAEL at an exposure level such that there is up to a 10x safety margin for human studies. The completion of this study is critical to an IND submission to the FDA to support subsequent clinical trials."}
{"text": "[unreadable] We aim to develop novel non-invasive and reiterative tests of nerve (small) fibre damage and repair in human diabetic neuropathy. We will quantify corneal nerve fibre degeneration and regeneration in diabetic patients compared with control subjects. We will assess circulating levels of the neurotrophin receptor p75 (p75NTR) as an index of small fibre stress/damage. Both measures will be validated against gold standard measures of neuropathic severity. A cross-sectional study will assess corneal nerve morphology and the p75NTR titre in diabetic subjects with a varying severity of neuropathy and non-diabetic control subjects. Moreover, subjects who have either recently undergone or are planned to undergo fascicular sural nerve biopsy will also have assessment of corneal nerve morphology and the p75NTR titre to define their exact relationship with peripheral nerve fibre degeneration and regeneration. Corneal nerve morphology and the p75NTR titre will be assessed in Type 1 diabetic subjects with painful neuropathy in a trial of strict glycaemic control using continuous subcutaneous insulin infusion, in order to assess the effect of near-normoglycemia and reduced glucose flux on these parameters. To further understand the relevant biology of the p75mR, and place its clinical assessment into a scientific frame of reference, we will undertake in vitro and animal studies. Tissue culture studies of neurones and Schwann cells will be employed to assess the effects of hyperglycemia, oxidative stress and NGF deficiency on expression, turnover and shedding of p75NTR. The role of these regulators specifically NGF therapy will be assessed longitudinally in STZ-rats in both peripheral nerve and in sensory ganglia to help further refine our understanding of the site of expression and shedding of p75NTR. The results of these studies will enable the introduction of two complimentary, non-invasive, and reiterative measures of small fibre damage and repair, thus enabling efficient, but accurate, assessment of treatment efficacy in human diabetic neuropathy. [unreadable] [unreadable]"}
{"text": "The objectives of this grant are to characterize the cellular immune responses to Epstein-Barr virus (EBV) and to define the immunoregulatory events important in successful control of EBV infection during acute infectious mononucleosis (IM). We will continue our studies to further characterize the in vivo EBV induced MHC-nonrestricted cytotoxic T-cells (anomalous T-killer cells). The T cell lineage of these anomalous T-killer cells will be established by looking for molecular rearrangement of the T cell receptor genes. We will determine whether these T-killer cells are directed against EBV antigens and test the hypothesis that they are in fact alloreactive T-cells directed at altered class I molecules. Anomalous T-killer cells will be cloned to further prove polyclonality and determine target cell specificities. We plan to investigate the immunoregulatory role of in vivo EBV induced activated helper/inducer T cells. We will use CD4 helper/inducer T cell monoclonals to identify functionally distinct subpopulations. These subpopulations will be functionally studied in assays to evaluate help and suppression of B cell and T cell function. Cloning studies will be attempted to look at function at the clonal level. The long-term objective of our laboratory is to better understand the immunoregulatory events which occur during acute EBV infection. This knowledge will be used to understand immune defects which predispose individuals to fatal infection and lymphoproliferative disorders."}
{"text": "The availability of the Transgenic Mouse Shared Resource enables our CCSG Research Base Investigators to conduct versatile, cutting-edge research with a battery of sophisticated genetic techniques for manipulation of the mouse genome to create models for studies of cancer for incisive in vivo mechanistic investigations of the fundamental processes involved in the etiology of tumor development. Transgenic mice carrying new or novel genes are created by microinjection of DNA into the pronuclei of fertilized eggs and knock-out mice lacking specific genes of interest are created by homologous recombination in embryonic stem cells followed by injection into blastocysts to create chimeric mice for breeding to homozygosity. The high degree of conservation of most sequences in genomes of humans and mice makes using mouse genetic manipulation technology to create models of human cancer pathogenesis extremely useful. These approaches are remarkably powerful in cancer research particularly in the analysis of oncogenes, metastasis, cell-cycle control, tumor suppressor genes, and in the crafting of cancer model systems for developing new treatment regimens, and methods for drug testing and tumor imaging. The mission of this Shared Resource is to provide the highly technical aspects of manipulation of genes in embryos and mouse embryonic stem cells as a service to our Center members, allowing them to create the genetically manipulated mouse models they need. This is an outstanding example of specialized techniques, highly trained dedicated personnel, and expensive equipment can be accessed by researchers who could not reasonably expect to develop or obtain them on an individual basis. The UCSD CCSG Shared Resource provides cutting-edge, rapidly evolving services that adapt and drive the field of mouse genetics and continue to be very responsive to the vast creativity of the CCSG membership, fulfilling their needs for the most cutting-edge embryology and molecular genetics in the mouse."}
{"text": "Children with a parent diagnosed with substance abuse disorder (SUD) show increased risk for greater drug use, delinquency, and their engagement in health-risking sexual behavior, suggesting maladaptive choice processes with great social and public-health costs (e.g., Kirisci et al., 2007; Reynolds et al., 2007; Tarter et l., 2002). Little is known, however, about the mechanisms that underlie the decisions to engage in these behaviors. We propose to conduct hypothesis-driven secondary data analyses to test a conceptual model that links parental SUD and childhood dysregulation to the development of poor decision-making skills and increased incidence of health-risking behaviors later in life. We will leverage data collected through the Center for Education and Drug Abuse Research (CEDAR; NIH# 5P50 DA05605), using its unique longitudinal dataset that includes validated measures of transmissible liability for SUD risk (Kirisci et al., 2009; Vanyukov et al., 2009), a battery of laboratory-based measures of decision-making competence (DMC; Parker & Fischhoff, 2005), and adult assessments of substance abuse, antisocial behaviors, and sexual behavior. We intend to address five main research questions in the proposed project. First, we plan to identify classes of developmental trajectories for psychological regulation. We will conduct analyses to characterize different patterns of intra-individual change in psychological regulation from preadolescence to emerging adulthood. Second, we plan to refine the measurement of DMC, which can be defined as adherence to normative, rational standards when making decisions (Parker & Fischhoff, 2005) by expanding the construct to encompass excessive risk-taking and insensitivity to expected value. Third, we will examine the associations between family and parenting variables on subsequent DMC and health-risking outcomes later in life. Fourth, we will examine the effects of developmental trajectories of psychological regulation on DMC. While research has linked self-regulative processes to advantageous decision making, such studies have largely relied on cross-sectional designs, and are thus silent on the progression from development of self-regulation to adolescent decision making. Finally, we will test the extent to which poor DMC can predict future health-risking behaviors, such as drug use, criminal behavior, high-risk sexual behavior, and inter-partner violence."}
{"text": "The Vision Science Research Center of the School of Optometry at the University of Alabama in Birmingham has now been in existence for nine months. During the first three months, a great deal of time and effort was spent recruiting and hiring the various technical and administrative support personnel, purchasing supplies and equipment and establishing the procedures and guidelines under which the Center would operate. During the next six months, the technical staff was trained, most of the equipment and supply purchases were received and the different CORE Support Modules began to meet the needs of the participants. Establishment of Vision Science Research Center at the University of Alabama in Birmingham has had an interesting effect. Given the concentration of vision scientists here, other departments on campus are becoming increasingly interested in hiring new faculty with vision research interests. A perfect example of this is the recent hiring of Dr. Michael Friedlander by the Department of Physiology. The Department of Physiology and others are finding that high quality vision scientists are attracted to Birmingham by the existence of the Vision Science Research Center and the individual investigators that are presently here. Given that several departments on campus are in the process of increasing their faculty, we are hopeful that even more vision scientists will be attracted to Birmingham and the Vision Science Research Center."}
{"text": "The International Association of Fire Fighters (IAFF) is requesting 664,757 dollars during the initial budget period and 3,529,286 dollars for the total project period of the DOE Nuclear Weapons Complex Cooperative Agreement. Emergency personnel responding to incidents related to the DOE complex face health and safety challenges involving radioactive and other hazardous materials. Since 1994, an average of 2,200 responders have been injured at hazardous materials incidents annually. Many more suffer serious health effects from toxic exposure associated with fire fighting and EMS response. The effective remedy to combat these health effects is a flexible training program that emphasizes occupational safety and health and OSHA defined responder training as a key to effective emergency response. The IAFF proposes to continue to implement such a proven training plan. This effort relies heavily on an efficient Train-the-Trainer approach. It offers three new course formats which can be customized to the specific hazards faced by a given target audience; uses a combination of the Internet, advanced training technologies and regional programs; and emphasizes Integrated Safety Management (ISM). The estimated 425 annual attendees leave the course with the knowledge and the tools needed to implement this program in local fire/rescue departments in and around ten specified DOE sites, as well as other regions upon request. The IAFF is the only national organization serving professional fire fighters and enjoys longstanding training partnerships and access with fire/rescue departments across the US. Our training curricula is current, focused, and ready to be delivered. In addition, we have a highly regarded 100-member professional fire fighter/paramedic instructor team trained in using facilitation techniques and problem-based learning to reinforce responder safety and health. It is a state-of-the-art program with a focused safety and health message provided by experienced, committed instructors."}
{"text": "The focus of this research will be important neurobiological substrates of aggressive behavior in mice, and how genetic, developmental, and experiential determinants of aggression are mediated by specific neuronal systems. To study this problem systematically, an integrated multidisciplinary approach will be used to examine the behavior and neurobiology of lines of mice, derived from ICR foundational stock, which have been selected over 21 generations for high and low levels of aggression. Alterations in aggression induced by genetic, ontogenetic, and experiential factors will be related to specific neurobiological mechanisms. Preliminary data support the hypothesis that an important mechanism for genetically-induced behavioral differences is an alteration in dopaminergic activity in specific terminal regions. Thus, the aims of the proposed studies are: (1) To determine whether alterations in brain monoamine, particularly dopamine, activity in specific terminal regions mediate behavioral differences observed in mice selectively bred for high and low levels of aggression; (2) to determine whether developmental and genetic differences in aggression are due to alterations in the magnitude or temporal development of innervation of discrete catecholamine terminal fields; (3) to determine whether social experience alters the neurobiological mechanisms mediating genetic and developmental differences in aggressive behavior. Because there are qualitative and quantitative changes in aggressive behavior changes as a function of developmental period, and as a function of social experience, the activity of dopamine and other monoamine (serotonin and norepinephrine) systems in both high and low aggressive mice will be studied during ontogeny, using behavioral, pharmacological, neurochemical, receptor, and immunocytochemical techniques. A cosibial\\longitudinal design will be used that allows genetic, developmental, and experiential factors to be disentangled. Computer-supported observational methods (interactional analyses) will be used to quantify behavior, while high performance liquid chromatography (HPLC) and quantitative receptor autoradiography will be used to measure neurochemical and receptor changes in monoamine systems. Immunocytochemical techniques, using antisera to tyrosine hydroxylase (TH) and dopamine-beta-- hydroxylase (DBH), will be used to characterize the distribution of catecholaminergic fibers in specific brain regions of high and low aggressive mice. The proposed studies will provide important, new information about the neurobiology of aggression and timidity, and the brain mechanisms that mediate genetic, developmental, and experiential effects on social behavior."}
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{"text": "The goal of this research is to develop a culture dish (OxyDish R) that, together with oxyrase (tm), will support growth of clinically significant, anaerobic microorganisms on an agar-medium surface without using anaerobic bags, jars, or chambers. The use of oxyrase and this dish to grow anaerobes is intended to be transparent to the microbiologist and to the microorganisms. The microbiologist is intended to be able to work with most anaerobic microbes. The combination of oxyrase with the dish is intended to provide an environment that will reliably support the rapid growth of anaerobic microorganisms. No other environmental intervention or special equipment will be needed to grow clinically important microorganisms. The simplicity of the OxyDish is intended to enhance the efficiency of the clinical microbiologist working with anaerobic pathogens. This increased efficiency, together with elimination of special equipment supplies, will reduce the cost of working with anaerobic pathogens."}
{"text": "This application requests support for 2 physician training positions for 3rd and 4th year neonatology fellows, which will allow them to develop the knowledge, understanding and skills needed to pursue independent research careers in academic neonatology. Two major academic research paths will be available to trainees, and each track will be associated with a specific curriculum containing required course work: Track I. Basic Research, Physician-Scientist (laboratory research mentorship), and Track II. Clinical Research, Physician-Scientist (clinical research mentorship) which includes Health Care Policy and Medical Informatics mentorship components. This program with its two research tracks resides with the Division of Neonatology which includes: five clinical research physician-scientists (two of whom are Masters trained); two basic research physician-scientists; ten Ph.D.s; and the Neonatal-Perinatal Research Institute (NPRI). The NPRI includes sixty-five senior investigators, forty of whom are NIH funded, and draws upon the academic strength and depth found across Duke University needed to support the formal training for the program in developmental biology, clinical sciences and health care research. Extramurally funded research within the NPRI currently supports 4 broad areas of investigation: 1) early cardio/cranio/facia development; 2) neonatal lung development/repair; 3) neural injury and repair in the fetus and neonate; and 4) clinical neonatal research in conjunction with the NICHD Neonatal Network and the Duke Clinical Research Institute. In addition, this grant proposes a unique program with curricula for the study of Health Care Policy and Medical Informatics in the context of a medical research career. The requested funds will provide critical support for the research years of the neonatology fellowship, which currently trains six to seven fellows at Duke University Medical Center. The unique research training program with its separate Tracks will provide a structured learning experience tailored to the needs and interests of the individual fellow, with each fellow working with a senior scientist who will serve as mentor. Upon completion of the training program, the young physician-scientist will have the tools, knowledge, experience and confidence to compete successfully as an independent investigator."}
{"text": "The long-range goals of this proposal are to better understand the molecular neurobiological events that underlie the development of excessive alcohol drinking, and contribute to the long-range consequences of excessive alcohol drinking. The overall hypothesis to be tested is that changes in the expression of genes involved in neurotransmission, neuroplasticity, and intracellular signaling pathways within discrete regions of the extended amydala (E-AMYG) contribute to the development of excessive alcohol drinking. Excessive drinking is defined as sustainable blood alcohol concentrations (BACs) in the range of 100-150 mg% that are repeatedly attained over a chronic period. The overall hypothesis will be tested using selectively bred alcohol-preferring (P) and high-alcohol-drinking (HAD) rats. Micro-punch techniques will be used to obtain samples containing the nucleus accumbens shell (ACB-sh) and central nucleus of the amygdala (CeA). Changes in gene expression will be determined using Affymetrix microarrays. RT-PCR and in situ hybridization will be used to verify key findings from the microarray experiments. The excessive alcoholdrinking paradigm to be used will be the 'drinking in the dark multiple scheduled access' (DID-MSA) procedure. Time-course changes in gene expression will be determined following a drinking episode, and during the development of excessive alcohol drinking. The specific aims will be designed to determine changes in gene expression within the ACB-sh and CeA of P and HAD rats prior to, during initiation of, and following development of excessive alcohol drinking. The effects of chronic alcohol exposure and the development of excessive alcohol drinking are influenced by multiple genetic and environmental factors. This project will provide important molecular neurobiological information in discrete regions of the E-AMYG of genetically vulnerable subjects that could more comprehensively describe the complex inter- and intracellular events leading to the development, maintenance, and consequences of excessive alcohol drinking. Such information would be critically important for developing pharmacotherapeutic strategies to treat alcoholism and alcohol abuse. [unreadable] [unreadable] [unreadable]"}
{"text": "Continued research is proposed toward the development of a chemical mediated artificial pancreas. This system will deliver glycosylated insulin (G-Ins) in response to glucose concentrations, based on the competitive binding between G-Ins and glucose to a saccharide binding substrate. This application will focus on optimizing the components of the system and comprehensive in vitro and in vivo animal tests. The synthesis, characterization, and isolation of glucosyl, as well as fucosyl, mannosyl, and galactosyl saccharride derivatives of insulin will be performed. By expanding the range of insulin derivatives, improved physical-chemical properties (binding kinetics, glucose exchange rates, and permeation) may be achieved. The interaction of G-Ins with surface bound insulin receptors on an insulinoma cell will be evaluated. This in vitro model will assess the conformation or bioactivity associated with G- Ins through changes in cell growth rates and the amount of insulin secreted by the cell. The immunogenicity of the G-Ins will be investigated. Mice will be injected with G-Ins and the antibody titer to G-Ins will be determined. The central component of the self-regulated insulin delivery system is a substrate that binds both G-Ins and glucose. ConA was evaluated as a soluble monomeric protein, enclosed within microcapsules, and formed into microspheres. This proposal focuses on coupling ConA as a soluble high molecular weight oligomer. This new form will maintain the binding characteristics and not leak or permeate through the device membrane. Other saccharride binding substrates will be investigated particularly, hexokinase and glucokinase. These proteins strongly bind sugars and may achieve a greater sensitivity in G-Ins release to glucose levels. A composite membrane 'pouch' will be used to house the G-Ins/binding substrate complex. A heat sealable, 40K MWCO membrane coated with a thin dense biocompatible material will prevent the permeation of immunoglobulins from the peritoneum into the device and maintain suitable glucose and G-Ins diffusion rates. A vascular graft delivery device will also be developed. G-Ins and binding substrate will he housed in a casing surrounding a vascular graft fabricated from the same membrane as the pouch. Glucose will rapidly equilibrate into the casing and release bound G-Ins. In vitro tests of both systems will evaluate the release of G-Ins in response to glucose concentrations, binding capacity, and effective duration of the device. Both the pouch and the vascular graft systems (anastomosed to the common illiac vein) will be implanted in the peritoneum of pancreatectomized dogs for short and long term in vivo evaluations including: G-Ins levels in response to blood glucose, lowering of blood glucose, biocompatibility, in vivo duration, and decreased long term complication (glycosylated hemoglobin) by the self-regulated insulin delivery device. It is anticipated that by the completion of this application, a final prototype device aimed toward human clinical uses will be realized."}
{"text": "Recurrent interactions between neurons generate dynamic patterns of activity that serve as a substrate for behaviorally relevant computations. However, we do not yet have a principled framework for relating neural dynamics to neural computations. We have recently synthesized a theory that explains how low-rank recurrent neural networks may serve as a building block for computations. Our overarching goal is to integrate insights from this theory with behavior and electrophysiology in awake, behaving monkeys to establish a principled framework relating neural dynamics to neural computations. The project will start with reverse engineering low-rank network models that capture cortical dynamics in simple timing tasks. We then move systematically toward progressively higher rank network models that can perform timing tasks with progressively more sophisticated computational demands such as probabilistic inference of time intervals. We aim to create models that simultaneously succeed in performing task-relevant computations (i.e., behavior) and emulate cortical dynamics recorded in monkeys performing those tasks. We will use this iterative process to establish a principled framework relating neural dynamics to neural computations underlying inference. Finally, we will put this framework to test using a novel task that demands an unprecedented level of computational flexibility. RELEVANCE (See instructions): It has become increasingly apparent that the neurobiology of behavior in health and disease has to be probed at the level of populations of neurons. However, we do not yet have a rigorous and quantitative language for linking population neural activity to behavior. Our work combines primate electrophysiology with neural network modeling and aims to develop such a language through the mathematics of dynamical systems. The results hold promise for future translational research to diagnose behavioral symptoms of brain dysfunction in terms of their computational modules and the dynamic patterns of activity that support those modules."}
{"text": "Our study of the neurochemical mediation of amphetamine response is continuing with the use of the serotonin receptor blocker metergoline in conjunction with amphetamine. Five persons have participated in pilot studies related to this project. We have undertaken to use several agents that are reported to cause the release of adreno-corticotrophic hormone (ACTH) because of reported abnormalities of this hormone in affective illness. Thirteen subjects have participated in a pilot study of the opiate antagonist naloxone and five persons in a pilot study of the serotonin precursor tryptophan. A replication study of cholinergic REM induction is in progress. Four out of six bipolar patients have shown high sensitivity so far, as compared with 1 out of 7 controls. This is consistent with our previous findings, and with our hypothesis of increased cholinergic sensitivity associated with vulnerability to affective illness. We plan to extend this study to offspring of patients in the high risk study. A study of the effect of physostigmine on melatonin is underway. Four persons have participated in a pilot study."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This research is studying a dietary supplement called Inositol, which is part of the Vitamin B complex and is important for the development of the eyes. Specifically, this study is looking for the best dose of Inositol to give babies before they are ready to start taking feedings. Previous studies in premature infants have shown that blood levels of Inositol drop quickly in the first week after premature birth. Inositol is important for normal cell functioning and to help body organs grow and mature. Infants receive Inositol from their mother before they are born [through their bloodstream] and from their mother's milk after they are born. Inositol is now added to the formula that is fed to premature infants, because studies have shown that the Inositol levels fall gradually after the first week of birth if the infant is not eating, or is fed formula that does not have Inositol added. Studies have also shown that keeping steady levels of Inositol in premature infants may decrease certain eye and lung diseases. This study is looking at the level of Inositol in the blood after giving inositol through a vein when a baby is not ready to be fed breast milk or formula, because it is not known how much Inositol is the best dose. When the best dose of Inositol to give to premature babies is known, the investigators plan to do a large study to give Inositol from birth until a baby is full term. The FDA has not yet approved for Inositol to be given through a vein to infants. This study, and the large study to follow, will give information to the FDA to decide if they should approve Inositol to be given through a vein."}
{"text": "Voltage-gated sodium (NaV) channels are fundamental signaling molecules in excitable cells that determine the rapid influx of Na+ ions. Perturbation to the function of human cardiac NaV1.5 channels (SCN5A gene) can result in arrhythmias and in some cases death. Genetic defects in the SCN5A gene are a known cause of long QT type 3 syndromes, and are associated with other life threatening conditions such as Brugada syndrome and sudden infant death syndrome. For normal functioning NaV channels, changes in intracellular calcium ([Ca2+]i) lead to fast inactivation of NaV1.5. This process is known to involve the intracellular calcium sensing protein calmodulin (CaM), specific elements of the channel located near the C terminus, and the linker region between domains 3 and 4 (D3D4 linker). Recent studies have confirmed the D3D4 linker functions as the fast inactivation gate (FIG). In several instances, the above-mentioned mutations occur in, or are likely to alter interactions and functioning of CaM and the FIG, which is the focus of this work. Recent advances in the characterization of CaM-FIG interactions have identified mutations to specific residues that uncouple fast inactivation gating from [Ca2+]i dependence. However, the molecular basis for the action of the Ca2+ sensing apparatus on the FIG is currently debated and largely unknown. Several models have been proposed in the literature based on conflicting views, but no functional model has been generated that is able to reconcile all the available data. I hypothesize that changes in [Ca2+]i lead to remodeling of CaM and specific elements in the C-terminus of NaV1.5, which result in CaM interacting with the FIG (D3D4 linker) in a manner that is NOT described by previous models. Understanding the molecular details of CaM-FIG interactions are essential to the development of an accurate model that describes the Ca2+ and CaM-dependent modulation of NaV1.5 fast inactivation. Completion of this project will provide a detailed description of the highly complex mechanism that involves CaM and the D3D4 linker translating changes in [Ca2+]i into modulation of fast inactivation gating of the human cardiac sodium channel. This investigation will utilize a complement of biophysical, structural, and electrophysiological techniques to characterize interactions between CaM and the FIG that are essential to this regulatory mechanism. The results will be used to determine the relative extent of perturbation to NaV1.5 fast inactivation caused by mutations to the CaM-FIG complex. This information will aid in understanding the molecular basis of specific cardiac life threatening arrhythmias as well as lay a broad foundation for evaluating the potential use of small molecules to target the NaV1.5 [Ca2+]i sensing apparatus for the treatment of certain cardiac arrhythmia syndromes."}
{"text": "The role of RNA polymerase in the regulation of gene expression during growth and sporulation of Bacillus subtilis will be investigated. Since the sigma-43 form of RNA polymerase plays a major role during growth, early stationary phase, and catabolite repression, the following aspects of this enzyme will be investigated in detail: (1) the genetic organization and regulation of the recently cloned and sequenced sigma-43 operon will be analyzed by determining its promoter sites and RNA polymerase specificities, by testing various environmental and physiological conditions that alter transcription from these promoters, by determining the function of the first gene of the operon, and by modifying the promoter regions by site directed mutagenesis; (2) the role of sigma-43 enzyme in catabolite resistant sporulation and catabolite repression will be investigated by studying the relationship between the crsA mutation in the rpoD (sigma-43) gene and the spoO and non-sporulation related suppressor genes for crsA. The studies will involve the isolation of additional suppressor genes for crsA including those which are not related to sporulation. Glucose sensitive promoters will be isolated by a newly developed promoter expression probe plasmid and the effect of the crsA mutation will be tested on the promoters by in vivo and in vitro transcription studies. Those spoO genes which suppress crsA and which can be suppressed by crsA will be cloned and characterized. The effect of spoO gene products will be examined on transcription carried out by the wild type sigma-43 enzyme and the crsA mutated sigma-43 enzyme."}
{"text": "The use of cardiopulmonary bypass (CPB) in cardiothoracic surgery is associated with significant postoperative bleeding and pulmonary injury due to massive activation of plasma protease systems during extracorporeal circulation. Contact activation of the kallikrein- kinin system, intrinsic coagulation, the complement cascade and fibrinolysis during CPB all contribute to excessive post-bypass blood loss and an inflammatory response manifested by pulmonary dysfunction. The goal of this research is to discover a potent protein inhibitor of human factor XIIa and plasma kallikrein which can be used to prevent activation of the contact system during CPB in order to reduce blood loss and attenuate parameters of pulmonary dysfunction. Protein engineering techniques have been used to identify variants of the Kunitz protease inhibitor domain (KPI) of the human amyloid beta-protein precursor which are potent inhibitors of factor XIIa, plasma kallikrein and plasmin. In the Phase II portion of this research, a high yield recombinant expression system will be established to generate preclinical supplies of one KPI variant. The human KPI variant will be tested in a sheep model of cardiopulmonary bypass for its ability to significantly reduce post-operative blood loss and lung injury. PROPOSED COMMERCIAL APPLICATIONS A potent recombinant human protein inhibitor of factor XIIa, plasma kallikrein and plasmin would be administered prophylactically during surgeries requiring cardiopulmonary bypass to reduce postoperative blood transfusions and improve recovery times."}
{"text": "We propose to undertake a fundamental research program in mass spectrometry and combined gas chromatography-mass spectrometry (GC-MS) dealing with the analysis of selected classes of biologically significant compounds. Primary emphasis will be placed on the analysis of dopaminergic agonists such as apomorphine and related compounds, selenium amino acids and natural products isolated from plants exhibiting antitumor activity. We expect to develop correlations between fragmentation pattern and structure, and apply this information to the identification of metabolic products and novel compounds in naturally derived mixtures. Close interaction with clinicians in Boston area hospitals and other applied scientists at Northeastern University will ensure the relevance of the basic research effort and applicability of information obtained from it. Derivatization techniques will be examined for the vapor phase (GC-MS) analysis of the various types of compounds indicated above. In addition, we expect that derivative formation will be useful for the mass spectrometric differentiation of positional or stereochemical isomers, based on ionically induced functional group interaction. Chemical ionization (CI) mass spectrometry will also be employed in the structural analysis of selected classes of natural products (e.g., sesquiterpene lactones). We expect to utilize intermediates, available through synthetic programs currently in progress in the university, for the preparation of isotopically labeled compounds, to assist both in the interpretation of fragmentation patterns and for use as internal standards for quantitation by mass fragmentography."}
{"text": "3,4-methylenedioxymethamphetamine (MDMA/ 'ecstasy') is a popular \"recreational\" drug with considerable abuse liability including potential neurotoxicities directed toward the central serotonergic system. Although much attention has focused on MDMA's neurotoxic effects and its' ability to promote release of monoamine transmitters, little is known about how the drug affects the operation of neural circuits and brain function either at high neurotoxic doses or doses considered to be in the \"recreational\" range. For example, MDMA users report that one of the major pleasurable outcomes of ecstasy self-administration is enhanced tactile sensation, but there is no explanation for how the drug produces this desirable and much sought after sensory experience. Because of the potential for long term damage to the nervous system after MDMA ingestion, there is a pressing need to understand the neural substrates underlying the motivation for human self-administration of this agent. The long-term goal of the proposed research is to better understand the neurophysiology underlying ecstasy's effects. The immediate goal of the present proposal is to develop and validate procedures for evaluating the impact of MDMA on sensory signal processing in the VPM thalamus of intact rats. The project employs plasma level analysis of drug concentrations, and electrophysiological determination of VPM cellular function in intact anesthetized or waking rats. Multi-channel, multi-neuron extracellular recording, systemic drug administration, activation of afferent trigeminal somatosensory pathways, and computer based analysis of spike train data are used to assess the impact of MDMA on sensory signal processing. A significant feature of this multi-dimensional approach is that drug effects will be determined at acute and chronic doses that: 1) approximate human self-administration regimens, 2) produce known plasma levels of the drug, and 3) elicit measurable changes in monoamine efflux within sensory circuits of the brain. Understanding the relationship between MDMA administration, monoamine transmitter efflux, and the operation of the somatosensory system will provide a basis for understanding the neurophysiological mechanisms underlying the drugs' effects on tactile sensory perception, in particular; and neural circuit functions, in general. A detailed knowledge of MDMA's effects on cellular and neural circuit function including its effects on sensory neurophysiology is essential in order to provide the public with accurate information regarding the risks associated with recreational use of this popular compound and its derivatives. Furthermore, once established, these methods will be used in future studies to characterize MDMA actions in other brain networks (prefrontal cortex, limbic) and more sophisticated behavioral assays (sensory discrimination, self-administration, craving and re-instatement) as a means of further clarifying its abuse liability. Waterhouse, Barry D. PROJECT NARRATIVE ()3,4-methylenedioxymethamphetamine (MDMA/ 'ecstasy') is an increasingly popular \"recreational\" drug that poses a significant threat to the nation's health because of its: 1) adverse acute and chronic effects on behavior and physiological functions, 2) neurotoxicity toward selected neurotransmitter systems in the brain, and 3) overall addictive potential. Although much attention has focused on MDMA's neurotoxic effects and its' ability to promote release of endogenous transmitters, little is known about how the drug affects the operation of neural circuits and brain function either at high neurotoxic doses or doses considered to be in the \"recreational\" range. Because of the potential for addiction and long term damage to the nervous system after MDMA ingestion, there is a pressing need to understand the neural substrates underlying the motivation for human self-administration of this agent. The goal of the proposed project is to develop and validate procedures that will help us better comprehend the neurophysiological basis for ecstasy's effects in human drug users. [unreadable] [unreadable] [unreadable]"}
{"text": "Xenopus Nucleoplasmin (Np) and N1 are equally abundant chaperones found in oocytes and eggs. Np participates in nucleosome assembly and exchange reactions involving histone H2A/H2B dimers and DNA; whilst N1 functions as a chaperone specific for the H3/H4 tetramer. Therefore, N1 and Np may function together to promote nucleosome assembly in oocytes. DNA replication and transcription are sensitive to the local packing of nucleosomes, which may be modulated by histone acetylation/deacetylation, phosphorylation, DNA methylation, or a combination thereof. Hence, nucleosome assembly and exchange factors may play a critical role in these cellular processes. We propose to study the roles of Np and N1 in nucleosome assembly and in the decompaction of sperm chromatin after fertilization, by obtaining the first high resolution crystal structures of these chaperones, alone and complexed with their cognate histories. This will reveal the molecular architecture and function of the Np and N1-families, while providing insights into the extreme thermal and chemical stability of the Np pentamer. It was suggested previously that N1 and Np function sequentially to deliver core histones to DNA during nucleosome assembly. We have demonstrated that Np forms a decamer in solution which can be reconstituted with separately purified histories H2A/H2B and H3/H4, to form stable Np decamer-octamer complexes in the absence of DNA. Hence, the Np decamer with 522 point group symmetry may provide a docking ring for 10 H2A/H2B dimers and 5 H3/H4 tetramers, to form an Np decamer-octamer complex which utilizes their common 2-fold symmetry. We further suggest that N1 may establish an N1 dimeric cap on H3/H4 tetramers in cells, which then docks with Np decamer-dimer complexes to form a larger Histone Octamer co-Chaperone (HOC) complex. Overall, our data suggest a new paradigm for nucleosome assembly, in which pre-formed histone octamer-chaperone complexes and DNA are brought together in a concerted mechanism. To understand this process, the structures of the Np decamer-octamer and putative HOC complexes will be determined by electron cryo-microscopy and X-ray crystallography, in combination with biochemical studies of nucleosome assembly."}
{"text": "APOBECSG (A3G) and APOBECSF (A3F) are involved in various anti-viral protein and nucleic acid transactions. Important primary interactions include interprotein interactions, binding single-stranded nucleic acids (RNA and ssDNA), and neutralization by HIV Vif. The full elucidation of these intermolecular interactions is necessary to understand the mechanism by which these cellular proteins inhibit HIV replication. The role of oligomerization in their activity is an issue of great importance. These are interactions at the nanoscale level, and AFM is the method of choice capable to addressing problems listed above. AFM currently reached a level of development that enables this unique instrument to provide a wealth of important information to the characterization of biomolecular complexes at nanoscale. The topographic analysis of molecular system is the initial area of the AFM application for characterization of the structure and specificity of protein-DNA systems of a broad complexity. In addition to imaging, AFM is capable of direct measurements of molecular interaction within the system and research during this decade led to a dramatic progress in this area of the AFM applications. Our Preliminary Studies have already enabled us to provide the first images of A3G bound to ssDNA substrates. We further anticipate that the use of all modalities of AFM technology will help us detail these complexes, dissect the protein and chemical requirements, and extend key observations to ASF. To help achieve these overarching objectives, we will pursue the following three Specific Aims: Aim 1. Image A3 complexes at nanoscale resolution using AFM (Program Objectives 1 &2);Aim 2. Measure interactions within A3-ssDNA complexes using force spectroscopy (Program Objectives 1 &2) and Aim 3. Characterize A3-Vif interactions using AFM imaging and probing modalities (Program Objectives 3). The studies under this project are an integral component of our overall Program Project aiming to provide a comprehensive understanding of A3-AS3 A3-nucleic acid, and A3-Vif interactions. The nanoscale studies will provide a platform for evaluating any future therapeutics that function by leveraging the AS-Vif interaction."}
{"text": "ADMINISTRATIVE CORE SUMMARY/ABSTRACT The mission of Mayo Translational PKD Center (MTPC) is to advance the understanding and reduce the individual and societal burden of PKD. The overall goal of MTPC is to develop Core facilities to support existing and stimulate new PKD research by Mayo and non-Mayo investigators and to facilitate the translation of basic research breakthroughs into improvements in clinical practice through the implementation of clinical trials. Since July 2010, the Administrative Core of the MTPC has: i) provided strong strategic and administrative oversight in the newly established PKD Research and Translation Core Center; ii) fostered the interaction and synergism of three, heavily used MTPC Biomedical Research Cores (Molecular Genetics and Biomarker Core, Model Systems Core, and Human Imaging Core), chosen as the most effective to achieve the Center's goals and building on the strength of the Mayo investigators; iii) strengthened the Internal Research Base and added a strong External Research Base of PKD investigators; iv) attracted and nurtured new PKD investigators through a dynamic Pilot and Feasibility Program; v) adopted software tools and developed metrics to monitor the performance of the Biomedical Research Cores and the Pilot and Feasibility Program; vi) implemented a vigorous Scientific Enrichment Program; and v) established relationships with internal and external partners to facilitate MTPC member access to resources. The Administrative Core leadership, provided by the Director and Co-Director, in conjunction with the Operations Committee, Executive Committee, Internal Advisory Committee, and External Advisory Committee, will continue to 1) Promote cooperative, successful scientific interactions among the multidisciplinary internal faculty and an extended faculty from 39 other institutions; 2) conduct periodic membership surveys to determine the needs of the internal and external members; 3) conduct periodic reviews of the Services provided by the Biomedical Research Cores of the MTPC to ensure that cutting edge technologies replace those no longer needed; 4) promote the communication and coordinate the activities of the three Biomedical Research Cores of the MTPC to ensure that they synergistically enhance the research capabilities of the members; 5) conduct periodic reviews of Core Use and involvement in Center Activities to modify the Research Base membership as needed; 6) provide budgetary and administrative management necessary for efficient, cost-effective accomplishment of Center goals and allocate resources commensurate with member needs; 7) ensure a successful Scientific Enrichment Program including weekly seminars, monthly conferences, periodic workshops, annual mini-symposia, visiting faculty program, mini-sabbaticals and communications via electronic media; 8) continue a Pilot and Feasibility Program funded equally by this grant and by the Mayo Foundation under the direction of the Pilot and Feasibility Program Director and the External Advisory Committee; 9) stimulate interest in PKD and provide career development opportunities for junior investigators through the NIH Summer Undergraduate Research Fellowship, NIH T32 training grant (T32) and other funding sources and by creating a forum for their research updates; 10) continue and expand existing cost-effective, scientific interactions between MTPC and the NIDDK-funded C-SiG, the Mayo CCaTS, and other NIH funded Centers."}
{"text": "The human body contains a diverse community of bacteria, a majority of which is found in the gastrointestinal (GI) tract. Alterations in the GI microbiota have been correlated to diseases such as obesity and irritable bowel syndrome. Secretory diarrhea exhibits alterations on the GI community, and up to 60% of people traveling from developed to developing countries acquire a form of secretory diarrhea know as travelers' diarrhea (TD). Enterotoxigenic E. coli (ETEC) and Norovirus (NV) are the leading cause of bacterial and viral TD, respectively. ETEC expresses two toxins, heat labile toxin (LT) and heat stable toxin (ST), which ultimately lead to secretory diarrhea. The mechanism of noroviral diarrhea is not well understood. Previous studies have shown that an etiologic agent is not identified in as many as 50% of TD cases, suggesting that travel alone may play a role in TD. Presumably, TD causes a shift in bacterial GI communities; however, this has not been studied. The goal of this proposal is to use 16S rDNA metagenomic sequencing and analysis methods to investigate how TD caused by ETEC and NV affects the gut microbiome and to identify shifts in the gut microbiome associated with foreign travel. In Aim 1 I will complete a retrospective study to examine how ETEC-TD alters the gut microbiome by comparing the GI microbial populations in stool samples from healthy travelers to samples from individuals with ETEC-TD and to samples from individuals with unidentified pathogen-TD. I will also develop a PCR assay to rapidly and reliably detect ETEC in human stool samples. Aim 2 will investigate how the microbial populations in the gut shift during NV-TD. In this retrospective study I will compare paired samples where the first sample was collected during an acute diarrheal episode and the second sample, collected from the same individual, was collected after amelioration of diarrheal symptoms. This will allow me to determine how the gut microbiota shifts after an acute episode of NV-TD. In my third Aim, I will complete a prospective study, which seeks to determine the effect of travel on the gut microbiome. I will compare bacterial populations from stool samples, collected at various time points, from healthy individuals traveling to Guadalajara, Mexico. Stool samples will be collected prior to travel, at 7, 14, and 21 days after arrival in Mexico, and upon return to the United States, which will permit me to identify specific changes within the gut microbiome associated with travel to a developing country. This proposal outlines the necessary first steps in understanding how TD affects the gut microbiome. Results from my research could potentially lead to development of preventative treatments and novel therapeutics against TD. )"}
{"text": "Respiratory syncytial virus (RSV) infection is the most common cause of respiratory disease leading to hospitalization in children. Preterm infants are especially susceptible to severe RSV infection. Respiratory epithelium is an initial site of RSV infection and epithelial cells along with alveolar macrophages (AM) and dendritic cells (DC) are vital to innate and adaptive immune responses. However, the extent of innate immune gene expression by epithelia and AM-DC in preterm infants can be variable/limited. The hypothesis is that: Reduced innate immunity by respiratory epithelia and AM-DC preterm enhances susceptibility to RSV infection This hypothesis is based on the facts above and our preliminary data in lambs demonstrating limited expression of surfactant proteins A and D (SP-AD), sheep beta-defensin-1 (SBD-1), and Toll-like receptor 4 (TLR4) preterm. It will be tested in preterm lambs which have close similarities with human disease including susceptibility, lesions, and innate immunity. Specific Aim 1 compares expression of key innate immune genes (SBD-1, SP-AD, TLR4) and protein/peptide production in vivo as well as AM-DC cytokine expression in pre- and full-term lung. It also tests the hypothesis that limited epithelial cell proliferation and/or differentiation pre-term underlie(s) the mechanistic basis for limited SP-AD, SDB-1 expression and tests this by comparing transcriptional activity, protein/peptide production in pre- and full term cultured cells with or without cell proliferation and differentiation. Specific Aim 2 tests the hypothesis that SP-AD, SBD-1 and AM-DC responses to RSV are less at pre- than full-term in the lamb model and in vitro with cultured epithelial cells. A second hypothesis, that increased cell proliferation and/or differentiation of epithelia protects against RSV infection, will be tested in vitro with treatments from Aim 1. The extent to which SP-AD, SBD-1 directly prevent RSV infection will be tested with RNAi assays. Specific Aim 3 tests the hypothesis that yet other innate immune genes expressed with cell proliferation/differentiation prevent RSV infection. This Aim uses gene expression profiling of primary polarized human lung cells and a respiratory epithelia-specific probe set (Unigene) that is the most well-annotated and defined gene target set to date. It also identifies ovine orthologs of human genes and test for impaired/reduced expression preterm. This team combines veterinary and human medical expertise to attain the goal of this project: to discover the reason(s) for inadequate expression of SP-AD, SBD-1 and other innate immune genes as well as AMDC responses at preterm that predispose to RSV infection. The work is significant because it discovers the underlying basis for RSV susceptibility preterm and mechanistic approaches to enhance innate immunity. [unreadable] [unreadable] [unreadable]"}
{"text": "Fatty acid amide hydrolase (FAAH) is the enzyme predominantly responsible for the catabolism of several fatty acid amides (FAA), including the endogenous cannabinoid N-arachidonoyI ethanolamine (anandamide), the sleep-inducing agent oleamide, the food-suppressing compound N-oleoylethanolamine (OEA), and the peripheral pain-suppressing agent N-palmitoylethanolamine (PEA). The creation of genetically engineered mice that lack this enzyme (i.e., FAAH(-/-) mice) has provided a powerful model to evaluate the function of this enzyme. These mice exhibit a CBI-mediated reduction in pain sensitivity, accompanied by substantial increases in endogenous anandamide levels compared to wild type mice. In the studies proposed in this application, we will evaluate the in vivo effects of a series of highly selective and reversible FAAH inhibitors. These FAAH inhibitors provide complementary tools to further our understanding of the physiological functions of the FAAH/FAA system. This project will employ pharmacological and behavioral methods to address three Specific Aims: 1) To determine the role of endocannabinoid-metabolizing enzymes in acute and chronic pain; 2) To determine the role of endocannabinoid-metabolizing enzymes in cognition and emotion; and 3) To determine the role of endocannabinoid-metabolizing enzymes in morphine reward and withdrawal. RELEVANCE (See instructions): The endogenous cannabinoid system regulates a broad range of neurophysiological processes. Elucidation of the enzymes that regulate endogenous cannabinoids and their mechanisms of action may lead to the identification of new therapeutic targets for the treatment of human disorders such as chronic pain, depression, and anxiety."}
{"text": "The postnatal development of both the optical and neural visual systems is dependent on visual experience. Visual experience is defined by the information available in the retinal images in the two eyes. The goal of the proposed research is to extend our previous examination of retinal image quality in one eye during infancy to full binocular viewing conditions. We will examine human infants' visual experience in the context of image clarity and image alignment, which are primarily defined by accommodation and vergence responses and their interaction. These studies will document the emergence of the interaction between accommodation and vergence and their role in the development of refractive and accommodative strabismus. There are three specific aims: i) To understand the normal maturation of the relationship between accommodation and vergence with emmetropisation and growth of the distance between the eyes. ii) To determine the relative bias towards accommodation or vergence accuracy during the critical period of human development. iii) To understand the effects of accommodation and vergence behavior on visual experience of infants and children with high hyperopia or strabismus. PUBLIC HEALTH RELEVANCE: This project will determine how young infants and children manage apparently conflicting focusing and eye alignment demands during typical development. It will also investigate why some children develop refractive or accommodative strabismus while others, with apparently matching visual systems, do not. The goal is to develop intervention strategies to prevent this strabismus and associated amblyopia."}
{"text": "Nira Pollock, MD, PhD, is currently an instructor of medicine and infectious diseases (ID) at Beth Israel Deaconess Medical Center (BIDMC). The candidate's long-term goal is to develop an independent patient- oriented research career focused on the development and evaluation of new diagnostic tests for infectious diseases. Dr. Pollock's interest in diagnostics emerged from her strong combined basic science and clinical training and was focused by her ID specialization. Recognizing the rise of global tuberculosis (TB) case rates in the era of HIV and the limitations placed on TB control efforts by the weaknesses of currently available TB diagnostics, the candidate has chosen to focus her research and clinical efforts on TB. Under the guidance of two senior Harvard-affiliated TB researchers, Dr. Antonio Campos-Neto and Dr. Edward Nardell, she has initiated a promising TB diagnostic development project and has prepared a multi- disciplinary career development plan which will allow her to develop into an independent translational researcher with an exceptionally strong grounding in both ID and clinical microbiology. For her project, Dr. Pollock will work to develop a new urine antigen detection test for diagnosis of active TB disease. Her goal is to create a fully non-invasive, point-of-care, rapid test which would be useful in resource-limited settings, thereby making a substantial contribution to patient care and TB control. After completing pre-clinical development, she will evaluate the new test by integrating testing into an established clinical study of TB in Peru, run by Partners in Health. This will allow her to assess test performance for routine diagnosis of active pulmonary TB in adults, as well as to gain preliminary data about test utility for diagnosis of TB in children and for monitoring treatment efficacy. Dr. Pollock will combine her research program with a structured, comprehensive didactic curriculum in clinical research methodology at the Harvard School of Public Health. Finally, she will continue to develop her expertise in clinical microbiology through further practical experience in the BIDMC microbiology laboratory, recognizing that the clinical microbiology laboratory provides an ideal setting for applied infectious diseases diagnostics research. RELEVANCE (See instructions): The goal of this project is to develop a new, rapid, simple, and highly accurate urine test for diagnosis of active tuberculosis (TB). This test could dramatically change how TB is diagnosed world-wide, and thus could help prevent the spread of this highly contagious and often deadly disease."}
{"text": "Recurrent HSV-2 shedding is a major risk factor for HIV-1 transmission. Recent data indicate that HSV-2 is present up to 40% of days in the periphery. The factors that control HSV-2 shedding in the skin are incompletely understood. One contributor is likely HSV-2-specific CDS T-cells, which will be studied in Project 1. This Project will focus on the expression of antiviral genes in the skin and correlating this with shedding of HSV-2. We hypothesize that a net antiviral state, as measured by the mRNA levels of interferon-stimulated genes (ISG), will correlate with the re-appearance of HSV-2 DMA after the healing of symptomatic HSV-2 lesions. Because circulating HSV-2-specific CDS T-cells over-express CXCR3, and mice with lesions in the CXCR3 axis have accelerated HSV-2 pathogenesis, we further hypothesize that the CXCR3 ligand chemokines, CXCL9, 10, and 11, will correlate with the infiltration of HSV-2-specific CDS cells (Project 1) and pDC, and negatively with detection of HSV-2 DMA and mRNA. Plasmacytoid dendritic cells (pDC) infiltrate HSV-2 lesions in animals and humans (our data), react strongly with HSV-2 via TLR9 to secrete IFN-a, and are required for defense against HSV-2 in mice. Substantial heterogeneity in pDC reactivity to HSV-2 has been observed, as have rare patients with pDC numerical or functional deficiencies and severe HSV infections. pDC likely contribute to the net IFN-driven antiviral state in lesions and the anti- HSV drug action of resiquimod. We propose blood-based studies to investigate the hypothesis that pDC number of function correlates with HSV-2 shedding rates, using subjects from the Clinical Core who are studied in Project I. Aim 1 will correlate ISG and interferon-producing cells with HSV-2 DNA/mRNA detection in human skin biopsies. Aim 2 will focus on the chemokine receptor utilization of HSV-2-specific CDS T-cells. Aim 3 will correlate pDC number and function in blood with HSV-2 shedding and clearance rates. The results may assist in designing vaccines for HSV-2 that elicit cells with the appropriate homing strategies, and clarify the mechanism of action of innate immunity modifiers as local therapy for HSV-2."}
{"text": "Technological developments in the field of genomics now afford the opportunity to define the complete sequence of an individual's genome in a rapid and affordable manner. Such whole genome sequencing and its simpler corollary, whole exome sequencing (WES), have already established themselves as powerful research tools. The natural next step in the evolution of this technology is its direct application in the clinical arena. However, while such technology holds considerable clinical promise, tremendous challenges exist in applying it and deriving practical benefit to patients. In this proposal we outline a highly interdisciplinary approach to identifying, confronting and overcoming the major challenges which must be met in order to implement deep sequencing technology in clinical medicine. Aim 1 will explore the use of WES as a diagnostic tool in the care of a broad array of patients, evaluate its performance, identify critical clinical characteristics which can guide its application and measure the impact of such information on patients and providers. Aim 2 will tackle one of the most pressing challenges in the clinical application of WES: the inevitable generation of collateral or bystander information. Educational materials will be developed to enable patients to make decisions about appropriate return of results and the impact of collateral information will be assessed at the level of the provider, laboratory and patient. The third major challenge in clinical implementation of genomic medicine, how do deal with vast amounts of information, will be addressed by Aim 3. A clinically oriented binning structure will be created and refined for classifying, storing and transmitting data within practical categories so as to make sense of the large amounts of data generated. Finally, as we stand on the cusp of genomic medicine, we must ensure that all have access to its benefits. Thus, Aim 4 will pursue clinical WES in traditionally underrepresented populations to identify special opportunities and challenges in the clinical translation of this new tool to the broadest possible population. Our ultimate aim is to establish a set of best practices to guide future implementation of robust genomic technologies for the real and practical betterment of human health."}
{"text": "A notable determinant of human intellectual capacity is the enormous size and complexity of our neocortex. The neocortex forms during embryogenesis and then expands during fetal development when progenitors differentiate to populate the cortical plate. Defects in brain growth and morphogenesis result in a host of neurodevelopmental disorders, neuropsychiatric diseases, and intellectual disabilities. A key step towards understanding the normal and abnormal functions of the brain thus lies in defining the mechanisms driving neocortical growth. Progress towards this goal has been made though the identification of functionally distinct neural progenitor populations, most prominently ventricular radial glia (vRG), intermediate progenitor (IP), and basal/outer radial glia (bRG) cell. These classes of progenitors are common to both rodents and humans. However, recent studies have proposed that the neocortical enlargement and complexity seen in humans may result in part from a substantial increase in the genesis of bRG and IP cells that is not seen in rodents. Remarkably little is known about the mechanisms behind this human-specific expansion. Our preliminary experiments implicate Foxp transcription factors as important components to this process. Foxp1 and Foxp4 are expressed in the human neocortex as vRG cells transform into bRG and IP cells, and altering Foxp1 and Foxp4 functions in mouse changes cortical development in a manner suggesting that they play pivotal roles controlling the production of bRG and IP cells respectively. In this proposal, we will determine the function of Foxp proteins in both mouse and human cortical development. In Aim 1 we will characterize the expression of Foxp proteins in the developing mouse and human neocortex. In Aim 2, we will determine how manipulation of Foxp functions alters the generation of bRG and IP cells, and the overall size and structure of the cerebral cortex. Lastly, in Aim 3 we will define the genomic targets of Foxp proteins mediating their contributions to neocortical growth."}
{"text": "The purpose of the NHANES (National Health and Nutrition Examination Survey) Initial Epideomiologic Follow-up Survey was to conduct a longitudinal study of 14,407 adults originally surveyed in 1971-1975, to investigate subsequent health and mortality outcomes. Respondents have been traced and re-examined. Additional information was obtained from hospital records, the National Death Index and death certificates. The NHANES Initial Follow-up Survey is essentially complete. The purpose of this intramural project is to examine the relationship of chemopreventive and nutritional factors and cancer in this representative population. This study provides an opportunity to examine these factors and potentially confounding or modifying factors in a prospective fashion, and to examine the effectiveness or toxicity of some dietary agents which are currently of great interest for cancer prevention. The relationship of baseline vitamin use, biochemical or nutritional measures and subsequent health status will be examined. In addition, descriptive data and trends in potential risk factors or protective factors over time will be examined. A continued follow-up for this cohort is expected to begin in 1985."}
{"text": "Human retinal pigment epithelial (RPE) cells accumulate fluorescent lipofuscin granules with age and this phenomenon has been implicated in several age-related degenerative retinopathies. The standing hypothesis of lipofuscin accumulation implicates lipid peroxidation processes in the formation of indigestable fluorescent (460 nm peak emission) residues which are sequestered and concentrated in tertiary lysosomes of the cellular phagolysosomal system. In preliminary studies utilizing human RPE, blue-emitting flurophores were not found within the lipofuscin granules as this hypothesis predicts. Rather, at least two yellow-emitting compounds of unknown identity have been extracted and isolated from purified lipofuscin granules. Yellow-emitting fluorophores are not normally considered indicative of lipid peroxidation byproducts, but their presence is more in keeping with subjective descriptions of yellow fluorescent emissions observed by fluorescence microscopy. I have further isolated at least two age-related blue-emitting fluorophores from cellular sources outside the lipofuscin compartment, and another compount which is present only in aged human RPE and which produces an intensely fluorescent compount upon reaction with ninhydrin. The aims of the present proposal are to utilize thin layer chromatography (TLC) to isolate and purify these compounds from human RPE extracts and to characterize and classify or identify them by means of corrected spectrofluorometry and uv-vis, infrared, nuclear magnetic resonance and mass spectrometry. Standardized high performance TLC and quantitative densitometry will be used to track the age-related appearnce and accumulation of these compounds in relation to one another in RPE from a wide age-range of human donors, and to survey a range of species in search of appropriate animal models. Fluorescence assays will be developed and deployed in further subcellular fractionation studies on the acute effects of the lipofuscinolytic drug, centrophenoxin, on these compounds. These studies will serve to focus the direction of future research and will yield new information essential for the reformulation of the hypothesis of lipofuscin formation and accumulation, and of our understanding of aging phenomena in postmitotic cells in general."}
{"text": "The purpose of this study is to (1) quantify the recovery of labeled CO2 in breath during a primed constant infusion of sodium 13C bicarbonate in human pregnancy, during fasting and in response to feeding and (2) to examine the effect of advancing gestation on these parameters."}
{"text": "Acanthamoeba keratitis is a painful and debilitating infection of the eye caused by an opportunistic amoeba that occurs ubiquitously in nature. From the discovery of this disease in 1973 until 1984, there were only 11 published cases. At present, an estimated 20 new cases are confirmed per month and the majority are associated with contact lens wear. Both awareness and incidence of the disease are thought to be increasing. The success of medical treatment is inconsistent and repeated surgical intervention often is required. There is considerable confusion over identification of clinical isolates. Cell morphology and affinity for specific antisera are the criteria used for identification, but these approaches often give ambiguous results. There is an urgent need for methods to detect small numbers of amebas and to identify the infectious organisms reliably. This project proposes the use of small subunit ribosomal RNA genes (18S rDNA) as a basis for development of genus-, species-, and strain- specific oligonucleotide probes suitable for laboratory and clinical identification of isolates. This one-year pilot project will focus on sequencing of 18S rDNA isolated from amoeba strains associated with human eye infections. Nuclear rDNA will be amplified by the polymerase chain reaction and then sequenced. Sequences will be determined using oligonucleotide primers, primer extension, and dideoxynucleotide chain termination. rDNA sequences then will be analyzed to determine the extent of sequence variability among strains and searched for subsets of sequences that would be suitable targets for probes with different levels of specificity (i.e. genus, species, or strain). Development of clinical probes would occur in subsequent projects if suitable sequence variability is discovered in this study."}
{"text": "The mechanisms mediating kidney damage in diabetes likely stem from chronic hyperglycemia; however, the steps involved are not completely understood. Our main hypothesis is that a high ambient glucose level, independent of genetic and hemodynamic factors, exerts a powerful influence on renal cell growth and extracellular matrix biosynthesis in tissue culture. An exciting finding has been the elucidation that Transforming Growth Factor-Beta (TGF-Beta) is an autocrine mediator of many of the renal effects of high glucose. The specific objective of this proposal are to focus on two major aspects of the effects of high glucose on renal glomerular mesangial cells: the modulation of key proteins which regulate transcription, synthesis and bioactivity of TGF- Beta; and the high glucose-induced perturbations in growth factor- mediated cellular signaling and the role of different protein kinase C isozymes. In vivo correlates will be sought by examining kidney expression and cell localization of TGF-Beta in experimental DN in rodents, and attempt to intercept renal TGF-Beta in vivo using neutralizing anti-TGF-Beta antibodies. The significance of these studies is that they will provide a detailed mechanistic understanding of the harmful effects of high glucose concentration on the kidney, and also pave the way for designing innovative means of prevention of DN, even when glucose control is suboptimal."}
{"text": "In this revised application for a Mentored Patient-Oriented Research Career Development Award (K23), Dr. David Kimhy will pursue training and research in the cognitive and neurobiological mechanisms of stress regulation and psychosis in schizophrenia. The goals of the training and research plan are integrated around the hypothesis that cognitive coping strategies mediate the link between stress and psychotic symptoms during recovery from psychosis in schizophrenia. Under the sponsorship of Dr. Dolores Malaspina, the training plan capitalizes on the resources of NYSPI/Columbia University, combining formal coursework, direct supervision, and clinical research experience and training. The plan involves mentorship and didactic instruction in key areas including: 1) psychosis research in schizophrenia using of Experience Sampling Method (ESM), 2) clinical methodologies for assessment of stress-regulating systems, 3) data analysis using multilevel linear modeling, and 4) cognitive-behavior therapy for psychosis (CBTp). Current methodologies have limited ecological validity to establish causal links between momentary changes in stress and psychosis. Dr. Kimhy will study simultaneously the real-time physiological and psychological correlates of psychosis using ambulatory assessment of cardiac autonomic regulation (Heart Rate Variability; HRV) synchronized with data collected using Experience Sampling Method (ESM) with Palm handheld computers. The research plan involves simultaneous probing of momentary psychotic symptoms, subjective stress, HRV, and cognitive coping strategies. Assessments will be conducted before and after pharmacological and CBTp treatments of psychosis. This methodology translates basic science into use in real-time, real-world environments (in-vivo and in-situ), allowing for the assessment of psychosis as part of the ebb-and-flow of daily functioning. Understanding these interactions will advance our understanding of the mechanisms of recovery from psychosis in schizophrenia. The proposed training and research plan will provide Dr. Kimhy with the knowledge, training, and experience necessary to develop as an independent researcher focusing on the neurobiological and psychological mechanisms of stress regulation and psychosis in schizophrenia. [unreadable] [unreadable] [unreadable]"}
{"text": "We propose to build a multidisciplinary Zebrafish Cardiac Development Research Center with a collaborative group of zebrafish developmental biologists, zebrafish cardiac physiologists, and experts in chromatin structure, genome-wide gene network profiling, bioengineering and bioinformatics. We will investigate gene regulatory networks at distinct steps in development that control normal cardiac development at multiple molecular levels. Several diverse perturbations of cardiac development will serve as \"inputs\" to assess changes in cardiac development gene regulatory networks, including genetic mutants and morphants, embryological cell lineage manipulations and pharmacological treatments. The \"outputs\" will be analyses of chromatin structure that regulates transcriptional programs, epigenetic modifications including DNA methylation and chromatin marks that contribute to transcriptional activation (H3K4me3) or transcriptional repression (H3K9me3, and H3K27me3), genome-wide gene expression profiles (including mRNAs, microRNAs, SINE RNAs, other small RNAs) and transcriptional regulators involved in the patterning, morphogenesis and physiology of cardiomyocytes during development. Bioinformatics comparisons of normal and a large number of aberrant profiles within zebrafish will uncover molecular signatures of cardiac developmental defects. This multi-layered molecular profiling of cardiac development has not been performed in any organism. As new candidate genes or other perturbations (including pharmacological or environmental) arise within the Cardiac Development Consortium and Pediatric Cardiac Genomics Consortium, they will be incorporated into this multi-layered molecular profiling program to rapidly obtain genome-wide molecular signatures. Our infrastructure will make these datasets readily accessible to the consortia for trans-species comparisons to uncover conserved molecular signatures relevant to human developmental heart defects. RELEVANCE (See instructions): Obtaining multi-layered molecular profiles of cardiac developmental defects will reveal the underlying causes of cardiac developmental defects, with the long term goal of applying these insights to treatment of children with heart defects."}
{"text": "Patients with systemic lupus erythematosus have been found to have disturbances of cell-mediated immune responses. Alterations of B and T lymphocytes as well as monocyte function, natural killer cell and interferon responses, have been observed both in patients with SLE and certain of their relatives. The major goal of these studies is to further characterize the types of immunological distrubances in SLE and to elucidate the mechanisms which lead to alterations of immune regulation and are responsible for the pathogenesis of this disease. The ability of immunosuppressive drugs to alter cell mediated immune function and possibly to re-establish more nearly normal immunoregulation is being actively studied."}
{"text": "TRANSGENIC AND CHIMERIC MOUSE CORE: Director-N. Cooke The Transgenic and Chimeric Mouse Core Facility (TCMF) of the University of Pennsylvania has been in operation since February 1992 under the continuous co-direction of Dr. Nancy Cooke and Dr. Stephen Liebhaber and under the day-to-day technical direction of Dr. Jean Richa. The initial components of the TCMF were assembled from financial contributions of the School of Medicine and resources from the laboratories of Drs. Cooke and Liebhaber in 1989 with subsequent phased expansions in facilities, capabilities, personnel, and services. Over the past five years the Core has generated a wide range of transgenic and gene-targeted mouse lines that have proven to be powerful tools for the study of the molecular biology and physiology of Type I and Type II diabetes, obesity, and developmental and mechanistic bases for a wide variety of other disorders of endocrinology and metabolism. The Core laboratory is located in a pathogen-free microbiologic barrier facility in the basement of the Clinical Research Building. A primary function of the TCMF is to provide a centralized laboratory that will generate infectionfree strains of mice carrying transgenes or targeted genetic alterations of specific interest to individual projects in the DERC. The centralization of these technically demanding procedures in the Core laboratory results in enhanced efficiency and significant cost reduction for each project. During the current project period the TCMF has grown significantly in service offerings and its facilities have been improved and expanded. A major increase in the throughput potential of the Core has been achieved by the doubling in the size of the Core microinjection laboratory, funded by the School of Medicine. This expansion has doubled the capacity in line with the continuing increase in demand for services. In addition, the Core is now finalizing a new Cryopreservation Facility for the long-term storage of embryos and sperm. This addition to the Core will be opening in the summer of 2006. This newly configured, secure facility is off-site from the main Core laboratory, and has been established by funds from the School of Medicine. Additional services that have been established during the current funding period include sperm cyropreservation, intracytoplasmic sperm injection (ICSI), DNA diagnostics, and integration with the School of Medicine gene knock-out service. These services have substantially increased the utility of the Core to Center members. The Core will continue to maintain the highest level of quality service and will continue to acquire new techniques and expertise to maintain services at a 'state-of-the-art' level."}
{"text": "The overall goal of our research is to understand the molecular and cellular events that lead to cancer. Our lab in particular studies the actions of a family of proteins called STATs, which are transcription factors. Transcription factors are proteins that bind to DNA and regulate the expression of genes, which then go on to determine the phenotype and behavior of the cell. Originally, all genes were thought to code for proteins, but it is now clear that certain genes are not made into proteins. This type of gene, called a microRNA because of its relatively short length, was only recognized fairly recently. We now know that microRNAs act by inhibiting the conversion of protein-coding genes into proteins. Since their discovery, microRNAs have been found to play a part in all aspects of cancer development and progression. At present, very little is known about microRNA genes regulated by STATs and their relevance to cancer. Recently, we have identified a novel microRNA whose expression is controlled by STATs. Our proposed research will investigate the functional significance of STAT regulation of this microRNA as it relates to cancer. In our first set of experiments, we will initially characterize where STATs bind to the DNA relative to the microRNA. This will reveal how the microRNA is molecularly regulated. Next, we will assess if regulation of this microRNA is intact in cancer cells. Since the microRNA may have anti-cancer effects, we believe that STAT regulation of the microRNA goes awry in cancer. Lastly, we will analyze the causes for dysregulation of the microRNA, which could be genetic (a change in the DNA sequence) or epigenetic (other properties of the DNA change, making it harder to read). In our second set of experiments, we hypothesize that the microRNA helps to sensitize cancer cells to a stimulus, called TRAIL, that is capable of selectively killing cancer cells. First, we wll enhance or inhibit the activity of the microRNA and see if that increases or decreases sensitivity to TRAIL, respectively. Next, we will test if the microRNA acts by inhibiting a specific survival pathway. We believe that enforcing the activity of this pathway will rescue cells with enhanced activity of the microRNA. Lastly, we will test the involvement of specific genes in the actions of the microRNA."}
{"text": "Objectives: Providers and patients face barriers to accomplishing the complete communication needed to make complex decisions together about diabetes treatments and self-management tasks. Providers often have limited time and patients often have limited resources. Peer mentoring models have been found in two recent VA research trials to be more effective than usual care, financial incentives, and nurse care management in improving glycemic control in high-risk Veteran patients with diabetes. While peers can be trained in effective approaches to support other Veterans' self-management behaviors, such coaches necessarily lack diabetes and diabetes medications content expertise to help Veterans better share in treatment decisions and goal-setting with their health care providers. Accordingly, in a recent research study we developed and tested a tailored, interactive diabetes and diabetes medication information tool that outreach workers can use to facilitate discussions with patients. Such tools can enhance the sustainability and effectiveness of coaching programs to better prepare patients to set self- management goals, action plans, and to discuss treatment options with their providers. Because this intervention addresses barriers to disease management for chronically-ill patients, physicians, and case managers, the study may have broader impact on management practices for other chronic illnesses. Project Methods: 348 diabetes patients with poor glycemic control (A1c>8.0% or A1c>8.5% if older than 70) will be recruited from the Detroit VA and randomized to either Peer Support with TEC or Peer Support alone. All participants will be assigned to one of 87 peer coaches, who also are Detroit VA diabetes patients who previously had poor glycemic control but are currently in good control. Each peer coach will be assigned about 4 participants on the basis of race and approximate age. Peer coaches will undergo training in motivational interviewing-based counseling approaches and instruction in use of the iPad-platform decision aid tool. After their baseline assessment, participants in both arms will receive information on their lab and blood pressure values and will be randomized to one of the two study arms. Participants randomized to the control group will be scheduled for an initial visit wit their coach. The coach will then help them list questions and concerns they wish to discuss with their health care provider, practice raising their questions and concerns and develop an action plan to address barriers to self-management they have identified. Participants in the TEC arm will be scheduled for an initial visit with their coach to review the decision aid, which has incorporated their personal baseline data. The coach will then provide the same intervention that the Control group receives. During the next six months the coach will call their assigned peers once a week to provide support for their action steps. These calls will be placed using a confidential IVR system that connects the callers without sharing phone numbers. The research will measure changes in HbA1c, BP, patient-centered outcomes, mediators and moderators of intervention effects and cost-effectiveness."}
{"text": "Both DNA replication and centrosome duplication are semi-conservative replication processes that must be coordinated to occur once-and-only-once per cell cycle. Recent findings have suggested that both cyclin dependent kinases and the SCF and APC ubiquitin ligases conspire to regulate both DNA replication and centrosome duplication. Biochemical studies show that the conserved Cdc14 family of phosphatases are selective for dephosphorylating substrates of cyclin-dependent kinases. We have recently discovered that a form of the human Cdc14 phosphatase also has a critical role at the centrosome. Our preliminary studies show that the two known forms of human Cdcl4, called Cdcl4A and Cdcl4B, localize to the centrosome and nucleolus, respectively, We have mapped targeting sequences directing each form to these sites and find that there are sequences that target Cdcl4A and B to their respective sites, but that both proteins partition between centrosomes and nucleoli and that this partition is affected by nuclear import and export signals. Importantly, perturbing the normal function of Cdcl4A by overexpression strongly disrupts centrosome duplication and causes mitotic catastrophe (including chromosome missegregation, a failure to reform nuclei following mitosis, and a block to cytokinesis). Thus, Cdcl4A may play a critical role in mitotic progression and genome stability. Supporting this idea, we find that in Xenopus eggs, the Xenopus Cdcl4 protein is important for mitotic exit. These studies will further test the role of Cdc 14 in mitosis and genome stability. Our specific alms are: (1) to test the role of Cdc 14 and cycin-dependent kinases in specific biochemical steps in the centrosome duplication cycle; (2) to map the centrosomal targeting sequences of Cdcl4A and to use these mutants to determine whether Cdcl4 has additional roles in centrosome maturation or maintenance and later steps in mitosis; (3) to study the specific biochemical requirements for Cdc 14 in mitotic exit using an in vitro system for mitotic exit in Xenopus egg extracts; and (4) to purify and identify Cdcl4A and B interacting proteins using an affinity purification procedure and mass spectrometry to sequence the associated proteins. By purifying proteins associated with both wild type and catalytically inactive (\"substrate-trapping\") mutants of Cdcl4, we hope to identify both Cdcl4 regulators and substrates. These aims will both map Cdc 14-regulated pathways and begin to define the critical Cdk and Cdcl4 targets for regulating steps in mitosis and genome stability."}
{"text": "Abstract: Emerging and established viral diseases take an enormous toll on human health. Current treatment approaches are unlikely to halt epidemic spread of many viruses, notably HIV-1, due to prohibitive costs of treatment (i.e. access), compliance issues, rapid viral mutation, and the influence of hard-to-reach high-risk viral 'superspreaders'. We propose to shift the treatment paradigm toward developing Therapeutic Infectious Pseudoviruses (TIPs) that require the pathogen to replicate. TIPs would transmit along a pathogen's normal transmission route, reaching precisely those high-risk populations that most require therapy. TIPs co-opt wild-type virus packaging elements, decreasing disease-progression in vivo and reducing disease transmission on a population scale. We have demonstrated that an anti-HIV TIP could mutate with equal speed and under evolutionary selection to maintain its parasitic relationship with wild-type virus, thereby overcoming viral mutational escape. Since TIPs replicate conditionally (i.e. piggyback) treatment compliance and cost issues are eliminated. A precedent for the safety of TIPs exists in the oral polio vaccine (a live-attenuated vaccine) which exhibits limited spread and is being used in the polio eradication campaign. To develop candidate TIPs we will capitalize upon our expertise in HIV-1 transcriptional circuitry. We discovered that HIV-1 exploits stochastic gene-expression to control entry into a dormant state (proviral latency). By targeting a cellular gene (SirT1) essential for viral feedback, we have biased HIV-1 toward dormancy and diminished reactivation. We will exploit this innovative strategy of forcing viruses into dormancy by utilizing our single-cell imaging methods to conduct high-throughput imaging screens for therapeutic candidates that promote viral latency. Next, these candidate TIPs will be analyzed in novel microfluidic chemostats that maintain homeostatic infection and allow viral evolution in an in vivo-like setting. By integrating these approaches with predictive models, we will develop a revolutionary therapy to halt the spread of HIV/AIDS and other infectious diseases. Public Health Relevance: Emerging and established viral diseases are major health concerns. Many viral diseases lack effective treatments or preventative vaccines and even when available these treatments are unable to halt epidemic spread due to viral mutational escape and the presence of infectious superspreader individuals. Clearly, new and more effective antiviral strategies are needed and this proposal presents a multi-pronged approach to identify and develop an innovative new antiviral approach."}
{"text": "Project Summary: Core A The Administrative Core provides for essential administration, communication, and compliance responsibilities of the Exploratory Grant. It acts to promote the integration, support the Program Director and Program Administrator, organizes meetings of the Program faculty and staff, and facilitate participation in national external activities. The Administrative Core is directed by the Program Director, Dr. Standaert. He is assisted by the Center Administrator, Ms. Katherine Belue. The Core will be responsible for the coordination of the Program. Although the investigators do have established collaborative interactions, achieving the integration needed for a successful P50 Udall application will require a higher level of integration and interaction among the PI?s, their laboratory trainees and staff, and the broader community of neuroscientists and immunologists who may participate in a P50 application. The approach to coordination will include electronic communication (video conferencing and a web-based content management system for document sharing, messaging, and calendaring) as well as in person meetings. These will include biweekly meetings of the project investigators and their team. The Core will support an annual ?mini-retreat? to assist in expanding collaborations beyond the initial Exploratory Grant investigators and achieving the scope required for a successful P50. The Core will oversee sharing, regulatory compliance, and financial management. The Core will establish a web site, to communicate the activities of the Exploratory Program and maintain ties with our community partners. Training is a key component of our plans for a future Udall Center plan. All three project PI?s have current pre- and post-doctoral fellows, and close relationships with important training. We will use the support from the Exploratory Grant to strengthen these connections and promote the participation of trainees and faculty at all levels in the activities of the Exploratory Grant."}
{"text": "Mycobacterium ulcerans is the causative agent of Buruli ulcer, a severe persistent skin infection which has been recently designated an emerging infection in West Africa. The disease has a unique pathology. Despite extensive tissue damage and the presence of a heavy bacterial load, there is little acute inflammatory response to the organism. A single Buruli ulcer may cover 15 percent of a person's body surface; the only cure is surgery and skin grafting. A polyketide-derived macrolide toxin designated mycolactone has been identified in M ulcerans. Macrolides are produced as secondary metabolites by soil bacteria and fungi. They have enormous pharmaceutical value as cytostatins, immunosuppressants, antifungal agents, antihelminthic agents and antibiotics. Mycolactone is the first macrolide identified from a pathogen. Evidence suggests that mycolactone is responsible for most of the pathology in Buruli ulcer. Mycolactone-mediated phenotypes include cell cycle arrest, immunosuppression and death via apoptosis. Neither the genetics of mycolactone synthesis nor its mechanism of action are known. The goals of this proposal are: 1) to clone and sequence genes for mycolactone biosynthesis, 2) to construct mutants defective in mycolactone production, and 3) to begin characterizing events in the cellular pathways involved in mycolactone mediated cell death and immunosuppression using micro-array gene-expression technology. Results from these studies should have a significant impact on the treatment and prevention of Buruli ulcer as well as provide insight into potential role of polyketides in other mycobacterial diseases such as tuberculosis. In addition, this work may provide useful insight into macrolide cell biology."}
{"text": "The goals of the University of Iowa as members of the Cancer and Leukemia Group B over the next five years are: 1) to sustain our pattern of increasing accrual to CALGB trials; 2) to continue to develop programs in both clinical and basic science areas at the University of Iowa which will lead to innovative clinical trials for the group; and 3) to add further affiliates in rural areas in Iowa so that state-of-the-art cancer care can be brought to these communities too small to support cancer programs. Over the last four years of the grant, our accrual went from 98/year to 148/year. Our stated goal in the last grant was to accrue approximately 100+ patients per year. In the first three months of 1992 we have accrued 52 patients (a rate of 208/yr). We hope to average 175 pts/year in this grant period. Current or soon to be open CALGB trials initiated from the University of Iowa include: 1) subcutaneous IL-2 in advanced small cell lung cancer with assay at the University of Iowa of sIL-2 receptor and LAK cell generation; and 2) the use of stem cells to allow multiple cycles of intensive chemotherapy without autologous bone marrow transplant. Basic science programs with potential for future CALGB trials are: 1) the development of bispecific monoclonal antibodies to lymphoma cell antigens; 2) studies in vitro of HMG-CoA reductase inhibitors to block the farnesylation and geranylation necessary for RAS expression; and 3) the use of PCNA as an intermediate marker for the development of neoplasia in the lung. Medical oncologists from the University of Iowa are now serving Mount Pleasant, Iowa and Muscatine, Iowa. Affiliates are going in Mason City, Davenport, and Waterloo. We are exploring further outreach programs in towns too small to ever support a fulltime oncologist and affiliates in larger towns to bring CALGB programs to underserved rural areas of the state. This will allow accrual of minority populations in towns such as Muscatine and the poor in rural areas served by these programs. As the physicians carrying out the care will be University of Iowa faculty, we will be able to provide quality care and data in these areas."}
{"text": "Two cortical pathways originate from primary core areas of auditory cortex: a ventral pathway subserving identification of sounds, and a dorsal pathway, which was originally defined - similar to the visual system - as a processing stream for space and motion. We have recently proposed that this dorsal pathway should be redefined in a wider sense as a processing stream for sensorimotor integration and control (Rauschecker, 2011). This broader function explicitly includes spatial processing but also extends to the processing of temporal sequences, including spoken speech and musical melodies in humans. In this project, we will test the expanded model of the auditory dorsal stream by training rhesus monkeys to produce fixed sound sequences on a newly designed behavioral apparatus (monkey piano). By pressing a lever the monkey will produce a musical tone of a specific pitch; by pressing several levers in succession, the monkey will produce a melody. After a monkey has learned to reliably play the same melody, we will perform functional magnetic resonance imaging (fMRI) of auditory-responsive brain regions in the awake monkey while it listens to the learned self-generated sequence. Control stimuli include melodies the monkey has been passively exposed to by listening to another monkey play for the same amount of time, and novel melodies that the monkey never heard before. Preliminary data suggest that areas activated by the self-generated melody include a region in inferior parietal cortex as well as one focus each in dorsal and ventral premotor cortex. The locations of activated regions will guide subsequent electrophysiological recording with linear microelectrode arrays (LMAs). Each recording site will be tested with the same sequences. Next we will record neuronal responses in premotor cortex to passive listening of the sound sequences and compare them to neuronal activity obtained when the monkey actively produces the sequence with and without sound. Finally, we will add video of a monkey playing the sound sequence on the monkey piano and study multisensory interactions along the dorsal stream using fMRI and LMAs. In particular, responses in caudal auditory belt and parabelt will be compared with those in inferior parietal and premotor cortex in simultaneous recordings. Our studies, using alert monkeys trained in a behavioral task, will contribute to the understanding of unified principles of perception and cognition across sensory systems and their interactions with the motor system. Investigating the auditory dorsal stream in a nonhuman primate will provide valuable information about the evolution of speech and music in humans. Our studies are highly relevant for higher-order processing disorders of audition and speech, such as dysarthria, apraxia of speech, aphasia and specific language disorders which involve inadequate coordination between sensory and motor systems. The results will also improve our understanding of disorders of sensory-motor integration, such as ataxia, which may be caused by stroke or neurodegenerative disease, thus, leading to better therapies and rehabilitation strategies."}
{"text": "Project Summary/abstract Herpes simplex virus (HSV) persists for life in peripheral neurons in the form of a latent infection. In response to neuronal stress, the virus reactivates from latency to permit reinfection. Reactivation is associated with significant disease. For example, replication in the cornea following reactivation results in keratitis. Transmission to the central nervous system following reactivation can result in herpes simplex encephalitis (HSE). Without treatment, HSE has a fatality rate of 70%, and even with treatment, many survivors exhibit long-term sequelae. Although anti-viral drugs are available that limit HSV productive replication, no therapies target the latent stage of infection to prevent reactivation and there is no vaccine against HSV. Therefore, our long-term goals are to understand how HSV responds to neuronal stress and develop strategies to prevent reactivation occurring. Our lab and others have shown that the mechanism by which viral gene expression initiates during reactivation is distinct from de novo infection with the virus. We have found that a neuronal stress pathway resulting in activation of c-Jun N-terminal kinase (JNK) triggers changes to the viral chromatin and permits reactivation. Specifically, the histones associated with viral promoters maintained a modification associated with heterochromatin (H3K9me3) but also became phosphorylated on H3S10 in a JNK-dependent manner. Using both a primary neuronal model of HSV-1 latency that we have developed and mouse models of infection we will determine how activation of JNK permits viral gene expression to be induced during reactivation. By performing ChIP-seq and shRNA knock-down of candidate proteins, we will examine how JNK gets recruited to viral promoters and identify additional cellular proteins involved in HSV-1 reactivation. We will also determine how JNK signaling overcomes the H3K27me3 repressive histone medication to permit reactivation from genomes associated with this modification. Finally, we will examine the mechanism that ATRX restricts HSV-1 reactivation and test the hypothesis that genomes associated with ATRX are non- permissive for reactivation. These studies into the intimate interaction between the latent viral genome and initiation of a neuronal stress response are especially significant as they provide mechanistic insight into how the virus undergoes reactivation. Because the virus has likely co-opted cellular pathway to achieve reactivation, we will also uncover process that are important for the host response to neuronal stress. Importantly, by understanding the very earliest events in HSV reactivation, our long-term goals are to develop therapies that target the latent genome and make it unresponsive for reactivation."}
{"text": "Engagement of the multicomponent antigen receptor in T cells (TCR) results in rapid activation of a protein tyrosine kinase pathway. A major TCR- associated protein tyrosine kinase is ZAP-70, a protein that binds to the activated TCR. Under conditions of TCR activation, the ZAP-70 bound to the TCR is itself tyrosine phosphorylated and activated. We have expressed this enzyme using a baculovirus system and have developed a purification scheme enabling us to obtain sufficient ZAP-70 for enzymatic analysis. Our studies of the mechanism of ZAP-70 activation includes further analysis of mutants of the enzyme that lack critical tyrosine residues. Expression of these mutants in T cells that lack endogenous ZAP-70 leads to the conclusion that phosphorylation of this enzyme is critical to its regulation. Additional studies focus on a substrate of tyrosine kinases in T cells, p120cbl, a proto-oncogene, which may be involved in the Ras pathway in T cells. Earlier studies demonstrate that this protein can be found in a complex with the linker protein Grb2. We have mapped the interaction and have demonstrated that the Grb2-p120cbl complex is regulated by TCR activation. Engagement of the Fc receptor for IgE on mast cells also results in p120cbl tyrosine phosphorylation. In these cells we have demonstrated that the Syk protein tyrosine kinase is primarily responsible for this phosphorylation. Moreover, Syk and p120cbl can be found in a complex whose function is under investigation."}
{"text": "Most oral leukoplakic lesions represent hyperkeratotic epithelium (benign), dysplasia, or squamous cell carcinoma. The lesions must be biopsied and examined microscopically for diagnosis. But, morphology alone does not answer the question about which non-invasive lesions will progress to cancer. The uncertainty of whether a given lesion will progress to cancer or not often compromises the use of the best therapeutic option which is complete excision because of the complications of surgery. So, the question to be answered by the proposed research is, \"Will a given leukoplakic oral lesion likely progress to cancer?\" The research will begin to answer the question by examining the genomic sequences of pp32r1 (GenBank AF008216) present within existing biopsy specimens. In a pilot sequencing survey of the pp32r1 gene in tobacco-associated, oral, leukoplakic lesions, the principal investigator found a mutant, growth-accelerating pp32r1 gene in an oral squamous cell carcinoma. The first specific aim of the project is to amplify and sequence the pp32r1 gene within formalin-fixed, paraffin embedded specimens of oral leukoplakia. The basic hypothesis is that certain neoplastic cells within those lesions contain mutations encoding non-conservative amino acid substitutions within residues 136-172. These mutations define malignant cells long before they clonally enlarge and form a clinical cancer. Another question concerns the discrete molecular activities of mutant, carcinoma-associated PP32R1 proteins as they accelerate cellular growth. The pp32r1 gene is a member of a family of genes that encode proteins with myriad activities. All the family members contain N-terminal domains with leucine-rich repeats that form adapter sites for protein-protein interactions. And, all the family members contain extremely acidic C-terminals that form alternative adapter sites for protein-protein interactions by ionic forces. Another common physical characteristic is that other family members, particularly PP32, often appear in nature bound to other intracellular proteins. It is thought that mutant, carcinoma-associated, growth-accelerating PP32R1s differentially bind intracellular proteins in comparison to wild-type PP32R1s and other members of the PP32 family. The resultant abnormal macromolecular complexes change cellular homeostasis in ways that accelerate growth. The second specific aim of the project is co-immunoprecipitation of PP32R1 binding proteins, separation of the binding partners by 2-dimensional (2D) gel electrophoresis, and identification by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Identification of the intracellular protein ligands will provide a specific functional context for mutant PP32R1S."}
{"text": ": Structural biology and, in recent years, structural proteomics have yielded tremendous insight into protein mechanism and function. However, a high percentage of proteins remain off-limits to high-throughput structure determination methods. For example, solving structures of membrane-bound proteins is a difficult and idiosyncratic art. Furthermore, proteins susceptible to aggregation are simply not amenable to current methods for structure determination. The first long-term goal of the research proposed here is the development of a high-throughput method for converting insoluble proteins, both membrane-bound and oligomerization-prone, to soluble proteins upon which the powerful tools of structural biology can be brought to bear. An equally important second long-term goal is to elucidate and understand the characteristics of protein structure leading to aggregate and membrane bound states. Determining the structures of previously unattainable targets will expedite the development of therapeutics for a host of diseases, such as disorders resulting from amyloid fibril formation, a specific type of protein aggregation. Specifically, the experiments proposed here focus on the caveolin-1, a key regulator of signal transduction. Caveolin-1 binds to a large number of different cellular proteins, and can inhibit key enzymes, including protein kinase A (PKA) and endothelial nitric oxide synthase (eNOS). Such activities allow selections for functional, yet more soluble, caveolin-1 variants. As both an aggregation-prone and membrane-associated protein, caveolin-1 provides an ideal system for the planned experiments. In essence, one series of experiments will uncover molecular determinants for both aggregation and membrane binding. In the first specific aim, the determinants of solubility, aggregation, and membrane-binding will be investigated during experiments aimed at engineering soluble variants of caveolin. The structure of the soluble variant will be determined by solution phase NMR in the second specific aim. This structure will be compared to a structure determined by solid-state NMR of an aggregated variant of caveolin. Structural insight will be then guide mutagenesis experiments aimed at testing the mechanistic basis for protein aggregation and membrane-binding. [unreadable] [unreadable] [unreadable]"}
{"text": "Abstract. According to the CDC, at least 2 million people per year in the U.S. develop illness arising from antibiotic-resistant bacteria, and there are at least 23,000 resultant deaths annually. Certain patient populations, such as surgery and trauma patients, are especially susceptible to infections with opportunistic microorganisms and receive aggressive antibiotic therapy. The development of new antibacterial therapies - to be used either in combination with current antibiotics or as an alternative to antibiotics - would reduce complications associated with a wide range of diseases and infections. This project will explore a novel approach to antibacterial therapy through inhibition of hydrogen sulfide (H2S) production. Over the last decade, H2S emerged as an endogenous gasotransmitter produced by mammalian cells. Recent work shows that H2S is also produced by bacteria. Importantly, bacterial H2S has been identified as a novel mechanism that protects the bacteria against antibiotic induced, oxidant-mediated killing in vitro. Whether or not bacteria produce H2S as a defense against the immune system is not known. Our preliminary data show that inhibition of either host or bacterial H2S improves bacterial clearance in vivo. Therefore, H2S-producing enzymes may be novel targets for antibacterial therapy. Since H2S also has multiple physiological roles, it is critical to understand the regulation and relevance of specific host and bacterial H2S-synthesizing enzymes during host- pathogen interactions. This project will address these questions through the following Specific Aims: Aim 1. To identify the contributions of specific H2S-synthesizing enzymes to the production of H2S that mediates bacterial and host responses during infection. Tissue H2S levels, expression and activity of the various H2S-synthesizing enzymes will be measured before and during infection. Mice deficient in H2S-synthesizing enzymes will be compared to wild type mice. Infections with wild type versus H2S-deficient strains of bacteria will elucidate the relative contributions of bacterial- versus host-derived H2S. Patterns and potential enzymatic regulators of constitutive and infection-associated H2S levels will be identified. Aim 2. To determine the roles of H2S in host susceptibility to infection and bacterial resistance to antibiotic therapy. The functional relevance of specific enzymes to infection responses will be determined in two models of infection: S. aureus pneumonia and E. coli abdominal sepsis. H2S-deficient knockout mice will be infected and survival, bacterial clearance, local and systemic inflammation, organ dysfunction, and immune cell activation will be measured and compared to that in WT mice. The same outcomes will be measured after infection with H2S-deficient strains of bacteria. Resistance of H2S-deficient and wild type bacteria to oxidative killing by the host will be examined. Finally, the efficacy of antibiotic treatment in combination with host or bacterial H2S-deficiency will be evaluated. This project will identify specific sources of bacterial or host-derived H2S to be pharmacologically targeted in the future as a fundamentally novel approach to treat bacterial infection."}
{"text": "Many clinical and pathological studies have suggested that the hypertrophied left ventricle may have an inadequate coronary circulation in the absence of disease of the epicardial coronary vessels. Thus, this question has obvious clinical implications for patients with many forms of heart disease. The primary objective of this proposal was to study the effects of left ventricular hypertrophy induced by a pressure load (Goldblatt hypertension) on the coronary circulation. The four major investigations proposed were: 1) study maximal coronary vasodilation; 2) response of the coronary circulation to treadmill exercise; 3) transmural lactate levels in the left ventricle; and 4) reactive hyperemia response in the coronary circulation. In all studies, dogs with pressure induced left ventricular hypertrophy were to be compared with controls. The effects of left ventricular hypertrophy and maximal coronary vasodilitation have been evaluated. Our general conclusion has been that the maximal dilator capacity of the coronary circulation is decreased by about 40-50% in the hypertrophied left ventricle. This is not the result of an increase in systolic compressive forces secondary to systemic hypertension since a similar abnormality was found in dogs with left ventricular hypertrophy who had been relieved of their hypertension. This suggests that left ventricular hypertrophy is associated with an architectural abnormality in the coronary circulation."}
{"text": "Four large multi-generational Tourette's Syndrome families are being studied. Identification of a biological marker or demonstration of a genetic linkage to a marker locus is being pursued. Data is being collected for documentation of both genetic and environmental factors important for the expression of TS and OCD."}
{"text": "The overall objectives for the proposed MBRS program at North Carolina Central University are: 1)to substantially improve and strengthen the research capabilities and competitiveness of science faculty; and, 2) to increase the number of underrepresented minorities seeking careers in biomedical research. To fulfill the first objective, NCCU's MBRS program seeks to build upon existing and emerging research strengths of the University. In this renewal, five subprojects are submitted from the Department of Biology, and one subproject is submitted from the Department of Chemistry. The request includes support for 6 Principal Investigators, 17 students, 8 research technicians, and 1 secretary. Among these projects are common threads of health related research problems which will foster collaborations and sharing of biomedical research ideas and technologies. The cover areas of cancer biology, molecular biology, microbiology, mycology, environmental health and bio organic chemistry. Four of the six subprojects are new MBRS projects and two are competitive continuations of existing MBRS projects. This program continues to be enriched by interdepartmental and interinstitutional collaborations that foster research productivity and assist in alleviating isolation often felt by researchers at minority and/or non research-intensive institutions. Improved productivity will be made evident y measurable increases in faculty/student publications, presentations at seminars and meetings, in patents, faculty research honors, and the acquisition of additional non-MBRS funding to support research and research training activities. This program will be enriched by the proposed MBRS seminar series. The second broad objective involves enhancing the student participation program. By insuring appropriate mentoring and guidance, students should improve their productivity, visibility, and accountability. Students will be involved in state-of-the-are research projects, in presenting their data in seminars and at meetings, in co-authoring publications, and in establishing linkages with students and faculty involved in other research programs at NCCU and at collaborating institutions and expected outcome is that this program will continue to motivate more minority students to pursue terminal degrees and careers in biomedical research."}
{"text": "Project Summary/Abstract During meiotic prophase, homology recognition between partner chromosomes is coordinated with the assembly of a proteinaceous scaffold, the synaptonemal complex (SC), along the length of the chromosome pair (synapsis). Interhomolog recombination progresses within the context of assembled SC, and many interhomolog recombination events rely on SC for their proper distribution and/or completion. Our lab investigates SC structure and how SC assembly is coordinated with meiotic prophase chromosomal events. Recently we demonstrated that In S. cerevisiae, at least some components of the SC remain dynamic during meiotic prophase, even after chromosomes are fully synapsed. Moreover we found that recombination sites maintain a different SC dynamic as compared to other sites on the SC. Furthermore, our preliminary studies show that SUMO, which localizes to SC structures, has a mutually dependent relationship with the coiled-coil Zip1 protein to assemble SC on meiotic chromosomes. With pioneering imaging studies using super-resolution light microscopy, we have also shown that SUMO localizes to Zip1 N termini, at the center of SC structure. Finally, the lab has isolated zip1 alleles that assemble on chromosomes independent of canonical regulators. Each zip1 allele represents a single amino acid substitution at the extreme C terminus of Zip1 and may reveal aspects of the mechanism underlying Zip1 assembly. The purpose of this grant proposal is to 1) use our established genetic and microscopy tools to define the molecular architecture and dynamics of all known synapsis proteins within the SC, and 2) to isolate point mutant alleles of synapsis initiation proteins and Zip1 in order to identif molecular features of each protein responsible for their function in synapsis and other meiotic prophase chromosomal events."}
{"text": "The Structural Basis of APOBEC Functions The APOBEC (Apolioprotein B mRNA-editing Enzyme Catalytic polypeptide) family of cytidine deaminases is a family of enzymes that deaminate cytidine residues of DNA/RNA. APOBEC proteins possess significant cellular functions and anti-viral activity, which partially accounts for the intense attention to this the field of research in recent years. APOBEC proteins are found only in vertebrates and APOBEC3 (Apo3) proteins are found only in primates. By binding and deaminating DNA/RNA, APOBEC enzymes achieve remarkably diverse cellular functions. For example, APOBEC1 (Apo1) edits the mRNA of a protein involved in lipid metabolism; AID plays a key role in somatic hypermutation for antibody maturation; APOBEC2 (Apo2) may play a regulatory role for heart muscle development; and Apo3 proteins, especially Apo3G, can restrict important viral pathogens, including Human Immunodeficiency Virus (HIV) and Hepatitis B Virus (HBV), and retro-element mobility. As a result, a novel approach to HIV therapy focuses on utilizing the potent anti-viral activity of Apo3G and Apo3F. Our long-term goals are to understand the structural/functional relationship for APOBEC cellular function and their anti-viral activity. Our specific aims are to extend our prior success in the structural characterization of APOBEC proteins to the studies of the structural basis of APOBEC's functional mechanisms, including their antiviral activity, with particular focuses on Apo3G and Apo3F. The research will provide valuable information for understanding the molecular details of the APOBEC enzyme family and for the potential drug development to provide therapy for HIV, immune diseases and other diseases related to APOBEC function or malfunction. PUBLIC HEALTH RELEVANCE: Understanding The Structural Basis of APOBEC Functions The Apolioprotein B mRNA-editing enzyme catalytic polypeptide (APOBEC) family of cytidine deaminases are found exclusively in verterbrates. APOBEC nucleic acid deaminases modify genes by deaminating cytosines in mRNA coding sequences and in ssDNA. Their critical biological roles include lipid metabolism, humoral immune response, and potential regulations of developmental process of certain human organs and reproductive system. Additionally, these enzymes can inhibit the replication of retroviruses, such as the human immunodeficiency virus (HIV) and hepatitis B virus (HBV), and retrotransposons. The important beneficial mutational ability of APOBEC proteins can become detrimental to the stability of genome if their activity is not tightly regulated. The understanding of the structural basis of the molecular mechanisms of APOBEC function, which is still poorly understood, bears scientific significance and direct health relevance. We propose to study the structure/function of this important APOBEC deaminase family, with focuses on APOBEC3G and 3F (Apo3G and 3F) proteins and their interactions with cellular and viral ligands, using mainly structural biology, assisted by biophysics, molecular biology, and functional biochemistry."}
{"text": "Develop an in vitro test for sarcoidosis."}
{"text": "ABSTRACT Pancreatic cancer (PC) is one of the deadliest cancers with a 5-year survival rate of 7%. Notable racial/ethnic differences in PC incidence have been observed, with U.S. blacks having 30% higher rates compared to whites. The rates of PC in Native Hawaiians and Japanese Americans have been rising in past two decades and have surpassed those of whites, but no study has been conducted to identify the cause(s) of this rising incidence. In the large prospective Multiethnic Cohort Study (MEC; N>200,000) we observed highly significant differences in PC incidence across racial/ethnic populations, with African Americans having 39% higher rates compared to whites. Native Hawaiians are observed to have the highest rates in the cohort that are 74% higher than whites while rates in Japanese Americans are 32% higher and rates in Latinos are similar to whites. We expect that inter-ethnic differences in risk factor prevalence are likely to explain the observed ethnic differences in PC incidence. However, to date, few studies have included African Americans and no studies have included Japanese Americans, Native Hawaiians, or Latinos; thus, the factors underlying PC disparities remain undefined. The goal of this study is to identify factors that explain racial/ethnic disparities in PC incidence, particularly the excess risks observed in African Americans, Japanese Americans, and Native Hawaiians. We hypothesize that in addition to known risk factors, host genetic factors and unknown non-genetic factors contribute to the observed racial/ethnic differences in PC incidence. To test our hypothesis, we will leverage the well-characterized lifestyle and genetic data of the MEC, a long-standing ethnically diverse prospective cohort of >200,000 African American, Latino, Native Hawaiian, Japanese and white participants established in the early 1990's in California and Hawaii. The MEC is uniquely positioned to address racial/ethnic disparities in PC incidence, with >2,100 incident cases of PC diagnosed over >20-years of follow-up and with detailed lifestyle and exposure data collected in a consistent fashion amongst all populations to permit valid ethnic comparisons. Our specific aims are: 1) To quantify racial/ethnic-specific associations of known and potential/suspected risk factors with PC incidence in African Americans, Japanese Americans, Latinos, Native Hawaiians, and whites; 2) To determine whether the risk factors confirmed or discovered in Aim 1 contribute to the observed ethnic differences in PC incidence; 3) To characterize the association of known common genetic variants with PC risk in African Americans, Japanese Americans, Latinos, and Native Hawaiians and build a quantitative risk model to compare the distribution of genetic risks across populations associated with these marker alleles. By leveraging the existing resources/infrastructure, this study will efficiently and cost-effectively examine multiple factors that may contribute to racial/ethnic disparities in PC. Because of the lack of effective treatment and low survivorship, identifying modifiable risk factors is critical in reducing PC burden; this is even more crucial for minority populations who are at greater risk and are likely to have limited access to care."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The 90-kDa ribosomal S6 kinase 2 (RSK2) is important serine-threonine kinase broadly expressed in response to various growth factors. RSK pathway is a key regulator of cancer cell proliferation. In humans, RSK2 gene mutations are manifested in Coffin-Lowry syndrome characterized by severe psychomotor retardation. Mechanism of activation of RSK2 is still unclear, and many contradictive reports are published. Elucidating the molecular structure of RSK2 is essential for understanding its function. RSK2 belongs to the family of unusual serine-threonine kinases that contain two distinct kinase domains connected by a linker region. First, we focused on the regulatory C-terminal domain of RSK2 (CTD), activation of which resulted in activation of full length protein. Recently we determined the X-Ray structure of the isolated CTD RSK2 at 2.0 [unreadable] resolution and successfully published it (Nature Structural &Molecular Biology, 2008). The structure revealed a C-terminal autoinhibitory [unreadable]L-helix which was embedded in kinase scaffold and pre-determined kinase inactive conformation. We suggested a mechanism of activation by interaction with up-stream kinase, ERK, which would displace the autoinhibitory helix from its position resulting in the re-arrangement of conserved Glu500 residue."}
{"text": "The protocols involved in this project are as follows: 02-M-0120, 03-M-0175, 88-M-0131, 92-M-0174. This report includes work arising from the following protocols: NCT00030147, NCT00060736, NCT00001231, and NCT00001322. In addition to the established increased risk of first onset depression during the perimenopause, there is preliminary evidence of estradiols antidepressant efficacy in perimenopausal depression (PMD). Indeed, observational studies report the emergence of depressive symptoms after the discontinuation of estradiol therapy (ET) in 5-10% of women. The role of estradiol -either declining or low levels - in the precipitation of PMD is unknown, largely due to the associational and indirect nature of the evidence linking ovarian function and depression. Correlative studies with plasma FSH or response to ET do provide indirect evidence of the relevance of changes in reproductive hormones to mood disturbances. However, even prospective epidemiological studies cannot test the estradiol withdrawal hypothesis since perimenopausal changes in the secretion of several hormonal and metabolic factors could confound the effects of estradiol withdrawal. In other studies of the role of ovarian steroids in affective disturbance, changes in ovarian steroids (in the context of otherwise normal levels) have been shown to directly trigger depression, but only in a subset of women with histories of mood disorders linked to reproductive function. In this study, we directly examined the hypothesis that declining ovarian function estradiol withdrawal - underpins depression occurring during the perimenopause. We further sought to examine whether the response to estradiol withdrawal differed in women with a past PMD compared with those without any past depression. In this second study, we determined if sudden, blinded withdrawal of ET would precipitate depressive symptoms and if it would do so differentially in those with a history of PMD. Our findings support both predictions. Estradiol withdrawal precipitated depression in women with a history of PMD, but no depressive symptoms were seen after estradiol withdrawal in women with no past history of PMD. Further, depressive symptoms did not emerge in those women with past PMD who were maintained on ET. Importantly, the recurrence of depressive symptoms in women with past PMD occurred in the absence of differences in several measures that could influence mood, including baseline clinical characteristics (other than PMD), the severity of daytime/nighttime hot-flushes, and plasma hormone levels after withdrawal. The lack of depressive symptoms in the control women despite identical hormone manipulation (and similar levels of hot-flushes and plasma estradiol levels) demonstrates that estradiol withdrawal differentially impacts CNS function in some women so as to render them susceptible to depression. This suggests that perimenopausal changes in estradiol can trigger depression, but only in the susceptible subgroup. These observations, in the context of similar plasma reproductive hormone levels, suggest that normal changes in ovarian estradiol secretion can trigger an abnormal behavioral state in susceptible women. There are several mechanisms by which changes in estradiol might mediate the observed effects on mood. First, estradiol modulates the activity of virtually every system implicated in the pathogenesis of depression, including regulation of neurotransmitter synthesis and metabolism, stress axis activation, neuroplasticity (including regulation of BDNF), epigenesis, and immune system activation. Second, estradiol signaling through estrogen receptor (ER) beta reverses depressive-like behavior in animal studies. Third, reward responsiveness, which is disturbed in depression, is modulated by estradiol in both rodents and humans. Finally, of particular note, discontinuation of long term ET in postmenopausal women was accompanied by decreases in medial frontal and temporo-occipital metabolism. Thus, through local signaling or network-level dysfunction, particularly in fronto-limbic regions, estradiol withdrawal could precipitate affective dysregulation. What remains unclear is the reason for the differential susceptibility to the mood destabilizing effects of estradiol withdrawal. Of interest in this regard is the recent identification of increased sensitivity to estrogen regulation among transcripts that were differentially expressed in women who developed postpartum depression. Alternatively, basic science studies report the de novo production of estradiol from androgens in brain regions involved in mood regulation (e.g., hippocampus). Thus, it is possible that women who did not develop depressive symptoms after ET withdrawal have sufficient CNS aromatase activity to prevent the development of depressive symptom during declining estradiol secretion. The investigation of these possible mechanisms will be the focus of future studies. One possible source of the susceptibility to developing perimenopasual depression is an abnormal hypothalamic pituitary-adrenal (HPA) axis. Dysfunction of the HPA axis is a frequent accompaniment of depression, and HPA axis function is modulated by both aging and ovarian steroid secretion. To date, only a few studies have evaluated basal (but not stimulated) HPA axis function in women with perimenopause-related depression, with findings suggesting differences in adrenal androgen secretion but not basal cortisol secretion in PMD compared with controls. We examined HPA axis function in depressed and asymptomatic perimenopausal women using the combined dexamethasone-corticotropin releasing hormone (Dex/CRH) test. In contrast to recent suggestions that declining ovarian estradiol secretion (and in turn alterations in neurosteroid synthesis), preliminary results suggest that abnormalities of HPA axis function do not distinguish perimenopausal women with depression from those without depression. Thus, unlike depression in premenopausal women, HPA axis dysfunction is not a frequent accompaniment of depression during the perimenopause. These findings suggest that reproductive endocrine-related mood disorders are not uniformly associated with the HPA dysregulation and could reflect underlying pathophysiologic processes that are distinct from those reported in women with non-reproductive-related depressions. Finally, we continue our longitudinal investigation of the onset of depression during the perimenopause. Over sixty women have completed this study that involved measuring early follicular phase blood samples, daily and weekly symptom self-ratings, and formal in-person interviews every six months during the transition from regular menstrual cycle function (premenopausal) until one year postmenopause. Women participate in this study for an average of five years (range = 3-10 years). Preliminary results confirm previous observations of a clustering of depressive episodes during the late menopause transition a stage of reproductive aging associated with estradiol withdrawal. Moreover, we have observed that depression occurring during the perimenopause,( but not other events related to the perimenopause including menstrual cycle irregularity, aging, or vasomotor symptoms) is accompanied by significant declines in measures of quality of life, social support, and personal disability."}
{"text": "Multiple Sclerosis (MS) is an inflammatory, demyelinating disease of the Central Nervous System (CNS) which likely involves an autoimmune mechanism directed against self-myelin associated antigens. There is substantial evidence suggesting that B cells are involved in at least one mechanism of MS pathogenesis. However, both functional and molecular analysis of the immunoglobulins (Ig's) produced by clonally expanded CSF B cells has been limited. We hypothesize that a subset of clonally expanded B cells in the CSF of MS patients is involved in at least one mechanism of MS pathogenesis by producing antibodies that bind to myelin associated antigens. In order to identify complete Ig rearrangements that may play a role in the autoimmune activities evidenced in MS patients and test for their antigenic specificity, we intend to define the Ig repertoires in the CSF of MS patients in order to identify Ig's from clonally expanded B cells. We then plan to determine the antigenic specificity of these unique Ig rearrangements by cloning both the heavy and light chain segments into an expression vector, isolating the resultant Fab fragments, and testing them for their antigenic specificity. We can also use PCR to track the persistence of these clones in the original MS patients. Moreover, we can determine if other MS patients have the same clonally expanded B cells in their CSF. These studies provide the necessary foundation to initiate and assess the potential role of B cells in at least one mechanism of MS pathogenesis, and ultimately will allow us to explore whether generating specific immunotherapies directed against these clonally expanded B cells in the future is warranted. [unreadable] [unreadable]"}
{"text": "The purpose of this proposal is to work collaboratively with the CDC and other SB clinics to build a disease specific national registry to improve care for individuals with SB. Proposed Aims: As part of the national network of participating SB clinics, the Greater San Francisco Bay Area SB Partnership will: a) Recruit and collect standard data on at least 125 SB patients for each of three years of the project using standard forms;b) Submit the de-identified data to a central repository using a web based electronic medical record (EMR) system;c) Adopt the EMR for ongoing clinical use;d) Participate in the national Coordinating Committee to promote improvements to the registry, propose research questions, develop an analytical plan, and promulgate changes to clinical practice standards based on data findings. Background: SB is the most common permanently disabling birth defect, with an estimated 70,000 individuals living with the more significant forms of this neural tube defect. Relatively little is known about how SB care varies by location, and currently, not much research is occurring related to SB. Consequently, the evidence based on which the current treatment of SB occurs is weak. It is anticipated that the establishment of a National SB Patient Registry (spearheaded by CDC, the SB Association, and Agency for Healthcare Research and Quality) will result in improved quality of care for all individuals with SB, and will identify and support opportunities for collaborative research. The proposed Greater San Francisco Bay Area SB Partnership is a collaboration between two high quality SB programs with very diverse patient populations: one at Children's Hospital &Research Center Oakland and the other at the University of California at San Francisco Benicoff Children's Hospital. Together, the sites follow 255 diverse patients in their clinics from newborn to age 21;about one half of these patients are Latino. Methods: Following protocol established by the CDC, the Greater San Francisco Bay Area SB Partnership will attempt to recruit all newborns, children, adolescents, and young adults to participate in the registry during their visits to SB clinic. Data will be collected on each patient for a minimum of three years using standard forms. Non-identifiable data will be entered into a web based EMR maintained by CDC. The project's Coordinating Committee will determine research topics and data analysis."}
{"text": "Arsenic is a carcinogen, a Superfund toxic compound, and a common drinking-water contaminant. The Environmental Protection Agency (EPA) has identified 1,300 sites on its National Priorities List (NPL) and arsenic has been found in at least 781 of these sites in the USA. Exposure may occur by a variety of pathways including inhalation of dusts in air, ingestion of contaminated soil or water, or through the food chain. The primary mode of exposure in non-occupationally exposed populations is drinking-water contamination from either alluvial deposits or industrial contamination. The precise relation of arsenic to cancer has not been established at low exposure levels. In addition, arsenic toxicokinetics in humans remains poorly understood. Thus, we propose to assess exposure and skin-lesion risk, as well as arsenic dosimetrics in a population with a wide range of arsenic exposure via drinking-water contamination. The proposal builds upon a small study in which a case-control study has been initiated. This proposal will use the necessary resources to analyze biomarkers of exposure and susceptibility and for statistical analyses of both a case-control study of skin lesions and a repeated-measures biomarker dosimetry study. The proposed studies in Bangladesh will assess this risk in a population with a wide range of exposures - from low to very high. Together, these data will add substantially to the existing risk assessment information by elucidating both early and late outcomes alter arsenic exposure, and the influence of susceptibility traits at all levels of exposure. This project is relevant to the overall aims of NIEHS in several ways: First, we will examine a range of health effects of an important environmental carcinogen. Second, we will define human biomarkers of exposure, early effects, and genetic susceptibility to arsenic exposure. Third, we will examine exposure-response relationships for arsenic-induced non-malignant skin lesions as well as for skin cancers."}
{"text": "Nonsteroidal anti-inflammatory drugs (NSAIDs) display striking antineoplastic activity, although toxicity resulting from cyclooxygenase (COX) inhibition and incomplete protection from disease progression limits their use for cancer chemoprevention. Preliminary studies suggest that the mechanism for the chemopreventive activity of a highly efficacious NSAID, sulindac, does not require the inhibition of COX-1 or -2 and that cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) inhibition is responsible for its tumor cell growth inhibitory activity. We hypothesize that it is feasible to chemically modify sulindac to block COX inhibitory activity, while enhancing tumor cell growth inhibitory activity by increasing selectivity for cGMP PDE. In support of this hypothesis, we have synthesized a prototypic amide derivative of sulindac sulfide that does not inhibit COX-1 or -2, yet displays high potency to inhibit colon tumor cell growth and cGMP PDE in vitro. Sulindac sulfide amide (SSA) displayed adequate oral bioavailability and appeared to be less toxic than sulindac in mice. Using a xenograft model involving human colon tumor cells, SSA displayed comparable in vivo antitumor efficacy as sulindac, albeit at high dosages. These studies demonstrate the feasibility of uncoupling the COX inhibitory activity from the growth inhibitory activity of sulindac and supports further research to develop additional derivatives that are more potent and/or bioavailable than SSA. The following specific aims are proposed: 1) prepare a series of sulindac analogs to optimize for potency, selectivity, and oral bioavailability, 2) evaluate in vitro activity, 3) determine the mechanism of action and identify the target cGMP PDE isozyme, and 4) evaluate in vivo chemopreventive efficacy. Our goals are to identify a safe and efficacious derivative of sulindac for cancer chemoprevention and to critically test the hypothesis that cGMP PDE is a target for cancer chemoprevention. This multidisciplinary research proposal is anticipated to result in a new drug candidate for cancer chemoprevention, and in particular, patients with familial or sporadic adenomatous polyposis who are at high risk of disease progression. Nonsteroidal anti-inflammatory drugs (NSAIDs) display striking cancer chemopreventive activity, although toxicity resulting from cyclooxygenase (COX) inhibition and incomplete efficacy limits their clinical use for patients at moderate to high risk of disease progression. Preliminary studies suggest that the mechanism for the antineoplastic activity of sulindac does not require COX inhibition and that cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) inhibition is responsible for its tumor cell growth inhibitory activity. We hypothesize that it is feasible to chemically modify sulindac to block COX inhibitory activity, while enhancing tumor cell growth inhibitory activity by increasing selectivity for cGMP PDE. Our primary goal is to identify a novel sulindac derivative as a clinical candidate for patients with either familial or sporadic adenomatous polyps who are at high risk of disease progression. A secondary goal is to critically test the hypothesis that cGMP PDE is a target for cancer chemoprevention and involved in carcinogenesis. This multidisciplinary research proposal may lead to a novel strategy for colon cancer chemoprevention."}
{"text": "The primary goal of the conference for which funding is being requested will promote the marketing of safe, fresh and accurately labeled shell eggs and egg products through the development of uniform, science based and coordinated state and federal regulations. The conference is designed to achieve this goal by providing educational segments related to current egg safety and quality issues and the opportunity for state, federal and industry representatives to interact and share information and ideas. The conference will also provide a forum to present specific issues which will be referred to the NERO committees comprised of specialists in that area. The committee will review the information and provide specific recommendations for the most effective and cost efficient method to achieve the desired results. The conference will address new rules for S. enteritidis, label requirements for egg, scientific information related to campylobacter in eggs as well as other presentations related to food safety. The conference will be held at the Florida Mall Hotel in Orlando, Florida from March 13 - 16, 2005."}
{"text": "Connexin 32 (Cx32), is a gap junction protein found in the paranodal loops and Schmidt-Lantermann incisures of myelinating Schwann cells. Mutations in the gene encoding the human gap junction protein lead to CMTX, the X-linked form of the clinical condition known as Charcot-Marie - Tooth disease, a group of inherited peripheral neuropathies. Some mutations in Cx32 appear to allow for appropriate membrane targeting and formation of functional channels, some may lead to altered cellular processing, and others prevent Cx32 synthesis entirely. Mutations in Cx32 may be important in determining the processing and biophysical behavior of the Cx32 ion-channel. I hypothesize that mutations in Cx32 found in patients with CMTX, lead to the loss of ability of Cx32 to provide a vital cellular communication pathway. Various mutations may lead to this loss-of-function by altering the biophysical properties of the pore, or by affecting cellular processing of the protein or its ability to organize into functional channels. To evaluate these possibilities: 1) I will determine the biophysical properties of the channels formed by connexins containing mutations corresponding to those found in patients with CMTX; channels will be expressed in Xenopus oocytes or transfected cells; and 2) I will observe the cellular trafficking of wild-type and mutant forms of Cx32 in real time, in living cells, using an EGFP-tagged form of the molecule. The work described in this proposal will provide the applicant with an opportunity to acquire the necessary intellectual and technical skills to be an independent investigator in translational neuroscience research."}
{"text": "Functional nerve stimulation (FNS) is a means to restore lost function to patients with spinal cord injury. By directly stimulating the nerve, it is possible to maximize muscle contraction and minimize injected charge. An effective neural electrode must meet the following criteria: 1) The electrode must be able to activate any subset of the neural axon population, either deep or surface, selectively and independently and; 2) The electrode must not cause significant damage to the nerve. Current nerve cuff designs place electrodes around the nerve. These exhibit some degree of selectivity, but, cannot provide adequate functional control since it is not possible to selectively activate portions deep in the nerve without stimulation of the surface of the nerve. Intraneural electrodes have been developed to provide access to the deep portions of the nerve. Two of the current intraneural approaches are regeneration electrodes and wire electrodes. The regeneration design places a silicon substrate penetrated by holes between two severed ends of a nerve. The axons then regenerate through the holes. Wire electrodes are electrical wires threaded through the neural fascicles, penetrating the epineurium and a second protective layer, the perineurium. Both the threaded wire and regeneration designs provide selective stimulation of neural regions, but produce significant damage to the fascicles and axons. We propose a new design which combines the advantages of the standard nerve cuff and intraneural electrodes. This new device slowly places electrodes inside the nerve, but between the fascicles without perforation of the perineurium. The interaction takes place over the course of several days by taking advantage of the mechanical energy stored in the cuff prior to installation. Preliminary experiments have shown that the slow rate of penetration causes minimal damage and trauma to the nerve while still placing electrodes deep in the nerve. The aims of this proposal are 1) to show that chronic damage from the electrode is minimal, 2) to determine the selectivity of the design for electrical stimulation of the sciatic nerve in the cat, 3) develop finite element model of fields generated within the nerve and use this model to optimize the electrode design, and 4) to develop a electrode that utilizes currently available silicon technology to maximize the selectivity and function of the electrode. The end-product of this project will be an electrode capable of producing selective activation of neural regions by placing multiple contact points within the nerve with minimal axon damage. The same electrode could then be used for recording neuronal activity such as sensory signals and this Slowly Penetrating Interfascicular Nerve Electrode (SPINE) could, therefore, become a general purpose neural cuff design capable of both selective activation and recording."}
{"text": "The long-range goal of this research is to isolate and characterize all of the components necessary for reconstitution of hormonally regulated adenylyl cyclase system. Using the liver glucagon-sensitive adenylyl cyclase systems as a model system and by monitoring principally specific binding of labelled glucagon and adenylyl cyclase activity, we specifically propose during the next 5 years: (1) Purify the catalytic unit of the liver adenylyl cyclase system in a form that is either sensitive or insensitive to stimulation by nucleotide, and study its subunit composition. (2) Develop the necessary methodology to purify the glucagon receptor from liver and initiate its purification. (3) Identify and purify key regulatory components of the system: a Guanyl nucleotide regulatory component associated with the catalytic subunit using Gilman's reconstitution assays with membranes from AC- S49 lymphoma cells as means of identifying the component once separated from the catalytic subunit of the system; and b. Brain stimulatory factor(s). (4) Continue exploring the kinetics of this system with respect to nucleotide and hormone stimulation with the aim to further delineate its regulatory aspects and to identify possible additional components involved in normal functioning of adenylyl cyclases."}
{"text": "Attended NCRR/NIH session to edit a workshop report on ~Technologies for the Future~, May 15, 1996. Consulting with various personnel on the progress/nuances of 2~PE scanning microscopy."}
{"text": "Description: (Applicant's Description) Since its origin in Fall 1996, the Pharmacology Core has steadily grown into being an important CCC resource for several investigators. Its mission is providing expertise in pharmacological sciences, including kinetics, dynamics, biopharmaceutics and physical pharmacy. Pharmacokinetic support includes drug absorption, metabolism, distribution, elimination, bioactivation and drug-drug interactions. Pharmacodynamic support includes molecular effects of drug exposure, and in vitro toxicity testing. Biopharmaceutic support includes the influence of dose and dosage form on pharmacokinetics. Physical pharmacy support includes production of (approximate dose) prototype forms of drugs for preclinical studies and clinical forms for IND studies. Enhancements of research by the Core take the form of study design, data acquisition, data analysis, and interpretation of results, including technical writing for manuscripts and grant proposals written by CCC investigators. This core also developed and oversees the Quality Assurance Plan for tracking and handling clinical pharmacology specimens throughout the Institute. Key features of this Core are the scope of services provided, its integral role in clinical specimen handling, existing collegial relationships, direct consultation for requesting investigators, and the interests and commitment of its staff. Pharmacological contributions benefited 5 Programs, including Developmental Therapeutics, Breast Cancer, Molecular Biology and Genetics, Prostate Cancer and Protease Programs."}
{"text": "Description: (Applicant's Description) This facility was previously part of the joint Transgenic/Histology facility. It was designed for the efficient processing, sectioning and examination of tissue from mouse and other species, and provides expert advice and assistance in slide preparation. It serves all groups using mutant mouse strains and is available to all other groups. A consultant veterinary pathologist provides expert review of slides. Support comes from this CCSG (equipment and one full-time technician) and from individual users (personnel and full cost supplies). Professor Tyler Jacks has supervisory responsibility for the facility. His research program centers on the generation of mouse models of human cancer, and he and his laboratory have performed extensive histological analysis of tumor models as well as developmental phenotypes in the mouse."}
{"text": "The mechanism of muscle wasting in AIDS is not known. It is the hypothesis of this proposal that wasting in AIDS is associated with increased production of glutamine by muscle. Glucocorticoids, by stimulating muscle proteolysis and glutamine synthetase activity, are a key contributing factor to wasting. Therefore, pharmacological blockade of glucocorticoid receptors might prevent or alleviate a significant component of the wasting syndrome. The proposed studies will employ muscle biopsy specimens from human AIDS patients, as well as mice with a persistent retrovirus infection (LP-BM5), the manifestations of which simulate many features of human AIDS. The specific objectives are: (1) to characterize muscle wasting in AIDS; (2) to evaluate the mechanism of changes in glutamine synthetase and glutaminase activities and glutamine production in muscle from human AIDS patients and LP-BM5- infected mice; (3) to determine alterations in glucocorticoid receptors in muscle from human AIDS patients and from LP-BM5- infected mice and the mechanisms underlying such changes; and, (4) to assess antiglucocorticoid therapy for muscle wasting in LP-BM5- infected mice. The proposed studies are expected to contribute to a better understanding of biochemical mechanisms underlying muscle wasting in AIDS, provide useful biochemical markers for clinical assessment of early wasting, and lead to the development of specific treatments for this devastating conditions."}
{"text": "The Epithelial Na+ Channel or ENaC is rate limiting to sodium absorption in renal epithelia. Channel activity sets the final urinary sodium concentration and determines in part sodium excretion. Channel activity affects body sodium and water balance and therefore blood pressure. This channel exists at the plasma membrane largely in an electrically inactive pool. Recently, it was demonstrated that this inactive pool can be activated by cleavage of two of the channel subunits by serine proteases. Thus, this mode of channel regulation affords ENaC expressing renal epithelia an enormous capacity to rapidly respond to changes of sodium load, renal filtration, and body sodium balance. Activation of silent channels by proteases is proposed to involve the removal of short inhibitory domains from the channel's alpha and gamma subunits. However, our data clearly demonstrate that non-cleaved channels can be active, while some cleaved channel subunits do not form active channels. We demonstrate that Po can be regulated by multiple mechanisms that include channel processing, membrane lipid rigidity, channel partitioning into lipid rafts, and interaction between inhibitory domains of the ENaC subunits. We propose that multiple interactions between the ENaC subunits determine channel Po. Cleavage of the channel subunits provides a mean of removing or altering some of these interactions and leads to channel activation. We also propose that channel subunit interactions affect subunit processing, membrane partitioning and stability. The unifying hypothesis is that unprocessed channels are stable at the membrane but inherently inactive unless conditions exist to reduce subunit-subunit inhibition, and that moreover, proteolysis activates or primes the channels for activation by permanently removing such inter-subunit interactions. Proteolysis then predisposes the channel subunits for internalization unless processed subunits are protected in raft membrane domains. To address the mechanism of control of channel activity, we develop new tools that utilize 1) engineered cleavage sites 2) a recently identified constitutively ENaC subunit (epsilon) 3) homology mapping of the channel to the crystal structure of ASIC1 and 4) analysis of channel partitioning into membrane domains. Our data will pave the way for understanding channel Po regulation and every downstream process that further modify this Po. PUBLIC HEALTH RELEVANCE: We examine the mechanisms that control the spontaneous activity of the renal epithelial sodium channel. The activity of this protein is important in determining kidney salt excretion and consequently salt retention by the body and blood volume and pressure. Understanding the spontaneous activity of this protein would help understand the basis of salt sensitivity of blood pressure and is important in designing therapy for individuals with high blood pressure."}
{"text": "Cytochrome P-450IIE1 is a constitutive, hepatic enzyme that has been implicated in the activation of pro-toxins and pro-carcinogens. Studies with animals have shown that P-450IIE1 can be significantly induced by a class of compounds that include ethanol, acetone, and isoniazid, as well as by abnormal metabolic states such as fasting, obesity, and diabetes. The major hypothesis to be tested in this proposal is that humans exposed to the same class of chemicals, or with a similar altered metabolic state, will have elevated levels of P-450IIE1 enzyme and can be identified with an in vivo catalytic probe. Four compounds (enflurane, chlorzoxazone, 4-methylpyrazole, and acetone) have been identified as potential probes based on literature reports and preliminary results. The utility of probes based on literature reports and preliminary results. The utility of probe candidate(s) will be validated by: a) determining the in vitro catalytic specificity of the probe towards human liver P-450IIE1, b) identifying an in vivo metabolic clearance parameter that reflects intrinsic P-450IIE1 catalytic activity, and c) determining whether the in vivo clearance parameter predicts direct measurements of hepatic P-450IIE1 levels in normal and isoniazid-treated populations. Once developed, the in vivo probe will be used to study the inducibility of functional P-450IIE1 enzyme in people with Type I and Type II diabetes, the morbidity obese, and obese people experiencing rapid weight loss. If P-450IIE1 levels are significantly elevated in selected human populations, those persons will be at greater risk for developing the carcinogenicities and cytotoxicities thought to be related to P-450IIE1 catalytic activity. This proposal will provide a safe and facile method for their identification."}
{"text": "In earlier work, hydrogen enchange methods developed in this laboratory were used to study structure and dynamics in a number of necleic acid systems. More recently we have adapted the method of D. Cross (absorbance-detected H-D exchange in stopped-flow) to the study of large nucleic acids and have used this to study base-pair opening reactions in synthetic double helices and also the ethidium-DNA interaction. The stopped-flow approach promises a remarkable advance in such investigations; data collection is very much faster, extension of time resolution to msec makes accessible the important early-time exchange region, and it appears possible now to study nucleic acids while complexed to major amounts of protein. We want to bring these capabilities to fruition. This will involve: completing the calibration of the various necleotide parameters for UV-detected H-D exchange, extending our previous studies of opening reactions to other synthetic duplexes and DNA, and attempting to gain information on the structure of the open form. In addition we want to see what can be learned about DNA in chromatin and RNA in ribosomes."}
{"text": "Increased susceptibility to infections including tuberculosis (TB) is a major cause of morbidity and mortality in diabetes. Despite its clinical importance, this phenomenon has received little basic research attention. We will investigate TB resistance in mice using models of type 1 and type 2 diabetes. Diabetic and non-diabetic control mice will be challenged by low-dose aerosol infection with Mycobacterium tuberculosis (Mtb). We will characterize TB susceptibility with parameters of survival, bacterial load, and lung leukocyte recruitment. The basis of susceptibility will be evaluated by characterizing cytokine expression and by testing macrophage, dendritic cell, and T cell functions. We will investigate whether advanced glycation end products (AGE), which have been linked to diverse complications of diabetes, are responsible for impaired host defense. We will also evaluate the potential role of other biochemical mechanisms implicated in diabetes complications including polyol pathway flux, hexosamine pathway flux, and over-production of diacylglycerol with activation of protein kinase C. Hyperlipidemia is a common co-morbidity in diabetes that exacerbates diabetic vasculopathy. In preliminary studies we found that hypercholesterolemia also increases TB susceptibility. We will explore similarities and differences in the effects of diabetes and hyperlipidemia on protective immunity, and we will characterize TB susceptibility of mice with combined hyperlipidemia and diabetes. This project will identify specific deficits in protective immunity caused by hyperglycemia and hyperlipidemia, and may provide new insights to critical parameters of host defense against TB. Understanding the mechanisms of susceptibility will inform the development of treatments to reverse this diabetes-related complication. At the same time our model will be used to test the impact on protective immunity of novel treatments for diabetes co-morbidities such as statins, aminoguanidine and pyridoxamine. While our focus is on TB, the new knowledge generated by this project will have broad relevance to other infections associated with diabetes and will further basic understanding of the interplay between immunity and metabolism. This project studies how diabetes weakens the body's ability to fight tuberculosis. Learning more about the harmful effects of diabetes on the immune system will suggest new ways to prevent and treat infections that are a major problem in people with diabetes."}
{"text": "[unreadable] [unreadable] This application is the competing renewal for the ongoing postdoctoral training program at Drexel University College of Medicine focused on the biological bases of nervous system disorders. The objective of the program is to provide trainees with the perspectives and skills needed to prepare them for independent careers in neurobiological research. We request support for four fellows, who will have either the Ph.D. or M.D. degree. The program will provide opportunities for multidisciplinary research projects in the laboratories of both basic and clinical scientists and exposure to a wide variety of experimental and clinical issues. The program is broadly-based, and consists of four focal groups that are highly interactive with one another. The first group focuses on cellular and developmental neurobiology; the second group on systems and behavioral neurobiology, the third group on spinal cord and regeneration; and the fourth group on neuroengineering. There are a total of 10 senior level faculty members who will act as the primary training faculty. There are an additional 8 junior level faculty members who will also act as trainers, under the supervision of the senior faculty. The entire participating faculty, both senior and junior, is funded by the NIH, well published, and active in both research and training. In addition, there is an abundance of clinical researchers at three different hospitals within our university system who will participate in the training of the fellows. In addition to the primary relationship between a trainee and the mentor, there are a number of activities that ensure a broad exposure to the issues and techniques of modern neurobiology. These include courses developed around the four focal groups, journal clubs and seminars, research conferences and shared laboratory facilities. The progress of each trainee will be formally reviewed regularly and monitored constantly, through research meetings and seminars. This process assures full access to the various disciplines and perspectives available in the program. [unreadable] [unreadable]"}
{"text": "The principal aims of this project are to 1) identify state-independent alterations in corticotropin releasing hormone (CRH)-mediated physiological processes that can be utilized in the identification of biologically vulnerable individuals as an essential step for future molecular genetic approaches to unipolar depression, and; 2) to determine abnormalities in the regulation and actions of CRH in depression that are relevant to the susceptibility and intrinsic pathogenesis of this disorder, as well as to abnormalities in growth, reproduction, metabolism, and immunity, and hence to long-term health. In our search for a state-independent marker, we employ three protocols that intensively study the HPA axis; a) assessment of HPA responses to a naturalistic stressor (exercise at 70% and 90% V02max [Ex90, Ex70]); b) deconvolution techniques to assess instantaneous ACTH and cortisol production rates; and c) analysis of pulsatile hormonal secretion utilizing non-linear dynamics and low dimensional stochastic chaos. Our assessment of HPA responses to Ex90 in patients with seasonal affective disorder revealed a state-independent hypothalamic CRH deficiency similar to what we have previously observed in other syndromes of atypical depression. We have identified a sub-group of controls that shows HPA hyper-responsivess and GH hyporesponsiveness to Ex90, and increased trait anxiety. In a study of 20 consecutively admitted patients with affective disorder, we found a clinically significant decrease in the density of trabecular bone in association with decreased plasma osteocalcin levels (a potent osteoblastic factor), as well as a significant increase in intra-abdominal fat mass. These defects often persisted for months or years after resolution of the depressive episode and are potentially attributable to CRH mediated alterations in pituitary-adrenal, growth hormone, and gonadal steroid secretion. We also found that core CRH responsive elements in the POMC promoter have high homology to key elements of the genome of viruses (human immunodeficiency virus and cytomegalovirus) as well as to oncogenes and genes involved in the encoding of immune mediators such as IL-1 converting enzyme; thus, CRH could potentially influence susceptibility to infectious, neoplastic, or inflammatory diseases. In experimental models of cerebral ischemia in the rat, we have also found that CRH acts as an endogenous neurodegenerative agent that enlarges the area of infarct, while the IL-1 receptor antagonist serves as an endogenous neuroprotective agent potentially protecting the organism from the consequences of ischemia or other profound stressors."}
{"text": "The general transcription factor P-TEFb, consisting of Cdk9 and cyclin T, strongly stimulates RNA polymerase II elongation. It is also a host cell cofactor for Tat activation of HIV-1 transcription. Accumulating evidence suggests that Tat and the TAR RNA, located at the 5'end of all viral transcripts, not only recruit P-TEFb to the HIV-1 LTR but also cause the activation of Cdk9 kinase. For general transcription of cellular genes, data obtained during the current funding period indicate that P-TEFb is recruited to chromatin templates by the bromodomain protein Brd4. In addition, a major reservoir of nuclear P-TEFb is sequestered in the inactive 7SK snRNP. Further analyses indicate that in response to Ca2+signaling, P-TEFb is released from 7SK snRNP upon the dephosphorylation of the conserved Cdk9 T-loop by PP11 and PP2B. The dephosphorylated P- TEFb is preferentially bound by Brd4, which recruits it to the transcription pre-initiation complex. As the phosphorylation of Cdk9 T-loop is essential for P-TEFb activity, the T-loop is expected to undergo rephosphorylation by an as yet unidentified Cdk activating kinase (CAK) at a later stage in order to restore full activity to P-TEFb. Given that P-TEFb is essential for productive HIV-1 infection, the objective of this proposal is to examine how the various modes of P-TEFb regulation exerted by its associated factors, a putative Cdk9-specific CAK and the HIV-1 Tat/TAR will impact HIV-1 transcription and replication. Proposed are experiments to investigate whether the expression and activity of various P-TEFb-associated factors can be manipulated to control HIV-1 replication and latency. A combination of targeted investigations and comprehensive, unbiased screens will be employed to identify the Cdk9-specific CAK and elucidate the mechanism and functional significance of its phosphorylation of P-TEFb. To determine the mechanism of Tat/TAR activation of P-TEFb, the impact of Tat/TAR on phosphorylation status of the Cdk9 T-loop at different stages of HIV-1 transcription, the possible existence of novel components within the Tat-TAR-P-TEFb complex, and the ability of TFIIH and TAF7 to inhibit P-TEFb activation will be examined. These experiments will offer an exciting opportunity to identify novel factors that contribute to the activation of P-TEFb and HIV-1 transcription and provide fresh insights into how P-TEFb stimulates transcription of both HIV-1 and cellular genes. A better understanding of the mechanism by which P-TEFb controls HIV-1 replication and latency and the versatility of Tat/TAR in modulating this process will be informative toward the identification of new targets for anti-HIV therapy. PUBLIC HEALTH RELEVANCE: The Cdk9-cyclin T1 heterodimer of P-TEFb is a host cell cofactor for Tat activation of HIV-1 transcription. The proposed experiments will allow us to investigate how the P-TEFb-dependent HIV-1 transcription and replication can be controlled by: (1) the various P-TEFb-associated cellular factors;(2) a putative Cdk9 activating kinase;and (3) the HIV-1 Tat protein and TAR RNA that can do more than simply recruiting P-TEFb to the viral LTR. These experiments will lead to the identification of novel factors that contribute to the activation of P-TEFb and HIV-1 transcription and provide fresh insights into how P-TEFb controls transcriptional elongation of both HIV-1 and cellular genes. A better understanding of the mechanism by which P-TEFb regulates HIV-1 replication and latency and the versatility of Tat/TAR in modulating this process may lead to the identification of new targets for anti-HIV therapy."}
{"text": "A collaborative project between the Microbial Interactions Section and the Department of Anaerobic Bacteriology at Virginia Polytechnic Institute and State College was initiated April 1990. The subgingival floras of 20 patients exhibiting frank AIDS and 20 HIV seropositive patients suffering from gingivitis are being enumerated and identified to determine whether: (1) any changes in the normal flora occur following the viral infection; and (2) the gingivitis is associated with a typical gram negative flora, respectively. Twelve subjects, six from each category, have been examined thus far. Qualitatively, the microbial flora appears to be much the same as that seen in HIV uninfected individuals, with slightly more Gram positive bacteria present. However, both the Gram positive and Gram negative bacteria present do not exhibit the same growth characteristics observed previously. In general, they are more difficult to cultivate and show abnormally long lag periods, or refuse to grow at all, when transferred from medium to medium. Internal controls reveal that this is a reproducible trait of oral bacteria from HIV-infected individuals."}
{"text": "Terminated, 9/12/2001. Survival following radiotherapy for glioblastoma remains limited to 9 to 12 months. Agents which inhibit DNA repair represent a novel class of radiosensitizers which are currently being explored in the treatment of Glioblastoma. Hydroxyurea (Hu) is a recognized radiosensitizer which is not commercially available in an iv preparation for continuous infusion. In vitro data supporting the various possible mechanisms of radiosensitization including DNA repair inhibition, suggest that this agent would be most effective if a treated tumor were exposed to steady rather than bolus concentrations. In addition, its rapid half life and clearance would require frequent oral dosing to achieve steady state concentrations. We are thus administering Hu as a continuous IV infusion. Hu is synergistic with other repair inhibitors such as Ara-c, Ara-a, and Pentoifylline (PTF). PTF is a methylxanthine analog with hemorrheologic activity which is a potent radiosensitizer and repair inhibitor in vitro. In this study, Hu is being given via continuous infusion with PTF in a phase I dose escalation of PTF to patients receiving twice daily radiotherapy for glioblastoma."}
{"text": "Biophysical studies are planned on human and porcine neurotubule assembly and disassembly. Kinetics will be characterized in vitro by electron microscopy and optical mechanism of assembly. Assembly and disassembly of twisted tubules, which have been found only in the abnormal human brain, will be studied using protein isolated from autopsied brain from patients with senile dementia, Alzheimer's disease or Guam- Parkinsonism dementia. Major goals are to understand the mechanism of action of colchicine and vinblastine on microtubules, the possible regulatory role of Ca and Mg ions on microtubule assembly and the difference between neurotubules and twisted tubules."}
{"text": "Catecholamine systems, operating at the interface between the mind and the body, exemplify three ways the brain regulates homeostasis -- via neurotransmitters (norepinephrine, NE), hormones (epinephrine, EPI), and autocrine/paracrine factors (dopamine, DA). We conducted investigations in the areas of clinical neurocardiology, catecholamine neurochemistry, and novel catecholaminergic systems. 6-[18F]fluorodopamine ([18F]-6F-DA) positron-emission tomographic (PET) scanning led to a new pathophysiological classification of primary chronic autonomic failure. Patients with pure autonomic failure (PAF) or parkinsonism and autonomic failure had cardiac sympathetic denervation, whereas patients with the Shy-Drager syndrome had decreased or absent post-ganglionic sympathetic neural outflows to intact nerve terminals. 13N-ammonia and [18F]-6F-DA PET scanning detected perfusion or sympathoneural abnormalities in the affected limbs of patients with sympathetic dystrophy (RSD). Patients prone to neurocardiogenic syncope had decreased cardiac NE spillover, and patients with hypertrophic cardiomyopathy (HCM) had intact sympathetic innervation of the hypertrophic myocardium. Clinical neurochemical studies linked specific catecholaminergic phenotypes with genotypic abnormalities in Menkes' disease, familial dysautonomia (FD), and L-aromatic-amino-acid decarboxylase (LAAAD) deficiency. Treatment with L-DOPA as a dopaminergic pro-drug produced beneficial natriuretic responses in patients with congestive heart failure. Plasma DA-sulfate levels were found to depend on dietary factors and on production of endogenous DA in the gastrointestinal tract."}
{"text": "The focus of the experiments of this proposal are the processes by which pigeons learn matching to sample (MTS) and the conditions that lead to different ways that pigeons learn MTS. A newly developed technique will be used to measure if-then rule learning in the MTS task. Another newly developed technique will be used to measure relational-concept learning during acquisition of the MTS task. Tests are proposed to manipulate and confirm the presence of configural learning when evidence indicates that the whole MTS display is learned as a configural pattern. Other evidence suggests that in certain conditions the MTS task may be learned by combinations of these different types of learning. Enhancing and inhibitory interactions between these different learning processes in MTS will be studied. Configural learning will be made more difficult by increasing the number of displays to be learned and by making the display less likely to be configured as a unitary pattern by the pigeons. These manipulations of configural learning. Other manipulations will test the durability of relational-concept learning and explore the type of learning that replaces it. This research will extend our knowledge of how tasks are learned, problems solved, and conditions that lead to either higher-order concept learning or stimulus-specific learning."}
{"text": "Fluorescence Spectroscopy Core (Millar. Director; Pond. Manager) Overview The Fluorescence Spectroscopy Core provides state-of-the-art instrumentation and expertise to support a wide range of fluorescence spectroscopic measurement techniques, including steady-state, time resolved and single-molecule methods. These resources will support the development of new imaging technologies to visualize the assembly and disassembly of virus-host complexes and to identify homogeneous complexes suitable for structure determination."}
{"text": "If more were known about the role of diet in the cause of colon cancer, then specific dietary interventions could be recommended that would reduce the incidence of colon cancer. It has been observed that hyperplasia of colon epithelium precedes neoplasia and that the cell proliferation rate of colonic epithelium is correlated to carcinogen-induced neoplasia. Thus, nutritional modification of cell proliferation rate in the colon is hypothesized to modify the carcinogen-induced progression of normal eptihelium to the neoplastic state. To test this hypothesis we have designed experiments to give answers to the following specific questions regarding the progression of 1,2-dimethyl-hydrazine (DMH) treated colon eptihelium to the overt neoplastic state. 1. Is the reported prevention of DMH-induced hyperplasia in the colon, which was caused by administration of total parenteral (intravenous) nutrition ITPN) at isocaloric intake levels, permanent or reversible? If hyperplasia does not return after TPN treatment, is colon tumor incidence reduced by such TPN treatment? 2. Does a hypercaloric diet given to DMH-treated rats enchane colonic hyperplasia, and increase tumor incidence? 3. How does the manipulation of the non-nutritive fiber content in the diet, the caloric content of the diet, the chemical composition of the diet, one at a time while holding all other variables constant, affect interactions between such nutritional factors and carcinogen-included colonic neoplasia? Briefly, the experiments call for dietary modification either during, or after, or during and after an eight-week course of DMH treatment to rats. Groups of DMH and non-DMH treated rats will be sacrificed at times after the beginning of the carcinogen administration. To monitor the progression of colon carcinogenesis, the epithelium will be examined for: cell proliferation activity and crypt structure using histologic examination, intracellular content of sodium using X-ray microanalysis procedures, and also for the incidence and size of all visible tumors. The data will be subjected to appropriate statistical analysis. The findings should give insight into the interactions between nutritional factors and colonic carcinogenesis and should lead to nutritional concepts of prophylaxis against colon cancer. We will be on schedule with our original proposed experimental protocol by the end of this year and will follow the original proposed schedule of experimentation closely this next year."}
{"text": "The major allergen (designated as Par h 1) was purified from the pollen of Parthenium hysterophorus (a major cause of allergic rhinitis) using anion exchange chromatography and HPLC. The purified protein was homogenous on SDS-PAGE and revealed a single N-terminal amino acid (lysine) upon Edman degredation. Par h 1 was a glycoprotein, rich in glycine and proline, and with an estimated molecular weight of 31 kd. Isoallergens were observed with pI values from 4.3 to 5.2. The carbohydrate moieties on Par h1 appeared related to its IgE binding ability. The N-terminal 91 amino acid sequence of Par h 1 shows 81% identity with a protein from sunflower anther and the hydroxyproline-rich region of Par h 1 is 30-40% identical to similar stretches in extensins. IgE antibodies in the sera of Parthenium allergic individuals cross-reacted with a 50 kd hydroxyproline-arabinose-rich extensin from potato tuber. Thus, it appears that a group of soluble plant glycoproteins related to extensins, have carbohydrate-containing IgE binding epitopes that may contribute to allergenic cross-reactivity among specific pollens and foods."}
{"text": "The overall goal of the Administrative Core (AC) is to provide oversight, coordination and integration of Center activities. The specific aims are to: 1) Coordinate and integrate the scientific aims of the Projects and Cores, track and evaluate progress and outputs, and ensure successful completion of all Center aims; 2) Manage resources to assure the needs and priorities of every Project and Cores are met; 3) Act as coordinating center by convening meetings of Project, Core, and subcontract investigators, organize External Advisory Committee, ensure timely translation of research findings, prepare Center-wide reports, and interface with NIEHS and EPA project officers; 4) Evaluate the progress and success of the career, development plan, with specific focus on the career development of Faculty Development Investigator; 5) Ensure quality control of all stages of research namely study design, establishment of standard operating procedures and study protocols, secure sample and data transfer and management, data analysis, and accurate interpretation and translation of research findings to academic, government, healthcare and community stakeholders. The AC will operate under the direction of the Core co-Leaders, Consultant, and Pediatric Health Specialist in collaboration with Leaders of each Project and Core. The AC will ensure coordination of Center activities, manage resources across Projects and Cores, identify the most efficient use of infrastructure and communication and resolve needs as they arise, in order to increase the efficiency and productive output of the Center. AC Leaders will interact with the External Advisory Board to ensure the merit and value of all UM-CEHC elements to accomplish the overall Center aims. The Center Manager will prioritize coordination with the Community Outreach and Translation Core to optimize outreach and research translation to community stakeholders. Finally, the AC will oversee career development and training of New Investigators, particularly the UM-CEHC Co-investigator designated as the Faculty Development Investigator. Career development and training activities will draw upon extensive institutional resources and will be fully integrated with the Center's Research Projects and Cores."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. There has been no change in the scope of this project. The success of islet transplantation is hampered by the high rate of islet cell death and dysfunction after isolation. Therefore, the repair of islet damage from the isolation process and the opportunity to maintain islets long term in vitro as a new islet resource would represent significant advances and lead to a more widespread use of islet cell transplantation. Successful utilization of bone marrow in repairing skin, neuron, heart, and muscle injury led us to propose that bone marrow could offer a potential solution to these challenges. In our preliminary studies using co-cultures of whole bone marrow with islet, bone marrow was shown to increase islet function/survival (more than six months), stimulate islet growth and generate long-term insulin producing tissue in vitro. We hypothesize that specific subpopulations of marrow cells may be responsible for these findings. We have also hypothesized that extracellular ATP, ATP receptor (purinoreceptor P2XR), and interleukin 1beta (IL-1beta) are involved in bone marrow-induced repair of islet injury. In this project, we plan to identify whether multiple or single specific lineage marrow cells contribute to islet reconstitution. We will examine whether these reconstituted islets have sufficient function and vascularization in vivo as determined by transplantation into NOD/SCID mice. Finally, we will investigate whether bone marrow modulates ATP, its receptor P2XR, IL-1[unreadable] and its downstream pathways. This project will have benefits for current islet transplantation protocols and will provide insight into the mechanisms of islet cell death and regeneration."}
{"text": "Of the body's fat depots, visceral adipose tissue (VAT) specifically has been shown to strongly correlated to metabolic disorders and diseases such as hypertension, diabetes and heart failure as compared to subcutaneous adipose tissue (SAT) or overall obesity. Furthermore, reduction of VAT is associated with a reduction of metabolic risk factors. Determination of VAT is therefore of high clinical importance, however, current methods to determine VAT are inaccurate, costly, or inconvenient. To meet the need for a more accurate, low cost, easy to use, portable device suitable for the convenient determination of an individual's VAT and obesity in general, PhiloMetron proposes to utilize ultra wideband radar (UWR). Accordingly the objective of this Phase 1 proposal is to assess the ability of UWR to determine the amounts of abdominal body adipose tissue, in particular to quantify SAT and VAT, in model systems. Upon successful completion of these Phase I studies, planned Phase II studies will include validating the use of the technology in the determination of abdominal fat, including VAT, in obese or overweight individuals. PUBLIC HEALTH RELEVANCE: Most of the major chronic diseases impacting the US healthcare systems are being driven by the obesity epidemic. There is a need to create a clinically effective weight management program that can be used to lose and maintain a person's weight to limit the long-term economic and societal impact. These weight management plans need efficacious diet and exercise plans to successfully lose and maintain weight. Currently, weight is the most often used physiological measurement to determine success of the patient's diet and/or exercise, but is a grossly insensitive measurement. More importantly, determination of regional body fat depots is showing increasing importance in obesity and wellness management however weight determinations are incapable of distinguishing between these fat depots. Of the body's fat depots, visceral adipose tissue (VAT) specifically has been shown to strongly correlated to metabolic disorders and diseases such as hypertension, diabetes and heart failure as compared to subcutaneous adipose tissue (SAT) or overall obesity. Furthermore, reduction of VAT is associated with a reduction of metabolic risk factors. Determination of VAT is therefore of high clinical importance, however, current methods to determine VAT are inaccurate, costly, or inconvenient. The objective of this Phase I proposal is to establish a foundation for the use of a potentially breakthrough technology for the measurement of VAT."}
{"text": "The overall objective of the Clinical Laboratory Core will be to provide support for all laboratory needs of the clinical trials described in Projects 1 and 2 of this Program Project. This will include the collection of biological specimens from study participants, facilitating clinical laboratory testing needed for patient management and assessment of drug safety and tolerability, routine storage of biological samples for future use, malaria specific laboratory testing from blood specimens, and the collection and process of placental samples including histopathology. The clinical laboratory core will also be responsible for supporting the collection of biological samples for the immunology studies described in Project 3 ofthe Program Project. Performance sites will include a phlebotomy center and clinical laboratory within our study clinic located on the Tororo District Hospital (TDH) campus, a placental histopathology laboratory located near our study clinic on the TDH campus, and the Joint Clinical Research Center (JCRC) referral laboratory where clinical laboratory testing will be performed. The specific objectives oithe Clinical Laboratory Core will be as follows: Objective 1: To collect, transport and store all biological specimens from study participants. Objective 2: To perform all malaria diagnostic testing. Malaria microscopy will be performed by experienced technicians including double reading of blood smears for quality control purposes. Malaria microscopy results will be available in real time for patient management. A PCR based assay will be available for the detection of asymptomatic parasitemia using dried blood spots collected on filter paper. Objective 3: To collect placental specimens and perform histopathology studies. The gold standard for classification of placental malaria is based on histopathological assessment of placental tissue collected at the time of birth. Our research center has established local capacity for the collection, processing, and staining of histopathology specimens for the diagnosis of placental malaria, one of the primary outcome measures for the studies outlined in this Program Project. RELEVANCE (See instructions): The success of the proposed Program Project, which includes two randomized clinical trials and supporting immunology studies, requires a strong Clinical Laboratory Core that includes proper collection of biological specimens, transport of specimens to our reference laboratory, and performance of on-site laboratory testing to ensure that study participants are managed properly and study outcomes accurately assessed."}
{"text": "The objectives of the Developmental Projects Program are to ensure a continual renewal of high-quality scientific endeavors in the DF/HCC Prostate Cancer SPORE and to fund efforts that will complement or enhance the overall quality of the DF/HCC Prostate Cancer SPORE. In general, the Developmental Projects Program has funded established investigators. Based on our review, we will be mindful of including more junior investigators. This Program will rely on the infrastructure created by the Administrative, Evaluation, and Planning Core (Core A) to: 1. Solicit applications and/or identify novel prostate cancer research projects 2. Evaluate these projects for funding 3. Fund innovative developmental projects 4. Re-evaluate projects for possible transition into full project status 5. Evaluate the success of the program"}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To reduce high blood glucose levels and sustain glycemic control levels among low-income, inner city adults receiving care at a local, federally qualified community health center Objective: To measure six quality indicators to assess quality improvement including four performance measures: HbA1c testing, lipid profiles total cholesterol, LDL, HDL, and triglycerides, retinal examination, and monitoring nephropathy and two quality indicators- glycemic control HbA1c level 7% and lipid control LDL 100 mg/dl. Key Findings Process measures are not being documented as stringently as they should be. This makes it difficult to determine whether or not a provider is giving adequate care and attention to disease management in this population of patients. Our main outcome measure is glycemic control. The majority of patients for each audit had an HbA1c value above 9% at the time of testing. This suggests to us that the efforts to reduce HbA1c levels could be improved. Challenges: There are some inherent challenges with the progression of this project. They are listed as follows: West End Medical Center is currently undergoing the implementation of an EMR system. The implementation process is projected for completion late September, 2006. Inevitably, this will cause some of the timelines for the audits and nurse-mediation component to shift if the implementation process does not go smoothly. Information entered into the EMR system will be provider driven. Consequently, this will affect the audit and feedback component of the project due to the fact that not all of the providers have decided on how much information should be entered into the system. Fortunately, the project can proceed without the aid of the EMR. We will proceed as if the EMR were never to have been implemented and the clinic was operating with a paper based medical record system. Change in investigators with Dr. Mayberry's departure."}
{"text": "The purpose of this core is to provide a centralized facility in which all of the various animal procedures outlined in the Program Project may be performed. Personnel experienced in implementing murine models of cardiovascular disease in a rigorous and reproducible manner will staff the core. The main responsibilities of the core will be the performance and analysis of the following murine models: 1. Myocardial infarction 2. Peritonitis 3. Carotid arterial injury 4. Bone marrow transplant"}
{"text": "This study will investigate the histopathology and histochemistry of normal human orbicularis muscle and orbicularis muscle injections of botulinum toxin, to weaken the muscle on a temporary basis. This will extend previous research in the histopathology of vertebrate extraocular muscles injected with botulinum toxin to human muscles and provide data correlating clinical and electromyographic effects of botulinum toxin injections with histology."}
{"text": "PROJECT SUMMARY/ABSTRACT Melanoma is the deadliest form of skin cancer and is one of the most difficult cancer to treat. Therefore, the underlying mechanism of melanomagenesis and melanoma development demands intensive study. Recent findings identified that palmitoylation of melanocortin-1 receptor (MC1R), primarily mediated by the protein-acyl transferase (PAT) ZDHHC13, is essential for activating MC1R signaling and prevent melanomagenesis. Preliminary studies also revealed that APT2 is the major depalmitoylating enzyme of MC1R and is harmful for metastatic melanoma patients. In this proposed study, I aim to elucidate the in-depth molecular mechanism by which ZDHHC13/APT2-regulated palmitoylation repress melanoma development and progression, and test the hypothesis that ZDHHC13/APT2-regulated palmitoylation is essential for melanomagenesis and melanoma metastasis in vivo. During the mentored K99 phase, I will elucidate the underlying regulatory mechanism of ZDHHC13 in melanocytes by using new developed ZDHHC13 transgenic mice. I will also characterize the effects of depalmitoylation inhibition in melanomagenesis by targeting APT2. During the independent R00 phase, I plan to determine the role of ZDHHC13/APT2-regulated palmitoylation in melanoma metastasis in vivo and identify whether inhibition of depalmitoylation improves survival by suppressing metastasis. Furthermore, I will need additional trainings during the award period in melanoma mouse model, targeted therapy, as well as professional skills which will contribute to my long term career goal. In summary, successful completion of my proposed studies will bring novel insights into the roles and molecular mechanisms of ZDHHC13/APT2-regulated palmitoylation in melanoma development and progression, which can be translated into new intervention and treatment strategies. Receipt of this award will allow me to expand my research plan and serve as a platform for me to receive additional trainings, thus prepare myself becoming an independent principal investigator in the field of melanoma research."}
{"text": "The goal of this project is to map and identify genes for herditary deafness. Linkage analyses are being conducted in several large pedigrees segregating non-syndromic and syndromic forms of deafness. If linkage to the known syndromic, DFNA (dominant) and DFNB (recessive) loci in large families are excluded, we initiate genome-wide screens. This strategy has allowed us to map new deafness loci such as DFNA20, DFNA27, DFNA28 and DFNA36. The chromosomal map locations of these novel deafness genes are then refined prior to initiating positional cloning strategies to identify the genes responsible for the hearing loss. Recently we have identified genes for DFNB12, DFNA28, Usher 1D and usher 1F. Additional families with dominant and recessive modes of inheritance with profound congenital or progressive hearing loss are being ascertained with the goal of mapping and cloning additional novel genes that are necessary for hearing and/or maintenace of the auditory system. We are also asscertaining families, mapping loci and identifying genes for Usher syndrome. The defining clinical features of Usher syndrome are hearing loss and progressive retinopathy."}
{"text": "The purpose is to measure neutrophil production and function in healthy, young and elderly subjects in their normal basal state and after stimulation with two doses of granulocyte colony stimulation factor."}
{"text": "I am a surgical oncologist dedicated to caring for patients with complex malignancies and to developing novel immunotherapeutic approaches for metastatic liver cancer. Early in my medical training, I developed a passion for immunology and host-tumor interactions. After graduating from the New York University (NYU) School of Medicine with the top academic record in my class, I trained in general surgery at NYU and Bellevue. I spent two years as a research fellow with Dr. Ronald DeMatteo at Memorial Sloan-Kettering (MSK), where I gained valuable experience in immunologic research and published papers on liver immune cells in Hepatology and the Journal of Immunology. During my clinical surgical oncology fellowship at MSK, I studied immune infiltrates in liver metastases, work published in the Annals of Surgical Oncology and featured in Nature Reviews Clinical Oncology. I was recruited to Roger Williams to join a strong immunotherapy program and build a lab focused on investigating how suppressive liver immune cells may contribute to the development and progression of liver metastases. My K08 proposal is a critical step in my career development as the project represents a fusion of my basic liver immunology background with a therapeutic platform, genetically modified or designer T cells (dTc), we are presently using in clinical trials at Roger Williams. The associated training plan, as noted below, will provide me with important educational opportunities to facilitate my transition toward independent funding. Environment - The Roger Williams Medical Center (RWMC) is an ideal environment for my academic growth and development. Dr. Richard Junghans, my mentor, is a well established immunologist with a rich experience with immunotherapy trials and dTc. I also work with Dr. Vincent Falanga, the RWMC Chief of Dermatology, as he shares valuable resources obtained through their COBRE grant. As the RWMC is of modest size, my laboratory and career development are top priorities for the institution. I receive a tremendous level of support from th administration and my Division Chief and Cancer Center Director, Dr. N. Joseph Espat. This enables me to have the necessary protected time and maintain a high level of focus on my laboratory work. Development and Training Plan - I have assembled a mentoring team of well respected experts to monitor my progress, support my research, and promote my career development. Each member of the team has successfully competed for NIH funding and offers a particular area of expertise that meshes well with my proposal. Dr. Junghans will be the primary mentor, and his experience with immunotherapy and T cell biology will be invaluable. The three co- mentors will make important contributions as well. Dr. Espat, an authority on liver metastases, will ensure that the work and publications maintain a translational focus. My mentor from MSKCC, Dr. DeMatteo, will provide important insight and critique from the vantage point of an expert in liver immune cell biology. Dr. Alfred Ayala at Brown, with whom I have recently begun to collaborate, is another well respected expert in liver immunobiology. I have also included translational medicine and immunology course-work at Brown to expand my knowledge base in critical areas. Research Plan - Based upon the work I have published to date, I hypothesize that suppressive liver immune cells limit the effectiveness of T cell based therapies for eradicating liver metastases. T regulatory cells (Treg) and myeloid derived suppressor cells (MDSC) are likely contributors to liver tolerance. Our lab has spent over one year optimizing our murine model of CEA+ colorectal liver metastases and the immunologic assays described in the proposal. My first specific aim focuses on elucidating the mechanisms by which Treg and MDSC may limit anti-CEA dTc function in the liver. I have designed the experiments to focus on each component of the model separately, including the Treg or MDSC, dTC, and immune receptor express by dTc. In addition, contact-dependent mechanisms such as the programmed death-1 pathway and secreted factors will be studied. For the second specific aim, specific strategies for blocking the suppressive interactions between Treg or MDSC an dTc will be tested in vivo. We hope some of our immunologic manipulations will be translatable into the clinical arena."}
{"text": "In addition to long known contributions from particular MHC class II molecules, there is growing appreciation that in both humans and NOD mice some MHC class I variants also play an essential role in autoimmune type 1 diabetes (T1D) development by mediating pathogenic CD8 T-cell responses. The overall goal of this renewal application continues to be dissection in NOD based mouse models of the mechanistic basis for MHC class I restricted diabetogenic CD8 T-cell development, and use of this information to identify potentially clinically translatable means to attenuate such effectors. While the H2g7 MHC haplotype encoded Kd and Db class I molecules are essential to T1D development in NOD mice, they are common variants also characterizing many non-autoimmune prone strains. This suggested H2g7 MHC class I molecules aberrantly mediate diabetogenic CD8 T-cell responses in NOD mice through interactions with some of the many other disease susceptibility (Idd) genes characterizing this strain. Aim 1 will test the hypothesis based on preliminary mRNA transcript profiling and congenic truncation analyses that a hyper-expression variant of the NFkB inhibitory Nfkbid gene located within the previously identified Idd7 locus is an important contributor to the failure of diabetogenic CD8 T-cells to undergo thymic negative selection in NOD mice. Similarly, epidemiological studies indicate that in humans certain common class I molecules such as HLA-A2.1 can aberrantly contribute to T1D development also likely through a genetically contextual process. Indeed, we found that when expressed in the context of the NOD genome, human HLA-A2.1 molecules mediate diabetogenic CD8 T-cell responses. HLA-A2.1 restricted diabetogenic CD8 T-cells in this NOD background stock primarily recognize two peptides each derived from the pancreatic ss cell proteins insulin (INS) and islet specific glucose-6-phosphatase catalytic subunit related protein (IGRP). Immunological tolerance can be efficiently induced to antigens bound to autologous leukocytes by the cross-linking agent ethylene carbodiimide (ECDI), and such an approach is in a clinical trial as a possible multiple sclerosis intervention. However, there are many hurdles to cell based therapies, and possible T1D intervention approaches can only be considered in humans already at a late prodromal stage of disease development. Therefore, to broaden potential clinical translation, Aim 2 will test the possibility supported by new preliminary data that treatment with synthetic microparticles bearing appropriate ECDI coupled INS and/or IGRP autoantigenic peptides can exert late disease stage T1D protective effects in NOD-HLA-A2 mice, and/or enables reversal of established disease by pancreatic islet transplantation. Finally, epidemiological evidence implicates B39 as a potentially highly potent diabetogenic HLA class I variant in humans. Thus, Aim 3 will assess whether transgenically expressed B39 molecules mediate diabetogenic CD8 T-cell responses in NOD mice, and if so, identify cell autoantigens displayed by this class I variant, and test their capacity to serve as broadened disease intervention reagents."}
{"text": "PROJECT SUMMARY The overarching hypothesis of this proposal is that kisspeptin may explain many of the changes in glucose metabolism in pregnancy: high levels of kisspeptin during normal pregnancy amplify insulin release to maintain normoglycemia, and relative hypokisspeptinemia may underlie the pathophysiologic defects in gestational diabetes mellitus (GDM). The initial steps to examining this overarching hypothesis are to 1) define the effect of kisspeptin (at pregnancy levels) on insulin physiology in non-pregnant women and 2) to determine if kisspeptin levels predict the development of GDM. Given the complexity of factors in pregnancy that regulate metabolism and insulin physiology, these initial studies are designed to isolate the effect of one factor: kisspeptin. This proposal utilizes gold standard (hyperglycemic and hyperinsulinemic euglycemic clamps) and physiologic (mixed meal tolerance test) methods in randomized, placebo-controlled and blinded clinical trials to assess kisspeptin?s impact on insulin secretion and insulin sensitivity in non-pregnant women. Going further, the grant utilizes comprehensive specimen banks with longitudinal pregnancy samples and patient outcome data to measure kisspeptin levels across pregnancy and to determine if kisspeptin levels early in pregnancy can predict the development of GDM. This grant will refine our understanding of the impact of hyperkisspeptinemia of pregnancy on insulin and incretin physiology and development of GDM. This application details a comprehensive five-year training program for mentored career development in patient- oriented research. The Applicant proposes research, including independent clinical trials, specifically constructed to provide focused training pregnancy physiology and in the mechanisms of insulin physiology. To achieve this goal, she has chosen mentors with complementary expertise: Dr. Stephanie Seminara is an expert in kisspeptin physiology with a strong background in human physiology research, and she is Chief of the Reproductive Endocrine Unit at Massachusetts General Hospital (MGH); Dr. Patrick Catalano is an expert in pregnancy physiology and gestational diabetes, and he is a Professor of Obstetrics and Gynecology at Tufts University School of Medicine and Principal Investigator in the Mother Infant Research Institute; Dr. Jose Florez is an expert in physiological mechanisms in the development of diabetes, and he is Chief of the Diabetes Unit at MGH. This mentoring team will position the Applicant well to launch a successful independent investigative career in metabolism, with a focus on pregnancy and the influence of reproductive hormones, a key NICHD research priority area. The Applicant's career development plan entails rigorous coursework and seminars, hands-on practical experience, and close guidance from scientific advisors with diverse scientific expertise. Collectively, the experience gained during this award will serve as the foundation for the Applicant's independent, academic career as a physician-scientist with expertise in translational research in human metabolic disease."}
{"text": "This is a multi-institutional, Intergroup III trial examining the potential chemopreventative effect of Finasteride for prostate cancer. Study subjects who accrue can have no evidence of prostate cancer clinically. Enrolled participants randomize to either placebo or the study drug (proscar 5 mg daily), which is administered in a double-blind fashion. Participants are followed with yearly digital rectal exams and serum PSA values. All PSA samples are sent to a central lab for processing, and elevated values are evaluated with biopsy. any cancer detected during the study results in patients being taken off study. The follow-up period will continue for 7 years, after which all study participants will undergo trasnrectal unltrasound-guided biopsy, and tiddues form the biopsy will be processed at a central pathology site. Differential rates, if any in the prevalence of prostate cancer between the two study groups will be recorded."}
{"text": "D-alanyl-D-alanine peptidases catalyze the crosslinking of peptidoglycan inthe final stages of bacterial cell wall biosynthesis. The DD-peptidases are the targets for B-lactam antiobiotics(penicillins and cephalosporins). The structure of the enzyme from Streptomyces R61 has been determined to 1.6A resolution (Crystallographic R-factor = 0.153). We are now studying complexes of the native enzyme with drug molecules and substrate analogs. These studies will be facilitated by access to the Cambridge Structural Database."}
{"text": "This project follows our discovery of ganglioside presence in the nuclear envelope (NE) of neural cells, and the functional association of GMtwith a Na-Ca exchanger in the same membrane. In potentiating the exchanger activity, GM1 was shown to bind to this protein with unusually high affinity, a phenomenon that was not shared by these molecules in the plasma membrane. One goal of the proposed studies is to elucidate the molecular basis of this unusual association by determining which of the several known exchanger isoforms has the ability to (a) traffic to the NE and (b) bind GM1. Various isoforms of the exchanger will be transfected into Jurkat cells, which we have found do not normally express the GM1-exchanger complex in the nucleus, and the NE examined for evidence of such expression. Exchanger isoforms having this ability will be compared for common structural motifs that appear to promote high affinity binding (likely to include basic amino acids); the structures will be altered through site-directed mutagenesis to determine whether such exchangers, following transfection, have lost the ability to sequester GM1 in the NE. We will test the hypothesis that the GM1-exchanger complex occurs in the NE in association with ganglioside GD1a and neuraminidase, both of which were also shown to occur in the NE. The presence of these 2 molecules could constitute a supply mechanism to ensure sufficient GM1 for potentiating the exchanger. The functional role of the GM1-exchanger complex, postulated to remove elevated nuclear calcium, will be tested by comparing nuclear calcium changes in cells which do and do not have the GM1-exchanger complex. The functional role will also be tested in vivo with knockout mice lacking GM1, which were found to be highly susceptible to kainate-induced seizures. Demonstrated attenuation of such seizures by administered LIGA-20, a membrane-permeant derivative of GM1, will be further studied by determining the role of nuclear GM1/exchanger in modulating the seizure mechanism."}
{"text": "Exercise Testing & Training Facility utilization only. Epidemiological studies suggest that low levels of physical activity and physical fitness are strong and independent risk factors for cardiovascular disease, cancer and all-cause mortality. Inactivity in the United States appears to be a major health problem that is of comparable magnitude to cigarette smoking, obesity, high blood pressure, and high blood cloesterol. There are over 40 million adults in the US whose sedentary habits put them at considerably increased risk of morbidity and mortality from several diseases. Exercise has been found to be a very strong predictor of successful weight management once weight loss has been achieved. No studies have compared changes in risk profiles and body composition in people treated by diet plus lifestyle activity vs. diet combined with a traditional exercise program. We hypothesize that patients who are randomized to diet-plus- lifestyle activity will maintain both weight losses and improved risk profiles significantly better than the subjects who receive the diet plus traditional exercise training because the former participants will continue to implement their increased lifestyle activity strategies into their daily schedules. The purpose of this study is to examine whether a lifestyle activity program helps patients maintain weight losses and adhere to the activity program after a supervised weight loss program. Fifty obese women will be randomly assigned to either: 1) control group, 2) diet plus lifestyle activity, or 3) diet plus traditional exercise, i.e, combined strength training and aerobic exercise in a supervised setting. We hypothesize that the women who receive the lifestyle treatment will achieve better weight maintenance and continue to pursue lifestyle activity after the supervised program is over."}
{"text": "Human milk oligosaccharides (HMOs) have been thought to play a role in the development of specific intestinal flora in breast-fed infants for many years. Nowadays it is known that they are also potent inhibitors of bacterial adhesion to epithelial surfaces (initial stage of the infection process). Oligosaccharides are not hydrolyzed in the upper small intestine and reach the large intestine intact, where they serve as substrates for bacterial metabolism. Thus, HMOs are considered as the ''dietary fiber'' of human milk. Another characteristic of oligosaccharides is their proposed ''anti-infective effect''. This role is achieved thanks to their capacity to inhibit the adhesion of bacteria to the epithelial surfaces, thereby playing an important protective role against infection in the gastrointestinal, respiratory and urogenital tracts by direct and indirect mechanisms. Therefore, HMOs have antimicrobial activity and may be useful in treating and/or preventing specific enteric bacterial and viral infections. However, the road to convert HMOs into pharmaceuticals or nutritional substances has been blocked by the lack of pure, single component oligosaccharides from human milk in quantities large enough for scientific investigation, as well as preclinical tolerance and safety studies and for safety and clinical testing in populations that are exposed to gastrointestinal pathogens. Therefore, this proposed research program aims to develop practical processes to produce HMOs on multi-gram to kilo-gram scales. Since it was repeatedly reported that 2-linked fucosyloligosaccharides exhibited more antimicrobial activity than non-2-linked fucosyloligosaccharides, we will choose five 2-linked fucosyloligosaccharides such as 2'-FL, LNF-I, 2H-antigen, LDFH-I and Ley as our main targets. Moreover, non-2-linked fucosyloligosaccharides LNF-II and LNF-III will provide us the opportunity to confirm the observation of higher antimicrobial activity for 2-linked fucosyloligosaccharides. In addition, the non-fucosylated oligosaccharide LNT and LNnT will provide control experiments to evaluate the effect of fucose in oligosaccharides. Over the past 14 years, the Wang lab has been developing enzymatic oligosaccharides synthesis. We have invented and further developed the superbeads and superbug technology for large scale oligosaccharide production. The most efficient approach for oligosaccharide synthesis is to follow the natural carbohydrate biosynthetic pathway where oligosaccharides are assembled together by specific glycosyltransferases using individual sugar nucleotides as building blocks. These building blocks are themselves biosynthesized and recycled from individual monosaccharides through a series of biosynthetic enzymes. For small to medium scale synthesis of oligosaccharides, Wang has developed simple solid phase synthetic systems by immobilizing all the necessary biosynthetic enzymes onto a so-called superbeads. These beads function as stable and versatile synthetic reagents, which can be used to synthesize a variety of glycoconjugates in cell-free systems. For large-scale production, Wang superbug essentially transfers the entire natural biosynthetic pathway into an E. coli strain. The approach includes cloning each enzyme along the biosynthetic pathway and connecting the genes of these enzymes together to produce an artificial gene cluster. A recombinant E. coli transformed with such a gene cluster is then used to produce the oligosaccharide through fermentation and purification. Thus, the superbeads and superbug approaches will be used in this program to produce the 9 oligosaccharides. Specifically, there are four aims: Aim I: Production of HMOs by immobilizing multiple enzymes (superbeads). This involves investigation on the necessary microbial glycosyltransferases, development of superbeads for UDP-GlcNAc, UDP-Gal and GDP-Fuc production, and combination of the glycosyltransferases with sugar nucleotide production to produce oligosaccharides. Aim II: Production of HMOs by recombinant E. coli (Superbug), which involves combination of the biosynthetic pathways of these HMOs into one or several recombinant E. coli strains. Aim III: Production of HMOs by GRAS (Generally Recognized as Safe) yeast cells. This new system will provide safer production system for HMOs synthesis. Aim IV: Characterization of the oligosaccharides through systematic biomedical and microbiome approaches in collaboration with other specialized laboratories, also in our own lab, with the advantage of multi-gram or kilo-gram scale neutral human milk oligosaccharides produced from this project. It is expected that the biosynthetic technology developed in Aim I - III will be transferred to biotech company(s) (such as the biotech startup Carbogene USA LLC which specializes in large scale oligosaccharide production) and GMP processes will be developed to produce the oligosaccharides in quantities large enough for preclinical studies of tolerance and safety, as well as for safety, dose-ranging, and efficacy trials in infants and children who are at high risk of exposure to gastrointestinal pathogens."}
{"text": "Responsibility for Planning and Evaluation is vested in the Georgetown Lombardi Comprehensive Cancer Center (LCCC) Director and Executive Committee (EC). Since the last review, LCCC has undertaken an extensive review and overhaul of its planning and evaluation processes and refined its overall Strategic Plan. Director Weiner, Deputy Director Atkins, 6 Associate Directors (ADs), and a MedStar Georgetown Cancer Network Representative form the EC, which oversees Center-wide strategic and operational initiatives, discusses overarching goals of LCCC related to mission and comprehensive cancer center status, reviews metrics for each area overseen by the ADs, approves membership, oversees Developmental Funds, and fosters collaborations across Research Programs and disciplines. An Internal Advisory Committee (lAC) meets semi-annually to provide advice on integration with other components of the University and MedStar Health. The annual External Advisory Committee (EAC) meeting is supplemented with individual EAC members' guidance via conference calls and on-site visits. In-depth external reviews have evaluated progress of specific aspects of LCCC (clinical research, Shared Resources, Administration, biostatistics and investigator-initiated protocols). Research Program Leaders meet monthly to review Program themes, vet new and continued Program membership, and discuss needs for Shared Resources and promising opportunities for collaborative interprogrammatic research. Program meetings allow in-depth research discussion, refinement of program themes and goals and progress toward meeting them, discussion of correlative science for clinical trials and encourage incorporation of health disparities into ongoing research. Identification of opportunities for collaboration (multi-investigator studies, translational and transdisciplinary projects) is high priority for all planning and evaluation activities. Within the Shared Resources, individual Advisory Committees meet annually to review metrics and update and prioritize goals. User satisfaction is monitored by surveys. Frequent clinical leadership meetings ensure metric review and update. Administration is monitored through external review, a faculty liaison group and user surveys. Retreats have focused on health disparities; the population science merger; development of therapeutic trials; transdisciplinary, translational projects; and Administration. These actions have led to LCCC's strategic objectives to 1) advance transformative cancer research, 2) reduce the impact of cancer and diminish disparities in our catchment area, 3) lead high-impact clinical research, and 4) ensure long-term growth, vibrancy and stability ofthe research enterprise."}
{"text": "The long-range goal of this project is to increase the percentage of Hispanic women who receive cervical cancer screening and who are in compliance with recommendations for follow-up after an abnormal screening. The objective of this proposal is to integrate existing theoretical models in the development and pilot-testing of a scale measuring how Hispanic women construct meaning regarding cervical cancer prevention screening. In addition to this scale, demographic and psychosocial information will also be included. The rationale for the proposed research is that this theory-driven and culturally-appropriate questionnaire can be used as a tool to determine factors that predict cervical cancer screening among Hispanic women, which, in turn, will assist in the development of tailored interventions to increase cervical cancer screening in this population. We are well suited to undertake the proposed research because we have assembled a multidisciplinary team with the scope and breadth of expertise in the areas of public health, health psychology, cancer control, Hispanic culture, measurement, and qualitative research. Given our long-standing relationship with a Hispanic grassroots organization and other agencies serving the Hispanic population in the Memphis area and our experience working with this population, we feel confident that we can recruit a large number of Hispanic women. Once we complete the work proposed in the R03 application, we expect to be well-positioned to develop and submit an R21 application to propose the validation of the questionnaire, as well as pilot-testing and feasibility-testing of a theory-driven, culturally appropriate cervical cancer prevention intervention targeting Hispanic women. The specific aims are: (1) To test and integrate components of the existing theoretical models (primarily the PEN-3 model and components of the Theory of Planned Behavior and Social Cognitive Theory) to determine contextual variables associated with cervical cancer screening among Hispanic women of reproductive age through a series of 20 focus groups with Hispanic women between the ages of 18 and 42; (2) To develop a theory-driven and culturally appropriate questionnaire to assess contextual variables (and their social constructions) that contribute to cervical cancer prevention screening among Hispanic women based on the results of Aim 1; (3) To psychometrically evaluate the culturally appropriate questionnaire in a random sample of Hispanic women (N= 300) based on the results of Aim 1 and 2."}
{"text": "The aims of this project are to (1) to understand the molecular mechanism for depression of synthesis of enzymes involved in polysaccharide synthesis that occurs in capR, capS, and capT mutants (2) to understand the molecular mechanism of control of cell division as it is specifically related to the point mutations in the E. coli K12 gene designated capR (1on). The capR mutants, besides overproducing polysaccharide synthesizing enzymes, are also sensitive to UV, X-rays and ozone. Upon UV or x-ray exposure they form long filaments without crosswalls and these forms are nonviable. The capR ion gene specifies a protein (as determined by genetic studies) that appears to be a repressor or controls repressors that bind to many sites on the E. coli chromosome to stop synthesis of enzymes of polysaccharide synthesis. The capR ion protein may function similarly to control UV and x-ray sensitivity. We intend to isolate the capR ion protein. We have obtained the capR gene on a tetracycline resistance plasmid using tetracycline as a selective marker and combining DNA molecules in vitro with restriction enzymes and DNA ligase. We can amplify the capR ion protein in each cell to permit us to isolate the protein. Using genetic techniques we will also identify the postulated DNA site (the \"UV operon\" for structural genes) of capR ion protein action that is responsible for causing a UV and x-ray sensitive phenotype in capR (ion) mutants. Recent results indicate the gal operon, in addition to being controlled negatively by the classic galR repressor and positively by cyclic-AMP and cyclic AMP receptor protein, is also independently regulated negatively by the capR protein. This is the most complex control system available that can readily be investigated at the molecular level and we will pursue this subject in conjunction with those described above. The capR gene also effects bacteriophage lambda transcription and committment to lytic growth, and we are pursuing this fact."}
{"text": "This project will develop X-ray tubes generating mono-energetic radiation in the energy region useful for mammography (17-30 keV). Earlier work has demonstrated significantly reduced dose in such examinations by using fluorescent radiation at higher energies than the 17.5 keV characteristic radiation of molybdenum. By proper choice of energy, adequate contrast can be maintained while the dose is substantially reduced. However, to date the production of clinically useful intensities has been hampered by thermal loading of the fluorescent anode. This problem will be overcome through the use of new materials and techniques for anode fabrication. Anode properties including radiant output per unit charge, spectral distribution, loading factors, phantom penetration, etc. will be studied under operating conditions with a demountable x-ray tube and with radiation detection equipment that includes a high resolution spectrometer interfaced to a minicomputer for data manipulation. The ultimate goal will be the production of a new fluorescent-anode X-ray tube which will give substantially reduced doses in clinical mammography."}
{"text": "The cellular and molecular events in limb development are being studied in mouse embryos. The techniques used require both histologic analyses and biochemical assays. The primary morphological tool is Golgi staining which permits studies of cell polarity as the Golgi apparatus occupies the pole of the cell from which material is secreted. Studies to date have shown that the epithelium is always oriented away from the underlying mesenchyme. The mesenchyme beneath the epithelium shifts its polarity on days 12 to 13 (the time of chondrogenesis) to point primarily toward the epithelium. The stimulus for this shift has not been identified. The types of collagen and proteoglycans in the developing limb are also being determined. We have found that three small molecular weight hydroxyproline-containing compounds are present during limb development. These are much more abundant than large molecules like collagen which also contain hydroxyproline. These morphologic and biochemical studies are also being carried out on mouse limbs malformed by the dominant gene Dh (dominant hemimelia). It is proposed that the single mutant gene malformation will interrupt primarily one aspect of limb morphogenesis. By comparing different hereditary limb malformations one would expect to find primary effects on different aspects of the same process. Specimens from human limb malformations are being studied to see if they contain abnormal or immature types of collagen."}
{"text": "Summary Stroke is the leading cause of disability in the US, and, apart from physical therapy, therapeutic options to reduce this burden are non-existent. Following a stroke, cell death and glial scarring leads to the formation of a non- regenerative stroke cavity surrounded by the regenerative peri-infarct region. We are interested in understanding how bioengineered therapeutics can synergize with pro-repair programs active in the peri-infarct region to promote tissue regeneration in the stroke cavity and thereby improve neurological recovery. We have previously engineered a dual-acting angiogenic hydrogel that, when injected into the stroke cavity, can reduce glial scarring and promote vascularization to allow axonal infiltration. Although achieving brain repair in the stroke cavity is remarkable, these results were achieved in young mice with un-impaired neuroplasticity. We believe that to bring this technology closer to clinical translation, we must be able to show similar brain repair and behavioral improvement in more clinically relevant animal models, like aged mice with decreased neuroplasticity. In this application, we aim to identify our angiogenic hydrogel?s mechanism of action and optimize its formulation and to utilize this new formulation in a stroke models with decreased neuroplasticity. The hydrogel is composed of hyaluronic acid functionalized with cell-binding integrins and loaded with clustered vascular endothelial growth factor (VEGF) and heparin nanoparticles. Heparin is a known anti-inflammatory agent that potentially acts by breaking the inflammatory cycle between macrophages and astrocytes following stroke. We will use design of experiment methodology to determine the composition of these three factors (integrins, clustered VEGF, and heparin nanoparticles) that leads to substantial brain repair (Aim 1) and assess how the hydrogel components modulate the pro-repair environment by analyzing temporal changes in proteins, mRNA, and immune cell populations following stroke (Aim 2). Using the improved formulation, we will also evaluate the angiogenic hydrogel?s ability to promote neurological regeneration and functional recovery in more rigorous animal models, specifically aged mice treated immediately following cerebral ischemia and young mice with cerebral ischemia treated as the plasticity window is closing (Aim 3). Overall, we aim to deepen our understanding of how bioengineered therapeutics synergize with endogenous pro-regenerative programs and thereby improve behavioral outcomes following stoke in more difficult-to-treat cases."}
{"text": "Extracorporeal shock wave lithotripsy (ESWL) has been used routinely for the treatment of kidney stone patients for almost two decades. Despite the widespread clinical application, the underlying physical mechanisms of stone comminution and tissue injury in ESWL have not been fully understood yet. In fact, although some progress has been made recently on the development of better lithotripters and treatment protocols for stone fragmentation, these new machines have also resulted in an increase in both treatment imes and soft tissue injuries; leaving the original Dornier HM3 lithotripter as the gold standard for ESWL. A possible reason for this lack of improvement is that the great majority of ESWL studies have been focused primarily on experimental approaches while, in contrast, there has been a very limited development in realistic and accurate mathematical models that can capture the essential physics of shock wave propagation, focusing, cavitation, and their interactions with renal calculi and surrounding tissue. Our goal is to develop, and experimentally validate, such a comprehensive model where all the important mechanisms in ESWL will be accounted for and coupled together. The model will include effects deriving from pure shock propagation, absorption, streaming, cavitation and bubble dynamics, as well as those associated with the elastic stresses generated in the stone. It will also be fast and accurate in simulating realistic ESWL treatments without the need of supercomputers. The relevance of this research to public health is that, once developed, this model could significantly advance our basic knowledge of how ESWL works, it could provide considerable guidance for improving the treatments' efficacy and safety of existing systems, and it could aid in the design of the next generation lithotripters. [unreadable] [unreadable] [unreadable]"}
{"text": "Better Understanding of the Neuroprotective Mechanisms of Korean Ginseng in Stroke It has been postulated that the determinants of neuronal cell death in acute and chronic neurodegenerative conditions are mediated by free radical damage. Ginseng has been reported to be neuroprotective and a potential preventive medicine, but the underlying cellular mechanisms are still unclear. Our preliminary results prompted us to focus our attention on Korean Ginseng and test the hypothesis that the transcriptional factor Nrf2 could participate in the overall Ginseng's neuroprotective function. HO, which cleaves heme (a prooxidant) to form biliverdin/bilirubin (antioxidants), carbon monoxide (a vasodilator), and iron (a prooxidant) has been shown to play a protective role in oxidative stress, ischemia, inflammation, and hypertension. Although HO2 is constitutively expressed, HO1 is inducible. Consequently, a possible way to increase HO levels to achieve neuroprotection may be to induce HO1. Of the compounds tested in our preliminary experiments in primary neuronal cultures, Ginseng was one of the most potent HO1 inducers. Our results also indicate that pretreatment of neurons with Ginseng is sufficient to provide neuroprotection, suggesting that co-treatment during oxidative stress is not necessary. This neuroprotective effect was abolished by a protein synthesis inhibitor, and was greatly reduced by an HO inhibitor. These preliminary results implied that specific induction of HO1 could be a mechanism by which Ginseng exerts its preventive neuroprotective actions and motivated us to propose that some of the neuroprotective effects attributed to Ginseng could be mediated through HO1 induction and the associated beneficial actions of heme degradation. Recently, we and others have described Nrf2 has as a key regulator of inflammation and redox homeostasis. In Aim 1, we will determine anatomical and behavioral outcomes following ischemia in wild type mice pre-treated (acutely or chronically) with Ginseng, determine whether the beneficial effect is sustained with aging, and test whether these effects are attenuated in Nrf2 knockout mice. In Aim 2, we will determine whether Ginseng induced characteristic changes in brain cells, and start addressing in which cell types these Nrf2 changes occurs. Together, these in vivo results will help us determine whether oral consumption of a standardized Ginseng extract could be beneficial, and which cells can be most associated with the Ginseng preventive brain mechanistic pathways by which Ginseng would provide the cell/brain with resistance to acute debilitating neurodegenerative conditions."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Apoptosis plays an essential role in the development and homeostasis of metazoans. The genetic characterization of programmed cell death (apoptosis) in C. elegans identified four proteins, CED-3, CED-4, CED-9, and EGL-1, that collectively control the onset of apoptosis. CED-3 is a caspase and its activation depends on CED-4. CED-9 antagonize the function of CED-4 through an unknown mechanism. CED-3, CED-4, and CED-9 form a hetero-oligomer. To elucidate the mechanisms of cell death control in C. elegans, we crystallized the ternary complex of CED-3/CED-4/CED-9. We plan to determine the structure by molecular replacement or by MAD."}
{"text": "This application establishes the Models for Integrating Juvenile Justice and Treatment (MIJJT) Research Center in collaboration with The National Criminal Justice Drug Abuse Treatment Research System (CJ-DATS) under RFA-DA-02-011. MIJJT provides the opportunity to engage in collaborative, rigorously conducted, multisite service delivery and evaluation research involving drug-abusing juvenile offenders. Empirically, the studies that have been conducted on adults in justice settings have shown that the most consistent effects occur when there is a continuum of primary treatment and aftercare. This is likely even more critical for juvenile justice clients who are generally unemancipated and present with multiple problems in addition to substance abuse. Adolescent substance abuse and the rising juvenile crime rate have been acknowledged as two of the nation's greatest concerns and, though their etiology is diverse, they are inextricably linked. Despite numerous education and prevention initiatives, adolescent drug abuse and its sequelae of juvenile crime, risk for HIV infection and school failure remain major juvenile justice, mental health, public health and public safety concerns. The MIJJT Research Center will complement the aims of the NIDA CJ-DATS by bringing comprehensive focus to the issue of adolescent substance abuse, juvenile justice and treatment by incorporating a spectrum of agencies and treatment providers in Ohio and Pennsylvania, including minimum to maximum secure facilities and community-based programs. The potential sample assures the inclusion of a broad range of youth, including females as well as males, those with co-occurring mental disorders, HIV risk populations and members of various racial/ethnic groups. The proposed studies aim to address the multidimensional needs of adolescents who enter the criminal justice system, the multivariate, interrelated structures of provider agencies that respond to these youth, and the range of services that are required to effect positive outcomes. In accordance with the CJ-DATS objective to provide a stable infrastructure for research to improve drug abuse treatment for individuals with drug abuse or addictive disorders who are incarcerated or transitioning from jail or prison to the community, the MIJJT investigative team proposes research concepts that follow the trajectory of an adolescent's juvenile justice and treatment experience. Study 1 proposes a CBT intervention in the treatment setting; Study 2 addresses the relationship of the parole and probation officer to the adolescent; and Study 3 focuses on the youth's reintegration into the community, particularly the role of the family in facilitating and participating in a successful integration. The collaborative energy that has facilitated the development of the current proposal reflects the support of the collectivity of the participants--research, administrative, and clinical."}
{"text": "Spinal cord injury (SCI) is among the most disabling conditions affecting wounded members of the U.S. military. Unfortunately, there has been no effective treatment available for SCI patients. It is, therefore, an urgent medical need to develop novel repair strategies to mitigate the devastating nature of SCI and to translate them clinically to improve quality of life of our veterans with SCI. Recently, a novel lipid signaling pathway, namely the cardiolipin (CL)-cytochrome c pathway, that control cell death/apoptosis has been identified. CL is a structurally unique dimeric phospholipid localized in the inner mitochondrial membrane where it is required for optimal mitochondrial function. CL is a preferred oxidation substrate in neuronal injury, is the only phospholipid in the mitochondria that undergoes early oxidation during apoptosis, and is an early target of reactive oxygen species (ROS) attack. Alteration of CL has been associated with mitochondrial dysfunction in a variety of pathological conditions. Using mass spectrometry-based lipidomics for the first time in SCI, we have generated preliminary data showing CL peroxidation and loss after SCI. Remarkably, XJB-5-131 (XJB), a novel mitochondria-targeted antioxidant, administered at 30 min post-SCI significantly reduces tissue damage and improves behavioral recovery in adult rats. These data strongly suggest that CL alteration is a key mechanism that mediates injury-induced cell death and tissue damage. However, the role and mechanism of CL alteration in SCI remain unclear. Here, we hypothesize that CL alteration, including peroxidation and loss, is a central process that mediates spinal cord secondary injury, and that restoration of CL level may lead to neuroprotection and recovery of function after SCI. Using a rat spinal cord neuronal culture system in vitro and an adult rat thoracic contusive SCI model in vivo, we will determine 1) whether CL alteration induces mitochondrial dysfunction and neuronal death and whether such detrimental effects can be reversed by a novel mitochondrial targeted antioxidant XJB; 2) the molecular role of CL alteration in the signaling pathway of neuronal apoptosis after SCI and whether such CL alteration is sufficient to mediate secondary SCI; 3) whether abnormal mitochondrial dynamics also play a role in CL alteration-mediated cell death; and 4) an optimal dose and therapeutic time window of XJB on neuroprotection and functional recovery after rat contusive SCI."}
{"text": "The great diversity of techniques used in Cancer Research demands equally diverse reagents and facilities. While the preparation of growth media is not technically difficult, the scale of our needs requires an efficient centralized facility. The media preparation needs for the Program will be provided by the Media Preparation Facility. This facility will prepare both liquid and solid media for the growth of bacteria and yeast, plus tissue culture media, salts, and buffers. In addition, sterile glassware and supplies will be provide for the Projects in the Program."}
{"text": "Many factors contribute to the current epidemic of obesity. Although estrogen (E2) status is not commonly recognized as a determinant of obesity risk in women, there is strong evidence from large randomized controlled trials that E2-based hormone therapy (HT) attenuates weight gain by about 40% in postmenopausal women. Importantly, there is also strong evidence that E2 attenuates abdominal fat accumulation, a fundamental component of the Metabolic Syndrome. The global aim of the proposed studies is to evaluate potential mechanisms by which E2 deficiency accelerates fat gain and abdominal fat accumulation in women. The first aim is to determine the effects of short-term (7 d) and chronic (5 mo) sex hormone suppression on resting energy expenditure (REE) and fat gain. It is hypothesized that a) REE will be reduced in response to short-term hormone suppression, as observed in a pilot study; b) the decrease will persist with chronic hormone suppression, promoting fat gain; and c) E2 add-back therapy will mitigate these responses. Whereas a dampened REE may cause fat gain in the E2-deficient state, it would not explain the disproportionate accumulation of abdominal fat that occurs. Altered hypothalamic-pituitary-adrenal (HPA) axis activity leading to cortisol excess causes abdominal fat accumulation (e.g., Cushing's syndrome) and there is evidence that E2 attenuates stress-induced HPA axis activity. Therefore, the second aim is to determine the effects of sex hormone suppression on stress-induced HPA axis activity and abdominal fat accumulation. It is hypothesized that stress-induced HPA axis activity will be a) amplified during sex hormone suppression; b) attenuated by E2 add-back; and c) a determinant of abdominal fat gain. These aims will be accomplished by using gonadotropin releasing hormone (GnRH) analog therapy, with placebo or E2 add-back, to determine the effects of E2 on REE, HPA axis activity, and fat gain and distribution in 100 healthy young women. Abdominal obesity purportedly accounts for 20% of coronary events in men but 48% of events in women, underscoring the value of learning the mechanisms of abdominal fat gain in women. Until menopause, women are largely protected against abdominal obesity, implicating a role for sex hormones. Recent findings that benefits of HT may not outweigh the risks in some women does not negate the importance of identifying mechanisms by which E2 attenuates fat gain and abdominal fat accumulation. [unreadable] [unreadable] [unreadable]"}
{"text": "The generation of rapid or saccadic eye movements requires a transformation from the visual input falling on the retina to the pulse of activity to the eye muscles to move the eyeball to a new position. The areas in the brain of primates (an excellent animal model of the human saccadic system) have been identified throughout regions of the cerebral cortex and brainstem so that in outline the entire pathway from visual input to eye movement output is now known. What is not know is what signals are conveyed from one region to the next and what transformations in that signal are made at each stage of processing in the brain, and until this is known, a circuit within the brain for the control of behavior can not be understood. We have addressed this issue by determining which neurons in two cortical areas, the frontal eye fields, and the posterior parietal cortex, are the output neurons to the brainstem center for the control of saccades, the superior colliculus. By electrically stimulating in the colliculus, we can activate the axons of neurons in cortex that reach to the colliculus (antidromic activation) and thereby see which neurons are the output neurons from cortex and what is the nature of the signal (visual, visuomotor, motor) that they carry. About three quarters of the antidromically activated neurons in the parietal cortex respond to the visual stimulation that could be used to guide a saccade. In addition, these neurons were active as the monkey prepared to make a saccade to a visual target indicating that the neurons going from this region of cortex to the brainstem were carrying both a visual and a movement related signal. This signal is similar to that carried by one set of neurons in the colliculus thought to be at the earliest stage of processing in that structure, the buildup neurons. In contrast, when the same experiment was done with neurons in the frontal eye field, more neurons tended to have a clear burst of neuronal activity before the onset of the saccade. These neurons were more like the neurons in the colliculus that are close to the output of that structure, the burst neurons. Thus the major output of the two areas of the cerebral cortex is different and may represent the input to different stages of signal processing in the superior colliculus, a possibility that we are now investigating. In addition to the signal going to the colliculus from cortex, we have also identified one from colliculus to the frontal eye fields (orthodromic activation), and found that all of the neurons in the frontal eye fields that receive this signal have visual activity. Thus the neurons that project out of the frontal eye fields to colliculus tend to carry a motor signal while those that receive information from the colliculus have a visual signal."}
{"text": "The importance of the mismatch repair system in humans is evidenced by the fact that inheritance of a defective allele in one of several mismatch repair genes will, with high probability, lead to cancer, usually colorectal cancer. There is increasing evidence that some of the most important substrates for the mismatch repair system involve damaged DNA, including types of damage likely to be produced by normal cellular processes, especially oxidative processes in the cell. The overall goal of this proposal is to examine the interactions of mismatch repair proteins with DNA lesions and mismatches likely to be found in the cell as a consequence of normal endogenous processes. These experiments will help to test the hypothesis that mismatch repair is important for preventing mutations due to damaged DNA. The specific aims of this proposal are to determine the interaction of yeast and human MutSalpha with substrates containing specific damaged nucleotides, to determine the effect of MutLalpha on the binding of these lesions by MutSalpha, and to examine mismatch repair, in vitro, of substrates containing these various lesions."}
{"text": "A major reason for failure of a biomarker to act as true disease surrogate endpoint is the lack of a direct association between the biomarker and the underlying mechanism of the disease. Fibrillar beta-amyloid (AB) plaques are the main pathological hallmark of Alzheimer's disease (AD), although its role as a causative mechanism in AD is controversial and continues to be disputed. The clear association of soluble AB oligomers to neurotoxicity in animal studies indicates that measuring soluble AB oligomers may hold more promise as a true surrogate biomarker for the clinical symptoms of AD. The lack of specific ligands that can differentiate soluble AB oligomers from fibrillar AB forms both in vitro and in vivo has hindered studies aimed at testing the correlation between soluble AB oligomers and plaques and/or the relative clinical value of measuring different forms of AB species. Our group discovered a fluorophore, 68B-3, that can selectively bind soluble AB oligomers and has no measurable binding to AB fibrils or monomers. The primary aims of this study are to characterize and validate our initial discovery on 68B-3, and evaluate the value of measuring different forms of AB species in clinical samples and in a transgenic model of AD. We will determine the correlation between levels and location of soluble AB oligomers to disease progression. While the affinity of 68B-3 to synthetic soluble AB oligomers is low, it may serve as a useful tool in providing novel information in biological samples. Furthermore, it may be optimized by chemistry to yield derivatives with better properties. Another aim of this study is to understand the mechanism of binding of 68B-3 to soluble AB oligomers. This is an important first step to improving its binding affinity. It is our intent to make 68B-3 available to researchers once we have validated its uses and once we have demonstrated the robustness of our preliminary data. Selective small molecule probes such as 68B-3 will not only serve as potential tracers for non-invasive imaging, but may be used in ex vivo postmortem studies to compare the regional distribution and temporal profile of soluble AB oligomers and plaques. Such information would enable the correlation of neuronal loss with the spatial location of soluble AB oligomers and thus may accelerate the establishment of a true surrogate for the diagnosis of early AD. PUBLIC HEALTH RELEVANCE: The proposed research is directed toward the validation of our initial discovery using 68B-3, a novel small molecule fluorophore that selectively targets soluble beta-amyloid (AB) oligomers and not fibrils or monomers, and evaluation of the value of measuring different forms of AB species. Completion of this study will be important to investigate the regional distribution and temporal kinetics of soluble AB oligomers compared to fibrillar AB in postmortem tissues and animal models of Alzheimer's disease. This would also provide powerful insight into understanding the role of soluble AB oligomers in the pathogenesis of Alzheimer's disease and the clinical value of measuring levels of soluble AB oligomers."}
{"text": "This project involves the measurement of prostaglandins and phospholipids in adipose tissue and liver, with a special regard to the interrelationship of these compounds in lipolysis and antilipolysis. Studies are progress, studying the relationship of the levels of the prostaglandin E series (measured by thin-layer chromatography and radioimmunoassay) and the different classes of phospholipids (measured by thin-layer chromatography) in situations within the cells of increased and decreased cyclic AMP levels. Levels of cyclic AMP are increased by exposing the isolated cells to epinephrine or norepinephrine, and cyclic AMP levels are lowered by the use of the hormone, insulin. It is hoped by these maneuvers to better understand the role and interrelationship of these compounds to this metabolic process. In addition, it is hoped that a better understanding of the physiology of these compounds will also be forthcoming."}
{"text": "Summary of Work: Phenolphthalein, a constituent in many laxatives, showed clear evidence of carcinogenic activity in 2-year feeding studies in rodents recently completed by the National Toxicology Program (NTP). About 3% of the older U.S. population uses phenolphthalein-containing laxatives on a daily basis. We will investigate the relation of phenolphthalein-containing laxative use with risk of ovarian cancer and adenomatous colorectal polyps in humans. We have established collaborative agreements with the principal investigators for two ongoing case-control studies of risk factors for ovarian cancer. At our instigation, these investigators have inserted into their questionnaire a 2-page addition that ascertains use of specific phenolphthalein-containing laxatives. Each study will yield about 300 cases and 300 controls, and the data will be ready for analysis in 1999-2000. Data collection for two case-control studies of risk factors for adenomatous colorectal polyps have been completed. Data on laxative use were available for both studies. We have examined laxative use in relation to risk of polyps in these data. The association of polyps with use of all laxatives and phenolphthalein- containing laxatives was specifically explored after taking into account dietary and other risk factors for colorectal adenomas. Results from the two studies were inconsistent with each other but overall offered little support for an association between phenolphthalein- containing laxative use and risk of polyps. - Phenolphthalein, Laxatives, Ovarian cancer, Adenomatous colorectal polyps, Epidemiology, Case-control study - Human Subjects: Interview, Questionaires, or Surveys Only"}
{"text": "We propose to develop a novel DNA chip specifically for high-throughput analysis of promoter hypermethylation in primary tumors. It is now clear that abnormal DNA methylation frequently occurs in multiple promoter CpG islands in cancer cells. Increased density of CpG methylation is known to alter local chromatin structure within a promoter , resulting in transcriptional silencing of the corresponding gene. At present, bisulfite DNA sequencing is considered to be the\" gold standard\" for marring methylated CpG sites within a gene promoter. This method, as well as other related techniques, has provided important insights into the functional relationship of promoter methylation and transcriptional silencing on a \"gene-by-gene\" basis. Such approaches, however, have given limited pictures of complex epigenetic alterations in cancer and are restricted in throughput for routine clinical applications. In this proposal, we build on the approach of microarray techniques by developing a novel method, called Methylation-Specific Oligonucleotide (MSO) microarray, for high-throughput methylation analysis. Our goal aims at generating MSO chips in which thousands of short oligonucleotides are tethered to glass slide surfaces. These oligonucleotides specifically designed and tested are capable of differentiating methylated and unmethylated CpG sites at the specific locations of a promoter. Test (tumor) and reference samples are bisulfite-treated, PCR-modified products that may contain different pools of DNA fragments due to the hypermethylation status of the tumor genome. These DNA samples are co-hybridized to an MSO chip and quantitative differences in DNA methylation are determined by two-color fluorescence analysis. Distinct from the existing microarray technologies, the MSO approach allows simultaneous analysis of the anatomy of multiple promoter CpG islands in reference to the role of DNA methylation on gene silencing. In addition, this MSO chip can be directly applied to determine molecular signatures of different tumor subtypes. In the pilot R21 phase, we will apply a prototype MSO chip to analyze a small panel of tumor samples and determine its reliability in detecting DNA methylation. In the R33 phase, based on the experience we gain in the pilot study, we will design a full-fledged MSO chip for a comprehensive analysis of promoter methylation in multiple tumor types. An advanced computation system will be developed to handle a large set of methylation data and patients' clinicopathological information and to support heuristic queries. The MSO assay will furnish digital profiles of promoter hypermethylation for individual tumors and offers an alternative to cDNA microarray approaches for molecular classification of different cancer subtypes."}
{"text": "DESCRIPTION (provided by candidate): Through this award, a researcher with a background in physics and acoustics will receive additional training and conduct research in diagnostic ultrasound. Structured training in clinical research practice will be provided through a sequence of courses, especially related to training in biostatistics and human disease. Training in current quantitative research methods will be provided through the research portion of this proposal, which examines the accuracy of nonlinear propagation computer models when applied to tissue mimicking media. Recent progress in numerical modeling of nonlinear acoustic fields has created efficient algorithms to simulate pulsed fields from array transducers. However, few results of acoustic field parameters have been reported for measurements conducted in media with tissue-like properties. A newly developed, stable tissue-mimicking liquid (TM) will be used to test the accuracy of these efficient algorithms that include nonlinear propagation effects. The hypothesis of this proposal is that numerical models that incorporate nonlinear propagation effects will provide accurate representation of the acoustic fields of diagnostic ultrasound devices. The results will be useful to improve simulations of ultrasonic fields that are used to develop and optimize ultrasonic imaging applications and in the determination of acoustic output indices. Measurements of the acoustic field amplitude propagating in TM liquid will be compared to results of numerical models to address three specific aims: 1) Comparison of numerical models and measurements in TM liquid with attenuation coefficient of 0.30 dB/cm/MHz to assess accuracy of current derating scheme. 2) Investigation of proposed methods of \"linearizing\" acoustic output methodology by conducting measurements at low output levels and extrapolating to high output levels to avoid nonlinear effects. 3) Assessment of accuracy of numerical methods for models related to imaging: TM liquids with greater attenuation coefficient, including 0.5 and 0.7 dB/cm/MHz, obstetrical, and other tissue models."}
{"text": "The specific aims of this project are: 1. To determine the feasibility of assaying the marrow and peripheral blood progenitor cell (PBPC) fractions of breast cancer patients enrolled on S9623 for the presence of malignant cells; 2. To determine the feasibility of quantitating the breast cancer cell content of the marrow and PBPC fractions using immunocytochemistry with anti-breast cancer monoclonal antibodies and reverse transcriptase polymerase chain reaction (RT-PCR) for cytokeratin-19; and, 3. To determine the true incidence of tumor cell contamination of hematopoietic fractions of Stage II/III breast cancer patients with 4-9 axillary lymph nodes. The bulk of our preliminary breast cancer detection data was generated with fresh marrow and PBPC specimens obtained from University of Colorado breast cancer patients. Over the next two years, the current study will determine the feasibility of conducting similar detection studies with patient specimens shipped overnight from the many cooperative group institutions. If this feasibility study is successful, we will be able to determine the incidence of breast cancer contamination in the hematopoietic cell fractions of patients enrolled in the first two years of the study. We will then seek funding for two additional years to continue to assay the hematopoietic cell specimens of patients enrolled on S9623 for the remainder of the study, which is projected to accrue patients for a total of four years. Clinical outcomes, including disease-free survival rates as well as sites and patterns of relapse, will be correlated with the breast cancer detection assay results. Alternatively, if we are unable to successfully assay the specimens shipped from outside institutions, such additional funding will not be sought. The major aspects of the tumor detection assays which the current feasibility pilot will address include: assessment of the quality of the assays performed on specimens shipped overnight; assessment of outside institution compliance; assessment of the true incidence of breast cancer in the hematopoietic cell fractions of Stage II/III 4-9 nod-positive breast cancer patients; and, will compare the sensitivity of the immunohistochemical and molecular assays for breast cancer."}
{"text": "BACKGROUND: Although about 7 million people in the US population use marijuana at least weekly, there is a paucity of scientific data on persistent neurocognitive effects of marijuana use. OBJECTIVE: To determine if neurocognitive deficits persist in 28-day abstinent heavy marijuana users and if these deficits are dose-related to the number of marijuana joints smoked per week. METHODS: A battery of neurocognitive tests was given to 28-day abstinent heavy marijuana abusers. RESULTS: As joints smoked per week increased, performance decreased on tests measuring memory, executive functioning, psychomotor speed, and manual dexterity. When dividing the group into light, middle, and heavy user groups, the heavy group performed significantly below the light group on 5 of 35 measures and the size of the effect ranged from 3.00 to 4.20 SD units. Duration of use had little effect on neurocognitive performance. CONCLUSIONS: Very heavy use of marijuana is associated with persistent decrements in neurocognitive performance even after 28 days of abstinence. It is unclear if these decrements will resolve with continued abstinence or become progressively worse with continued heavy marijuana use."}
{"text": "Muscle protein synthesis, muscle carbohydrate metabolism, and the effects of insulin and growth hormone in myotonic dystrophy are under study using insulin clamps, forearm perfusion, and stable isotopes."}
{"text": "Mycobacterium avium-intracellulare complex organisms are the most common cause of mycobacterial lung disease other than tuberculosis and are the leading cause of morbidity and mortality in the HIV-infected AIDS patients. Infections caused by M. avium patients with AIDS often go untreated as existing antibiotics are ineffective in adequately controlling these infections. This necessitates the need for novel molecular targets for chemotherapy. Replication of the bacterium leading to its multiplication is one of the necessary steps to establish an infection. Initiation is the first committed step in the replication, and the replication process is believed to be regulated at the level of initiation. Thus, understanding the basic steps involved in initiation of DNA replication in M. avium will help define important molecular targets against which new generation drugs can be developed. Initiation of replication occurs once per cell cycle at a specific site on the chromosome called oriC. Initiation is believed to be triggered by the interactions of dnaA with its recognition sequences present in oriC called the dnaA-boxes. These interactions are thought to facilitate recruiting of other proteins resulting in the completion of initiation. This research proposal focuses on identification and characterization of the oriC and Dna protein of M. avium. To obtain oriC, chromosomal DNA fragments of M. avium that support autonomous replication when present in nonreplicative plasmids will be identified, cloned and their nucleotide sequence will be determined. Sequential deletions from both the 5' and 3' regions will be carried out to identify the minimal DNA region that is essential for oriC activity. Site directed mutagenesis will be carried out to identify the putative DnaA boxes that are essential for oriC activity. The ability of M. avium oriC to function as autonomously replicating sequences in other bacteria will be determined. The dnaA gene will be over-expressed, the gene product will be purified and its interaction with the wild type and mutant sequences will be investigated. Antisense dnaA oligonucleotides that target to the dnaA mRNA to affect the expression of M. avium dnaA gene will be explored in an effort to evaluate the role of the M. avium gene. Using oriC plasmids and cell free extracts, an in vitro replication system will be established. The ultimate long-term goal of these experiments is to utilize the knowledge thus gained to develop rational drugs that affect the initiation of replication thereby preventing growth and resulting M. avium infections."}
{"text": "The goal of this project is to develop much-needed new tools for study of the Kallikrein-Kinin system. This system appears to play a critical role in regulation of blood pressure, counteracting the pressor effects of the renin-angiotensin system, and as a principal mediator of the inflammatory response. We have developed a new assay system for kallikreins, and will use it to try to find inhibitors of kallikrein, particularly of urinary kallikrein, which appears to be the critical enzyme in blood pressure regulation. The potential inhibitors will be analogs of the natural substrate of the enzyme. Potential inhibitors of bradykinin at its receptors will be sought among analogs of bradykinin modified so as to increase binding affinity and decrease intrinsic activity. Peptides will be synthesized by the solid phase method and purified by standard techniques. Biological activity of the potential kallikrein inhibitors will be determined in our kallikrein assay using fluorescence for quantitation and HPLC for characterization of products. Biological activity of kinin analogs as agonists and antagonists will be determined in rat uterus, guinea pig ileum, and rat blood pressure assays. Binding affinity of the kinin analogs will be determined by measuring their ability to compete with 125I-Tyrosine1-kallidin for receptors on renal membranes. Studies to define completely the solution conformation of bradykinin will be completed, and conformational changes in the bradykinin molecule in combination with antibodies and membrane receptors will be studied with fully enriched 13C-bradykinins."}
{"text": "Considerable evidence demonstrates that pain disproportionately affects racial and ethnic minorities in the United States, especially African Americans (AAs), who experience frequent, severe and disabling chronic pain compared to their non-Hispanic white (NHW) counterparts. These racial and ethnic disparities extend to many chronic pain conditions, but are perhaps best documented in people with osteoarthritis (OA). Previous research revealed that AAs with OA experience more frequent and severe clinical pain and disability and show a quantitative sensory testing profile suggesting impaired pain inhibition and enhanced generalized pain facilitation compared to NHWs. In addition, previous findings demonstrated higher pain catastrophizing (a tendency to negatively evaluate one's ability to cope with pain and to respond to anticipated or actual pain in a heightened negative cognitive and emotional manner) among AAs compared to NHWs. While multiple factors inevitably contribute, cognitive and affective processes and ethnic group differences in central pain processing represent potentially important determinants of greater clinical pain among African Americans. Despite its pervasive negative effects, no neuroimaging study to date has experimentally manipulated pain catastrophizing and measured cerebral activity during experimentally-induced pain to determine the neural mechanisms whereby catastrophizing impacts pain responses. Moreover, the extent to which the influence of pain catastrophizing on these central pathways contributes to ethnic group differences in the experience of pain remains unexplored. Therefore, the overall goal for this mentored career development proposal (K22) is to elucidate the neural mechanism involved in pain catastrophizing and its influence on pain processing in different ethnic groups. Primary training goals for the current proposal are to: 1) develop a comprehensive knowledge base in neuroimaging techniques, methodological designs, data acquisition, data analyses, and interpretation of findings; 2) expand knowledge of advanced pain assessment skills including quantitative sensory testing and clinical models of pain; 3) Obtain expertise in principles of experimental design and statistical methodology utilized for biomedical research, including neuroimaging studies; and 4) enhance translational research skills to function as an independent investigator. Study 1 (Phase I) will determine whether pain catastrophizing contributes to ethnic group differences in pain-related brain function, clinical pain, and pain sensitivity among AAs and NHWs with knee OA. Study 2 (Phase II) will characterize the impact of an anti-catastrophizing manipulation on central pain processes and pain among AAs and NHWs with knee OA. The proposed career development plan extends from the PI's prior work on pain catastrophizing and mechanisms of pain processing, and will also provide the neuroimaging training and expertise to propel a promising young investigator at the intersection of pain and neuroscience research."}
{"text": "PROJECT SUMMARY/ABSTRACT The majority (>60%) of clinically important drug targets in humans are either integral or peripheral membrane- bound proteins, including G protein-coupled receptors, ion channels, cytochrome P450 enzymes, and P- glycoprotein. Partitioning of drugs within the lipid cell membrane or membranes of subcellular organelles are known to affect their interactions with such membrane-bound proteins, influencing their efficacy and fate in the body. While membrane interactions can result in better efficacy, selectivity and longer duration of action, the excessive membrane accumulation of drugs causes undesirable toxicities through off-target interactions. Therefore, merely increasing the lipophilicity of drugs to increase potency for membrane-associated targets is often detrimental in terms of the overall drug profile. Thus, the knowledge of quantitative estimate of membrane distribution, preferred location (depth), orientation, and conformation of drug molecule within the bilayer is crucial in understanding its target binding kinetics, onset and duration of drug action, and disposition. Our central hypothesis, formulated based on extensive literature and our preliminary data, is that application of this knowledge to lead optimization in rational drug discovery will result in improved efficacy, selectivity and safety. Hence, the long-term goal is to understand the ?specifics? of the structure-membrane interaction relationship and apply this knowledge to rational design of new therapeutics aimed for membrane-associated targets. To this end, the overall objective of this application is to develop, validate, and apply an integrated in silico approach for fast and accurate prediction of membrane partitioning characteristics, combining all-atom MD simulations and a fragment-based continuum solvent model. The objective of this project will be accomplished by two specific aims: (1) Develop an ?integrated in silico approach? for fast and accurate prediction of percentage distribution, most preferred location, orientation, and conformation of drugs in phospholipid bilayer. The current continuum solvent model will be recalibrated using MD simulation results of 27 structurally diverse chemicals with experimentally known bilayer locations to obtain optimized solvatochromic fragment constants for bilayer distribution prediction. (2) Determine whether the membrane-partitioning characteristics of 17 clinically relevant ?2-adrenergic receptor (?2-AR) agonists and antagonists, quantified by the integrated approach, correlate to their experimental association rates (kon) to the ?2-AR and thus affect their onset and duration of action. This study is innovative because it uses atomistic details of membrane-drug interactions from combined molecular dynamics (MD) simulations and our1 continuum solvent model to understand the structural determinants of receptor-binding kinetics of the studied short- and long-acting anti-asthmatic drugs with diverse structural and physicochemical properties. This proposed study is significant because the expected results will fundamentally advance our knowledge on how specific membrane-drug interactions can be exploited in drug discovery of new therapeutics binding to any membrane-associated receptors, enzymes, and transporters with optimal efficacy and safety."}
{"text": "This project consists of two parts: the synthesis of peptides of biological interest, especially those with enzymatic activity; and the modification and improvement of solid phase peptide synthesis. We are developing resin supports of improved stability and protecting groups that can be selectively removed. Several side reactions are under study and ways to eliminate them are being devised. Methods to monitor the coupling and deprotection step, especially the picric acid method, are being developed. We are using the synthetic approach to study structure-function relations in various hormones and enzymes. Bibliographic references: Detection and Prevention of Urethane Acylation During Solid Phase Peptide Synthesis by Anhydride Methods. Merrifield, R.B., Mitchell, A.R. and Clarke, J.E., J. Org. Chem. 39, 660-668 (1974); Synthetic Approaches to the Study of Proteins. Merrifield, R.B. and Hodges, R.S., in \"Proceedings of the International Symposium on Macromolecules\", Rio Janeiro, July 26-31, 1974, E.B. Mano, ed., Elsevier, New York, 1975, pp. 417-431."}
{"text": "The threat of terrorist attacks involving radioactive material and the potential for radiation accidents require the development of improved treatment strategies for victims of radiation exposure. Hematopoietic cells are highly sensitive to radiation damage, and their loss after radiation exposure results in lethal infections. Development of treatment that prevents immune damage from radiation and reconstitutes immune function after radiation exposure would be a significant advance. The aim of this project is to study four promising, clinical-grade cytokines that are likely to significantly improve immune reconstitution after lethal dose irradiation in the well-established dog model. The four cytokines are fms-like tyrosine kinase-3 ligand (Flt3 ligand [FL]), keratinocyte growth factor (KGF), interleukin (IL)-7, and transferrin (Tf) given alone or in combination either before or after exposure to lethal doses of total body irradiation (TBI). All four cytokines have anti-apoptotic activity after gamma irradiation and have direct or indirect beneficial effects on lymphocytes and other immune cells. We will study these cytokines in the dog since (1) the dog model of radiation exposure and hematopoiesis has been highly predictive of human clinical outcomes, (2) there is extensive preliminary data with cytokines for radioprotection of the dog after acute radiation, (3) all four of the human cytokines we have proposed are cross-reactive in the dog, and (4) these cytokines have been studied in humans and are in various stages of clinical development. The goal is to achieve survival of dogs after an otherwise lethal dose of irradiation with sustained immune reconstitution without hematopoietic stem cell (HSC) transplantation. In Specific Aim 1, cytokines will be given after TBI and in Specific Aim 2, cytokines will be given before and after TBI. In Aim 1, we will give 500 cGy TBI and treat dogs with G-CSF plus each study cytokine. In this model, a radioprotective cytokine is defined as achieving significantly improved survival compared to G-CSF alone. In the subsequent experiments, the TBI dose will be successively increased by 100 cGy increments and dogs will be treated with a combination of radioprotective cytokines. The primary endpoint is recovery of hematopoiesis and survival beyond day 30. The secondary endpoint is immune reconstitution. Upon study completion, we will have identified the optimal cytokine treatment and the highest dose of TBI that can be reliably survived without HSC support."}
{"text": "PROJECT SUMMARY (ABSTRACT) Helicobacter pylori is a Gram-negative bacterium that colonizes the gastric mucosa of humans. Although most H. pylori-infected persons remain asymptomatic, potentially serious sequelae of infection include gastric adenocarcinoma, duodenal ulceration, gastric ulceration, and gastric lymphoma. Gastric cancer is the second leading cause of cancer-related death worldwide, and H. pylori has been classified as a type I carcinogen by the World Health Organization. One of the major secreted proteins of H. pylori is a toxin known as VacA. VacA causes multiple alterations in gastric epithelial cells, and inhibits activation and proliferation of T lymphocytes. Most cellular effects of VacA are dependent on its ability to form anion-selective membrane channels. There is a high level of genetic variation among vacA alleles from unrelated H. pylori strains, and the encoded VacA proteins exhibit marked differences in their ability to cause alterations in human cells. The molecular basis for the observed differences in activities is not yet completely understood. A large body of literature indicates that H. pylori strains containing certain forms of vacA (termed s1, i1, or m1) are associated with a higher risk of gastric cancer or peptic ulcer disease than are strains containing other forms of vacA (termed s2, i2, or m2). Thus, VacA is considered to be an important H. pylori virulence factor. The long-term goals of this work are to understand the mechanisms by which H. pylori infection can lead to disease, to understand the basis for variation in clinical outcomes among H. pylori-infected persons, and to develop effective means for prevention and treatment of illnesses associated with H. pylori infection. The specific aims are (i) to investigate VacA structural features that are required for intracellular toxin activity and membrane channel formation, (ii) to analyze differences in functional properties of VacA proteins encoded by different H. pylori strains, and (iii) to identify and analyze host cell components that are required for VacA cytotoxicity. Methods will include cryo-electron microscopy, crystallography, molecular genetics, and analysis of gene trap and shRNA libraries. This work is relevant not only for the study of H. pylori-associated diseases, but will also increase our understanding of bacterial pore-forming toxins, chloride-conducting membrane channels, beta- helical passenger domains secreted by an autotransporter pathway, and protein targeting of mitochondria."}
{"text": "Patients identified as having non-A, non-B hepatitis will be followed as long as possible. Liver biopsy will be obtained when the SGPT is elevated for more than 6 months and then as indicated thereafter. Biopsy material will be saved for fluorescent studies when reagents for non-A, non-B hepatitis become available. All patients developing non-A, non-B hepatitis following open heart surgery will be followed with serial studies of SGPT to determine the incidence of chronic liver disease and to compare this incidence with those having type B hepatitis. BIBLIOGRAPHIC REFERENCE: Alter, H.J., Seeff, L.B., Kaplan, P.M., McAuliffe, V.J., Wright, E.C., Gerin, J.L., Purcell, R.H., Holland, P.V. and Zimmerman, H.J.: Type B Hepatitis: The infectivity of blood positive for e antigen and DNA polymerase after accidential needlestick exposure. New Engl. J. Med. 295: 909-913, 1976."}
{"text": "Central to synaptic transmission are the family of ionotropic neurotransmitter receptors, which are responsible for the rapid responses to neurotransmitters in nerve and muscle. In this proposal, a class of receptors that are members of this family, the neuronal nicotinic acetylcholine receptors (AChRS), will be studied. As the site where nicotine binds in the brain, these receptors are responsible for nicotine addiction and may also play a role in neurodegenerative diseases such as Alzheimers disease. Neuronal AChRs are composed of a number of subtypes as classified by their diverse pharmacology and distribution. Further evidence of the diversity has been the cloning of a large number neuronal AChR subunit isoforms. While neuronal AChRs are found throughout the central and peripheral nervous system, neither the subunit composition nor the function of single nAChR subtype is known. The neuronal alpha-bungarotoxin binding receptor (BuTxR), a neuronal AChR subtype, has been selected for study because recent studies suggest that these receptors are a Ca2+ influx pathway, which at presynaptic sites stimulates neurotransmitter release and underlie nicotine addiction. The first objective is to examine whether BuTxRs are homomers composed only of alpha7 subunits or heteromers composed of subunits in addition to alpha. In addition, the number of BuTx sites per BuTxR will be determined. The next objective is to begin to define the function of BuTxRs. Towards this objective, we will test how permeable BuTxRs are to Ca+2 and if Ca2+ entering through BuTxRs contributes to secretion in PC12 cells. We will also address why so few BuTxRs functional in our undifferentiated PC12 cells and test the hypothesis that the number of functional BuTxRs are regulated. The last part of the grant is concerned with questions about BuTxR expression. Heterologous expression of alpha7 homomers in mammalian cells is poor to nonexistent. In contrast, alpha7/5HT3 chimeric subunits readily express as homomers in mammalian cells. Using alpha7/5HT3 chimeric subunits, we will determine alpha7 subunit regions that prevent homomer formation. In PC12 cells,different subunit processing at folding events will be characterized and their effect on BuTxR expression tested. Finally, we will further characterize intracellular BuTxR subunit pools, which appear to be precursors of the cell-surface BuTxRs."}
{"text": "DESCRIPTION: This proposal is for continued support for a program of research that has explored the relationship between visual structure and attentional functioning in mentally retarded and nonretarded individuals. Because basic properties of multi-element stimulus arrays are intrinsically relational and are embedded in ubiquitous environmental contexts, the ability to detect interstimulus relations like similarity/dissimilarity is essential for adaptive attentional functioning. Although children who have low mental ages (both mentally retarded and young nonretarded children) typically exhibit markedly deficient performances on tasks that demand relational responding, the present investigators have demonstrated that appropriate modifications of the visual array, designed to increase the perceptual salience of relevant stimulus relationships, can rapidly facilitate the performances of mentally retarded individuals in such experimental contexts. Informed by the findings obtained concerning the effects of perceptual structure on stimulus detection, the investigators are now proposing to extend their approach to several new paradigms, including (a) an integration of oddity and match-to-sample methodologies, and (b) assessing the visual processing abilities of mentally retarded individuals at the critical preattentional stage. This \"front-end\" perceptually-based approach of \"guiding\" attention is based on manipulations that have been used successfully in much prior research, particularly that of the current investigators. The proposed studies involve an examination of a range of experimental contexts that share common perceptual variables (e.g., symmetry and contiguity), and assess the relative contribution of these variables to attentional and preattentional processing in individuals with and without mental retardation. The findings of the proposed studies are expected to inform both applied and theoretical issues with respect to the attentional functioning of individuals with mental retardation. The proposed experiments include an integration of oddity and match-to-sample methods (i. e., oddity-based comparison arrays) with implications for generalized performances and extension of that paradigm to the investigation of set size and spatial contiguity in the matching/oddity framework. In a match-to-sample paradigm, stimulus variables including stimulus structure, symmetry of stimulus pattern, and adjacency of elements will be investigated and an analysis of the relationship between stimulus inspection time and stimulus variables will be conducted. Experiments on visual search are proposed that address stimulus variables of element contiguity and symmetry, and are followed by an investigation of guided search. The remaining experiments proposed utilize random dot kinematograms to evaluate stimulus organization variables in motion-defined forms, as well as the influences of training on detection and motion direction discrimination."}
{"text": "Project Summary The understanding of basic DNA repair mechanisms is crucial, as altered regulation of these systems are observed in cancerous cells and also allow for antibiotic resistance in prokaryotes. In E. coli, two major mechanisms of transcription-coupled nucleotide excision repair (TCR) are responsible for removing bulky lesions in DNA that transcribing RNA polymerase encounters- the Mfd-mediated ?forward translocation? model and the UvrD-mediated ?backtracking? model. In this project I will use a genome-wide assay called XR-Seq to further understand E. coli TCR across the genome by determining locations of the genome repaired by each pathway. Further, I will perform NET-Seq in -/+ UV conditions to correlate transcription of both the sense and antisense nascent transcripts to TCR. Together, these studies will provide important information concerning the two known TCR pathways in E. coli by helping us understand how they may work together or separately across the genome and how their absence impacts genome integrity. Ultimately, this work will contribute to further defining the mechanisms of important repair processes that occur in prokaryotes and provide a guideline for future genome-wide studies of other important DNA repair processes."}
{"text": "Epithelial ovarian cancer is the leading cause of death in women with gynecologic cancer. Although it represents only one-fourth of all new cases of gynecologic cancer, it causes over one-half of all deaths due to these diseases. Memorial Sloan-Kettering Cancer Center (MSKCC) is an institution with extensive clinical and laboratory research facilities. The Gynecology Service of the Department of Surgery in collaboration with the Breast/Gynecology Service of the Division of Solid Tumor Oncology of the Department of Neurology, the Department of Pathology, the Department of Epidemiology and Biostatistics and the Immunology Program of the Sloan- Kettering Institute with their principal interest being directed towards ovarian cancer. Since approximately 140 new cases (both untreated and previously treated) of ovarian cancer are seen annually, an adequate patient population exists. For these reasons, a program project to develop new therapeutic strategies for the treatment of epithelial ovarian cancer has been developed. The aim of this program project is 1) to conduct phase I and II clinical trials of innovative chemotherapy regimens 2) to collect a bank of ovarian cancer tissues and body fluids, 3) to evaluate the symptomatology of cancer pain and to assess quality of life as related to therapy, 4) to perform pharmacokinetic studies of new monoclonal antibodies against ovarian cancer antigens in animal models and humans with the aim of being able to target ovarian cancers for diagnostic imaging and radioisotope therapy, and 5) to study in a systematic fashion the immunology and immunochemistry of ovarian cancer in order to characterize malignant and normal ovarian and mesothelial tissues and develop a better group of monoclonal antibodies for clinical use. Based on initial collaboration, the working structure of this program project has been developed. Progress to date warrants funding of a program project to expand this research and carry it forward."}
{"text": "The goals of this program are to improve California State University's (CSUN) research capacity and infrastructure and to increase minority participation in basic biomedical research. The majority of the students who attend CSUN are ethnic minorities (39.8% are underrepresented minorities; University enrollment 31,312, fall 2004). There will be a direct effect on enhancing minority participation in science. Improving research capacity of faculty and students: This proposed MBRS SCORE program includes 18 full SCORE proposals and 3 pilot SCORE proposals. The proposed projects are from diverse fields including theoretical and materials physics, cellular, physiological, genetic, and developmental biology, chemistry and biochemistry, disease processes and social and neuropsychology. Improving the research infrastructure: Cutting-edge research requires instrumentation that is expensive and complex. The successful completion of projects included in this proposal requires specialized instruments. We are requesting support to purchase equipment that will bring the DNA sequencing facility up-to-date and to begin its transformation into a Center for Cellular, Genetic, and Molecular Analysis. Meeting the specific outcomes presented in the following project proposal will fulfill the mission of the MBRS SCORE program which is to \"....significantly improve the research capabilities of minority and minority-serving institutions....to increase the number of underrepresented minorities participating in biomedical and behavioral research.\" The funding provided by the NIH for the CSUN SCORE program will improve the quality and quantity of biomedical research conducted in the laboratories of the PIs and in the affected departments. We expect that 12 of the proposed SCORE PIs will arrive at a point in their research careers after this round of SCORE that they will be able to successfully compete for R01-type of research support. The students, many of whom are underrepresented in basic biomedical sciences, in these departments will have the opportunity to become engaged in top-notch research that will be conducted in SCORE-supported laboratories. [unreadable] [unreadable] [unreadable]"}
{"text": "This is an application for partial funding of the Summer Conference on Physiology and Pathophysiology of the Splanchnic Circulation, to be held under the auspices of the Federation of American Societies for Experimental Biology (FASEB), from July 24 to July 29, 1993, at Copper Mountain, Colorado. The participants will be limited to 150 basic and clinical scientists, who will be selected on the basis of their expertise and interests. There will be 9 major scientific sessions, each with 4 to 5 oral presentations by the experts in the field and open discussion. In addition, there will be 4 poster sessions, which will allow junior participants to present their data and discuss them with the experts in the field. The topics to be discussed will be recent advances in the following subjects: 1) non-adrenergic, non-cholinergic regulation of gastrointestinal circulation; 2) imaging technology for study of the vascular system; 3) endothelial cell biology; 4) role of leukocytes in modulation of gastrointestinal inflammation; 5) ischemia/reperfusion and tissue injury; 6) systemic toxins of gut origin; 7) nitric oxide and the splanchnic circulation; 8) vasomodulators of endothelial cell injury; 9) hepatic circulation and heterogenous liver injury. The areas of controversy, uncertainty and agreement in these fields will be defined. The conference will provide a unique opportunity for interaction among basic scientists (physiologists, pharmacologists, etc.) and clinical scientists (gastroenterologists, surgeons, etc.) with an interest in the splanchnic circulation and gastrointestinal and hepatic diseases. The interactive environment should stimulate collaborative research efforts and help to identify new and more productive directions for future research on pathophysiology, pathogenesis, and treatment for gastrointestinal and hepatic diseases."}
{"text": "To determine the safety and optimal dose of DNA/liposome compound when injected directly into colorectal cancers which have spread to the liver and to transfer this gene to the cancer cells in the liver thereby making them susceptible to destruction by the normal immune cells of the patient."}
{"text": "The National Medical Association (NMA) is the nation's oldest and largest organization representing African American physicians and health professionals in the United States. It was founded in 1985 and is the collective voice of more than 25,000 African American physicians and the patients they serve. The NMA extends equal right and privileges to all physicians. This meeting is targeted to clinical practitioners, academic researchers and professionals interested in reaching a large population of minority physicians. The NMA Ophthalmology Section meets at the annual meeting. The meeting is targeted to clinical practitioners and research clinicians and fellowship trained ophthalmologists. Subspecialty scientific sessions are given over the annual meeting with a Symposium targeted on the first day which offers issues that are cross-sectional in their appeal to ophthalmology. The Sunday Symposium allows the top 7 awardees residents/medical students to present their scientific papers. The presenters are chosen from the scientific committee in the early part of the year for the Rabb-Venable Outstanding Research Award. The presentation of honorable mentions and winner of the Rabb-Venable awards is conducted on Sun. & Mon. evening at a dinner symposium. The winner of the Rabb Venable Award will go to ARVO the following year. The awards will increase by 3 awards a year over the next 5 years. The scholarship funds are supported by the pharmaceutical industry. The deadline for applications is Jan. 31st of the convention year. This grant allows the student/resident awardees to present their research, attend the grant writing session given by the NIH at the NMA meeting, interact with the NEI representative and travel expenses to the annual meeting. The submitted abstracts are encouraged to be in any of the subspecialties of ophthalmology emphasizing the latest developments in understanding, diagnosing, preventing and treating the many sight-threatening diseases and conditions which affect minority patients as well as the general population. This is the premier eye meeting for minority physicians in the U.S. Today's NMA fights for better medical care and opportunities for all Americans. The NMA is on the frontlines of evolving issues that affect the profession, including: burgeoning health care costs; the eroding autonomy of physicians; and the caring for uninsured patients who need immediate medical attention. These are many of the issues that affect our patients today. [unreadable] [unreadable]"}
{"text": "The goal of this proposed collaborative R01 randomized controlled trial is to test the effectiveness of a novel intervention, the Treatment Initiation and Participation (TIP) program, on depression treatment adherence and depression outcomes among geriatric primary care patients. TIP is a brief, psychosocial intervention aimed at reducing the multifaceted personal barriers to adhering to depression treatment. Adherence is a challenge across the lifespan, but among older adults with depression, this challenge is compounded by medical and psychiatric co-morbidity, medical regimen complexity, and skeptical attitudes towards mental illness and its care. For older adults, the effect of stigma on seeking care for mental health issues such as depression is particularly strong. The decision to begin treatment for depression entails both countering the ageist notion that depression is a normal outgrowth of aging and confronting the stigma of mental health treatment particularly prevalent in this cohort. The treatment gap created by non-adherence in later life is becoming an even more prominent issue as the nation's demographic profile shifts. The proposed RCT will be conducted with diverse community samples from two geographically complementary primary care centers (Ann Arbor, Michigan and New York City). The study will recruit 260 older adults who have been newly prescribed antidepressant medication by their primary care physician, and randomize participants to receive either the TIP intervention or usual care. To test the study hypotheses, research assessments will be conducted at study entry, and at 6, 12 and 24 weeks after enrollment. If the proposed intervention is useful in improving antidepressant adherence, it has the potential to decrease the deleterious effects of untreated depression in a growing number of older adults. As a brief manualized intervention, TIP-PC is designed to fit easily within primary care practices and to be delivered by non-MD staff. PUBLIC HEALTH RELEVANCE: Depression and its treatment in later-life presents many challenges for the growing number of older adults in our society; beyond personal suffering, untreated depression worsens the outcomes of many medical illnesses and increases the risk for falls, cognitive decline, and death. Older adults experience many barriers that interfere with their choice or ability to follow the treatment their doctor recommends. The purpose of this research is to test a personalized and flexible primary care-based program designed to help patients experience successful depression treatment."}
{"text": "RNA-directed DNA polymerase (reverse transcriptase) is found in animal oncornaviruses and some human cancer cells. The avian myeloblastosis virus (AMV) DNA polymerase is the most readily available and best studied such enzyme and has been shown to have an unusually high degree of infidelity of transcription. Examples of this infidelity include random initiation, premature termination, a double strand reaction, incorporation of noncomplementary bases, a slippage reaction and inability to transcribe certain homopolymers. These phenomenon have not yet been studied with the human enzyme, in part because of the limited amounts of enzyme purified. By doing a large scale purification of RNA-directed DNA polymerases from human malignant tissues, it will be possible to do a number of studies on these enzymes which have already been done by us with the AMV DNA polymerase, compare the human enzyme to the AMV DNA polymerase, and compare enzymes from patients with different cancers. If the human enzyme behaves qualitatively the same as AMV DNA polymerase, studies with AMV DNA polymerase can be extrapolated to the human enzyme. If there are differences, these studies will be the start of detailed investigations of the human enzyme which may lead to more specific therapy of cancer."}
{"text": "Bacterial cells have evolved multiple regulatory mechanisms to ensure that their subcellular structures and organelles, such as pili and flagella, are assembled and positioned correctly. Our goal is to determine how the core components of a conserved regulatory system, and their interactions, have been adapted to serve the particular needs of different organisms. In Caulobacter crescentus, the protein components of this regulatory system (PodJ, DivJ, and PleC) follow defined patterns of subcellular localization that contribute to asymmetric cell morphology: organelles specifically develop at one pole but not the other. The regulatory components are conserved in Sinorhizobium meliloti, a related bacterium that induces nodule formation in plant roots during symbiosis. However, S. meliloti cells are morphologically symmetric compared to C. crescentus. We will examine how these conserved components function in S. meliloti to regulate the assembly of surface organelles and how this regulation affects host-microbe interaction. (1) We will first determine the localization patterns of the regulatory proteins and the phenotypes of null mutants that lack the proteins. (2) We will then detect and analyze changes in the mutants' transcriptional profiles to see if the proteins control expression of similar genes in different species. (3) We will also assess whether the mutations disrupt interaction between the bacterium and its hosts, preventing effective nodulation. In addition to these research objectives, the principal investigator aims to achieve the following developmental objectives under the guidance of a senior scientist: (4) establish an independent research group, (5) enhance mentoring skills, and (6) improve the quality of research. [unreadable] [unreadable] Relevance to Public Health: The proposed research is expected to reveal the driving principles of a regulatory pathway that exists in multiple bacterial species, including pathogens. Components of the pathway may serve as targets for new antimicrobial compounds. [unreadable] [unreadable] [unreadable]"}
{"text": "Ischemic brain injuries are leading causes of morbidity and mortality to the aging population, but current therapy is poor in part because of our limited understanding of pathogenic mechanisms leading to neuronal loss. A critical trigger of the injury process seems to be acute energy loss, leading to membrane depolarization, excessive release of the excitatory neurotransmitter glutamate and neuronal Ca2+ accumulation. A large and persistent Ca2+ rise (Ca2+ deregulation) seems to be indicative of neuronal death. Recent evidence implicates critical contributions of another divalent cation, Zn2+, which is abundant in the brain and is normally very tightly regulated. However after ischemia or prolonged seizures, free Zn2+ accumulates in neurons, and observations that Zn2+ chelation is protective implicates a role in neuronal death. Culture studies have revealed that exogenously applied Zn2+ can enter neurons and accumulate in mitochondria and powerfully disrupt their function. However, little is known about mechanisms of injury caused by the accumulation of endogenous Zn2+ in native brain tissues. The proposed project thus seeks to address the following hypothesis: Accumulation of Zn2+ in hippocampal pyramidal neurons contributes critically to the initiation of ischemic neuronal injury, in part via deleterious interactions with mitochondria. Preliminary studies indicate that endogenous Zn2+ accumulates in pyramidal neurons in hippocampal slices subjected to oxygen glucose deprivation (OGD), prior to detectable Ca2+ accumulation, and that the Zn2+ appears to enter mitochondria and contribute to the induction of Ca2+ deregulation and cell death. Aim I will apply fluorescent imaging techniques (using both single cell and bulk loaded indicators) to acute hippocampal slices to examine Zn2+ accumulation in CA1 neurons during OGD, examine its interactions with mitochondria and determine its contributions to Ca2+ deregulation and cell death during acute OGD, and the subsequent reperfusion period. This key aim will seek to provide the first rigorous examination of the above hypothesis, and examine a range of interventions that may offer protection while helping to elucidate the sequence of events involved in the triggering and expression stages of injury. Aim II will use a range of approaches to determine the sources and routes of the injurious Zn2+ accumulation. These issues of where the Zn2+ comes from are complex, yet crucial to development of optimal interventions. Aim III will use organotypic slice culture models to examine roles of Zn2+ in the triggering of delayed neurodegeneration (up to 3 days after the OGD), in order to examine downstream injury processes and test therapeutic interventions that may offer protection when delivered well after the ischemia. It is hoped that these studies will provide new insights as to the sequence of events involved in the triggering of ischemic neuronal injury which will lead to new and effective neuroprotective strategies."}
{"text": "In vivo imaging of cellular and molecular structures in the intact brain provides a powerful tool for wideranging investigations in normal physiology, or in experimental models of disease processes. We have recently developed methods using a light microscope-based technique, multiphoton microscopy, to image microscopic structures in the brains of living transgenic mice over periods of months. Multiphoton microscopy utilizes a near-infrared laser for excitation of fluorophores deep within scattering tissue, with high spatial and temporal resolution. The spatial resolution of this imaging technique is about 1micrometer, several orders of magnitude better than other in vivo techniques, like PET, or MRI. In this application, we propose to develop new techniques that will provide important in rive readouts for biological imaging. This research will also lay the groundwork for development of contrast reagents suitable for use in human brain imaging with PET or MRI. We will develop, in Aim 1, techniques for high-resolution, in vivo imaging of structural reporters in the brain. We will investigate procedures to image individual neurons and microglia with high spatial resolution in the intact brain. In Aim 2, we propose to develop imaging techniques that exploit functional reporters in these living cells in the brain. Development of these molecular imaging techniques will build upon techniques accomplished in Aim 1. We have been using an experimental, transgenic mouse model of Alzheimer's disease that develops senile similar to those found in patients with Alzheimer's disease (AD). Our imaging techniques have allowed us to image the senile plaques in vivo in these mice with high spatial resolution. We will apply our new imaging techniques to this mouse model and address important questions that will provide insight into the pathophysiology of this disease. Our current techniques, however, rely on invasive procedures to gain access to the brain for imaging. In Aim 3, we will develop new techniques for non-invasive, in rive detection of senile plaques. New techniques using nearinfrared contrast reagents, and IR-sensitive detectors will allow non-invasive detection of plaques in the intact animal, and may also lead to clinically relevant diagnostic procedures for AD patients. In summary, the proposals outlined in this application will lead to generally applicable new techniques for cellular and molecular imaging in the intact brain."}
{"text": "This application seeks further support for the development of a wayfinding system specifically for building interiors of the facilities of public accommodations (e.g. office buildings, transportation facilities, hospitals, etc.) that will increase accessibility of those facilities to visually impaired users and serve as an environmental rehabilitative intervention for vision loss. This computerized system, called Pathfinder, interacts with users using artificial speech, voice recognition, electronic tactile touch tablet, and a high contrast visual CRT display. Embodying principles of universal design and intended to be useful for all users, including fully-sighted, the system will also increase accessibility for wheelchair users and hearing-impaired persons. Phase I results demonstrated feasibility with blind users unfamiliar with one floor of a Manhattan office building, and showed that the system is capable of training users with no experience with visual or tactile maps on its use without human intervention. Phase II research will address further development of the physical design of the product, the user training module, the tactile and voice recognition interfaces, and the visual display for low vision. Additionally, computer programming tools for building map and signage design professionals will be developed and tested. The product, along with an instructional package will be commercialized in Phase III."}
{"text": "Reports on HIV treatment trials in primates rarely publish any statistical analysis. VacMan is user-friendly computer program intended for a virologist's use on Macintosh, IBM Windows, and SUN X-Windows. VacMan's windowing interface was made possible by the NCBI Coretools and Vibrant software packages. VacMan calculates statistics for in vivo and in vitro infectivity data. The VacMan's statistical techniques are based on Bayesian analysis, but have a novel means of statistical inference: Bayesian predictive distributions are used like classical direct probabilities to make decisions about null hypotheses. The resulting decisions agree remarkably well with virologists' opinions about primate trials, but are objective and quantitative. Quantitative assessment of primate trials leads to several unappreciated principles of effective animal use: dilutions other than 10-fold can improve titration accuracy; infecting titration animals at the lowest doses possible can lower challenge doses; and finally, challenging test animals in small trials with more virus than controls safeguards against false successes, \"reuses\" animals, and strengthens experimental conclusions. These principles take on greater significance when one considers that chimpanzees in HIV trials cost about $50,000 each to use, and are an endangered species."}
{"text": "Project summary/Abstract: Alzheimer?s disease (AD) is a devastating disease that affects more than five million people in the U.S. alone. Early neurodegeneration biomarkers can play an important role in clinical trials of AD, especially biomarkers that are non-invasive, inexpensive, and radiation-free. Our laboratory has recently developed and validated a technique, T2-Relaxation-Under-Spin-Tagging (TRUST), to measure the brain?s oxygen extraction fraction (OEF) and metabolism with MRI. The technique does not require any exogenous tracer, can be completed within 5 minutes on a standard 3T, has high test-retest reproducibility, and has been successfully evaluated in a multi-site setting. Mounting evidence suggests that OEF is a potential marker of neurodegeneration in AD. First, we have shown that patients with amnestic Mild Cognitive Impairment (MCI), a mild form of AD, have significantly diminished OEF. Second, cognitively normal elderly individuals with genetic risk to developed AD, e.g. APOE4 carriers, have reduced OEF. Finally, brain oxygen extraction and metabolism have also been found to be associated with cognitive dysfunction and tauopathy measured from the CSF. To validate the oxygen metabolism biomarker using gold-standard measures of neurodegeneration, human studies often require a long follow-up period and a relatively high cost. Therefore, it is logical to first conduct an exploratory/developmental (R21) study to demonstrate the plausibility of such hypothesis in animal models. Toward this goal, we propose a longitudinal study in AD mouse model using a novel oxygenation MRI technique, and validate its association with neurodegeneration as measured by histology. This study has two specific aims. Aim 1 is to determine cross-sectional and longitudinal characteristics of brain oxygenation and metabolism in AD mouse model relative to control mice. We will measure OEF and cerebral metabolic rate of oxygen (CMRO2) in a novel AD model that our co-investigator, Dr. Wong, recently developed. We hypothesize that cross-sectionally OEF will be lower in the AD mice compared to control mice, and the difference will be more pronounced at an advanced age. Longitudinally, OEF in the AD mice will show progressive decrease starting 6 months of age. Aim 2 is to validate the imaging biomarkers with histological measurement of neurodegeneration. The animals undergoing imaging will be sacrificed at three time points (a sub-sample at each time point) and histology will be performed to measure neuron count and tau burden. Imaging markers will be compared to these histological measures to determine the degree to which they can predict current and future neurodegeneration. Impact: Upon the completion of this study, we will have established a concrete relationship between imaging measures of brain oxygen metabolism and hallmarks of AD pathology such as tauopathy and amyloidosis, which will provide a strong foundation for human validation studies in a larger-scale project."}
{"text": "Newly formed blood vessels fail to mature into fully functional vessels in tumors due to the chronically angiogenic microenvironment. The functional impairment of these vessels hampers drug delivery, thereby diminishing the efficacy of antitumor therapies. Excessive vessel permeability associated with tumors also allows tumor cell invasion into the circulation facilitating metastatic spreading. Therefore, the ability to conrol vessel maturity in tumors provides a potential therapeutic opportunity. The long-term goal of this study is to understand the molecular basis for vascular maturation. Our recent studies uncovered an essential role of the small GTPase R-Ras in establishment of mature, functional blood vessels in tumors. Thus, R-Ras promotes normalization of the tumor vasculature. Now, the important new objective of this investigation is to determine the molecular pathway for R-Ras-mediated vessel regulation so that new molecular targets may be identified for controlling the tumor vasculature for therapeutic advantage. R-Ras enhances vascular integrity through regulation of VE-cadherin. Our recent studies also show that R-Ras attenuates VEGF signaling in endothelial cells by inhibiting VEGFR2 internalization upon VEGF stimulation. Furthermore, R-Ras not only promotes endothelial cell-pericyte association but also facilitates intercellular signaling between the two cell types via TGF-beta and Jagged1-mediated Notch signaling. These pathways promote endothelial cell quiescence and mural cell differentiation; therefore, they are important for vessel maturation. Based on these observations, we propose a hypothesis that R-Ras orchestrates these pathways to redirect nascent tumor vessel formation from an angiogenic sprouting/branching process to a maturation process. In this proposal, we will investigate the significance and precise roles of the R-Ras pathways during tumor vascularization. In Aim 1, we will identify and characterize the key signaling pathways mediating the R-Ras effects on endothelial cells and pericytes in a series of in vitro experiments using various culture/coculture systems. In Aim 2, we will validate the findings from Aim 1 in various animal models to determine the role of these mechanisms during the establishment of functional tumor vessels. The proposed studies will provide an important insight into the molecular basis for the vascular normalization phenomenon and its implications in tumor malignancy and therapies."}
{"text": "DESCRIPTION: The overarching goal of this application is to investigate the face recognition deficits of individuals with autism. Initially, this will entail documenting the behavioral as well as functional and structural cortical signatures associated with this impairment and, in doing so, contrasting face processing with that of other stimulus classes, such as objects or words. To understand the nature of the face impairment further, two additional lines of investigation will be pursued. In the first, the investigators will characterize the topography and function of striate and extrastriate visual areas in these individuals using functional magnetic resonance imaging of visual eccentricity and meridian maps. In addition, they will undertake analyses of the structural morphometry of regions such as fusiform and lingual gyrus to explore the neural substrate further. In the second, the investigators will explore the intermediate level vision of these individuals, with specific emphasis on their ability to derive configurations from local elements using sensitive behavioral assays. The investigators will also explore the developmental aspects of these perceptual processes by comparing the performance of young and older high-functioning autistic individuals, along with matched controls."}
{"text": "This project will study the mechanisms by which individual cells maintain control over their own internal calcium milieu while transporting substantial quantities of this element. Two calcium transporting epithelia, embryonic chick chorioallantoic membrane and the small intestine of rat and chicken, will be studied. Parallel studies will be pursued involving measuring calcium uptake and transport by intact tissue preparations either in vivo or in vitro, using conventional radioisotope counting procedures; and studies at the cellular level employing electron probe microanalysis and transmission electron microscopy. We intend to elucidate the mechanism by which actively transported calcium enters a transporting cell, is sequestered within the cell, transferred to another surface, and how the sequested calcium leaves the transporting cell. We hope to discover whether calcium in transit is complexed with phosphate or other counterion; and whether calcium can diffuse between adjacent epithelial cells, in a passive mannner, through junctional complexes."}
{"text": "The overall objective of this project is to explore the potential of nuclear magnetic resonance for providing detailed information about the structure and dynamic properties of enzymes, particularly those of high molecular weight. Enzymes specifically labeled with C13 or F19 nuclei will be obtained by in vivo incorporation of labeled amino acids or by in vitro chemical modification of intact enzymes with labeled reagents. Carbon-13 and F19 nuclei were chosen because their wide chemical shift range and the relative narrowness of their nmr signals should result in high resolution spectra. The labeled enzymes and their noncovalent complexes with substrates, inhibitors, and coenzymes will be studied by pulse nmr techniques. The enzymes to be studied include E. coli alkaline phosphate containing 4-fluorotryptophan or m-fluorotyrosine, horse liver alcohol dehydrogenase amidinated or guanidinated with a C13 enriched reagent, E. coli tryptophan synthetase alpha subunit iwht C13 enriched histidine residues, and E. coli aspartate transcarbamylase labeled with C13 in phenylalanine, tyrosine, cysteine or tryptophan side chains"}
{"text": "We have recently established several functional clones of normal rat anterior pituitary cells. These clones have exhibited good growth characteristics and have retained a normal diploid karyotype after 20-24 passages. One of these clones (2B8) appears to produce only prolactin since no other anterior pituitary hormones have been detected in the conditioned media. We propose to carry out baseline studies on the 2B8 clone using light microscopy, immunocytochemistry, transmission and scanning electron microscopy and time-lapse cinematography. Polyacrylamide gel electrophoresis will be carried out on conditioned media after incorporation of (3H) leucine into newly synthesized hormone. 2B8 cells will be implanted under the kidney capsule of immature, hypophysectomized rats in order to determine if the prolactin secreted is biologically active. The direct effect of selected steroids, hypothalamic hormones, neurotransmitters, and putative pineal hormones on prolactin synthesis and release from 2B8 cells will be determined. Finally, we will attempt to induce the transformation of 2B8 clonal cells by prolonged exposure to hydrocortisone. Hydrocortisone has been shown to markedly stimulate growth hormone secretion and inhibit the production of prolactin from GH3 pituitary tumor cells."}
{"text": "Recent studies suggest that surgical exposure of the sciatic nerve prior to assessment of nerve blood flow (NBF) may account for discordant reports of increased versus decreased NBF in diabetic and galactosemic rats. Regardless of whether NBF is observed to be increased or decreased, it is normalized by inhibitors of aldose reductase (ARI). To clarify these paradoxical findings, NBF was examined in rats (7-8 rats per group) fed normal chow (C), chow containing 50% galactose (G), or 50% galactose + the ARI zopolrestat (Z) at 100 mg/kg/day. After 3 weeks the rats were anesthetized and one sciatic nerve was surgically exposed (SE) without touching the nerve. The incision was immediately closed and 2 h later NBF (ml/g wet wt/min) was assessed in the SE nerve and in the contralateral unexposed nerve (UE) by injection o 10 um 46Sc-microspheres. In UE nerves NBF was 37% higher in G than in C. This increase was prevented by Z. In SE nerves NBF was 49% lower in G than in C and was virtually normalized by Z. These data indicate that 1) galactosemia increases NBF by 37% in UE nerves, 2) SE markedly increases NBF by 4 fold in C rats but only 1.5 fold in G rats, and 3) the reduced NBF in SE nerves in G vs C are largely prevented by ARI. These observations are consistent with numerous reports that vasodilatory responses to various stimuli are blunted by aldose reductase-linked mechanisms in diabetic rats. These observations provide an explantation for most previous discordant reports of increased versus decreased sciatic NBF induced by galactosemia and diabetes."}
{"text": "Defining the complex mechanisms of viral oncogenesis requires a broad understanding in at least three areas of research: molecular and cellular biology, immunology, and virology. It is the goal of the proposed training program to provide graduate students and postdoctoral fellows a challenging and enriched scientific environment in which both formal and informal training within these three broad, but interrelated areas can occur. The breadth of the scientific interests of the training faculty create a blend of research areas, many of which incorporate virus systems to dissect the molecular events that occur within the normal versus the cancer cell. The specific research programs include (a) molecular and cellular biology-regulation of cell growth and gene expression, oncogenes, signal transduction, and mechanisms of neoplastic transformation; (b) immunology-expression, oncogenes, signal transduction, and mechanisms of neoplastic transformation; (b) immunology; and (c) virology-molecular studies of virus replication, assembly, oncogenesis, pathogenesis, latency, and gene regulation using adeno, cytomegalo, hepatitis B, herpes simplex, papilloma, papova, and retroviruses. The trainers associated with the proposed training grant have their primary academic appointments within the Department of Microbiology and Immunology. However, each of the trainers is associated with multiple interdisciplinary graduate programs which cross departmental and college boundaries. Graduate students eligible for appointment on the training grant may be associated with one of several graduate programs including Chemical Biology, Cell and Molecular Biology, Genetics, Microbiology and Immunology, Molecular Medicine, and Neurosciences. The potential of the trainers to recruit students from such a variety of programs helps to insure that a significant pool of highly qualified students will be available for appointment to the training grant. The training program for graduate students and postdoctoral fellows includes a dynamic research environment that has excellent research and core support facilities and faculty research laboratories with productive and well funded research programs. The research and training environment is further enhanced by numerous interactions among the various laboratories as well as journal clubs, student seminars, and visiting scientist seminar programs. Finally, the research areas of emphasis within the training program provide a broad foundation on which a creative student and postdoctoral fellow can base a productive career in research in the area of citruses and cancer."}
{"text": "Dopamine (DA), norepinephrine (NE), and epinephrine (EPI) and their metabolites and dopamine-beta-hydroxylase (DBH) are present in human plasma and cerebrospinal fluid (CSF). Levels of these substances reflect the balance between their release and removal. The increases in levels of NE and EPI with standing, exertion, stress, drugs, administration, etc., are used to examine sympathetic neuronal responsivity. The responses and level of activity of the sympathetic nervous system in cardiovascular disorders (hypertension, hypotension) in alcoholism, a variety of psychiatric disorders, and after treatment with drugs are being examined. Dopamine-beta-hydroxylase (DBH) is the enzyme which converts dopamine (DA) to NE. NE and DBH can be measured in cerebrospinal fluid (CSF) and may be useful to estimate in disease in which a defect is suspected, i.e., essential hypertension, Shy-Drager syndrome, amyotrophic lateral sclerosis (ALS), enuresis and hyperactivity in children, and schizophrenia."}
{"text": "The process of cytoskeleton assembly and disassembly is essential for many critical phagocyte functions, including locomotion, phagocytosis, adhesion, egress from the vasculature, and cytokine production. Understanding the mechanisms by which cytoskeleton assembly is regulated and acts in turn to direct the functions of the phagocytic cell is at the core of understanding the mechanisms of augmentation of host defense at sites of inflammation. The molecular details of this regulation and it's influence on leukocyte activation has been difficult to study. This laboratory has developed a system which has significantly advanced the understanding of the role of the cytoskeleton in the mechanism of leukocyte activation. This lab has found that the pathway initiated by ligation of FcR by Ig6 in phagocytes increases [Ca2+]i by stimulating release of Ca2+ from intracellular stores through a novel IP3-independent pathway that requires the actin cytoskeleton. A specific actin-binding protein called l-plastin is required for this release of Ca2+. Our hypothesis is that l-plastin is an important link between cell surface receptors, such as FcR, and the cytoskeleton in phagocytes. In order to test this hypothesis, we will study the cell biology and biochemistry of l-plastin in phagocytes that naturally express the protein, as well as develop an in vitro transfection system to allow us to study the effects of altering or mutating l-plastin on l-plastin cell biology and biochemistry as well as IgG and integrin-mediated signal transduction. Actin binding, phosphorylation, and immunolocalization of l- plastin and IgG-mediated calcium responses and phagocytosis in phagocytic cells will be studied as models of normal l-plastin function. L-plastin function will be defined further by applying these assays to cell transfection systems using mutated forms of l-plastin. Cells that do not normally express l-plastin as well as mouse cell lines that express endogenous l-plastin will be transfected with the human form of l-plastin. The function of wild type human l-plastin in these cell lines will be validated by comparing l-plastin function and IgG-mediated responses in cells transfected with normal l-plastin and normal phagocytes."}
{"text": "This project involves studying the biochemistry and genetics of Drosophila melanogaster RNA polymerases to determine how the different forms of this enzyme are involved in the regulation of transcription and thus the development of eucaryotic organisms. The method of analysis will involve (1) locating in the D. melanogaster genome the structural genes coding for polymerases I and II and (2) biochemically isolating and characterizing these forms of the enzyme and any factors which may affect their activity. This information will be used as an aid in isolating temperature conditional (both heat and cold) mutants of polymerases I and II for studies concerning (1) the correlation of genetic data with biochemical data on the subunit structure of the polymerases, (2) determining how the forms may be related with respect to the subunit structure, (3) the regulation of the levels of polymerases I and II during development and in response to hormones, (4) the regulation of transcription and metabolism of hnRNA and rRNA, (5) the effect of inhibiting transcription on developmental processes."}
{"text": "[unreadable] Inadequate dietary intake assessment tools hamper studying relationships between diet and disease. Methods suitable for use in large epidemiologic studies (e.g., dietary recall, food diaries, and food frequency questionnaires) are subject to considerable inaccuracy, and more accurate methods (e.g., metabolic ward studies, doubly-labeled water) are prohibitively costly and/or labor-intensive for use in population-based studies. A simple, inexpensive and convenient, yet valid, dietary measurement tool is needed to provide more accurate determination of dietary intake in populations. We therefore propose a three-phase project to develop and test a new assessment tool called FIVR (Food Intake Visualization and Voice Recognizer) that uses a novel combination of innovative technologies: advanced voice recognition, visualization techniques, and adaptive user modeling in an electronic system to automatically record and evaluate food intake. FIVR uses cell phones to capture both voice recordings and photographs of dietary intake in real-time. These dual sources of data are sent to a database server for recognition processing for real-time food recognition and portion size measurement through speech recognition and image analysis. The user model will allow for enhanced identification of food and method of preparation in situations that images alone might not produce accurate results as the system learns through experience and adapts to the individual's food patterns. Objectives are to fuse existing voice and image recognition techniques into a system that will recognize foods by food type and unique characteristics and determine volume by film clip showing an image from at least two angles. The item identified will be matched to an appropriate food item and amount within a food composition database and nutrient intake computed. Researchers will be able to view the resulting analysis through an adapted version of the dietary analysis program, ProNutra. The proposed protocol will incorporate three discrete phases: 1) technology development, integration, and testing; 2) validity testing with a controlled diet (metabolic ward study); and 3) real-world utility testing. Validity of the data collected will be judged by how closely the nutrient calculations match the known composition of the metabolic ward diets consumed. FIVR has the potential to establish a method of highly accurate dietary intake assessment suitable for cost-effective use at the population level, and thereby advance crucial public health objectives. [unreadable] [unreadable] [unreadable] [unreadable]"}
{"text": "Current investigations are being conducted on the events leading to the formation of several types of cataracts. Diabetic or sugar cataract formation initiated by the enzyme aldose reductase is being studied. Methods for controlling the onset of these cataracts through the regulation of this enzyme are being developed. Hereditary cataract formation is also being studied in a strain of mice developed in our laboratory. Known as the Philly mouse, these mice develop osmotic cataracts by an as yet unknown mechanism."}
{"text": "Biliary atresia results from an inflammatory and fibrosing obstruction of extrahepatic bile ducts in young infants. Despite prompt diagnosis and surgical treatment, the disease progresses and leads to end-stage cirrhosis in most infants. The etiology and pathogenic mechanisms of disease are largely undefined. In the first period of this award, we searched for prominent molecular processes in livers of infants with biliary atresia and found a dominant interferon-gamma (IFN?)-rich proinflammatory circuit at the time of diagnosis. We then used a novel mouse model of experimental atresia to directly examine the role of IFN? in regulating the biliary atresia phenotype. This model displayed a similar proinflammatory process in the liver. Most notably, the in vivo loss of IFN?, completely prevented duct obstruction and improved long-term outcome. In this competing renewal application, we propose an overriding hypothesis that the pathogenic mechanisms of biliary atresia begin with an epithelial injury by the innate immune system and progress to duct obstruction by the activation of an exuberant adaptive immune response. This hypothesis will be tested in three closely related but independent aims. In Aim 1, we will determine the role of hepatic CD8+ T cells in neonatal injury of bile ducts. This will be done by investigating how CD8+ T cells engage cholangiocytes through recognition and binding to the MHC-I complex, and by dissecting the intracellular signals trigged by IFN? to render cholangiocytes susceptible to apoptosis induced by tumor necrosis factor-alpha. In Aim 2, we will establish the mechanisms by which hepatic NK cells injure the neonatal duct epithelium. Using a similar experimental approach, we will examine the mechanisms of NK cell-mediated cytotoxicity and how NK cells work in synergy with CD8+ T cells to induce epithelial injury and duct obstruction. And in Aim 3, we will define how hepatic macrophages trigger the innate immune response following a neonatal viral challenge that targets the bile ducts. In this aim, we will determine whether hepatic macrophages are targeted by rotavirus. In related experiments, we will also investigate the molecular mechanisms by which infected macrophages induce chemotaxis to neutrophils and, possibly, cytotoxicity to cholangiocytes. Upon completion, the proposed experiments will advance our understanding of the biological basis for experimental atresia and potentially identify new therapeutic targets to stop progression of disease and improve long-term outcome in children with biliary atresia. Project Narrative: This project studies biliary atresia, the most common cause of chronic liver disease in children and the leading indicator for pediatric liver transplantation in the United States. With the aim to determine key cellular and molecular mechanisms of disease pathogenesis, the proposed experiments will use a unique mouse model of neonatal biliary injury to dissect the biological processes regulating the injury and obstruction of extrahepatic bile ducts. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]"}
{"text": "The kallikrein-like enzyme activity of the toad bladder was measured after 24 hour incubations with either aldosterone 10 to the minus seven, vasopressin 10 to the minus seven, or Ringer's solution. There was no significant change in kallikrein-like acitvity after aldosterone or vasopressin incubation when compared with the control."}
{"text": "The above research project is concerned with certain aspects of the enzymology of nitrate metabolism, namely, a continuing investigation at the molecular levels of (a) in vitro assembly of assimilatory nitrate reductase by incubation of a cell-free extract of the Neurospora nitrate reductase mutant (nit-1), induced by nitrate, and that of other mutants, uninduced wild type and other molybdenum-containing proteins. It includes the purification and characterization of both the nitrate- inducible protein of the nit-1 mutant and the presumed molybdenum- containing component or cofactor (including its ultimate identification), (b) Neurospora assimilatory NADPH-nitrate reductase, elucidating its subunit and cofactor interrelationships both structurally and functionally, (c) the purification and characterization of nitrate reductase-related proteins of several non-allelic nitrate reductase Neurospora mutants, and (d) the purification and characterization of the enzymes involved in the stepwise reduction of nitrate to ammonia (i.e., nitrate assimilation) and in nitrate respiration (where nitrate replaces molecular oxygen as the terminal electron acceptor) including denitrification. In addition to its academic value, the data could be helpful in evaluating the relationship between nutritive value and yield with respect to the use of different nitrogen compounds in agricultural practice. It may also prove to have a bearing on the normal and abnormal metabolic states of mammaliam tissue in view of the known occurrence of the molybdenum cofactor in mammalian xanthine oxidase, liver aldehyde oxidase, and liver sulfite oxidase."}
{"text": "This research aims to study mechanisms of synaptic function with emphasis on the origins and locations of individual synaptic membrane polypeptides. By reacting epsilon-amino groups of lysines which are exposed on membrane surfaces with sodium boro (H3) hydride in the presence of pyridoxal phosphate it is possible to selectively label exposed peptide areas of intact synaptosomal membranes. Combining this technique with purification and subsequent gel electrophoresis of synaptosomal sub-fractions from chick brain allows classification of membrane polypeptides into four groups: those occurring entirely within the membrane, those exposed on both the internal and external faces, and those exposed only at one surface, either external or internal. The nature of the peptides around the exposed lysine residues will be characterized by labeling polypeptides eluted from the gels with I125, cleaving with trypsin or cyanogen bromide, and performing autoradiographic peptide mapping on the resulting peptide mixtures. Whether or not synaptic vesicles fuse with the external membrane will be investigated by comparing labeled gels and peptides of these two fractions. Distances between polypeptides in the membrane will be determined with bifunctional reagents with various bridge lengths between the functional groups. When basic parameters are defined synaptosomes prepared from synapses in differing states of activity will be examined. Alterations of membrane conformation and subsequent protein exposure may reflect neural activity. Synaptosomal membranes will be prepared from electrically stimulated brain slices and chick optic lobes that are either denervated (by enucleation) or receive minimal input (by eyelid suture). Comparing such preparations with normal synaptosomal membranes may reveal a relation between synaptic membrane structure and its functional use. Such data will have important implications for theories both of normal brain function and of the etiology of neurological and psychiatric illness."}
{"text": "Project 1 Determinants of disease progression and role of latent infection in transmission will study a series of factors that will help understanding why only a limited number of individuals infected with Leishmania donovani develop Visceral Leishmaniasis (VL) on the Indian subcontinent. Project 1 will also contribute to assess the role of asymptomatically infected individuals (those who do not progress to disease) in L. donovani transmission. This project will combine field and laboratory work. Two serological surveys of leishmaniasis infection will be done in high transmission areas to identify recent asymptomatic seroconvertors and controls in the study area (see Core C for details). Similariy, VL cases and controls (i.e healthy household contacts) will also be identified and included in the different studies. Project 1 is divided in 4 specific aims. Aim 1 will examine the association of HLA-type with seroconversion and disease progression controlling for several confounders identified in the previous risk factor studies. Aim 2 will use a recently developed quantitative PCR to assess parasite load in different subpopulations (i.e.seroconvertors and seronegative individuals) ahd assess its influence in progression to VL taking into account genetic and environmental factors. Aim 3 will examine the association between co-infections with other Neglected Tropical Diseases (NTD) and VL in two case-control studies. (1) An unmatched case-control study comparing Leishmania seropositives with the general population and (2) a matched case-control study comparing VL cases to community controls will be used to assess the effect of filaria- or geo-helminths-L. donovani co-infection on progression to VL. Finally, Aim 4 will contribute to study the role of asymptomatically infected persons by (1) validating a modified SLA-based Quantiferon to detect cellular immune response in asymptomatic individuals and (2) selecting individuals (peripheral blood mononuclear cells (PBMC) culture positive) to be included in the xenodiagnosis experiments (see Project 2 for details) . RELEVANCE (See Instructions): Results generated in this project will be of importance for individual as well as for public health. Identifying risk factors for progression from VL infection to disease may allow for secondary prevention. Evidence on the potential role of asymptomatic infections in L. donovani transmission may have major implications for the current VL control strategy."}
{"text": "Somatic stem cells residing in many organs are responsible for tissue maintenance and repair and also contribute to aging and cancer. While the role of stem cells and cell turnover is obvious in rapidly renewing tissues, their significance in the gastrointestinal neuromuscular compartment has only recently been recognized. The classic view of gastrointestinal motility disorders including diabetic gastroparesis is that they are due to impaired tissue function and degeneration of terminally differentiated cell types such as enteric neurons, smooth muscle cells and interstitial cells of Cajal (ICC). This is also reflected in current treatment modalities, which focus on symptom control and stimulation of residual function. However, these treatments are not curative and to a large extent ineffective. Recent studies demonstrating considerable plasticity of postnatal enteric neurons, smooth muscle cells and ICC indicate that this old view is untenable. We recently identified a stem cell residing in the gastric musculature of adult mice that can give rise to ICC (ICC-SC). This paradigm shift has opened entirely new opportunities to unravel the mechanisms of ICC maintenance and differentiation and to develop new, rational therapies to regenerate ICC networks from endogenous or transplanted tissue stem cells in these disorders. However, there are several barriers that must be overcome before this goal can be realized including a lack of understanding of the mechanisms that control ICC-SC self- renewal and differentiation, as well as the role of the tissue microenvironment in these processes. Therefore, the overall goals of the current project are to determine the key cell-intrinsic and cell-extrinsic factors that control ICC-SC proliferation and differentiation and to test pharmacological agents that target ICC-SC via these factors in preclinical disease models. Our first specific aim is to determine the role and mechanisms-of-action of polycomb group proteins as cell-intrinsic factors in the epigenetic control of ICC-SC maintenance and differentiation and to test the in vivo utility of indirect histone methyltransferase inhibitors to stimulate, through the inhibition of polycomb activity, the differentiation of transplanted and endogenous ICC-SC. Our second specific aim is to determine the role and molecular mechanisms-of-action of glycogen synthase kinase 3 (Gsk3) as an extrinsic factor in regulating ICC-SC through stem cell factor expression in smooth muscle cells and to determine the in vivo utility of Gsk3 inhibitors to reverse gastroparesis by supporting ICC-SC self- renewal and differentiation. We will use novel models and new technology developed in our laboratory including freshly isolated and cultured ICC-SC, transplantation of genetically labeled ICC-SC, and quantitative analysis of key stages of ICC development by flow cytometry. We will also employ state-of-the-art molecular biological and recombinant DNA techniques to study epigenetic control of gene transcription and mouse models for preclinical testing. The proposed studies will determine the mechanistic basis for, and set the stage for clinical trials of, novel pharmacological interventions for loss of ICC in motility diseases."}
{"text": "This project involves the application of microprocessor technology and improved man-machine interface methods to permit physicians and their associates to more directly communicate with computer record systems. A pilot study involving medical transactions input directly by practicing physicians is underway. The goal is to develop better ways to automate the essential physician contribution to the health care record that is used in both research and patient care."}
{"text": "Epidemiological studies provide compelling evidence for a link between low birth weight, arising either from fetal growth restriction, preterm birth, or both, and adult diseases such as coronary heart disease, Type 2 diabetes, obesity and stroke. In growth restricted pregancies the fetus often experiences periods of hypoxemia. We have begun examining the effects of hypoxia during specific develomental stages of pregnancy, and have preliminary data suggesting that 30 day old postnatal rats whose dams were hypoxic from day 12 to 15 of pregnancy, exhibit elevated plasma insulin, normal plasma glucose, reduced liver glycogen and glycogen synthase concentrations. These characteristics were not altered in postnatal rats exposed to prenatal hypoxia from either day 15 to 18 or day 18 to 21 of gestation. Based on these studies, our overall hypothesis is: Development stage-specific prenatal hypoxia is a key regulator of \"fetal programming\" setting the stage for the onset of insulin resistance, obesity and ultimately Type 2 diabetes. The specific hypothesis to be tested is: Prenatal hypoxia from fetal day 12 through 15 will alter postnatal metabolism resulting in insulin resistance in the young adolescent rat. To test this hypothesis we will determine if hypoxia during discrete windows of development induces insulin resistance at postnatal day 30. Time-mated rats will be exposed to hypobaric-hypoxia from day 12-15, day 15-18 or day 18-21. Offspring from these pregnancies will be compared to offspring from ad-lib fed controls, controls pair-fed during the window of hypoxia, and controls pair-fed during the window of hypoxia through parturition. Pups derived from hypoxic pregnancies, as well as those derived from the pair-fed, and continuously pair-fed controls will be fostered onto additional ad-lib fed dams at birth. At 30 days of age, rats will receive intravenous glucose or insulin tolerance tests, or will undergo euglycemic, hyperinsulinemic clamps to assess insulin resistance. Rats undergoing glucose tolerance or insulin resistance tests, following a 48 hr recovery, will be sacraficed for collection of liver, skeletal muscle and visceral adipose tissue. If peripheral insulin resistance is conferred, alterations in insulin receptor and GLUT 4 mRNA and protein concentrations will be examined. If insulin resistance is liver specific, we will focus on the insulin signalling cascade (e.g. insulin receptor, IRS's, PIS kinase, protein kinase B, glycogen synthase kinase and glycogen synthase), in liver. These studies will provide insight in to the role fetal hypoxia alone may play in the development of Syndrome X. [unreadable] [unreadable]"}
{"text": "PROJECT ABSTRACT With sepsis screening and increased compliance with evidence based, guidelines-driven standard operating procedures for early diagnosis and treatment of sepsis in surgical intensive care unit (ICU) patients, early mortality has been dramatically reduced and late-onset fulminant multiple organ failure has virtually disappeared. A new phenotype of chronic critical illness (CCI), however, has emerged in surgical ICU patients characterized by prolonged ICU stays, recurrent nosocomial infections, poor wound healing, progressive cachexia and manageable organ dysfunctions. We have coined the term persistent inflammation, immunosuppression and catabolism syndrome (PICS) to reflect the pathophysiologic hallmarks of this growing epidemic. Many of these patients (especially the elderly) are discharged to long-term acute care facilities where they often suffer an indolent death. We hypothesize that CCI characterized by morbid long-term outcomes is now a predominant clinical trajectory of surgical ICU patients who survive sepsis. The overall objective of Project #1 is to characterize the epidemiology of CCI in surgical ICU patients who develop sepsis and to determine its long-term consequences. The challenge is to return those surgical ICU patients who survive sepsis to a functional, productive life, and to reduce their burden to the healthcare system and to society through early interventions. We need to identify early, however, which patients are at highest risk for morbid long-term outcomes and might benefit from novel interventions. Project #1's main functions will be the following: ? Define the epidemiology and long-term consequences of sepsis in surgical ICU patients. Clearly, there is a compelling need to better understand the long-term consequences of sepsis in surgical ICU patients, especially those who progress into CCI who are at high risk for PICS. ? Identify clinical indices and biomarkers that can predict CCI in surgical ICU patients early (within 48 hours) after sepsis. These prediction models could help provide insight into underlying pathophysiology and design entry criteria in future trials. ? Identify clinical indices and biomarkers on day 14 in patients with CCI after sepsis that predict morbid outcome (defined as death or full functional dependency at 1 year). These findings could be used to gain insight into underlying pathophysiology by comparing novel biomarkers at earlier time points in patients at highest risk for morbid long-term outcomes versus those patients at lowest risk. A CCI score could then be developed as a composite endpoint in future interventional trials. To accomplish the above goals, Project #1 will perform, over 5 years, a single-site, prospective, longitudinal study of 400 adult surgery and trauma ICU patients who develop sepsis."}
{"text": "The ovum is surrounded by the cumulus, the corona, the zona and vitelline membrane. Capacitated rabbit sperm undergo the acrosome reaction during the passage through the cumulus. Enzymes released following the acrosome reaction or enzymes still bound to the vesicles composed of the plasma (PM) and the outer acrosomal membranes (OAM) hydrolyse the cumulus matrix and allow sperm to reach the corona cells and sperm which are left with a limiting inner acrosomal membrane (IAM) encounter the zona pellucida. The sperm binding to the zona and its subsequent penetration may involve a sequential action of binding to the zona and its subsequent penetration may involve a sequential action of acrosomal hydrolases bound to IAM. The fusion of spermatozoa with vitelline membrane probably involves phospholipases of the persistent PM and OAM on the equatorial segment (ES) and the post acrosomal region (PAR). The subsequent exocytosis of corticle granules may also be triggered by sperm enzymes. This sequence of events provides the rationale for this proposal. The overall objective of this proposal is to elucidate further the role of acrosomal enzymes in fertilization. Specifically, we will isolate and compare enzymes associated with acrosomes; determine the localization of key enzymes involved in sperm-egg binding; isolate acrosomal enzymes from rabbit spermatozoa and determine their effects on rabbit ova. After the extraction of acrosomal enzymes, sperm still contain nearly one-half of neuraminidase, one third of arylsulfatase, and one third of hyaluronidase. these enzymes can be extracted by further extraction of the denuded sperm with 1% Triton X-100 and 0.1 M CaCl2. This treatment also solubilizes the equatorial segment. We will follow this extraction procedure by studying simultaneous changes in sperm morphology at the ultrastructural level. In view of increasing importance of glycosyltransferase and sialic acid in gamete interactions, we will attempt localization of these and other acrosomal enzymes by immunofluorescence, radiolabeling, and cytochemical techniques. The enzyme purification will employ affinity chromatography on hydrophobic columns, high pressure liquid chromatography coupled with conventional methods. The role of acrosomal enzymes in sperm-egg binding, and the ovum penetration will be studied. Enzymes will also be tested on isolated zona."}
{"text": "A small amount of mercury is released from dental amalgam restorations and may be absorbed in the human body. Although restorations are believed not to be a health hazard to a large majority of patients, some concern remains because of the toxicity of mercury. The goal of this research is to gain a scientific understanding of the processes involved and to use the knowledge to minimize the release. The study will be performed in the laboratory using a simulated oral environment. The objective is to measure the rate of the processes, which are believed to play major roles in the mercury release: dissolution of mercury from a surface, which has been stripped of the protective film, repassivation of the amalgam surface after film stripping, and changes in the chemical form of mercury after dissolution in the liquids. The rates of these processes will be determined for representative dental amalgams and as a function of major variables, such as pH, temperature, and chemistry of the environment. The experimental techniques will include solution analysis by Atomic Absorption Spectrophotometry, and electrochemical measurements. The results of the study will identify the factors that have major roles in the mercury release, and provide quantitative information on the rates of the reactions involved. They will serve as a basis for a selection of measures to lower the release."}
{"text": "Cardiovascular calcification is a common consequence of aging, diabetes, hypercholesterolemia, hypertension, abnormal valve mechanics &inflammation, &chronic renal insufficiency. Once thought benign, the deleterious clinical consequences of calcific vasculopathy are now becoming clear;it is a key component of pathophysiology leading to CVA, Ml, and PVD -- with amputation and cardiovascular mortality portended by the anatomy and extent of calcific vasculopathy. The ability to cure or substantially reverse macrovascular calcification (MVC) represents an unmet clinical need. A better understanding of MVC initiation and progression is required. Osteogenic &inflammatory gene regulatory programs are activated in MVC-- variably involving adventitial, medial, intimal, and valvular tissues -- via mechanisms overlapping those that control bone physiology. We've shown that a pro-osteogenic program -- a \"feed-forward\" BMP2-Msx2-Wnt signaling cascade -- is activated in aortic myofibroblasts by diabetes and dyslipidemia. By contrast, PTH/PTHrP receptor agonists (e.g., teriparatide) suppress vascular myofibroblast calcification and aortic Msx2-Wnt gene expression. We now study the role and regulation of myofibroblast Msx2-Wnt signaling in MVC. In Aim 1, we use LDLR-/-.TOPGAL+ reporter mice (LacZ transgene under control of Wnt-responsive TCF/LEF element) to relate spatial activation of aortic Wnt signaling to diet-induced Msx2-Wnt expression and recruitment of osteoprogenitors from adventitial, medial, &valvular myofibroblasts. We also test if the enhanced aortic calcification and Wnt expression we observe in CMV-Msx2 transgenic mice activates aortic LacZ expression. Co-culture studies of primary aortic myofibroblasts will test if Msx2--activated paracrine Writ signaling is inhibited by PTH (1-34). In Aim 2, using TOPGAL mice, we test if PTH(1-34) inhibits canonical Wnt signaling in vivo. In Aim 3, we map Msx2 promoter protein-DNA interactions that convey transcriptional suppression to PTH(1-34) in myofibroblasts, emphasizing elements that mediate BMP2 and Wnt activation."}
{"text": "Obese patients are at increased risk for kidney disease due to circulation of pro-inflammatory mediators and underlying renal metabolic and hemodynamic disturbances, including glomerular hyperfiltration. These patients also have unfavorable lithogenic urinary metabolic profiles, such as low urinary pH, hypocitrituria, hyperuricosuria, that predispose them to kidney stone disease. Gastric operations, in particular Roux-en-Y-gastric bypass (RYGB), are increasing as an interventional strategy to facilitate weight loss in this population. Despite the theoretical renal advantages gained during weight loss for glycemic and hypertension control, up to 75% of RYGB patients will develop sustained hyperoxaluria post-operatively, and a subset will develop kidney stones, nephrocalcinosis, impaired renal function, or even renal failure due to oxalate nephropathy. Currently, the mechanisms behind these adverse renal events are unknown. Preliminary data from an obese rat model of RYGB surgery indicates that gastric bypass causes more glomerular injury, interstitial macrophage migration, increased osteopontin production than sham surgery or pair-fed obese controls. Therefore, we hypothesize that obesity is a low-grade inflammatory process that amplifies renal host responses to RYGB-associated hyperoxaluria. In this setting, glomerular, interstitial, and papillary cells are chronically exposed to high amounts of oxalate and/or calcium oxalate crystals, leading to the production of reactive oxygen species, oxidative stress, renal injury, and inflammation. The objective of this application is to provide a training environment for the principal investigator to examine the role of RYGB-associated hyperoxaluria in the development of renal injury and nephrolithiasis, with special focus on renal histology, transport physiology, tissue proteomics, and genomics. Our specific aims are: 1) To further characterize renal effects of RYGB surgery in a diet-induced obese rodent model versus controls by comparing metabolic profiles, pro-inflammatory mediators, histology, protein expression, and pathway-focused gene expression profiles;2) To further our understanding of RYGB induced hyperoxaluria by investigating segmental differences in intestinal oxalate handling and the effect of Oxalobacter colonization on urinary oxalate levels;3) To compare protein and gene expression profiles from renal papillary tips of human RYGB stone formers with and without Randall's plaque deposition. Understanding the mechanisms of hyperoxaluria following RYGB will provide insights into the pathogenesis of oxalate nephrolithiasis associated with obesity. My K08 program is structured to allow me to further explore relevant rodent models of obesity, to validate preliminary hypothesis generated by a recent minority supplement award, to enhance my understanding of oxalate transport within the gut and kidney, and then, most importantly, translate these efforts into mechanisms of disease within human renal tissue. PUBLIC HEALTH RELEVANCE: Morbidly obese patients who undergo Roux en Y gastric bypass (RYGB) may develop sustained post-operative hyperoxaluria that leads to kidney stones, impaired renal function with calcium oxalate deposition within kidney collecting ducts associated with inflammation and fibrosis, or even renal failure due to oxalate nephropathy. The objective of this application is the establishment of a training environment for the candidate to develop an animal model that explores gut and renal mechanisms of RYGB-associated hyperoxaluria in the development of renal injury and nephrolithiasis, with special focus on renal histology, transport physiology, tissue proteomics, and genomics. As a boarded urologic surgeon with a strong track record of publications in urologic stone disease, the candidate is professionally trained to develop a surgical model of stone disease and renal metabolism, and his K08 program is structured to allow him to further explore relevant rat models of obesity, to validate preliminary hypothesis generated by a recent minority supplement award, to enhance his understanding of oxalate transport within the gut and kidney, and to translate these efforts into mechanisms of disease within human renal tissue."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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)"}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "This project attempts to delineate the nature, frequency, and natural history of clinical manifestations among women heterozygotes for adrenoleukodystrophy."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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]"}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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]"}
{"text": "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]"}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "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]"}
{"text": "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."}
{"text": "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."}
{"text": "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."}
{"text": "DESCRIPTION: Induced pluripotent stem cell technology, which allows reprogramming of cell fate through ectopic expression of selected transcription factors, has opened the possibility to reprogram fibroblasts or other somatic cells directly into specific cell types. This direct reprogramming offers an alternative approach for generating lineages of interest without passing through a progenitor state. Similar to the pluripotent reprogramming by the Yamanaka factors, the process to convert fibroblasts directly to neuronal cells, hepatocyte-like cells, insuin producing-cells or cardiomyocyte-like cells requires the delivery of multiple transcription factors into the cells. Several vectors, such as retroviral-, lentiviral-, AAV-vectors or plasmids, have been used for this purpose with different levels of success, but with the potential of integration f genetic material in the host genome. Thus, there is a need for the development of a new viral platform to generate transgene-free direct reprogrammed cells. Here, we propose to develop a new RNA vector system based on a non-integrating human Paramyxovirus, measles virus (MV), to deliver the transcription factors needed for the direct reprogramming of fibroblast into insulin producing cells. We have developed a reverse genetic system allowing the production of a recombinant virus equivalent to the Moraten vaccine strain, which is currently used for vaccination in the US. From this viral genome, we have produced a one cycle vector and used it to express the Yamanaka factors for iPSCs reprogramming. We propose to modify these vectors and use them to express the factors required for direct reprogramming. We will test the hypothesis that our replication-deficient measles vector can be modified to express the transcription factors shown to be necessary for the reprogramming of adult somatic cells into functional insulin-producing cells. The cells will be then tested in a diabetic mouse model that will validate the functionality of the reprogrammed insulin producing cells. Achieving the proposed studies outlined here will lead to the development of a new polycistronic vector platform allowing direct trans-differentiation without genomic modification. These studies will open the perspective of using these vectors for future direct reprogramming in vivo."}
{"text": "Dietary restriction extends lifespan, improves mid-life vigor, and retards age-related disease in many species. Mechanisms underlying these benefits are not well understood. Defining the cellular pathways by which dietary restriction is initiated and maintained holds potential for combating obesity, diseases of old age such as cancer, and the general functional decline that accompanies aging. We have begun to investigate how microRNAs (miRNAs), small molecules that target partially homologous transcripts to block their translational expression, impact the \"quality of aging\" (healthspan) in the powerful experimental model C. elegans. In this animal, conserved genes can be readily manipulated to address specific hypotheses regarding their activities. Moreover, nearly all the C. elegans microRNA genes are likely to have been identified, deletions of most of these genes have been recently generated, and our preliminary work suggests several miRNAs impact lifespan and and/or healthspan phenotypes. In mammalian systems, miRNA expression profiles change with different metabolic conditions, although no studies to date have addressed changes in dietary restriction. The working hypothesis we propose to test here is that specific microRNAs exert significant effects on the initiation and/or maintenance of dietary restriction metabolism. If we identify these miRNAs and figure out how they work, we could suggest manipulation of their mammalian counterparts to improve healthy mid- and late-life. We will therefore exploit the considerable experimental advantages of C. elegans model to identify miRNAs that are differentially expressed in dietary restricted animals, and we will genetically test candidates for causative action in DR induction and / or maintenance. Because dietary restriction appears to transpire by a fundamental mechanism conserved from nematodes to humans and because virtually nothing is known yet of microRNA modulation of dietary restriction, our planned work should produce data relevant to human biology that pioneers a new area in dietary restriction research with implications for therapeutic development."}
{"text": "The broad objective of this interdisciplinary project is to elucidate the pathogenesis of human neuromuscular diseases. The Center comprises 17 independent investigators in 13 laboratories. In many projects basic scientists and clinical investigators work together. Basic research includes the dynamics of muscle activation and contraction, calcium regulation in muscle, physiology of human muscle fibers, ultrastructure of muscle, development of muscle, muscle culture immunocytochemistry, glycoconjugates, structure and function of acetylcholine receptor. Clinical investigation includes study of muscular dystrophy, myasthenia gravis, malignant hyperthermia, disorders of glycogen and lipid metabolism, hexosaminidase deficiency, and peripheral neuropathy."}
{"text": "The proposed CRECD Program at Charles R. Drew University (CDU), Mentored Postdoctoral Training in Translational Research, is a Phase II mentored clinical research and career development program designed to develop a diverse cadre of clinical and translational investigators who conduct innovative research on the underlying causes of diseases, in particular those diseases that disproportionately impact minority populations in the United States (e.g., HIV/AIDS, cancer, cardiovascular disease, diabetes, and mental health/psychiatric disorders), and to foster and facilitate professional development activities in clinical and translational sciences. Over the initial 5-year program period, the CRECD Program will accept a total of eight postdoctoral trainees at the junior faculty level at CDU, each for 3-year appointments, staggered in appointment periods. The CRECD Program will be embedded within the existing, extensive clinical research and training infrastructure at CDU, and thus will mutually leverage resources with other CDU research education and career development programs so as to achieve maximal cross-program synergies as well as efficiencies wrought from utilizing already-in-place education and training curricula and related resources germane to the CRECD agenda. However, the CRECD Program will also carve out a unique niche of training and career development opportunities within the broader CDU science-generating critical mass by (1) focusing exclusively on health disparities and mastery of community-partnered participatory methods in the research and research training agenda and (2) providing to each trainee a first-of-its-kind (at CDU and, by our estimate, nationwide) intensive and community-immersive mentoring configuration that includes CDU faculty from both the standard or conventional Academic Career track and the newly emerging Community Faculty track, called the Partnership for Equity and Equality in Health and Wellness. This intensive mentoring innovation directly emerges from, and formalizes and systematizes into a signature trainee mentoring configuration, a long and fruitful history of community-engaged research, training, and educational curricula at CDU. As an integrated research training and career development package, this approach is designed to bring social determinants of health to the forefront of molecular and clinical research. Ultimately, the goal is to increase the impact of community- academic partnered research and adoption of its evidence-based best practices through comprehensive dissemination of research findings so as to encourage and facilitate implementation of evidence-based treatment and prevention practices within health care organizations and the communities most in need of reliable access to high-quality care."}
{"text": "Mast cells and basophils, the central effector cells of immediate type hypersensitivity, will be purified from animals and humans. The mast cell and basophil content of preformed chemotactic mediators and enzymes and their release by immunologic method will be studied. The effect upon mediator release of permeant anions will be assessed and the regulatory neurohormone receptors identified and characterized. Chemotactic mediators released in vitro from isolated cells or in vivo in human disease with known mast cell involvement will be purified by physiochemical technique and characterized functionally with regard to target cell specificity for chemotaxis, chemotactic deactivation, and cell membrane receptor alteration. The effect of chemotactic factors and cells will be extended to in vivo models in animals. Taken together, these studies will expand the understanding of the biologic importance of chemotactic mediators in regulation of inflammatory events in human disease."}
{"text": "A diethylenetriamine functional stationary phase, capable of complexing transition metal ions, was developed and evaluated for use in HPLC separations. Synthesis involved evaporation of the trimethoxysilylpropyl- monomer from acetate buffered aqueous solution and derivatization at 200 degree C 0.55 torr. Reproducible surface coverages of 3.0 microgram moles/m-squared were obtained on 100-angstrom, 550 m-squared/g silica. Analytical columns were loaded with Cu(II), Ni(II) by frontal elution process; breakthrough volumes corresponded well with metal uptake determined by atomic absorption spectrophotometry. Buffered aqueous- organic mobil phases were used for separations of sulfonamide antimicrobials that included sulfa-diazine, sulfathiazole, sulfa-merazine and sulfa-pyridine. An empirical retention expression was developed that related chromatographic retention to: a)electrostatic interaction of the acidic sulfonamide function with the cationic stationary phase, b) secondary metal-ligand complexations involving the heterocyclic aromatic substituents of the solutes and c) solubilization of the relatively hydrophobic solutes in the mobile phase. Retentions were inversely proportional to concentrations of organic modifier and buffer and were generally directly proportional to eluent pH. An eluent of pH 7.3 phosphate buffer and 50% methanol was effective in minimizing electrostatic interactions while allowing retention based on metal-ligand interactions. This work was the topic of a Ph.D dissertation, entitled \"Electrostatic and Metal Interaction Chromatography of Sulfonamides\" that was defended and accepted pending editorial revisions on April 30, 1991."}
{"text": "Idiopathic pulmonary fibrosis a fibrotic interstitial lung disease characterized by a median survival of 3-5 years post-diagnosis but exhibits heterogeneous longitudinal disease progression. Recent studies of novel agents confirm beneficial effects on longitudinal change in forced vital capacity but inconsistent benefits on clinical endpoints or health status. Both agents are difficult to tolerate and are likely to be prohibitively expensive. Conducting clinical trials to assess clinical endpoints requires that larg numbers of patients are enrolled and followed for a sufficient period of time. The inability to rapidly recruit sufficient numbers of IPF patients means that many key clinical questions have not been addressed. Restrictive inclusion criteria ensure that patients enrolled in clinical trials often differ from those seen in clinical practice. There remains a critical need to use innovative, pragmatic study designs to identify well tolerated and inexpensive therapies which improve clinical outcomes in patients with IPF. Our group was the first to identify that an abnormal lung microbial community is independently associated with disease progression in IPF subjects. Additional preliminary data link this to a circulating gene expression signature of altered host response. Intriguingly, one investigative group has suggested improved clinical outcomes in IPF patients treated with trimethoprim/ sulfamethoxazole compared to a matched placebo. The totality of these data suggests that an abnormal lung microbiome interacting with genetic susceptibility in host response may be associated with impaired clinical outcomes in IPF. Our principal hypothesis is that antimicrobial therapy in IPF patients will improve clinical outcomes. Our long-term goal is to define patient-specific therapy in IPF. Using a pragmatic trial design CleanUP-IPF will remove many of the known obstacles to clinical trial enrollment in order to recruit a patient population that is highly representative of those seen in clinical practice. We anticipate demonstrating that: 1) a large, pragmatic study in IPF is feasible and will identify clinically meaningful endpoints beyond FVC; 2) anti- microbial therapy will improve clinical outcomes; and 3) genetically predisposed patients will experience differential response to therapy. CleanUP-IPF will revolutionize future studies in IPF and provide data that will alter therapeutic guidelines."}
{"text": ": The revised application focuses on complement activation induced by heparin-protamine complexes. A specific aim of the proposal is to study the relationship of complement activation induced by protamine neutralization of heparin with pulmonary dysfunction. The study will be conducted in patients undergoing first-time cardiopulmonary bypass for coronary artery surgery. A second specific aim is to correlate preoperative complement activation in the presence of heparin and protamine with the actual measurements of in vivo complement activation induced by protamine neutralization of heparin. A third specific aim is to determine whether excessive in vivo complement activation by heparin-protamine is dependent upon the phenotypic or functional differences in C4. Two hundred patients will be entered into the study. Pulmonary shunt fraction will be determined as will pulmonary vascular resistance. Red cell bound C3b/C3d will be determined. IgE and IgG antiprotamine antibodies will be detected using ELISA. Blood samples will be studied at four time points: 1. prior to sternotomy, 2. prior to bypass, 3. before protamine, 4. 10 minutes after protamine. Leukotriene B4 and thromboxane B2 will be determined on samples drawn from pulmonary artery and radial artery lines to represent pre-and post-pulmonary circulation samples. Other studies will be directed at determining the relationship between C3d binding to red cells and subsequent heparin-protamine induced complement activation. Preoperative blood samples will be assessed for C3d binding to red cells. These samples will be used in a series of in vitro assays to determine whether or not the differences in sera with respect to C3d binding are due to differences in the C4b binding to red cells. Statistical analysis will include a number of independent variables such as gender, body surface area, age, number of diseased vessels, number of grafts, heparin dose, time on pump, diabetes, Leukotriene B4 levels, thromboxane B2 levels, changes in C3a during bypass, changes in C3a and C4a during protamine infusion, etc."}
{"text": "This study is a phase II, placebo-controlled clinical trial to evaluate the safety and efficacy of orally administered SP-303, an inhibitor of ileal chloride transport, in patients with HIV-associated disease. Male and female patients with AIDS between 18-60 will be studied. Subjects will qualify for the study of they have a confirmed stool volume >200 gm and >3 liquid bowel movements during the 24-hour screening visit. Qualified subjects will next be randomized and orally adminstered either SP-303 or placebo for the next 96 hours (4 days), ruding which they will remain hospitalized in the GCRC."}
{"text": "The overarching aims of this Project are to synthesize the microtubule stabilizing natural product dictyostatin-1 and its analogs, and to evaluate the potential of these compounds as anticancer agents. In addition to the tight collaborative medicinal chemistry aspects of the work, the use of the new technique of fluorous mixture synthesis is also featured. The specific aims are: 1) Assignment of the structure of dictyostatin by total synthesis. Aim 1 is well advanced. We have recently completed the total synthesis of dictyostatin 1, and we now know its full structure, including absolute and relative configurations. It turns out that dictyostatin and discodermolide have the same configurations at all ten shared stereocenters. This structure determination has removed a major roadblock to development of dictyostatin as a potential anti-cancer agent. However, we still would like to learn how similar (spectroscopically, biologically) the isomers are with the same relative configurations at the three main fragments but coupled together in different pairings. This question will be answered by making multiple isomers by fluorous mixture synthesis. We have also nearly completed synthesis of the original Pettit structures, so this work will be finalized. 2) Synthesis of 0.35-1.0 g of dictyostatin for in vivo characterization. To accomplish this goal, the current synthesis must be improved, and a plan to streamline it by increasing convergency is outlined. 3) Synthesis of stereoisomers and analogs of dictyostatin for SAR studies. We will use the recently established synthetic route to make analogs by fluorous mixture synthesis. We plan to make stereoisomers, and to make simplified analogs with the goal of beginning to elucidate the structure/activity relationship. 4) Conformational modeling of dictyostatin and key stereoisomers. Multiconformational searching coupled with MM3 calculations in Macromodel will be used to predict conformational minima of key isomers. Initially, this data will be used to help interpret NMR experiments, and will serve as a basis for analog design as well as for more sophisticated modeling and docking experiments."}
{"text": "Hearing impairment/deafness is the most common sensory limitation in the U.S. An estimated 11 million individuals in the US are deaf or hard-of-hearing. It has been estimated that approximately 1 million Americans use American Sign Language (ASL) as their primary means of communication making it a distinct linguistic minority. This proposal seeks to improve clinical practice by creating a computerized, self-administered depression screener in ASL that is culturally and linguistically accessible to deaf individuals, an at-risk and traditionally underserved population. No current depression screeners have been shown to be valid for the majority of prelingually deaf persons who use ASL as their main communication mode. Studies have shown that depression occurs in higher rates among deaf persons than hearing persons. While the U.S. Preventive Services Task Force recommended systematic screening for depression in primary care clinical settings in 2002, deaf persons have not yet routinely had access to this preventive service. Providing primary care physicians with a depression screener that is culturally and linguistically accurate and can be self-administered via computer and can be used on iPads and iPhones with no further development, can greatly increase the chance that deaf persons with depression will receive proper diagnostic assessment and treatment which can substantially improve their quality of life. The Deaf Depression Screener will also improve scientific knowledge by providing a valid method for estimating the prevalence of depression among deaf persons in primary care which cannot now be accomplished due to the lack of a valid screening instrument."}
{"text": "This is a request for renewal of a Program-Project Grant to support research on the neural and humoral mechanisms regulating endocrine function. It also requests funds for continuation of the research previously supported by a grant to Drs. J. de Groot and M. Dallman (NS09538) for the study of brain-pituitary-adrenal interrelations. The research, which is carried out by the Program Director and faculty associates, faculty collaborators in other departments, postdoctoral fellows, graduate students, medical students and visiting scientists, is aimed at elucidation of the fundamental mechanisms that regulate the secretion of the endrocrine glands. Attention will be focused on extending current research on the regulation of renin secretion, with particular emphasis on the role of sympathetic nervous system in this process. The mechanism by which norepinephrine stimulates renin secretion will be studied in vitro. The factors affecting angiotensinogen secretion will also be studied. The relation of growth hormone to the maintenance of the secretory capacity of the zona glomerulosa of the adrenal cortex will be investigated. Research on the regulation of ACTH secretion will include studies to determine the site at which glucocorticoid hormones act to inhibit ACTH secretion. The inhibitory influence of norepinephrine-secreting neurons in the hypothalamus on ACTH secretion will be investigated further, along with the influence of these neurons and dopamine-secreting neurons on the secretion of growth hormone and prolactin in the dog. The effects of electrical stimulation of these aminergic neurons on the secretion of anterior pituitary hormones will be investigated. In addition, research on the neural mechanisms regulating the onset of puberty will be continued and experiments are planned to elucidate the effect of changes in endocrine environment on responses of single neurons in the hypothalamus and related parts of the brain."}
{"text": "Autoimmunity is caused by conspiring effects of genetic predisposition and environmental factors such as injury, infection and microbiome constitution. While multiple genetic loci affect susceptibility, in most cases each in isolation has only a small effect, suggesting that disease develops only when multiple risk-conferring alleles that function in concert are inherited by a single individual. We hypothesize that such a situation exists in autoreactive B cells where multiple SLE risk alleles encode molecules that appear to function in signaling pathways that function normally to limit/terminate antigen receptor signaling. In this application we propose to test this hypothesis, analyzing the functional interplay of this set of genes/protein and their risk conferring alleles. Future development and implementation of ?precision? medical approaches for treatment of autoimmunity will require an understanding of the mechanisms by which genetic variations conspire to increase disease risk, and research proposed here represents a critical first step to enable these efforts. A number of autoimmunity risk alleles encode molecules previously proposed to function as intermediaries in signaling pathways involved in regulation of B cell activation. As such they may be important in keeping autoreactive B cells from becoming activated and contributing to autoimmunity. In this application we request support to define the functions and functional interactions of proteins encoded by six genes, variants of which confer increased risk of autoimmunity. Previous reports indicate that B cell-targeted deletion of genes encoding SHIP-1, PTEN, SHP-1 or LYN, expression of PTPN22 (PEP-R619W), or increased expression of CSK, promote the development of autoimmunity. However the mechanism by which this occurs is unknown. We hypothesize that these proteins function as intermediaries in a bifurcating pathway in which final effectors are the inositol lipid phosphatases SHIP-1 and the tyrosine phosphatase SHP-1. Further, we suggest that both terminal effectors are required for maintenance of antigen unresponsiveness of anergic B cells. The studies will employ reductionist genetic models in which risk allele mimetic changes in expression/function of the proteins can be induced acutely in anergic B cells, and subsequent cell activation, proliferation, differentiation and autoantibody production monitored. Aim 1 will test the hypothesis that PTPN22, CSK and LYN act in linear pathways upstream of SHIP-1 and SHP-1, and that genetic variations that confer risk compromise anergy by undermining their regulatory function. Aim 2 will define the downstream consequences of acute introduction of risk allele mimetic conditions in terms of development of autoimmune disease, and will test candidate therapeutic kinase inhibitors. Aim 3 will translate findings, examining the role of SHIP-1 and SHP-1 phosphatases in maintenance anergy of human B cells. Proposed studies will provide important new insight regarding the in vivo lifestyle of autoreactive B cells whose anergy is compromised by autoimmunity risk alleles."}
{"text": "Although more than 200,000 children are treated annually in hospital emergency rooms for injuries incurred on the playground, our review of current teacher training materials reveals no interactive multimedia program targeting effective playground supervision. This project will develop a comprehensive set of multimedia materials to guide elementary school faculty in implementing a school-wide approach to positive and well-disciplined playground supervision. The Phase I prototype will teach playground supervisors effective behavior management techniques. PROPOSED COMMERCIAL APPLICATION: Multimedia educational software is a growing field, with schools comprising a large customer base. The individualization afforded by multimedia, combined with superior instructions design and remediation as needed, make this product a simple cost-effective way for schools to design and implement safe playground policies."}
{"text": "During the current period of funding, we have shown that the framework of the equilibrium-point hypothesis can be used to address issues of synergy organization. A novel method for analysis of the structure of variability of multi-element systems has been developed and tested experimentally. The next challenges are to generalize the applicability of these tools and approaches to different effector systems and tasks and to link them to physiological processes within the human body. If this is accomplished successfully, our understanding of the human motor function will reach a level when direct application of the presently theoretical and metaphorical notions to practical issue of motor disorders and rehabilitation becomes possible. Hence, our next plans include: (1) Testing the new method of analysis of the structure of variability as a tool for discovering performance variables that are selectively stabilized during a variety of motor tasks; (2) Studying the process of synergy evolution with practice; and (3) Approaching the central mechanisms of synergy organization using the method of transcranial magnetic stimulation. We plan to address the following specific hypothesis: 1). Within a synergy, variability of individual elements is structured so as to selectively stabilize functionally important variables; 2). Learning a novel multi-element task leads to the creation of a synergy reflected in changes of the structure of variability within the state space of elements and responses of the elements to stimulation of the corticospinal tract; 3). Organization of elements into synergies with practice leads to plastic changes reflected in task-specific patterns of finger force responses to transcranial magnetic stimulation; and 4). Quick corrections to perturbations are organized in a synergy-specific way, so as to minimize deviations of functionally important variables. Their qualitative pattern (timing of muscle activation) may be largely independent of the local joint kinematics while quantitative features reflect joint kinematics in accordance with the EP-hypothesis. Seven experiments with oscillatory and discrete force production are suggested to test the hypotheses using tasks of multi-finger coordination within a hand and multi-finger coordination between the two hands. We also plan to monitor changes in indices of synergy organization with practice of an unusual multi-finger coordination task."}
{"text": "Summary Chikungunya virus (CHIKV) is an emerging arthropod-borne virus for which there are no vaccines or therapeutics. Infection with CHIKV can lead to severe and chronic arthralgia. CHIKV displays wide tropism targeting the epithelial, endothelial, and myeloid compartments. The innate immune system is the first line of defense. Viral nucleic acids are sensed, leading to the activation of classical type I interferons (IFN) which induces hundreds of interferon stimulate genes (ISGs). In response, viruses including CHIKV, encode antagonists of interferon signaling to attenuate these antiviral activities. In addition, there is a growing list of basally expressed, effector proteins known as intrinsic factors, which play roles in controlling infection. Altogether, it is clear that there are additional antiviral regulators that play important roles in controlling infection, and in particular, there is less known about antiviral responses in non-hematopoietic cells which are the major targets of CHIKV. Moreover, while much of our understanding of antiviral effectors has been focused on antiviral proteins, there is an emerging literature that suggests that noncoding (nc)RNAs can also play an important role in controlling infection. Long noncoding RNAs (lncRNAs) are an emerging and abundant subclass of ncRNAs that are known to dynamically regulate transcription and translation and can control innate immune responses. The contribution of lncRNAs to innate defense downstream of viral infection is poorly understood, and there is nothing known during CHIKV infection. We hypothesize that lncRNAs play a regulatory role in the control of innate antiviral immunity against CHIKV. Therefore, we will characterize the role of lncRNAs in the innate immune response to CHIKV infection. We are beginning our studies in endothelial cells, as these are a target during infection and little is known about antiviral defenses in this tissue. In addition, we are particularly interested in cytoplasmic lncRNAs as there is less known about their functions, and CHIKV replication occurs exclusively in the cytoplasm. To achieve these goals, our specific aims will (1) Characterize lncRNAs that control CHIKV infection and (2) define the mechanism of action of one antiviral lncRNA we have validated (chikvLNC)"}
{"text": "We are studying the biological mechanism of action of hepatocyte growth factor (HGF). u(In vivo), HGF exists in both a single chain form and as a heterodimer. u(In vitro), HGF has been shown to be multifunctional since the protein can promote DNA synthesis as well as the ``scattering\" of cells. Experiments are under way to determine which parts of the protein are important for the biological effects of HGF. Programs available through the supercomputing facility will initially be used to develop subcloning strategies for the HGF cDNA and later used to assist in analysis of protein structure and function."}
{"text": "Central leptin resistance results in decreased downstream signaling to brain circuits that regulate food intake and energy expenditure (EE), thereby promoting hyperphagia and obesity. We and others have shown that the nonapeptide, oxytocin (OT), circumvents leptin resistance and elicits body weight (BW) loss in diet-induced obese (DIO) rodents, nonhuman primates and obese humans, by reducing both food intake and increasing EE. The discovery of recruitable brown adipose tissue (BAT) in humans has renewed interest in targeting BAT to elicit weight loss by increasing EE. OT neurons that project directly from the parvocellular paraventricular nucleus (pPVN) to the hindbrain nucleus of the solitary tract (NTS) are positioned to regulate energy homeostasis by reducing food intake and increasing BAT thermogenesis. In Specific Aim 1 we will test the hypothesis that OT-induced stimulation of sympathetic nervous system (SNS) outflow to interscapular (IBAT) contributes to its ability to elicit weight loss in DIO rodents. To test this, we will determine whether disrupting sympathetic activation of IBAT blocks the ability of fourth ventricular (4V) OT administration to increase EE and elicit weight loss in DIO rats. We will also determine if sympathetic outflow to both IBAT and white adipose tissue (WAT) mediates the effects of OT on EE by testing the extent to which pharmacological blockade of beta-3 adrenergic receptors (?-3-AR) impairs the ability of 4V OT administration to increase EE in DIO rats. In addition, we will determine if OT can be combined with low doses of the ?-3-AR agonist, CL316243, to increase EE and promote weight loss in DIO rats. Endpoints will include EE, IBAT temperature, norepinephrine turnover (NETO; marker of sympathetic activity), food intake, body composition (total and relative fat mass, lean mass), and BW. We anticipate these studies to establish a key role for SNS outflow to IBAT in the mechanism by which OT increases EE and elicits BW loss in DIO rats. In Specific Aim 2 we will test the hypothesis that in DIO rhesus monkeys, intranasal OT reduces BW and improves glucose tolerance and other metabolic parameters, in part, by stimulating SNS outflow to BAT. To accomplish this, we will determine if intranasal OT increases the temperature of axillary BAT (ABAT, the predominant BAT depot found in rhesus monkeys) at a dose that elicits weight loss in DIO NHPs. We will also determine the extent to which intranasal OT elicits weight loss by reducing body adiposity while sparing lean mass and identify if these effects are associated with improvements in glucose tolerance. We will further identify if intranasal OT may also increase SNS outflow to WAT by measuring changes in subcutaneous WAT temperature and uncoupling protein-1 protein expression in subcutaneous WAT relative to ABAT (pre- and post-intervention). If we find that intranasal OT increases EE and BAT thermogenesis in the NHP model, these studies will provide evidence for a role for BAT activation in how OT elicits weight loss in a translational model of diet-induced obesity. Moreover, these studies will identify intranasal OT as a potential treatment option to evoke weight loss and improve glucose tolerance in humans and direct future studies to address the extent to which intranasal OT may reverse obesity in humans by stimulating BAT thermogenesis."}
{"text": "Resource Core PROJECT SUMMARY / ABSTRACT The overarching goal of the Resource Core is to provide nonhuman primates in the quantity necessary to meet the needs of Somatic Cell Genome Editing (SCGE) awardees. The Resource Core is embedded within the established, comprehensive infrastructure provided by the California National Primate Research Center. Rhesus macaques and marmosets will be available for SCGE investigator studies to be conducted in the Genome Editing Testing Core and in order to generate data on efficiency, specificity, and safety of the technologies developed. The Resource Core will accelerate translation of genome editing technologies from studies in nonhuman primates into treatments for human diseases through the following Specific Aims: (1) Provide nonhuman primates and related capabilities for investigator projects, and (2) Receive, breed, and maintain reporter nonhuman primates. The Resource Core will provide at least 100 animals per year, which will include wild-type and reporter rhesus and marmosets. The Resource Core will ensure investigators have the quality and quantity of nonhuman primates necessary to meet their needs both through the provision of currently available animals (wild-type), and through procurement and expansion of reporter strains that will be provided to the Nonhuman Primate Testing Center from the SCGE Large Animal Production investigators. Paired with a robust Coordination Component and the Genome Editing Testing Core, and within the context of the infrastructure of the UC Davis California National Primate Research Center, the Resource Core will deliver the animals needed to comprehensively assess the promise of new genome editing technologies developed by SCGE awardees."}
{"text": "Short bowel syndrome (SBS) is characterized by severe malabsorption following massive small intestinal resection. Intestinal Lengthening Procedures (ILP) have been performed in children who fail to wean from Parenteral Nutrition (PN) within one year. Although ILP is associated with increased intestinal nutrient tolerance, most patients remain PN- dependent. Intestinal absorption has improved in adults with refractory SBS utilizing growth hormone (GH) and glutamine (GLN). Some adults have discontinued PN with this therapy, and the cost of medical care has been reduced. GH may act via insulin-like growth factor-I (IGF-I) to stimulate morphologic and functional adaptation. GLN is the primary fuel for small intestinal cells and may become depleted in catabolic states. GLN supplementation attenuates PN-induced mucosal atrophy in rats. The purpose of this protocol is to assess whether a 3-month course of treatment with GH and GLN modifies nutrient absorption in children who underwent intestinal resections."}
{"text": "Our understanding of the transcriptional regulation of cardiac development has improved greatly over the last decade, as many transcriptional regulators important for heart morphogenesis have been identified and characterized. It is also becoming clear that many of these regulators recruit a host of other protein cofactors that modulate gene expression in a cardiac specific fashion. One such family of transcriptional modulators important for cardiac development is the Friend of GATA (FOG) family. Both members of the FOG family have been shown to be expressed in the heart, interact with GATA factors, and be required for normal heart development. In this proposal, we demonstrate that both members of the FOG transcriptional modulator family contain a repression domain localized to their N-terminus that physically interacts with subunits of a large chromatin remodeling complex called the NuRD complex. Given their important role in gene regulation, it is not surprising that chromatin remodeling complexes might also be very important in regulating events during embryonic development. In this proposal, we outline research to further characterize these FOG/NuRD interactions and demonstrate their importance for mammalian cardiac development. Specifically, we propose to (1) characterize the subunits of the NuRD complex that interact with the FOG repression motif during cardiac development, (2) determine the functional significance of FOG-2/NuRD interactions during cardiac development, and (3) determine the functional significance of FOG-I/NURD interactions during cardiac development. The results of this work will lead to new insights into the transcriptional regulation of heart development, and may lead to a better understanding of the molecular basis of congenital and acquired heart disease."}
{"text": "The sodium/potassium adenosine triphosphatase (Na+,K+-ATPase) is the membrane protein that maintains the cell's Na+/K+ electrochemical gradient via the active transport of Na+ and K+ across the plasma membrane. Through coupling with other cation transport mechanisms, it is involved in the regulation of cell volume, differentiation and proliferation; ion/solute uptake in the kidney, liver, intestine; propagation of action potential of nerve, skeletal and cardiac muscles, modulation of synaptic action, and cardiac glycoside inotropy. It has also been implicated in cardiovascular hypertension, cardiac hypertrophy, brain edema, cystic fibrosis, and autohemolytic red cell membrane diseases. Its study is, therefore, important in basic and medical sciences. Molecular biology coupled with cell and protein biochemistry provides an incisive tool to the analysis of structure-function relationships and gene regulation of the Na+,K+-ATPase alpha- subunit. Recently we have characterized cDNA clones coding for three rat alpha-subunit isoforms alpha 1, alpha 2, and alpha 3). They are encoded by a multigene family that is differentially expressed in a tissue-specific and developmental manner. Of interest is their unique developmental expression in the heart. The alpha 1 isoform is constitutive; alpha 2 is feta/neonatal predominant; and alpha 3 is adult predominant. We have obtained the complete primary structure of alpha 1 and alpha 2 isoforms. This research proposes to study the molecular genetics of these cardiac Na+,K=-ATPase alpha-subunit isoforms, thus gaining insight into their role(s) in the heart during development and in pathology by aiming to: 1) complete the structural analysis of alpha 3 determining its complete primary structure, 2) establish a human tissue culture system to study the synthesis of the rat alpha-subunit isoforms, 3) define the functional differences of the presently isolated alpha 1, alpha 2, and alpha 3 isoforms, 4) assess the structure-functional significance of the putative ouabain- binding sites I and II via site-directed mutagenesis, and 5) study the Na+-pump's role(s) in cardiac development and cardiac hypertrophy by assessing the modulation of expression of the Na+- pump isoforms in rat by Northern blot, in-situ hybridization, and S1 nuclease mapping analyses. This research study will provide insight into the mechanisms of the Na+-pump's general and highly specialized functions. It will also pave the way for future research on precise mechanisms of Na+-,K+-ATPase structure- function relationships and gene regulation in cardiac development, hypertrophy, and cardiac-glycoside induced inotropy."}
{"text": "The Career Developmental Research Program exists to identify, support, and mentor candidates with promising independent careers in translational research. The Career Development Research Program also represents an opportunity to encourage new and established non-translational investigators, including women and minorities, to consider careers in translational research. In this manner the program ultimately serves as a means to expand translational research, as well as a source of translational projects and investigators for the SPORE Program itself."}
{"text": "DESCRIPTION: This application proposes the development of an interactive, video-based program targeted to children who do not engage in enough physical activity and their families. The program will help children learn how to be physically active, teach them basic behavioral skills needed to develop and maintain a physically active lifestyle and actively involve parents in the process. The program will be delivered by primary care pediatricians and will capitalize on their authority to motivate patient behavioral change. The combination of these elements represents a true innovation in physical activity promotion and primary care prevention of illnesses related to sedentary behavior. During Phase I, a prototype program will be developed to demonstrate the nature and quality of the materials, consisting of a rapid, self-administered screening measure for use by physicians, and videotape, parent guidebook and child- parent workbook materials for home use by families. The prototype will be tested with families and physicians for user satisfaction. Phase II will include a randomized clinical field trial of the programs efficacy. If effective in improving levels of child physical activity and teaches parents to support their children in this manner, it would represent a significant advance in child health promotion. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE"}
{"text": "As the HIV epidemic moves into its third decade it is increasingly a disease of older women who have age-related co-morbidities, and concomitant prolonged exposure to the virus, to ART, and to the metabolic consequences of both infection and therapy. The WIHS in general, and the Brooklyn site in particular, are ideally suited to address the most salient questions related to an aging female cohort. We propose three projects that capture important aspects of the HIV epidemic in aging women. Each project contains specific studies with their own sets of aims and hypotheses. Project 1 focuses on neurocognition, metabolic, and vascular factors in HIV and includes three studies: 1. Adipose tissue, genetic susceptibility, and neurocognition; 2. The HIV-Neuroimaging Initiative; 3. Vascular factors and neurocognition. Project 2 focuses on aging biomarkers in HIV and includes two studies: 1. Frailty-Physical, functional and neurocognitive aspects of aging; 2. Reproductive aging and telomeres. Project 3 focuses on behaviors and implementation science approaches to understanding and promoting successful aging in HIV. It includes three studies: 1. Life course transitions and care engagement; 2. An intervention to increase adherence among women who have experienced childhood sexual abuse; 3. An intervention to enhance smoking cessation. The Brooklyn site, the largest WIHS site, has an exceptional record in cohort retention as well as participation in WIHS leadership and substudies. We further energize our activities for WIHS-V by expanding our cadre of co-investigators and biostatisticians to include those with expertise matched to the aforementioned projects and studies. Dr. Minkoff, who has been chair of the WIHS EC for over a decade, will be joined in a dual-PI leadership role by Dr. Deborah Gustafson, a neuroepidemiologist who has worked with aging cohorts in Sweden and Argentina. Among the many new collaborators are Dr. David Keefe, Chair of OB/GYN at NYU and one of the nation's leaders in reproductive aging, and Dr. Richard Havlik who has served at the NIH in research related to aging. We will also collaborate with the Alzheimer's Disease Neuroimaging Initiative, led by Dr. Michael Weiner. These and other new investigators will join an array of established site investigators, including Dr. Tracey Wilson who heads the WIHS behavioral working group, Dr. Howard Crystal, a neurologist who has a WIHS-linked R01 and many others, to assure the scientific productivity of the site. We have also established a biostatistical core, with members whose expertise corresponds to our project needs, including Drs. Lanza and Yang (Penn State) and Dr. Wilson (SUNY) for analysis of the behavioral projects; Drs. Gustafson (SUNY), Nalls (Molecular Genetics Section, NIA, NIH), Donahue (UCSD) and Weedon (SUNY) for neurocognitive, neuroimaging, and genetic analyses; and Dr. Wu (Penn State) for telomere analysis. Given our exemplary track record to date, in conjunction with an innovative fusion of new and seasoned investigators, and an invigorated statistical core, we look forward to a continued productive relationship with our colleagues in WIHS."}
{"text": "Despite recent advances in biomedical sciences, treatment options remain limited for patients suffering from stroke or other forms of brain injuries. Endogenous regenerative capacities in the brain hold therapeutic promise for nervous system repair after disease and injury. Throughout embryonic and postnatal development, neural progenitors/stem cells give rise to differentiated neurons, astrocytes, and oligodendrocytes. While these progenitors are relatively abundant during embryogenesis, they become restricted to specialized regions in the adult brain. In contrast to the well-investigated area of adult neural stem cell biology, relatively little is known about the cellular and molecular mechanisms controlling multiciliated ependymal cell (EC) function. As a primary cell type lining the adult brain ventricles, ECs form an important part of the lateral ventricular neurogenic niche, but their roles in postnatal/adult neurogenesis have long been controversial: whether ECs represent a neurogenic source remains unclear. Beyond ciliary movements, we know relatively little about basic ependymal biology. Using a combination of mouse genetics, biochemistry, and multiphoton live-imaging, we plan to elucidate the steps necessary to induce and control ependymal cellular plasticity. Our proposal is centered on basic molecular discoveries: we found that expression of the Foxj1 transcription factor, necessary for EC development from radial glial progenitors, is kept inherently unstable in mature ECs. Moreover, continued Foxj1 expression is required to prevent mature ECs from de-differentiating back into a progenitor-like state. We found that this surprising intrinsic cellular instability of mature ECs can be controlled by inhibitor of NF-?B kinase (IKK) signaling, a novel pathway distinct from cannonical NF-?B control. We plan to further explore these unexpected observations by determining the following: 1) whether the non- canonical IKK signal transduction pathway in ECs integrates extracellular signals to determine EC phenotypic stability; 2) what are the molecular mechanisms regulating Foxj1 transcription factor stability and proteolytic degradation - signaling cascades required to initiate EC de-differentiation; and 3) whether molecular events initiating de-differentiation of ECs represent the first steps toward their neurogenic transformation. Tackling the poorly-understood basic molecular and cellular mechanisms regulating EC biology, and using them as a basis for solving a long-standing problem on ependymal contributions to postnatal/adult neurogenesis, should further our understanding of CNS regenerative capacities in health and disease."}
{"text": "Oxygenated derivatives of arachidonic acid (AA) produced by inflammatory cells act as potent mediators of inflammation and allergy. This study proposes: 1) that vegetarians who consume little preformed AA have lower AA in serum and cellular complex lipids; and 2) dramatic reductions in serum and cellular AA levels as well as eicosanoid biosynthesis can be achieved when AA and its metabolic precursors, such as linoleic acid, are largely removed from diets of omnivore volunteers."}
{"text": "Inhibitory interneurons crucially control information processing in neuronal networks, but their vast molecular and anatomical diversity has made it difficult to dissect their functional roles. The cerebellar cortex with its relatively simple architecture and few neuron subtypes constitutes an ideal model circuit to study interneuron function. In the cerebellar cortex, mossy fibers (MFs) relay sensory information to granule cells (GCs) that send their axons to the molecular layer to excite Purkinje cells (PCs). As the only interneurons of the cerebellar input layer, Golgi cells (GoCs) are strategically positioned to control the propagation of sensory information to the cerebellar output layer. Spontaneously active GoCs inhibit GCs with two distinct time courses: rapid phasic inhibition that narrows the time window for excitatory input integration, and persistent tonic inhibition that controls the gai of incoming signals. Moreover, GoCs are thought to mediate the slow oscillations observed in the GC layer prior to the onset of motor behaviors. Strong electrical coupling between GoCs permits these oscillations that coordinate large assemblies of GCs. Although it is well established that GoCs crucially determine the flow of information within the cerebellar cortex, less is known about the mechanisms that orchestrate GoC firing, regulate GoC activity, and dynamically control GC excitability. Contrary to current beliefs in the field, preliminary data supports the hypothesis that active GoC dendrites enhance electric coupling. This proposal thus seeks to determine the cellular mechanisms that enable GoCs to fire synchronously using two-photon calcium imaging, patch clamp electrophysiology, voltage imaging and array tomography. Preliminary results also suggest that GoC activity dictates dendritic calcium concentration. The planned experiments will therefore examine the functional relationship between GoC activity and dendritic calcium dynamics, and determine the consequences for synaptic plasticity of excitatory PF and MF input. Preliminary data indicates that activation of metabotropic receptors on GoCs suppresses firing, and that this suppression is accompanied by a decrease in tonic inhibition of GCs. With the help of electrophysiology and optogenetics, this proposal will therefore test the hypothesis that dynamic modulation of GoC firing rate controls GC excitability and MF input integration. Completion of the outlined work will elucidate the mechanisms that control integration of sensory information by varying the activity of a single interneuron subtype in the cerebellar cortex. It will also extend our general understanding of how inhibition governs computational processes in neural networks."}
{"text": "The presence of food in the gut produces both direct and indirect effects on the growth of the gastrointestinal mucosa. Becasue ingestion of foodstuffs leads to increased desquamation of the epithelium and changes in luminal or local nutrition, direct effects on growth are produced when food moves through the gut after a meal. Indirect effects of a meal include release of GI hormones, increased secretions and increased activity of the autonomic nervous system. There may be a common mechanism which mediates both types of stimulation. The polyamines are a group of ubiquitously distributed polycations which are required for normal cell growth and proliferation. Preliminary studies have shown that they are involved in the regulation of mucosal growth. These data demonstrate that 1) there is a proximal to distal gut gradient of polyamine metabolism, 2) the aliphatic amine, ethylamine, has direct trophic effects in the gut, 3) the trophic response to intestinal obstruction has, as a required event, the induction of polyamine metabolism. These observation may explain 1) the proximal to distal gut gradient of villus height and crypt depth, 2) the role of amines, both aliphatic and polyamines, as regulators of GI mucosal growth, 3) the mechanism whereby the hormones of the gut exert their trophic in GI tissues, and 4) the as yet unexplained adaptive response of the gut to intestinal surgery. It is proposed to study and define the role of amines as regulators of mucosal growth. Initial experiments are designed to localize the areas of the gut in which amine metabolism is important to growth. It will also be necessary to study amine metabolism in the bacteria of the intestinal microflora as amines secreted from these cells may have direct effects on mucosal growth. It will be determined whether the hormones of the GI tract induce polyamine metabolism and whether this step is required for expression of their trophic effects. Finally, the role of amines as mediators of the adaptive response to intestinal surgery will be defined in various animal models of postsurgical hyperplasia and hypertrophy. The importance of a thorough understanding of GI growth is underscored by the realization that many diseases of the bowel, e.g., Zollinger-Ellison syndrome, chronic gastritis, pernicious anemia and the growth of cancers of the alimentary canal, are accompanied by disorders of gastrointestinal growth."}
{"text": "DESCRIPTION (Principal Investigator's Abstract): The O-alkyl ether linkage in glycerolipids was originally discovered as a significant component of fish oils. As with the n-3 fatty acids, fish from the ocean are thought to be highly enriched in ether lipids. The aim of this proposal is to develop a better understanding of the nutritional significance and possible health-related benefits of ether-linked lipids in the diet. In one portion of the planned work, the investigators propose to conduct a systematic quantitative analysis of the amount, type, and acyl composition of ether-linked glycerolipids in fish, meat, and poultry products consumed in the American diet. The second aim of this investigation is to evaluate the influence of dietary supplements of ether lipids and its fatty alcohol precursor on: a) the levels of ether lipids and their n-3/n-6 acyl composition in rat tissues, b) various biological parameters (growth, blood pressure, and cellular responses of neutrophils and alveolar macrophages to proinflammatory agents), and c) de novo enzyme activities for the synthesis of tissue ether lipids involved as cellular membrane components or as bioactive mediators. The experiments described in this application would be the first comprehensive dietary study ever conducted with the glyceryl ethers. Such information is important to obtain in view of the important role that potent alkyl type glycerolipids such as PAF (e.g., 10 -11M PAF can aggregate platelets), alkylglycerols, alkylacetylglycerols, and alkylacylglycerols play as modifiers of signal transduction events and as factors that can affect cellular differentiation and the activation of macrophages."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Previous workers have proposed high resolution models for the docking of the myosin heads on actin on the basis of combined crystallographic and electron microscopy data from isolated proteins (Mendelson and Morris, 1997 PNAS 94:8533;Holmes et al. 2003 Nature 425:423). Another group also claims that the first step in the force generation is associated with a rearrangement of the myosin-actin interface, followed by the lever arm tilt, and that it is temperature-dependent (Ferenczi et al. 2005 Structure 13:131). The goal of this work is to collect small angle X-ray scattering (SAXS) data from muscle that may be used to check in vivo the prediction of the models for the acto-myosin docking and whether there is a temperature-dependent rearrangement of the myosin-actin interface. For this purpose, the most sensitive reflection in the pattern is the 2.73nm meridional reflection arising from the regular repeat of the actin monomers along the actin filament, which changes its intensity upon myosin attachment to actin. Preliminary modelling has shown that the reflection intensity is little influenced by the lever arm tilt but it is highly sensitive to the relative axial position of actin and catalytic domain of myosin. 2D patterns will be taken from muscle at rest and during isometric contraction at different temperatures (4 to 17[unreadable]C) up to 0.5 nm-1 in reciprocal space, in order to collect the actin-based 2.73nm meridional reflection and the 5.9nm and 5.1nm layer lines, also influenced by myosin attachment to actin."}
{"text": "Alcohol (EtOH) abuse and dependence continue to be significant public health problems. Thus, a better understanding of their neurobiology will facilitate the development of interventions targeting prevention and/or treatment of these major health issues. Emerging evidence indicates that many aspects of EtOH and drug dependence involve changes in glutamate transmission. Because glutamate transporter 1 (GLT1) is responsible for the uptake of the majority of extracellular glutamate, we tested the hypothesis that increased GLT1 function would attenuate EtOH consumption in alcohol-preferring rats (P rats). After P rats had been chronically exposed to a free choice of EtOH (15 and 30%) for five weeks, they were administered ceftriaxone (i.p.), a beta-lactam antibiotic known to elevate GLT1 expression, for five consecutive days. We found that ceftriaxone-treated P rats showed a reduction in EtOH intake for the duration of treatment as compared to rats that received a saline vehicle. The long-term effects of ceftriaxone, however, are unknown. Here, we will test the long-term effects of ceftriaxone and other drugs (GPI-1046 and MS-153) known to activate GLT1 in the attenuation of EtOH intake at two different time points of chronic EtOH exposure in P rats. We will also investigate the effects of GLT1 activation in EtOH-drinking behavior in Wistar rats as comparison control groups. Our working hypothesis in aim 1 is that an increase in GLT1 function, via up- regulation or activation, attenuates EtOH consumption in both P and Wistar rats. The EtOH deprivation effect, which we will employ, has been used to assess relapse-like behavior in P rats. In aim 2, our working hypothesis is that an increase in GLT1 function during withdrawal periods will reduce relapse-like behavior when animals are re-exposed to EtOH. The lowest tested dose of ceftriaxone (25 mg/kg), which did not show an increase in GLT1 expression, was also effective in reducing EtOH intake. These findings suggest that ceftriaxone may have other pharmacological effects on GLT1 or may act by another mechanism (a functional increase may involve a change in one of several mechanisms). Thus, we propose in aim 3 to determine the signaling pathways involving ceftriaxone or GPI-1046 in up-regulation of GLT1 expression. Moreover, we aim to determine the molecular mechanisms of action of ceftriaxone, GPI-1046 and MS-153, which may involve activation of GLT1 function through phosphorylation of key proteins. The findings generated from this proposal will provide ample information about the role of GLT1 in the regulation of EtOH consumption and will pave the path toward finding a potential therapeutic target for alcohol addiction."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Structural studies of bacterial signal-transduction protein complexes Histidine kinases have been attractive new targets for drug design to circumvent drug resistance in Gram-positive pathogenic bacteria. Our research is aimed at structures and functions of a multi-domainal, membrane-spanning histidine kinase and its response regulator from an essential two-component signal transduction system. Proposal: Histidine kinase of Gram-positive pathogenic bacteria Resistance to common antimicrobial drugs is a global phenomenon, and an increase in resistance has been observed for every major group of bacterial pathogens. There is an obvious need for identification of novel targets for development of new antimicrobials. Two-component systems are ubiquitous in bacteria and are central elements of the intricate regulatory pathways utilized by pathogenic bacteria to control a wide range of cellular processes including expression of virulence factors and resistance to certain antimicrobial. A paradigmatic two-component system consists of two proteins: sensor kinase and response regulator. The sensor kinases monitor environmental signals, and modulate functions of response regulators through phosphotransfer reactions. The response regulators usually contain an N-terminal receiver domain and a C-terminal DNA-binding domain. The receiver domain receives signals from histidine kinase through phosphorylation, which enables the DNA-binding domain to act as a transcription factor to regulate expression of certain genes. YycF (response regulator):YycG (histidine kinase) system is highly conserved in the low G+C Gram-positive bacteria including Bacillus subtilis, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and Listeria monocytogenis, many of which are human pathogens and pose significant threat on public health in the U.S. and worldwide. The YycF:G system is the only two-component system essential for cell viability known to date, and has been suggested as a prime antimicrobial target and subjected to small-molecule inhibitor developments. The YycF:G system has been implied in cell wall biogenesis. However, it is not clear what signal YycG senses and how the signal is relayed in the YycF:G system."}
{"text": "The use of antiepileptic drugs (AEDs) in pregnancy and early infancy poses special challenges and concerns, as even transient exposure to certain drugs during CNS development may impede subsequent neuronal and synaptic development. Several AEDs, including phenobarbital and phenytoin, when given in therapeutically relevant doses to rats during the early postnatal period, cause a pronounced increase in apoptotic cell death in several brain regions. Other drugs such as lamotrigine (in low or moderate doses) and levetiracetam are devoid of this effect. We have recently discovered a single exposure to phenobarbital, at the time of peak vulnerability to the propapoptotic action (postnatal day 7 in the rat), resulted In the suppression of the normal, developmental increase in the frequency of inhibitory post-synaptic currents (IPSCs) recorded via patch clamp from striatal medium spiny neurons (MSNs) in slices taken between postnatal day 10 and 14. We have also identified impairments in adult rotorod performance in rats treated at P7 with a proapoptotic dose of phenytoin, a reduction in prepulse inhibition (a measure of sensory-motor gating) and a reduction in seizure threshold to pentylenetetrazol In animals treated in the first postnatal week with phenobarbital. Each of these behavioral assays are sensitive to damage or altered function in the striatum. The proposed experiments will follow up on these exciting preliminary findings to determine if there is a consistent and predictive (potentially causative) relationship between AED-induced neuronal apoptosis and impaired maturation of I PSC frequency in the striatum and/or adult behavioral toxicitiy. This will be assessed by comparing AED treatments that are proapoptotic with those that avoid this toxicity. We hypothesize that AEDs that do not cause cell death will not cause impaired maturation of IPSC frequency in striatal MSNs, nor will they result in impaired rotorod performance, reduced seizure threshold, and impaired prepulse inhibition ofthe acoustic starle response. Moreover, the extent to which a neuroprotective treatment can prevent impaired maturation of IPSC frequency in striatal MSNs and/or behavior will be evaluated to test the hypothesis that neuronal death is necessary for this adverse functional outcome. The results ofthe proposed experiments will allow us to better understand the potential functional outcomes of exposure to AEDs in late gestation or early infancy, and the extent to which induction of excessive neuronal death is a valid marker of subsequent functional impairment. Furthermore, it will identify strategies to avoid deleterious functional sequelae of AED therapy during critical developmental periods. GOALS FOR KIRSCHSTEIN-NRSA FELLOWSHIP TRAINING AND CAREER I am seeking this fellowship tp support my dissertation research in Dr. Karen Gale and Dr. Stefano Vicini's laboratories, investigating the impact of neonatal anticonvulsant drug exposure. My goal for dissertation research is to develop a broad skill set of experimental techniques that will allow me to address scientific questions at the level of molecules, cells, networks and behavior. The complementary expertise of my mentors provides such exposure. A limited portion of this support is also requested to allow me to continue to teach (present lectures to graduate and undergraduate students, and continue to direct a course entitled, \"Diseases and Disorders of the Brain\". A key goal of my training is to complete and publish a series of focused studies on outcomes following drug exposure during development. My long-term goals are predicated on the broad training I am receiving at Georgetown. Following Georgetown I will seek post-doctoral training and then I hope to develop an independent, productive research program at an academic institution where I can ask and answer questions of interest at multiple levels of function, to maintain an active Involvement in teaching and mentoring, and to contribute to scientific discourse."}
{"text": "The broad objectives of this proposal are to understand the metabolic role and enzymatic mechanism of one of the most abundant folate enzymes, 10-formyltetrahydrofolate dehydrogenase (FDH). FDH converts 10-formyltetrahydrofolate to tetrahydrofolate in an NADP-dependent dehydrogenase reaction or in an NADP-independent hydrolase reaction thus regulating two of the major folate pools. It has been also proposed that the enzyme serves as an intracellular folate depot protecting folate coenzymes from oxidative degradation. The enzyme is a natural fusion of two unrelated proteins. The amino-terminal domain bears the folate-binding site and functions as a hydrolase. The aldehyde dehydrogenase like carboxyl-terminal domain works as the catalytic tool in the dehydrogenase reaction when the two domains are combined in one polypeptide. A hundred residue intermediate domain is a linker between the two functional domains required to bring them together to catalyze the dehydrogenase reaction. It is hypothesized that the hydrolase reaction of FDH although by itself is not of physiological significance, is an important and essential part of the FDH dehydrogenase mechanism. The FDH dehydrogenase mechanism is a combination of two sequential reactions, the hydrolase and aldehyde dehydrogenase. During the dehydrogenase reaction transfer of an intermediate product from the hydrolase domain of FDH to the aldehyde dehydrogenase domain takes place. The intermediate domain is crucial to bring two functional domains in correct orientation to allow the transfer. Another part of this project is based on the hypothesis that one of the major roles of FDH is to regulate de novo purine biosynthesis by controlling 10-formyltetrahydrofolate levels. The recent findings that FDH is highly down-regulated in carcinogenesis, apparently due to increased demand of cancer cells for purines, make the protein an important potential target in anticancer chemotherapy. The following specific aims are proposed to test the hypotheses. (1) To determine the role of the intermediate domain in the enzyme mechanism. (2) To characterize the folate binding site and to evaluate the hydrolase mechanism of FDH. (3) To crystallize and to resolve the crystal structure of the FDH individual domains and the full- length protein. (4) To elucidate the role of FDH in cellular metabolism. Site-directed mutagenesis and protein design approaches, enzyme activity assays, binding studies, crystallographic and immunochemical methods, mammalian cell expression, antisense oligonucleotide techniques, purine and folate assays will be used to achieve the goals of the project. The well known role of folate in prevention of megaloblastic anemia, vascular disease, neural tube birth defects and cancer make these studies particularly relevant."}
{"text": "The search for biological correlates of endogenous depression (ED) has been on for several years. Anomalous regulation of the hypothalamo-pituitary-adrenal (HPA) axis has been implicated using the Dexamethasone Suppression Test (DST). While there is active debate concerning the clinical usefulness of the DST, there is general agreement that some ED patients show defective HPA regulation, especially as seen in their baseline plasma cortisol and the lack of suppression after dexamethasone (DEX). In our previous funding period, we studied the pituitary component of the HPA axis in ED patients by measuring the peptide products from the ACTH/endorphin precursor. We have shown substantial pituitary regulation defects as reflected by plasma beta-endorphin (BE) assays in ED patients when compared to psychiatric controls. These studies revealed a partial concordance between BE and cortisol dysregulation. The combination of both measures after DEX reveals abnormalities in 70% of the ED patients. Based on the peptide and steroid measures we are proposing the possible existence of 4 subgroups of ED patients with different patterns of HPA dysfunction. The present proposal is aimed at testing these notions and further exploring the nature of the HPA defect. Studies on the effect of age, sex and time of testing in normals and psychiatric subjects will further explore variables contributing to the different patterns of response post DEX. Studies with CRF-induced release of ACTH and BE, metyrapone, cortisol fast feedback and biochemical characterization of the released peptides will be conducted to elucidate the locus and nature of the dysregulation. Finally, we will study patients in remission whose DST has normalized to determine if other HPA correlates reveal continuing abnormalities."}
{"text": "The Northeast Center for Research to Evaluate & Eliminate Dental Disparities (CREEDD) is committed to the elimination of oral health disparities through research, training and action. Our efforts will build on a strong foundation of early and continuous community engagement, community-based research interventions, integrated training and career development, broad dissemination of research findings, and targeted health policy initiatives. Based at Boston University, the CREEDD will maintain its current regional approach, with partnering institutions in Massachusetts, Maryland and Ohio. A diverse, multidisciplinary and multi-institutional team will implement community-based intervention research projects aimed at reducing early childhood caries (ECC). Our organizing theme is engaging non-dental care providers in oral health promotion, and extending the venues for oral health promotion to non-clinical care settings directly in health disparity communities. The two major community-based intervention research projects will be: Project #1 (D&IR Project) - Partnering with Community Health Centers to Prevent ECC (D&IR Project) Physician-delivered interventions, targeted at children aged 1 to 3 years old, with include fluoride varnish applications, patient-centered counseling, and systems-level changes to clinical information systems and clinical prompts used in order to include age-appropriate, oral health-specific anticipatory guidance items. Project #2 - Oral Health Advocates in Public Housing Resident Health Advocates (RHA), trained peer health advocates, will incorporate motivational interviewing and community oral health promotion activities into their ongoing health promotion efforts targeting caregivers with children from birth to 5 yrs old, aimed at caries risk reduction and lowering incidence of ECC. We will use a community-based cluster randomized study design, in Project #1 with community health centers (CHC) as the unit of randomization, with CHC located in Boston, Maryland, and southeastern Ohio; and in Project #2 with individual public housing developments in Boston as the unit of randomization. [unreadable] [unreadable] [unreadable]"}
{"text": "Targeted dendrimer-based nanodevices have shown excellent promise in both in vitro cell culture and in vivo animal studies as cancer therapeutics. However, each device must be custom synthesized for a particular set of targeting molecules, imaging agents, and desired therapeutics. We propose a unique solution to this limitation by developing single function dendrimer modules linked by complementary oligonucleotides. This allows targeting, imaging, and therapeutic dendrimers to be combined into multifunctional therapeutics simply by heating mixtures of these agents above the annealing temperature of the oligonucleotide duplex. The project will consist of five specific aims. Specific Aim 1 will involve the design and synthesis of complementary oligonucleotides conjugated to poly(amido)amine dendrimers or dendrons to achieve the desired structural topologies. The ability to construct complex devices will be assessed using well-defined targeting molecules (folic acid, her2 antibodies and RGD peptides), drugs (methotrexate, Taxol, cis-platin and doxorubicin), imaging agents (Gadolinium chelators and fluorescent dyes). Specific Aim 2 will characterize the self-assembled nanodevices using techniques including PAGE, HPLC, CE, Mass Spectroscopy, NMR, AFM, and NSOM. Specific Aim 3 involves testing the DNA-linked nanodevices for binding and internalization in vitro;the avidity and specificity of binding will be examined using CD, differential calorimetry and Biacore analyses. Devices carrying therapeutics will be tested for effectiveness at inducing cell death, and all devices will also be tested for inherent cytotoxicity. Specific Aim 4 employs animal models to assess the effectiveness of the dendrimer linked therapeutics to treat tumors in vivo. In addition, the biodistribution of the therapeutics will be assessed using radiolabeled material and a novel fiber optic probe that uses two-photon excitation with femto-second pulses. Finally, under Specific Aim 5 we will work with the NCI nanoparticle characterization lab in Frederick to make the materials developed in this program available to other investigators. This platform has the potential to revolutionize cancer therapeutics and facilitate \"personalized medicine.\" Lay description: We are designing a method and the tools for developing targeted cancer drugs that can be tailored to the needs of an individual patient. The physician can select various components and the components are then linked together like \"tinker toys\" to make a personalized medicine. This medicine would selectively target only the cancer, thereby avoiding the nausea, hair loss and illness caused by regular cancer chemotherapy."}
{"text": "We request continued support for a NINR-funded study on the effectiveness of an innovative parent training program (The Chicago Parent Program; CPP) developed in collaboration with a parent advisory council and used in childcare centers serving low-income families of color. The current study has been affected by issues common in prevention trials: low enrollment and attendance rates and the inability to include Spanish- speaking Latino parents. Historically, scientists have addressed these challenges by excluding non-English- speaking Latinos and instituting costly and unrealistic subject incentives, strategies that hinder the growth of prevention science for people of color and its translation to health care policy. The primary goal of the proposed study is to build upon the current grant by testing the effectiveness of two strategies for increasing participation rates among Latinos and maximizing the effects of preventive parent training for low-income families of color using methods that can be generalized beyond the research context. Using a randomized experimental design (n=450), we will test the effectiveness of (1) a Spanish-language version of the CPP and (2) discounting parents' childcare co-pay costs contingent upon attendance. Eight day care centers will be matched and randomly assigned to control and experimental conditions: (1) a 12-week intervention condition in which barriers to attendance are reduced but no financial incentives for attendance are offered (Standard), (2) a discount co-pay incentive condition in which barriers to participation are reduced and parents receive a discount on their weekly childcare co-pay bill contingent upon attendance (Discount Co- pay), and (3) a no-intervention control condition. Participation will be defined by enrollment rate, attendance, and parent engagement in the intervention. CPP effectiveness will be examined using multiple parent and child outcomes assessed up to 1 year post-intervention. The cost-effectiveness of using a discount co-pay strategy compared to the standard condition will also be examined. Relevance: The goals of this study are to use a real world approach to increase participation rates in a prevention intervention designed to promote positive parenting and mental health among low-income ethnic minority families of 2-4 year old children. Data from this study would lead to improved outcomes for parents and children during the first 5 years of life. [unreadable] [unreadable] [unreadable] [unreadable]"}
{"text": "Histone deacetylases (HDACs) are vital regulators of fundamental cellular events, including cell cycle progression, stem cell functions, cell fate determination, cell differentiation, and the pathogenesis of many diseases. As such, it is not surprising that protein acetylation is central to human diseases, as diverse as neurodegenerative disorders, cardiac hypertrophy, cancer, HIV infection, and more generally the process of aging. More importantly, small molecule HDAC inhibitors and activators are currently in clinical trials for the treatment of leukemia and lymphoma, solid tumors, neuromuscular disorders, metabolic disorders and other diseases. In addition, the Sirtuins, a subclass of HDACs, were fingered in yeast genetic studies as regulating lifespan, and these enzymes might be targeted by resveratrol, one of the components in red wine that has been linked to increased lifespan in humans. Therefore, a thorough understanding of HDACs is required, not merely for understanding the regulation of chromatin structure, gene regulation and protein function, but also because HDACs are intimately involved in normal and abnormal cellular processes that greatly impact human health. With the identification, isolation, cloning and functional characterization of 18 human HDACs (HDAC1- 11 and SIRT1-7) and many acetyltransferases in the past decade, the coming years will see a continued dramatic expansion in our knowledge of the biological roles of HDACs and protein acetylation. As the only conference dedicated to this field, this biannual meeting plays an essential role in bringing together approximately 44 basic and clinical scientist speakers to exchange information and develop new therapeutic avenues with approximately 120 participants from around the world. A primary objective is to transfer knowledge between basic academic researchers, clinical scientists, and pharmaceutical scientists to create efficiencies in understanding how they control human health and how these activities can be harnessed to fight a diverse slate of diseases. A second objective is to foster the development and interests of younger investigators to help support their career development. To accomplish these objectives, the meeting venue houses meeting space, dining, and rooms so that participants have ample time to move from formal presentations to informal brainstorming and collaborative discussions. In addition, the morning and evening oral presentation sessions include 2-3 talks from junior scientists selected from the submitted abstracts, which is important for career development and to encourage the next generation of HDAC scientists. There will be an emphasis through both the scientific and social programs on creating a global HDAC community. This meeting will be particularly timely because data from on a new generation of HDAC and Bromodomain drugs will be presented. Therefore, support is requested for the 4th biannual conference on the biology and therapeutic targeting of HDACs and Sirtuins, and their role in aging and disease to be held at Il Ciocco, Lucca, Italy, August 18-23, 2013 in conjunction with FASEB."}
{"text": "The molecules that control Retinoblastoma (Rb) phosphorylation called the Rb pathway consisting of D-type cyclins, cyclin dependent kinases (cdks) and cdk inhibitors (cdkis) as well as Rb itself are regularly disrupted in most cancer types including breast cancer leading to excessive phosphorylation (hyperphosphorylation) of the Rb protein. Hyperphosphorylation of Rb is known to promote proliferation and block apoptosis. Treatments that inhibit cdk activity have begun to exhibit promise in the clinic in several cancer types. Although Rb can be phosphorylated on 15 amino acid residues, very little is known about the contribution any individual phosphorylated amino acid has on the function of Rb in proliferation and apoptosis. In this project we propose to undertake the first investigation of Rb phosphorylation in proliferation and apoptosis utilizing a model of breast epithelial cells using three-dimensional cultures. This model system is employed to recapitulate the physiological context in which breast epithelial cells regulate cellular processes. We propose to target Rb hyper- phosphorylation by using a novel method of activating Rb-specific phosphatase activity in breast epithelial cells grown in 3D cultures. In this way we can determine the effect of dephosphorylation of Rb on proliferation and apoptosis in these cells. In addition, we will perform site directed mutagenesis of each Rb phosphorylation site to either alanine or glutamic acid to block phosphorylation or mimic phosphorylation at each site and evaluate the effects on proliferation and apoptosis in breast epithelial cells grown in the 3D culture model. Finally, we will clarify the functional significance of complex formation between Rb and the pro-apoptotic protein Bak that is regulated by Rb dephosphorylation in apoptosis. Thus, the proposed studies will elucidate the role of specific Rb phosphorylation sites involved in the regulation of proliferation and apoptosis in breast cancer and will yield useful information that could inform the development of future therapies that target Rb phosphorylation in cancer."}
{"text": "SUMMARY This study focuses on understanding the psychosocial impacts of genetic causal attribution in the epilepsies, a set of common neurologic disorders with significant psychosocial dimensions, including stigma, discrimination, reduced rates of marriage and reproduction, and reduced quality of life. Genetic research and clinical genetic testing in the epilepsies are advancing rapidly, and plans are underway to develop precision medicine approaches for clinical care. These developments reflect a strong emphasis on genetic causes of epilepsy, which is being communicated to patients in multiple ways. For some people with epilepsy, this emphasis will be welcome; for others, psychological and behavioral responses may be more complex. However, little is known about the psychosocial impact of genetic causal attribution on people with epilepsy. In previous research, we studied a unique set of >100 families containing multiple individuals with epilepsy, to assess their preferences for genetic testing, their beliefs about genetic causes, and the associations of these beliefs with psychosocial outcomes. We now propose to build on these studies to include a more diverse and representative sample of ~600 adults treated for epilepsy at our institution. These patients are being offered whole exome sequencing (WES) as part of a research program sponsored by Columbia's Institute for Genomic Medicine (IGM), and we will take advantage of this opportunity to study patients' decisions to participate or not participate in WES and the impact or receiving or not receiving genomic results. In Aim 1, we will assess the relations of genetic causal attributions to other illness perceptions (e.g., severity, persistence, treatability), and psychosocial (e.g., felt stigma, depression, anxiety) and behavioral (medication self-management) outcomes. Data analyses will test consistency with theoretical expectations under models of attribution theory, ?genetic optimism,? and genetic essentialism. In Aim 2, we will investigate the ways in which receiving or not receiving genomic results may influence illness perceptions, psychological well-being, and health-related behavior, through in-depth qualitative interviews with patients who receive epilepsy-related findings, receive secondary findings, have WES but do not receive findings, and elect not to have WES. In Aim 3, we will develop mechanisms to translate our findings into improvements in education about the role of genetics in the epilepsies. The results will clarify the beliefs and attitudes about genetics that underlie any adverse impacts of geneticization in epilepsy and provide a framework for education programs designed to ameliorate them, in order to maximize the benefits of genomic medicine."}
{"text": "Purification of individual chromosomes by flow sorting is often a first step in defining the structure of normal and abnormal human genes. The polymerase chain reaction drastically reduces the amount of sample required for isolation of chromosomal DNA, making it feasible to develop simpler, less expensive chromosome sorters. The performance of an instrument using air-cooled ultraviolet (UV) and blue helium-cadmium (He-Cd) laser light sources in flow cytometry of chromosomes stained with DAPl/olivomycin or Hoechst 33258/chromomycin A3 will be evaluated to determine whether the system can resolve the human karyotype as well as can existing commercial apparatus. As an alternative, several nucleic acid fluorochromes not previously applied to chromosome analysis and potentially usable in a large number of existing flow sorters will be examined as adjuncts or alternatives to stains presently used for bivariate flow karyotyping."}
{"text": "Retinoic acid (RA) is a metabolic derivative of vitamin A (retinol) that functions as a signaling molecule. RA is essential for eye development, but its action is poorly understood. RA signaling occurs when retinol is metabolized to RA which serves as a ligand for nuclear RA receptors that regulate gene expression. The enzymes controlling synthesis of RA during embryogenesis are now under investigation and such studies are providing new information on the mechanism of RA action during eye development. Studies on mouse embryos have demonstrated the existence of three retinaldehyde dehydrogenases differentially expressed in the eye that synthesize RA, i.e. RALDH1, RALDH2, and RALDH3. Investigations of Raldh1, Raldh2, and Raldh3 null mutant mice have uncovered eye defects, and further studies of these mice are beginning to reveal the mechanism of RA action. As the three Raldh genes are conserved in mice and humans, the null mutants we have developed are excellent mouse models for understanding the mechanism of RA action during human eye development. Evidence exists suggesting that RA deficiency caused by dietary vitamin A deficiency may be linked to the human eye defect known as ocular coloboma. The genetic studies proposed will provide information relevant to treatment of human eye diseases whose etiology involves genetic deficiency in RA synthesis and/or dietary vitamin A deficiency: We have found that the location of RA synthesis undergoes dynamic spatiotemporal changes during eye development, and that the location of RA action changes in synchrony. Raldh2/Raldh3 double mutant mouse embryos develop an optic vesicle, but this structure lacks RA synthesis and fails to invaginate ventrally to form the optic cup. Raldh3 null mutant embryos develop an optic cup but they display defects in closure of the optic fissure (coloboma). Raldhl null mutant embryos lack RA synthesis in the dorsal retina, but eye defects are not observed. However, Raldh1/Raldh3 double mutants display excessive invasion of perioptic mesenchyme anterior to the retina, thus revealing a function for Raldhl that is normally compensated by Raldh3 (and vice-versa). These findings have led to the hypothesis that RA controls eye morphogenetic movements rather than dorsoventral patterning of the retina as previously thought. The overall goal of this project is to determine the mechanism of RA signaling during eye development, particularly the gene networks regulated by RA in the eye. We will test the hypothesis that RA regulates eye morphogenetic movements of both the retina and the surrounding perioptic mesenchyme. These studies will be performed genetically using Raldh compound null mutant mice that are unrescued or rescued by various genetic or pharmacological methods. Specific investigations will focus upon: (1) RA control of cell shape and cell adhesion during optic cup formation; (2) RA-FGF antagonism during optic cup formation; (3) RA control of perioptic mesenchyme invasion following optic cup formation. [unreadable] [unreadable] [unreadable]"}
{"text": "Polymorphonuclear neutrophils must respond to multiple exogenous stimuli to proceed through the complex transformation from quiescence to activated phenotypes. The plasma membrane is the interface where this transformation must be initiated. Recent work has shown that plasma membranes are heterogeneous structures containing discrete regions, \"lipid raft microdomains\", enriched in glycosphingolipids, cholesterol, lipid-anchored receptors, and signaling molecules. Selective partitioning into lipid rafts appears to be a key mechanism for imposing organization on the distribution of proteins in the plasma membrane, thereby compartmentalizing discrete functions within the plasma membrane and, in essence, creating a syntax that translates otherwise dissociated signaling effectors into a meaningful language of signal transduction. The central hypothesis underlying this proposal is that the composition, structural integrity, and spatial distribution of lipid raft microdomains critically regulate key processes in neutrophil activation. Our preliminary studies of human neutrophils have shown that lipid rafts: i) spatially compartmentalize and selectively regulate neutrophil activation signaling, ii) selectively influence cellular responsiveness to proinflammatory agonists, and iii) regulate signal propagation during cell migration. The long-term objective of this project is to determine how the function and composition of lipid raft microdomains regulate the antimicrobial and proinflammatory functions of neutrophils. The short-term objectives are to: i) determine the mechanisms by which lipid rafts influence agonist-specific signal transduction, ii) determine how lipid rafts regulate the production and release of reactive oxygen intermediates, iii) determine how lipid rafts influence the expression, distribution, and function of beta2 integrins, and iv) determine how lipid rafts control cellular polarity and calcium signaling during non-directional and chemotaxin-driven migration. Hopefully, targeting the function of lipid raft microdomains will engender new therapeutic strategies for enhancing antimicrobial defenses and suppressing the deleterious effects of acute inflammation."}
{"text": "Project Summary Most people living with HIV (PLWH) in Malaysia also have an opioid use disorder (OUD), yet only a minority receive ART and OUD treatment, resulting in increased mortality and HIV incidence. The proposed pragmatic, multisite, implementation and effectiveness research will evaluate a strategy to improve HIV treatment outcomes (increased rates of patients on ART with virological suppression, improved treatment retention and ART adherence) for PLWH with OUD. Engaging 4 large regional HIV/AIDS treatment centers in Malaysia, with a stepped rollout of the study protocol across the study sites, offset by one year with a random order of the sites initiation, the study will evaluate barriers and facilitators for implementation of improved care model and will evaluate the comparative effectiveness of the model in a clinical trial. At each of the study sites, individuals testing HIV positive who also have OUD (n=4x70) will be enrolled to receive concurrent ART and MMT based on the usual care standards. Their patient level outcomes will be compared with individuals meeting the same inclusion criteria (n=4x70) and treated under the proposed improved model (post implementation evaluation). The usual care standard will consist of provision of ART and medical care for HIV and other medical HIV co- morbidities provided at the HIV/AIDS treatment center with an expedited and facilitated referral to a methadone maintenance treatment (MMT). The improved care model will include the usual care supplemented by continuing education and coaching of medical staff at HIV/AIDS and MMT clinics and by provision of additional peer-based counseling intervention focused on behavioral skills and strategies that patients can learn and master to achieve uninterrupted, long-term ART treatment participation while continuing OUD recovery through MMT. The primary outcome measure, rates of patients with virologic suppression (< 20 copies/mL) in the two care models will be assessed at 24 weeks. The secondary outcomes, also followed for 24 weeks, will include ART adherence measured by objective measures (tenofovir dried blood spots, clinic records) and self-report; decreased illicit opioid use measured by rates of opioid negative urine toxicology results and self-report; and improvements on other health-related and functional status outcomes. A statistically significant effect on the primary outcome and clinically meaningful effects on secondary outcomes favoring the improved care model are hypothesized. Concurrently at each study site, using implementation science mixed methods research tools and engaging key local stakeholders (treatment providers, patients, their families, and community activists), and evaluating clinical and healthcare data, the study will assess existing barriers (organizational, personnel, and community level factors) and uncover available resources and facilitators for a successful implementation of the improved care model. The research will provide critically important evidence for implementation of effective Seek-Test-Treat, and Retain models for PLWH and OUD throughout Malaysia and inform healthcare policy in other low to middle income countries and regions with limited healthcare resources."}
{"text": "The proposed studies are designed to define the alterations in tubular sodium, chloride and bicarbonate reabsorption that occur when the number of functioning nephrons is reduced. Using micropuncture and microanalytic techniques in the rat, we will determine the relative contributions of changes in proximal and distal reabsorption in maintaining sodium balance and extracellular fluid composition in this model of renal insufficiency. These studies will be carried out for the first time in animals in which the surgically induced plasma losses are replaced to maintain volume status and renal function at levels seen in awake animals. The relative roles of alterations in single nephron GFR, aldosterone, peritibular physical factors, and \"natriuretic hormone\" in producing the alterations sodium transport will be systematically examined. With respect to tubular bicarbonate transport in the remnant kidney, experiments will be conducted to examine the role of changes in filtered load chronic acid-base disturbances, and parathyroid hormone in producing the observed changes in reabsorption. These studies should provide new insights into the nature of the adaptive response of the kidney to nephron loss."}
{"text": "PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human diseases. The focus of research, which had been placed mostly on development of therapeutic agents, has shifted gradually towards its prevention. In this context, many studies have linked obesity and long-standing type-2 diabetes mellitus with PDAC development. These metabolic diseases are characterized by peripheral insulin resistance, hyperinsulinemia, increased IGF-1 and chronic inflammation. Previously, crosstalk mechanisms between insulin/IGF-1 receptors, G protein-coupled receptor (GPCR) and EGF receptor (EGFR) signaling systems have been identified that potently stimulate proliferation of PDAC cells harboring a KRAS mutations. Mitogenic crosstalk depended on the function of mTORC1, ERK and PKD and opposed by AMPK, a target for the antidiabetic agent metformin. The identification of the key downstream targets of this signaling network is of fundamental significance and major translational interest. The YAP/TAZ transcriptional co-activators are emerging as points of integration in the action of KRAS, GPCRs and AMPK, all signaling pathways highly relevant in PDAC. New preliminary studies demonstrate that YAP/TAZ activation is a crucial point of convergence in the crosstalk between GPCR and insulin signaling in PDAC cells. Importantly, lipid-lowering drugs of the statin family potently blocked YAP/TAZ activity, including YAP/TAZ/TEAD-regulated genes, such as CTGF, Cyr61 and NUAK2. Lipophilic statins, including cerivastatin, simvastatin and atorvastatin, strikingly inhibited colony formation of human and mouse PDAC cells. Statins inhibited PDAC colony formation acting synergistically with metformin. Further preliminary results in vivo show that oral administration of simvastatin attenuated the loss of intact acini and the development of pre-neoplastic lesions in the pancreas promoted by an obesogenic diet in conditional KrasG12D (KC) mice. Accordingly, the central hypothesis to be explored in Project 2 of this P01 is that the well tolerated cholesterol-lowering drugs of the statin family inhibit obesity-induced promotion of PDAC via inhibition of PKD/YAP/TAZ. The Specific Aims of Project 2 have been designed to investigate important gaps in current knowledge: 1) Characterize the chemopreventive effects of statins on the progression of PanINs and development of PDAC using the conditional KrasG12D model (KC mice) subjected to control or diet-induced obesity (DIO) and in KC mice carrying a homozygous deletion of the ob gene. 2) Identify a novel molecular mechanism by which statins inhibit YAP/TEAD signaling in mouse and human pancreatic cells. 3) Characterize the inhibitory effect of a low-dose combination of statin and metformin on the development of PDAC: a novel chemopreventive strategy. The studies proposed in Project 2 of this P01 will provide mechanisms and rationale for novel chemo-preventive strategies in obesity-related PDAC. Since statins and metformin are widely used Food and Drug Administration (FDA)-approved drugs, the mechanistic and preclinical studies proposed have the potential for rapid translation to PDAC and other obesity-associated cancers."}
{"text": "[unreadable] [unreadable] Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardio-degenerative disorder for which there are currently no established effective treatments. The identification of the disease gene in 1996 and the subsequent elucidation of the function of the encoded protein, frataxin, have opened the door to possible therapeutic approaches, including conventional high-throughput drug screening. This proposal describes a novel, complementary approach to the development of therapeutics for FRDA. We devised a method to construct a library that encodes random, short-hairpin-loop RNAs (shRNAs). This library can be used to identify shRNA sequences of therapeutic, therapeutic-targeting, and/or biological interest. The identification is based on functional selection, with effective sequences retrieved by PCR from cells that survive a particular condition or exhibit a predetermined phenotype. The design allows for hit-optimization of effective sequences, with re-selection for sequences with improved effects. Using primary FRDA fibroblasts, we developed screening and selection assays based on the critical role of mitochondrial dysfunction in the signs and symptoms of FRDA and on the sensitivity of FRDA cells to oxidative stress. With our live/dead selection assays, we can use our random shRNA-encoding library to identify shRNA sequences of benefit to FRDA cells. With our screening assays of mitochondrial function, we can confirm and prioritize these sequences. The primary objective of this proposal is to identify potential shRNA therapeutics. A secondary objective is to begin to understand the mechanisms of action of the shRNAs we identify. The Specific Aims are: 1. To identify shRNA sequences that allow survival under conditions lethal to primary FRDA fibroblasts but non-lethal to normal control cells. 2. To optimize the shRNA sequences identified in Aim 1. 3. To confirm and prioritize optimized shRNAs and begin to understand mechanisms of action. This proposal describes an approach to develop novel therapeutics for Friedreich ataxia using a random shRNA library. This approach has potential implications for the development of therapeutics for other genetic diseases, as well as for infectious diseases, and is therefore highly relevant to public health. [unreadable] [unreadable] [unreadable] [unreadable]"}
{"text": "It is proposed to investigate the immunological status of mice with mammary cancer and of mammary tumor-prone and -resistant mice of several inbred strains. Emphasis will be placed on the analysis of both cell- mediated and humoral immune response to viral and tumor antigens associated with mammary tumors as a model for immunological studies on human breast cancer. In addition, techniques and concepts developed in animal studies will be concomitantly applied to human breast cancer material in a study of antigens in cells derived from human breast cancer patients and antibodies to these antigens in the sera of these patients. Conventional immunological methods to be applied in the study include analysis of lymphocyte-mediated toxicity, blastogenesis determinations, lymphoid cell migration inhibition, cytotoxicity, immunofluorescence, immunoelectron microscopy, immunodiffusion, and radioimmunoassay. The goals of the proposed research are to determine the role of viral and tumor antigens in the immune response to breast cancer in mice and in women and to determine by these methods whether viruses similar or related to mouse mammary tumor virus may be associated with the human neoplasm."}
{"text": "A search for potential regulatory proteins among the components of mammalian cell chromatin is in progress, using high-resolution, two-dimensional polyacrylamide gel electrophoresis. Several hundred rare species (less than 10,000 copies/cell) have been found in HeLa chromatin, and it has been shown that these are not detectable in cytoplasm. Comparisons with the protein of other human cell types are underway. Similar studies are in progress with Friend leukemia (mouse) cells, comparing protein patterns of control cells and cells induced to form hemoglobin. Several possible regulators of globin gene transcription and/or post-transcriptional processing of globin mRNAs have been identified. Attempts to test these proteins for specificity of interaction with globin mRNAs and/or globin genes are planned."}
{"text": "The overarching goal of this proposal is to create and validate pragmatic tools to rapidly detect and define the mechanism of diuretic resistance (DR), allowing individualized therapy in patients with acute decompensated heart failure (ADHF). ADHF is the most common hospital discharge diagnosis among Medicare beneficiaries and accounts for more than half of all heart failure (HF) related expenditures. This epidemic of ADHF is primarily driven by fluid and sodium overload, leaving the loop diuretics as the most commonly used medications to treat and prevent ADHF. Unfortunately, a loss of response to loop diuretics, termed diuretic resistance (DR), is common and contributes to a vicious cycle of out of hospital fluid/sodium retention, incomplete in-hospital decongestion, followed by post-discharge re-accumulation of fluid/sodium and worsened outcomes. Despite its importance, the speed and fidelity with which we can diagnose DR is extremely limited. This results in potentially avoidable hospitalization of outpatients and wasted hospital days where effective diuresis does not occur in inpatients. Once recognized, tools to determine the mechanism for DR and thus individualize treatment are nonexistent. Although multiple mechanisms contribute to loop DR, two therapeutically distinct groups exist: (1) inadequate effect at the tubular site of action, whih requires treatment with increased dose or delivery of loop diuretic and (2) compensatory distal tubular sodium reabsorption, which requires treatment with sequential nephron blockade (i.e., thiazide diuretics). Our inability to differentiate these mechanisms leaves trial and error as the only method to treat DR, further delaying effective diuresis and exposing patients to medications with known toxicities when the empiric choice is incorrect. In the present study, we will enroll 200 ADHF patients and evaluate them longitudinally through key transitions in loop diuretic therapy (early into IV therapy, late IV therapy, after conversion to oral diuretics, and 5-7 days post discharge). Patients with significant DR during early IV therapy will be randomized to increased loop diuretic dose or add on thiazide diuretic stratified by the DR mechanism. The data generated through the above investigation will allow us to: (1) develop inexpensive and efficient tools to predict diuretic response in a reliable and timely manner; (2) understand the prevalence of therapeutically targetable mechanisms of DR using endogenous lithium clearance, a gold standard technique to query in vivo proximal tubular/loop of Henle sodium handling; (3) develop methodology to differentiate DR mechanisms using common/inexpensive laboratory tests; and (4) provide proof of concept that mechanistically tailored diuretic therapy can improve natriuresis. Our preliminary data suggests that diagnosis and phenotyping of DR can in fact be done with excellent accuracy (AUC =~0.9) using universally available urine/serum chemistries. At the conclusion of this research, our goal is to provide clinicians and researchers with a workable tool to accurately/rapidly diagnose and phenotype DR allowing individualized diuretic therapy for both inpatients and outpatients with heart failure."}
{"text": "Investigators who have achieved independent National Eye Institute (NEI) funding will be provided with additional shared support to enhance their own and University of Washington's capability for conducting vision research. Collaborative studies will be facilitated, and scientists will be attracted to research on the visual system by the presence of this shared support. A modular organizational structure will be maintained, with each module devoted to a specific activity that would be impractical or less efficient to support on an individual research grant. Each module will support a service or resource that enhances or facilitates the research efforts of a CORE group of investigators, each having independent funding. Some sharing of resources and services with non-NEI-funded collaborators and with investigators new to vision research will occur. Junior researchers will also have access to these facilities to improve their ability to attain independent NEI funding. Proposed modules include: Biochemistry/Immunology (B/l), Genotyping/Phenotyping (G/P), Morphological Imaging (M/l), and Psychophysics/Physiology (PIP). Areas of investigation include retinal and choroidal diseases, corneal wound healing, corneal diseases, lens and cataract, glaucoma, strabismus, amblyopia, visual processing, and ocular development. Specific disciplines that will be brought to bear on these problems include: behavioral studies, biochemistry, biostatistics, molecular biology, cell biology, proteomics, immunology, microscopy, microbiology, morphometry, neurophysiology, and pathology. This project will elucidate basic mechanisms that underlie the function of the eye and the visual system and apply this knowledge and other information to the solution of problems in vision and ophthalmology. Collaboration among investigators from the University of Washington and elsewhere will be promoted. This proposal will improve the effectiveness of funding available on individual research project grants."}
{"text": "At total body radiation exposures of 3-8 Gy, the predominant cause of death is the hematopoietic syndrome. Many of these deaths are preventable with rapid triage of victims for cytokine therapies and aggressive supportive care. Unfortunately current modalities for identifying patients at risk for the hematopoietic syndrome suffer from inaccuracy, high expense, long analysis times, and delayed diagnosis. Furthermore, none of these methods directly measures radiation damage to the bone marrow, nor do they indicate the existence of residual hematopoiesis, and most are not amenable to point-of-care in emergency conditions. Thus, there is a critical unmet need for a field-deployable diagnostic for use following a radiological incident to identify victims who will develop the potentially fatal ye often treatable hematopoietic syndrome. We propose to build upon a highly successful paradigm in clinical diagnostics, which is that injured tissues leak, shed, and/or secrete proteins into the plasma, where they are useful biomarkers indicating the extent of tissue injury. Precedents include the troponins in myocardial infarction, transaminases in liver injury, creatine kinase in muscle injury, and lipase in pancreatic processes. Accordingly, we hypothesize that following radiation-induced injury to the bone marrow, proteins are released from the marrow into the bloodstream, where they provide useful biomarker signals predictive of the onset and severity of the hematopoietic syndrome. We propose to identify these plasma biomarkers of radiation-induced marrow injury using an innovative approach incorporating targeted proteomic technologies that we have developed and validated in a biomarker discovery pipeline that will substantially increase our chances of success compared with traditional approaches by enabling the testing of a very large (unprecedented) number of plasma biomarker candidates. Aim 1. Using SILAC-labeled mice, identify proteins induced in the bone marrow in response to radiation, and subsequently test hundreds of these putative tissue injury markers to identify the subset that are stably elevated in the plasma post-exposure. Aim 2: Characterize candidate marrow injury biomarkers identified in Aim 1 with respect to their: a. stability in plasma over time, dose range, and dose rates b. correlation with clinical endpoints (indicators of hematopoietic syndrome) c. specificity for damage to the hematopoietic system and damage caused by radiation d. use across a heterogeneous population (pediatric, geriatric, gender, genetically susceptible) Aim 3. Determine which of the radiation biomarkers of marrow damage identified in the mouse are elevated in human blood following radiation exposure, and develop a point-of-care assay device that will form the basis of subsequent human clinical validation trials (beyond this proposal)."}
{"text": "This study will examine patterns of care and management of mental illness among displaced persons in the Republic of Georgia, with a focus on decision-making about care- seeking and types of informal strategies and services used. The study will take a mixed methods approach with semi-structured interviews leading to instrument development. Background: There is limited research on mental health concerns or access to mental health care in low and middle income countries, including among displaced populations. Care-seeking to mental health services is impeded by limited access and stigma of mental illness. The Republic of Georgia is a country in political and economic transition that has experienced significant displacement within its borders. Available research on the displaced population in Georgia suggests that they experience higher rates of psychological disorders and chronic disease. It is not clear how mental health problems among the displaced are conceptualized, addressed, and managed. There is a need to study care-seeking behavior to formal services, as well as to informal services and within social support structures. Available literature on mental health services does not clearly delineate care-seeking and management processes to informal services. Understanding such care-seeking will inform the development of prevention and service delivery interventions. A mixed methods approach allows for in-depth exploration and the development of an instrument that can be adapted to different programmatic settings with migrant and displaced populations. Methods: The study site is Zugdidi, an urban center in northwestern Georgia. The study population is protracted, internally displaced persons (IDPs) ages 18 and over residing in the community. In-depth, qualitative interviews will be carried out with 30-45 IDPs and will include modules on migration history, problem recognition, perceived need, and care-seeking behavior. Interview data will be used to construct an instrument to measure patterns of care and management. The instrument will be pilot tested with 165 IDP households. Local service organizations will be partnered with for future validation studies and hypothesis testing."}
{"text": "Many important biological processes are regulated by tyrosyl phosphorylation, which is controlled by the opposing actions of protein-- tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). Abnormal regulation of these pathways can lead to diseases such as cancer. A complete understanding of cellular and whole organism regulation by tyrosyl phosphorylation requires defining how specific PTKs and PTPs interact. Such understanding may lead to the development of new, more specific therapeutic reagents for treating human disease. The long range goal of this research program is to understand the biological functions and mechanism of action of the SH2 domain-containing tyrosine phosphatase SHP2 in hematopoietic cells. Previous studies by several laboratories, including our own, established SHP2 as a vital component of signaling pathways downstream of cytokine receptors, hematopoietic growth factor receptors and multi- chain immune recognition receptors. These studies suggested that a 97kD phosphotyrosyl protein was a major target/regulator of SHP2 action, but the identity of this protein and its functions had remained unclear. During the initial funding period, we identified, purified and cloned p97. p97 is a relative of Gab1 and Drosophila Dos, a regulator of the fly homolog of SHP-2; thus, we renamed p97 as Gab2. Analyses of the effects of wild type and mutant versions of Gab2 suggest that it is a key regulator of cytokine signal transduction and also participates in signaling downstream of other types of hematopoietic cell receptors. Our studies establish that SHP2 acts at more than one step in cytokine receptor signal transduction: one requires Gab2 binding and the other is Gab-2 independent. The Ciab2-independent pathway is required for MAP kinase activation, whereas Gab2, acting via SHP2, regulates a novel signaling pathway downstream of/or parallel to MAP kinase activation but upstream of cytokine-induced gene expression. Our recent studies show that Gab2 is recruited to cytokine receptors via a novel complex involving Shc, and then provides the major route to PI-3 kinase activation for those receptors that do not bind PI3K directly. We will elucidate the details of the Shc/Gab2 pathway in cytokine signaling. and determine what other pathways are regulated by Gab2/PI3K complexes. Next, we will define how SHP2 regulates MAP kinase activation in a Gab-2- independent manner and how the Gab2/SHP2 complex regulates immediate early gene transcription. We will characterize the hematopoietic effects of \"true-null\" SHP2 knockout mice and Gab2-/- mice to determine the physiological function of these molecules in vivo. These studies should yield new insights into how tyrosyl phosphorylation is controlled. clarify how specific PTPs contribute to regulation, and identify a new signaling pathway regulating cytokine-induced gene expression. Since abnormal regulation of cytokine signaling is implicated in leukemogenesis and some carcinomas, our studies may also suggest new targets for therapeutic intervention in human neoplastic disease."}
{"text": "1. We are studying the transmembrane channel-like (TMC) genes 1 and 2. We have generated mice segregating knockout (null) alleles of Tmc1 and Tmc2. We are characterizing their mutant auditory and vestibular (balance) phenotypes. Mice that are homozygous for knockout alleles of both genes are deaf and have abnormal vestibular function. Mice that are homozygous for the Tmc1 knockout allele are deaf. Mice that are homozygous for the Tmc2 knockout allele have normal hearing and balance. These results indicate that both Tmc1 and Tmc2 are required for normal vestibular function (balance), whereas only Tmc1 is required for hearing. We use LacZ reporter genes in the knockout mice to study where the genetic interaction might be taking place. The Tmc1 reporter is expressed in hair cells of all of the vestibular end organs whereas the Tmc2 reporter is expressed in hair cells of the cristae ampullaris. We are now using hair cell markers to determine if these genes are expressed in type 1 or type 2 hair cells, or both. We are collaborating with Dr. Charley Della Santina to measure vestibulo-ocular responses in the mice to determine if their abnormal balance and gait is due to abnormal function of the semicircular canals (cristae) or other parts of the vestibular system. [unreadable] [unreadable] 2. We used a yeast two-hybrid screen to isolate genes encoding proteins that potentially interact with TMC1. We narrowed the list to a few candidate genes implicated in vesicular trafficking and in antiapoptosis and cell survival. We are using a combination of approaches to determine which interactions occur in situ in hair cells.[unreadable] [unreadable] 3. We generated knockout mice for Tmc6 and Tmc8 to better understand the function(s) of Tmc genes and proteins. Mutations in human TMC6 or TMC8 genes cause epidermodysplasia verruciformis, a recessive disease resulting in chronic cutaneous HPV infections (papillomas) with increased susceptibility to non-melanoma skin cancers. We have done extensive RNA expression analyses to show that Tmc6 and Tmc8 are primarily expressed in lymphoid cells and tissues and lung and skin, and primarily during development. The homozygous knockout mice have no obvious phenotypic abnormalities, so we are collaborating with Dr. Paul Lambert to determine if these mice have alterations in their susceptibility or response to papillomavirus infection.[unreadable] [unreadable] 4. We are continuing our work to identify the gene mutated in the mouse Twirler strain. Heterozygous Twirler mice have inner ear malformations and obesity, whereas homozygous mice are born with cleft palate and die at birth. The critical interval containing the Twirler gene is approximately 750 kilobases and contains one known gene and two predicted genes. We have identified a probable mutation. We have generated a knock-in mouse line with this mutation to confirm its pathogenicity. We have completed a comprehensive characterization of the Twirler obesity and inner ear phenotypes. The obesity phenotype is associated with hyperphagia of unknown etiology and insulin-resistant diabetes mellitus. The inner ear phenotype is characterized by hypoplasia or dysplasia of the semicircular canals; the lateral canals are most severely affected.[unreadable] [unreadable] 5. Enlargement of the vestibular aqueduct (EVA) is the most commonly detected radiologic malformation in temporal bones of individuals with hearing loss. A significant proportion of EVA cases have been reported to be associated with mutations of the SLC26A4 gene, in which mutations cause Pendred syndrome. PS is an autosomal recessive disorder comprised of bilateral sensorineural hearing loss and a defect in the ability of the thyroid gland to organify iodine. EVA is a universal finding in the ears of affected PS individuals. PS is correlated with two mutant SLC26A4 alleles, and nonsyndromic EVA is associated with one or zero mutant SLC26A4 alleles. Based upon our data, we hypothesize that one or more other genetic or environmental factors may act alone or in combination with a single SLC26A4 mutation to cause EVA. We have completed a comparative genome hybridization-microarray analysis to search for deletion and duplication mutations of SLC26A4 that might cause EVA. We have also PCR-amplified and sequenced all noncoding conserved sequences in and around SLC26A4. We found not evidence for such mutations. [unreadable] [unreadable] We have completed segregation analyses of EVA to gain insight into the causes of EVA in patients with one or zero mutant alleles. The results are consistent with inheritance of a second recessive allele in the patients with one detectable SLC26A4 mutation, whereas the results for patients with no mutant alleles of SLC26A4 suggest one or more of the following: autosomal recessive inheritance of alleles at another locus, non-genetic causes, a complex etiology of two or more genetic and/or non-genetic factors, or a variety of different etiologies among different patients. These results yield recurrence risk estimates, dependent upon the number of SLC26A4 mutant alleles, that are very helpful for counseling patients and families with EVA.[unreadable] [unreadable] We analyzed our genotype and phenotype data to identify clinical features that guide molecular diagnosis or clinical prognosis. We comprehensively and quantitatively analyzed the thyroid phenotypes in our EVA cohort. Our study is the first to rigorously analyze and describe the ultrasonographic phenotype of the thyroid gland in EVA patients. Our results show that thyroid gland volume (goiter) is dependent upon the number of mutant alleles in pre-pubescent subjects whereas it is dependent upon age in post-pubescent subjects. Therefore goiter is a more specific sign of biallelic SlC264A4 mutations (Pendred syndrome) in children than in adults. We continue to observe a strong correlation of an abnormal perchlorate discharge test with two mutant alleles of SLC26A4. Our data provides a basis for the surveillance and management of the thyroid gland in EVA subjects.[unreadable] [unreadable] We have completed functional studies of several missense substitutions of SLC26A4 that have always or usually been detected as the single SLC26A4 variant in nonsyndromic EVA patients and shown that they appear to have wild type trafficking and functional activity, indicating they are benign polymorphic variants. These results have important implications for molecular diagnosis of EVA patients, as well as for categorizing patients according to SLC26A4 genotype for studies to identify other causes of EVA.[unreadable] [unreadable] 6. We ascertained a large North American family segregating progressive, nonsyndromic sensorineural hearing loss in a matrilineal/maternal/mitochondrial pattern of inheritance. We sequenced the entire mitochondrial genome in several affected individuals and have identified a rare mutation in the tRNA-Ser(UCN) gene. We analyzed the mitochondrial haplogroup associated with this mutation and compared it to that associated with this same mutation in a previously reported family. Our results show the haplogroups are different, and we did not find the mutation in haplogroup-matched normal control samples. Our study confirms the pathogenicicty of the tRNA-Ser(UCN) mutation in sensorineural hearing loss.[unreadable] [unreadable] 7. The research laboratory of Dr. Guy Van Camp (University of Antwerp, Belgium) discovered a family with dominant progressive hearing loss caused by the D572N mutation of TMC1, the same mutation causing hearing loss in a family we studied. We compared the mutation-linked genetic marker genotypes and haplotypes, and found they were different between the two families. This indicates that the mutations probably arose independently, not from a common founder.[unreadable] [unreadable] 8. We performed a genotypic survey for DFNB7/B11 deafness caused by TMC1 mutations in Pakistan. We identified several novel mutations and showed that TMC1 mutations account for approximately 3.4% of severe to profound, prelingual-onset deafness in Pakistan."}
{"text": "Conventional methods for treating chronic diseases such as HIV/AIDS have proven to be relatively inefficient both in terms of administration of therapeutic molecules and eradication of the underlying diseases themselves; while conventional antiretroviral therapy (ART) suppresses plasma viremia, low-level viral replication persist. Our recent findings have indicated that pigtailed macaques transplanted with gene-modified CD34+ cells expressing a membrane-anchored fusion inhibitor (mC46) develop infection-resistant CD4+ T-cells, maintain normal CD4+ T-cell levels, and develop an enhanced immune response against the SHIV-challenge virus resulting in a 300- to 1400-fold decrease in plasma viremia. Despite these very encouraging results, additional methods to further reduce plasma viremia to undetectable levels and/or potentially eliminate viral reservoirs will be required. Hence, we propose the development of a novel delivery system based on modifying hematopoietic stem cells (HSCs) or CD4+ T-cells in order to directly target viral reservoirs in vivo. The use of primary cells to deliver therapeutic peptides represents an innovative and ideal mode for systemically delivering therapeutic molecules throughout a patient's body. Furthermore, the ability of HSC-derived lineages to transverse both physiological and anatomical barriers represents a unique potential of this therapy. Low levels of engraftment would likely yield sufficient quantities of secreted antiretroviral peptides, while the efficiency f uptake would be greatly enhanced as there is a continuous source of the therapeutic molecules at sites typically thought to maintain persistent viral reservoirs. In order to deliver proviral targeting endonucleases to latent reservoirs, terminally differentiated primary cell such as CD4+ T-cells that migrate throughout lymphoid tissue represents the ideal vehicle. Hence, the goal of this study is to examine the feasibility of using genetically modified HSCs and CD4+ T-cells to secrete antiretroviral molecules to control viral replication and directly target latent reservoirsin vivo."}
{"text": "The storage and mobilization of lipid are fundamental cellular processes that are conserved throughout metazoan evolution and are present in virtually all mammalian cells. Intracellular FFA (FFAi) are normally kept under tight control since excessive FFAi are toxic. Indeed, excessive accumulation of intracellular FFA and FFA metabolites leads to cellular dysfunction, termed 'lipotoxicity', which is thought to be a major means by which obesity contributes to diabetes and cardiovascular disease. Thus, a mechanistic understanding of how cells assimilate, mobilize and channel FFA is an important biological question with broad implications for health and disease. The uptake and mobilization of FFA within cells appear to have important temporal and spatial domains. Nonetheless, investigation of the \"when and where\" FFA metabolism occurs within live cells has not been possible because intracellular FFA levels cannot presently be imaged. Therefore, the aim of the R21 application is to develop and implement an optical sensor of FFAi having high spatial and temporal resolution for biological applications. This sensor is designed to be used as a general cytosolic sensor, or can be specifically targeted to proposed subcellular sites of FFA uptake, mobilization and oxidation. We anticipate that a validated sensor will have wide applications and will lead to new insights into biology of cellular lipid trafficking. PUBLIC HEALTH RELEVANCE: The storage and mobilization of lipid are fundamental cellular processes that are present in virtually all mammalian cells. Intracellular FFA (FFAi) are normally kept under tight control and since excessive FFAi are toxic and can contribute to obesity-related diabetes and cardiovascular disease. Thus, a mechanistic understanding of how cells assimilate, mobilize and channel FFA is an important biological question with broad implications for health and disease. Evidence indicates that the uptake and mobilization of FFA within cells has important temporal and spatial domains, however, analysis of these dynamics has not been difficult because intracellular FFA levels cannot presently be imaged. Therefore, the aim of the R21 application is to develop and implement an optical sensor of intracellular FFA with high spatial and temporal resolution for biological applications. This sensor can be used as a general cytosolic sensor, or can be specifically targeted to key subcellular sites of FFA uptake, mobilization and oxidation. We anticipate that a validated sensor will have wide applications and will lead to new insights into biology of cellular lipid trafficking."}
{"text": "The overall objective is to develop an organoselenium compound with lowest toxicity and yet maximal chemopreventative efficacy against colon cancer, and to understand the mechanisms of cancer inhibition by this agent. Our studies indicate that 1,4-phenylenebis(methylene)selenocyanate (p-XSC) is superior to other organoselenium compounds against colon carcinogenesis. We hypothesize that the metabolism of p-XSC by glutathione leading to the formation of putative glutathione conjugate (p-XSe-SG) and/or metabolite of p-XSC, tetraselenocyclophane (TSC) are responsible for its chemopreventative activity. Initially, 40 and 80% MTD levels of these two agents and 80% MTD of p-XSC (parent compound) will be evaluated for their chemopreventative efficacy during initiation and post-initiation phases of AOM-induced colon carcinogenesis. Five week old male F344 rats will be fed high-fat diet or 40 and 80% MTD levels of organoselenium compounds and at 7 weeks of age, all animals except vehicle-treated groups will be administered s.c., AOM (15 mg/kg b.w., once weekly for 2 weeks) and one week later, animals on control diet will be transferred to organoselenium diets and those on organoselenium diets will be transferred to control diet and continued on this regimen until 50 weeks and sacrificed. Histopathological evaluation of colon tumors will be performed and organoselenium exhibiting highest chemopreventative index will be evaluated during promotion/progression phase. Groups of 5-week old male F344 rats will be fed a high-fat diet. At 7 weeks of age groups of rats in each dietary group will be treated s.c. with AOM as above. Fourteen weeks later, groups of animals on high-fat diet will be transferred to high-fat diet with and without organoselenium compound and continued on this regimen until termination of this study. Animals will be sacrificed at 30 and 50 (termination) weeks after second AOM injection. Colon tumor incidence and multiplicity among the dietary groups will be compared by appropriate statistical methods. The mechanisms of colon tumor inhibition by this compound will also be examined. These aspects will include measurement of colonic mucosal and tumor (a) glutathione peroxidase (b) cyclooxygenase (COX-1 and COX-2) isozymes (c) apoptosis, and (d) F/2- isoprostane as an indicator of lipid peroxidation and oxidative damage; and (e) DNA cytosine methylation and DNA methyl transferase during different time periods."}
{"text": "Telomeres are made up of repeat sequences of nucleotides at the ends of human chromosomes. The function of telomeres is to protect chromosomes from degradation and to maintain their structural integrity. Telomeres are analogous to a molecular clock reflecting the number of divisions a cell has undergone and cells with critically short telomeres are predisposed to enter senescence. Previous research suggests that telomere length is reduced in association with lifestyle and clinical factors that are common in study spinal cord injury (lac of exercise, obesity, chronic or recurrent inflammation from skin ulcers, and urinary tract infections) as well as in persons with cardiovascular and pulmonary diseases. These latter diseases are the most common causes of death in chronic spinal cord injury. By using telomere length as a molecular biomarker, it is proposed to study spinal cord injury as a disease state that promotes accelerated aging at the cellular level. It is hypothesized that greater central obesity determined by DXA scan and greater systemic inflammation assessed by plasma C-reactive protein and interleukin-6 will be associated with shorter telomere length, and that persons with shorter telomere length will have reduced pulmonary function. This pilot project will obtain preliminary data regarding these associations in 350 persons with chronic SCI, and assess longitudinal associations between systemic inflammation and telomere loss in a subset. The study of telomere length is significant since it may serve a biomarker of persons with chronic spinal cord injury most likely to develop of chronic cardiopulmonary disease and premature mortality. PUBLIC HEALTH RELEVANCE: Telomeres are a part of human chromosomes that function to protect chromosomes from damage. It has been proposed that telomeres are analogous to a molecular clock reflecting the number of divisions a cell has undergone. Cells with critically short telomeres ultimately undergo cell death. The proposed pilot study will obtain preliminary information regarding associations between reduced telomere length with greater central obesity, reduced pulmonary function, and increased blood biomarkers of systemic inflammation. It is proposed that the measurement of telomere length in persons with spinal cord injury will be a molecular biomarker that can be used to detect persons at greatest risk to develop reduced pulmonary function and cardiopulmonary diseases. This study is consistent with the Veterans Health Administration's commitment to providing longitudinal care to persons with chronic spinal cord injury and supporting research directed at understanding causes of disability and death."}
{"text": "The Central Core Media Laboratory will be responsible for the production of stringent quality-controlled sterile tissue culture and bacteriological media and buffers necessary for in vitro research by all laboratory researchers at the University of Texas M.D. Anderson Cancer Center. Numerous pathogen-free chemically defined solutions will be available for immediate delivery at lower cost than commercial products. The Central Core Media Laboratory will be located in the new Bertner Clinical Research Building, in T5.3964 and consist of approximately 1,300 square feet of laboratory space and 200 square feet of cold-room space. The high trained staff will offer personalized service tailored to each researcher's needs. Two research technicians will prepare solutions and perform quality-control checks. A facility manager will be responsible for performance evaluations, accounting procedures, and general supervision of the laboratory. The facility manager will report regularly to the project investigator. An oversight committee will meet quarterly to assist with the commission of the facility and in determining policy changes. This centralized resource will alleviate the duplication of personnel and valuable laboratory space by concentrating these services into one dedicated location."}
{"text": "We propose an innovative video teleconference hosted by the Brown University Center for Primary Care and Prevention and the University of Oxford Department of Primary Healthcare with the goals of: 1. Promoting evidence-based best practice models of cancer prevention though applied genetics by primary care physicians and 2. Advancing the role of the primary care scholar in translational genetics research. Our specific aims are to translate emerging genetic epidemiological and pharmacogenetics research into evidence-based practice models for primary care physicians by: 1. Fostering a broad understanding of the emerging role of the new genetics in primary care. 2. Promoting a culturally-appropriate skill set to identify, screen, and counsel patients with an elevated heritable risk of cancer and other illnesses. 3. Addressing key policy and ethical implications of genetics for primary care. 4. Identifying strategies for organizing primary care office system to promote the use of genetic technology. 5. Envisioning the new frontier of primary care genetics research from \"bench-to-bedside-to-society\". Trans-disciplinary experts from primary care, genetic epidemiology, behavioral medicine, and health policy will present an interactive trans-Atlantic forum to promote the development of key knowledge, skills, and practice behavior among practicing, community-based primary care physicians. This half-day conference will take place in Providence, Rhode Island, USA, with video simulcast to London, United Kingdom, and will serve as the nidus for synergistic collaboration in translational genetics research and education."}
{"text": "Glomerulosclerosis is the common pathologic process responsible for 90% of End Stage Kidney Disease (ESKD) associated with diabetes, hypertension, IgA nephropathy, Focal Segmental Glomerulosclerosis (FSGS), lupus and other glomerular diseases. Together these diseases cost approximately $18 billion per year in the US, and carry high morbidity and mortality. They are increasing in prevalence and are commoner in minority populations. They affect both children and adults, although ESKD is particularly a disease of aging with average onset of ESKD treatment at 64 years. The central hypothesis underlying this work is that glomerulosclerosis is caused by depletion of one of the glomerular cell types, the podocyte. During the prior grant cycle we developed a new transgenic model system in the rat where we can deplete podocytes by an amount and at a time of prior choosing. We show that podocyte depletion does indeed cause all the features of FSGS in a dose-dependant manner. Second we have developed a new method for counting podocytes in glomeruli. Third, we have shown how glomerular enlargement during aging on a high calorie diet causes relative podocyte depletion that predisposes to glomerulosclerosis, and how this can be prevented by calorie restiction. Because we know that hypertension is commonly associated with glomerulosclerosis and progression of glomerulosclerosis to ESKD, these new tools and concepts will be used to test specific hypotheses relevant to human glomerulosclerosis: Aim 1: Podocyte depletion itself causes systemic hypertension and mesangial expansion, an important pathologic component of early glomerulosclerosis in man. Aim 2: Test the hypothesis that limitation of the ability of the podocyte to increase in size makes the glomerulus more susceptible to glomerulosclerosis, while enhancement of podocyte's ability to increase in size prevents glomerulosclerosis, by using transgenes of the mTOR pathway of cell size control to modulate podocyte size. Aim 3: Validate the novel concepts of podocyte alteration in phenotype in response to stress that we have termed \"adaptation\" and \"decompensation\" in two rat models of glomerulosclerosis, with the underlying concept that understanding the biology of this process will allow us to develop markers for human podocyte stress as well as new approaches to treat and prevent podocyte stress before it results in loss of podocytes and consequent glomerulosclerosis. [unreadable] [unreadable] [unreadable]"}
{"text": "Corneal hydration and transparency are dependent on the ion and fluid transport properties of the corneal endothelium. Fluid transport from stroma to aqueous humor is apparently coupled to the net transport of Na and HCO3 and is sensitive to the pH of the bathing solution. A complete model for ion coupled fluid transport however, is not available. Since extra- and intracellular [HCO3] are determined by extra- and intracellular pH (pHo and pHi), it is reasonable to suggest that any model of HCO3 transport will require an understanding of pHi regulation. This notion is supported by work showing that additions of drugs which are known to influence pHi regulation (amiloride and DIDS) also inhibit endothelial fluid transport. This study proposes to identify and characterize the pHi regulatory and HCO3- transport mechanisms present in fresh and cultured bovine corneal endothelial cells by utilizing the pH sensitive intracellular fluorescent probe, BCECF. Cells will also be grown on filter supports to allow independent access to apical and basal surfaces in order to determine the locations of the HCO3 transport mechanisms and establish a complete model for transendothelial HCO3- transport. Transendothelial fluid secretion can also be measured across filter cultures so that the HCO3- transport mechanisms essential for fluid transport and the transport mechanism-that is rate limiting for fluid flux can be identified. A long term goal is to identify agents or conditions that stimulate HCO3- transport and test their ability to stimulate fluid secretion as well. Lastly, post-surgical specimens of Fuch's Endothelial Dystrophy will be obtained and the pH regulatory and HCO3- transport capacities will be tested in order to determine if and how pump function is deranged in this disease. Once the transport deficit is identified, the long term goal is to test drugs which stimulate it or drugs that inhibit an opposing transport mechanism in order to enhance fluid secretion in this diseased endothelia."}
{"text": "Defects within the insulin signaling pathways may lead to insulin resistance in peripheral tissues. The prevalence of obesity generally increases with age and is associated with insulin resistance characterized by an elevation in hepatic glucose production, impaired glucose tolerance and defects in the ability of the insulin receptor to transduce insulin signals. Cross-sectional studies have associated obesity and other components of the so-called metabolic syndrome with low-grade inflammation. Hence, better understanding of the complex interplay between proinflammatory cytokines and insulin signal transduction could lead to the development of effective strategies to reduce or delay the progression of complications associated with insulin resistance states, such as Type 2 diabetes. A strong positive correlation has been reported between adipose tissue levels of adipocytokines (e.g., interleukin (IL)-6, tumor necrosis factor (TNF)) and the increased secretion of acute phase proteins by the liver of obese subjects. IL-6 activates the Janus kinase/signal transducer(s) and activator(s) of transcription (JAK/STAT) pathway to elicit the synthesis and secretion of several hepatic inflammatory proteins. In response to IL-6, JAK induces tyrosine phosphorylation of STAT3 (pY-STAT3) at a single residue, followed by its nuclear translocation for the inducible expression of genes through binding to specific DNA responsive elements. In addition to tyrosine phosphorylation, STAT3 is serine phosphorylated at a single residue in response to IL-6, which enhances its transcriptional activation. We have recently completed a study showing that the antioxidant pyrolidine dithiocarbamate (PDTC) was protective against IL-6?induced JAK/STAT3 signaling in liver-derived human HepG2 cells and a primary culture of rat hepatocytes. We found that PDTC inhibited IL-6?stimulated phosphorylation and nuclear translocation of STAT3 by interfering with the inducible association of STAT3 with an adaptor protein. This reduction in STAT3 activity led to a sharp decrease in acute phase protein gene expression in response to IL-6 as determined by microarray analysis and Northern blot. Of significance, pretreatment of rat hepatocytes with PDTC selectively reduced the adverse effects of IL-6 on insulin-stimulated Akt phosphorylation. It is established that activation of Akt plays a major role in various biological processes triggered by insulin. It remains unknown whether PDTC can inhibit the development of insulin resistance in obesity and in aging, two chronic in vivo models of low grade inflammation. In a second project, we investigated the role of actin-binding proteins in the promotion of STAT3 signaling in response to IL-6. The IL-6-mediated increase in JAK/STAT activity is initiated at the level of raft plasma membrane microdomains of diverse cell types. Despite the fact that STAT3 has been shown to localize to the actin cytoskeleton, little is known about the involvement of actin-binding proteins in regulating the JAK/STAT signaling pathway. Therefore, we hypothesized that filamin A may be a component in STAT3 activation by IL-6. Human melanoma M2 cells that lack filamin A and filamin A-expressing M2A7 cells were used in a number of experiments to assess the kinetics of STAT3 phosphorylation, its nuclear translocation and transcriptional activity following IL-6 stimulation. Because Ser727 of STAT3 is located in a conserved Pro-Xaa-Ser/Thr-Pro site, which is recognized as a phosphorylation site for members of the mitogen-activated protein (MAP) kinase family, we have also defined which subgroup(s) of the MAP kinases is involved in regulating serine phosphorylation of STAT3. Our results show a functional role of filamin A in the modulation of IL-6-induced responses. While these cell lines are suitable for initial in vitro studies, they are surrogates for more physiologically relevant models. Toward this goal, functional studies in vivo will be carried out to address the potential relevance of filamin in the proinflammatory actions of IL-6."}
{"text": "The specific aims of this proposal are to investigate the mechanism of rejection of the murine ovarian teratocarcinoma that follows intraperitoneal injection of Corynebacterium parvum. We will test the hypothesis that the acute inflammatory response to the injection of microbial biologic response modifiers is capable of influencing tumor growth. The inflammatory neutrophil (PMN) is envisioned as initiating two distinct anti-tumor responses that occur sequentially. The first effect is direct tumor cell lysis induced through activation of the PMN by bacteria complexed to C'3b. Activation results in the release of reduced oxygen metabolites which lyse tumor cells within the peritoneal cavity. The second effect is the activation of tumor-cytostatic macrophages by inflammatory PMNs that have ingested bacteria. The long range goals of the proposal are to improve survival of patients with ovarian cancer. The use of the current animal model as a preclinical system for studying regional immunotherapy in human ovarian cancer is expected to lead to a better understanding of and offer insight into possible improvements in it for these patients. (TA)"}
{"text": "Nanotechnology Materials Core: Project Summary/Abstract One of the primary areas of focus in the Koch Institute is the development of new materials systems and biomedical devices for cancer prevention, detection and treatment. These systems underpin development of new approaches for biomarker detection, imaging modalities, and targeted therapies and vaccines. Center Members are exploring a wide range of materials to achieve these goals, and employ combinatorial development approaches to facilitate the discovery of new biomaterials and nanoformulations. State-of-the-art capabilities for high resolution and surface imaging and chemical and physical analysis are key tools to enable these studies. To fully characterize new materials, establish structure-property-function relationships, and facilitate subsequent rational design, we established the Nanotechnology Materials Core in 2012. In the previous competing renewal, the Core was approved for CCSG funding as an established Shared Resource. This Core integrates a range of advanced technologies including a state-of-the-art cryo-electron microscopy (cryo-EM) imaging suite, which enables high resolution analyses of soft biomaterials and cellular ultrastructure. In the current funding period, the Core added a significant number of new capabilities and modified existing services, including: new EM sample preparation instruments; new EM and correlative light and EM imaging systems; new physical/chemical characterization capabilities; and upgrades to existing instruments. To best capitalize on these acquisitions, CCSG Developmental funds were used to support two Cryo-EM Specialists. Core service usage by Center Members has increased, from 14% at the time of the last renewal to 43% of Center Members, who account for 79% of Core service use and include investigators from all three Research Programs. Core staff has established cryo-EM workflows that are customized for Center Member research needs, and we seek continued, partial funding to support and stabilize the Research Specialists? efforts. In the upcoming period, the Nanotechnology Materials Core will continue to offer a wide range of state-of-the- art services to support Center Member research programs, and will evaluate emerging capabilities in the context of Center Member needs and interests. A primary focus will be on refining new workflows to take full advantage of newly acquired instrumentation. Other planned initiatives include: developing resources to support advanced image processing, visualization, quantitation and machine learning, and collaborating with other Cores to develop a monthly workshop series to support Center Members? access to imaging technology platforms and data analysis. This Shared Resource is essential to the success of the Koch Institute mission and provides exceptional value to the CCSG. The requested CCSG budget for Year 49 is decreased by 40.6% over the Core CCSG budget for the current period (Year 48), reflecting the increased fraction of support from user chargebacks as Core staff is moved from CCSG Developmental funds to CCSG Core funds."}
{"text": "This Program Project is concerned with three general areas of neurobiological investigation each of which combines studies of structure and function in attempts to provide correlations useful in defining the elementary processes underlying the organization of complex neural activities. Studies will be carried out on neuronal development, interaction and organization. A wide variety of species and preparations will be employed in order to examine: 1) the basis for electrotonic coupling between neurons in systems where coupling is demonstrable and 2) the contribution of synaptic vs. ephaptic interactions in neuronal organizations subserving highly synchronized activities. Morphophysiological studies of developing neurons and synapses in tissue culture, and in vivo systems will be carried out with emphasis on the role of hormonal influences in altering developmental features. Electron microscope as well as histochemical studies of synapses will be carried out in conjunction with electrophysiological investigations of synaptic activities and developing neural systems in different vertebrate species. Studies of the mechanism of hormone actions on developing neurons and synaptic complexes will also be pursued. Neuroendocrine relations will be studied in other aspects of this Program and investigations will be continued on the organization of subcortical systems regulating sensorimotor activities in the mammalian brain."}
{"text": "The aim of this proposal is to use personal digital assistants (PDAs) to integrate psychosocial assessments and interventions (based on Social Cognitive Theory) for disordered eating prevention for at-risk college women. PDAs have been used in two distinct capacities: (a) an assessment device employing Ecological Momentary Assessment (EMA); (b) a device for delivering interventions. The proposed work is innovative because it combines the assessment and intervention capacities of PDAs. Participants will complete EMA reports, and micro-interventions (Mis) will be administered on PDAs based on concurrent EMA responses. Mis are brief treatment extensions delivered on portable devices. The Ml will be targeted to three specific experiences (eating, media exposure, negative mood) when women are most vulnerable to body image dissatisfaction (identified in preliminary research). Undergraduate women at-risk of developing disordered eating (n=144) will be randomly assigned to a CD-ROM intervention and EMA reports (EMA+CD), CD, EMA and micro-interventions (EMA+CD+MI), or EMA only (EMA) groups. All participants will complete baseline assessments of body image dissatisfaction, disordered eating, and psychological well-being. Six times daily for the following 1 week, participants will complete EMA surveys (including body image dissatisfaction, eating behavior, media exposure, and mood items) on PDAs. The EMA+CD and EMA+CD+MI groups will receive the Food, Mood, and Attitude CD-ROM intervention. One week later all participants will receive the PDAs again and complete the same EMA survey. The EMA+CD+MI group will also receive targeted. Mis consist of reminders to use skills learned in the CD-ROM and will be administered on the PDA immediately following the survey, and then 10 to 20 minutes following the EMA. All participants will complete 1- and 3-month follow-up assessments of disordered eating, body image dissatisfaction, and psychological well-being. The EMA only group will receive the CD-ROM intervention after completing the 3-month assessment. All participants will be given referral information if they wish to seek additional care. The public health significance of this research includes the innovative development of a methodology to increase the efficacy of eating pathology prevention programs (i.e., micro-interventions). These prevention programs will prove useful in reducing the incidence of eating disorders and have implications for other treatments and prevention strategies, by informing the development and implementation of treatment extensions more broadly. [unreadable] [unreadable] [unreadable]"}
{"text": "Epidemiological studies have consistently shown a linkage between individuals who chronically consume significant amounts of alcohol and hepatitis C viral (HCV) infection. The mechanism by which HCV is able to efficiently evade immune responses is still unknown. It is the purpose of this proposal to study the effects of chronic alcohol consumption on the expression of Toll-like receptors (TLR) and the function of hepatic plasmacytoid (p) dendritic cells (DC). This subset of DC has become of great interest as they are speculated to be primary responders in viral infection, producing large amounts of interferon-alpha upon stimulation. Studies on various TLR have shown that activation through these receptors can modulate the subsequent DC response in a distinct manner and thus influence the activation of T cells. We plan to investigate alterations in the expression and function of these receptors on pDC in established in vitro and in vivo mode s of culture-generated pDC and chronic alcohol consumption. If there are defects in the development of pDC or receptor expression as a result of chronic ethanol intake, it may be possible to promote an appropriate immune response through adoptive transfer of non-ethanol exposed culture-generated pDC. [unreadable] [unreadable]"}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Pathologic processes affect the concentrations of certain metabolites or neurotransmitters as well as local surroundings such as pH and blood flow. Glycine is a neurotransmitter that serves as an important inhibitory neurotransmitter in the central nervous system. Glycine-dependent synapses are found to be highly concentrated in the brain stem, retina, and spinal cord. The presence and location of glycine receptors suggest that glycine signaling might be a key player in the neural pathology of many motor and cognitive diseases;some notable examples include Amyotrophic Lateral Sclerosis (ALS), Parkinson's disease, hyperekplexia, and paroxysmal movement. Measurement of these factors invasively is difficult and may pose more harm to the patient than benefit. While magnetic resonance spectroscopy (MRS) has previously been used to measure neurotransmitter concentrations in the central nervous system (CNS), this technique lacks the adequate spatial resolution to be used in routine clinical studies. The ability to measure such aspects non-invasively at high resolutions would be a major breakthrough in the diagnoses of these and many other disorders. Chemical exchange between labile protons of proteins and water protons can make Magnetic Resonance Imaging (MRI), utilized mainly for the detection of bulk water signal, sensitive to information about the concentrations of endogenous proteins and their environments. Recently, a technique called Chemical Exchange Dependent Saturation Transfer (CEST), which uses the attenuation of bulk water magnetization through magnetization exchange with saturated labile protons, has been used to characterize properties of dilute labile groups. While CEST studies have explored numerous metabolites, there have been no studies demonstrating the CEST effect in glycine. Spin-lattice relaxation in the rotating frame (T1[unreadable]) is another contrast technique that depends on chemical exchange. There have been no studies to date using T1[unreadable] for contrast in the chemical exchange of protons. T1[unreadable] Chemical exchange effects vary quadratically with the static magnetic field. Therefore, T1[unreadable] potentially offer higher sensitivity at higher fields in probing exchange mediated interactions in nuclear spin systems. This higher sensitivity of T1[unreadable] MRI may enable detection of metabolites with very low concentrations (~1 mM) in the brain. We hypothesize that it is possible to quantify the CEST effect in glycine at higher magnetic fields (e3T). Also, that T1[unreadable] MR imaging has higher sensitivity to proton chemical exchange than the CEST method at ultra-high static fields. Finally, we believe that it is feasible to measure glycine in vivo in the spinal cord. These hypotheses will be tested by accomplishing the following specific aims: Aim #1: To measure the chemical shift of glycine in-vitro under physiological conditions using CEST imaging. Aim #2: To determine the effect that concentration, pH, static magnetic field, B1 field strength and saturation time have on the chemical exchange of glycine. Aim #3: To demonstrate, at varying static magnetic field strengths, that the chemical shift of glycine can provide contrast on T1[unreadable][unreadable]weighted images and that at ultra-high static fields it shows greater sensitivity than the CEST effect. Aim #4: To measure glycine concentrations non-invasively in-vivo in rat models using CEST and T1[unreadable][unreadable]weighted images."}
{"text": "The overall goal of the Assays and Reagents Core is to provide state of the art assay methods, reagents, advice, training, assistance, and service in ways that will increase the cost effectiveness, quality, and timeliness of research performed by principal investigators of eligible projects. Specifically, the Core will: obtain, inventory, and maintain adequate stocks of analytical reagents needed by eligible projects (this includes developing, purchasing in bulk, requesting gifts or chemically synthesizing immunogens, first and second polyclonal and monoclonal antibodies and antibody fragments, standards, other reference and quality control preparations, and tracers labeled with radioiodide, biotin or enzymes); characterize and test acquired reagents and make them available to eligible projects; meet the needs of low volume Users, individuals with inadequate laboratory support, and Users with other needs by providing high quality, cost-effective service assays on a samples-in, results-out basis; incorporate advanced analytical quality control procedures in all activities; maintain performance records, inventories, and documentation for all reagents and assay systems provided by the Core; provide timely information, assistance, and advice in utilizing the methods effectively; develop or otherwise implement improved, state of the art assay methods and analytical procedures that reduce the use of radioactive isotopes and provide increased specificity, sensitivity, reliability and ease of use; provide quality control assessments for assays and assay kits provided to eligible projects and; teach assay technology and immunoassay data processing, and provide training and instruction in radioactive and hazardous materials safety, in universal precautions for biohazards, in laboratory safety and sanitation, and in applicable regulatory compliance."}
{"text": "Cholesterol homeostasis is maintained through coordinate regulation of cholesterol uptake, esterification, synthesis, secretion, and its degradation to bile acids account for approximately half of daily net cholesterol elimination, while remaining 50% takes place almost entirely via biliary cholesterol secretion. Bile acid synthesis from cholesterol occurs via two pathways. The initial/rate-determining enzyme in the major \"neural\" pathway of bile acid biosynthesis is microsomal cholesterol 7alpha-hydroxylase (C7alphaH. The \"acidic\" pathway of bile acid synthesis is initiated by sterol 27-hydroxylase (S27H). Whereas C7alphaH is present almost exclusively in the liver, S27H is also present in many extrahepatic tissues, where its function remains unclear. The overall objective of the proposed studies is to better define the physiologic role of these key enzymes in the maintenance of hepatic and total body cholesterol homeostasis. More specifically, we propose a systematic series of in vivo and in vitro studies, in both liver and non-liver cells, using recombinant adenovirus to deliver CMV driven genes encoding C7alphaH and S27H. These studies attempt to combine the techniques of molecular biology with recombinant adenoviral gene transfer to determine the kinetic and molecular changes in the pathways of cholesterol homeostasis which follow selective over expression of the genes encoding C7alphaH and S27H . A clearer understanding of the mechanisms which maintain cholesterol homeostasis is needed to continue to lay the groundwork for the development of better therapies for cholesterol related disorders."}
{"text": "The major objective of the proposed research is to determine the residual effects of low level lead exposure on the fetal and infant rhesus monkey. Pregnant rhesus monkeys will be fed low levels of lead throughout gestation and their infants evaluated biologically and behaviorally during infancy, adolescence, and early adulthood. Similar studies will also be conducted on infant monkeys that are exposed to low levels of lead during the initial year of life. These studies will help to clarify the effects of lead exposure during early life on the subsequent development of the infant. In addition, maximum tolerable levels of lead administered over a given period to fetal (via the mother) and infant monkeys will be established. The methods that will be employed in evaluating these aminals will include behavioral studies that will emphasize social interaction and learning capabilities of these animals from birth to adulthood. The clinical studies conducted will be systematic hematological, radiological, and biochemical determinations throughout infancy, adolescence, and adulthood. After the completion of the clinical and behavioral studies histological and ultrastructural studies will be conducted on all of the major organs. In addition, total concentrations of lead in various tissues of the body will be determined."}
{"text": "Quantitative cytochemistry and morphometry will be used to characterize the ultrastructural and enzymic configuration of pseudostratified granulosa cells of preovulatory follicles in rats. The enzyme complexes studied will include delta 5-3 beta hydroxysteroid dehydrogenase, glucose-6-phosphate dehydrogenase and cytochrome P-450. Acid phosphatase will be studied to localize organelles associated with lysosomal activity and the naphthylamidase reaction will be used to measure lysosomal membrane permeability. The development of cytochemical characteristics unique to pseudostratified granulosa cells of preovulatory follicles will be followed throughout the estrous cycle. The ultrastructural characteristics that distinguish the pseudostratified granulosa cells of the preovulatory follicle will be established. Since the granulosa cells form an essential component of the preovulatory follicle as well as of the corpus luteum formed from it, the information from these studies could provide a basis for evaluating abnormal development and the formation of inadequate corpora lutea--conditions associated with infertility."}
{"text": "Antibodies play a critical role in the immune system for recognition of foreign intruders. Because of their excellent affinity and specificity, they hav also been exploited as therapeutic molecules and biotechnological components for sensing and assembly. Structures of antibodies in complex with their antigens can yield insight into biological phenomena or drug and disease mechanisms. However, structures of antibodies and antibody-antigen complexes can be difficult, time consuming, and expensive to determine. The proposed research focuses on the computational prediction of the structure of antibodies and antibody-antigen complexes. Computational approaches are particularly important because the repertoire of antibodies in a human patient is far too large for complete structural characterization by experiment. Prior work has isolated the most critical challenges: most of the antibodies in the human repertoire have hypervariable CDR H3 loops longer than that which is predictable using current loop methods; backbone conformational uncertainty and flexibility confound current docking methods; and no current method can quantitatively predict antibody-antigen binding affinities from structure. Thus, the first three aims of the project are to (1) develop new methods to predict the structure of long CDR H3 loops using statistics to identify likely turns, (2) develop flexible backbone docking routines using an expanded ensemble approach with a conformational web, and (3) develop methods to quantitatively predict protein-protein binding affinity using improved electrostatics treatments. Finally, the fourth aim will be to (4) use existng and proposed methods to predict structures of antibodies and antibody-antigen complexes for entire polyclonal antibody repertoires. Structures will be predicted for antibody repertoires determined from bone marrow plasma cells of mice immunized against ovalbumin (a food allergen) and enzyme C1s (a therapeutic target for autoimmune diseases and transplant tolerance). Ultimately, these studies will yield insights into immunology, molecular recognition, and design of protein-protein interfaces and vaccines. PUBLIC HEALTH RELEVANCE: Antibodies play a critical role in the immune system for recognition of foreign intruders, and are excellent therapeutic molecules because of their high affinity and specificity. This project investigates computational approaches to determine the three-dimensional structures of antibodies alone and in complex with their target antigens, including specific applications relevant to food allergies and a treatment for transplant rejection autoimmune disorders and inflammatory diseases."}
{"text": "These investigations are devoted to the development of noninvasive methods of accessing tissue structure and function. Two general techniques are being developed: nuclear magnetic resonance (NMR) and optical spectroscopy/imaging. Over the last year we have made the following developments in NMR technology: (1) We have further developed the theory and application of magnetization transfer contrast (MTC) to the study of biological tissues. This approach permits the imaging of the interaction rate between water and macromolecules in the body resulting in a unique form of high resolution image contrast and tissue characterization. The quantitative aspects of this approach, defining its frequency and power dependence, were established out-lining the methods of obtaining quantitative rate constant maps in vivo. (2) 3-H NMR studies have revealed that the interaction of water with macromolecules, the dominant source of contrast in tissues, is due to spin diffusion between the water and the macromolecules. (3) The molecular basis with macromolecules was evaluated which of the dipoler interaction of water demonstrated a unique role for hydroxyl groups in lipid bilayers. (4) The first human images using MTC were collected by adding a second radiofrequency channel to a standard clinical scanner. These images were 3-dimensional images of the knee revealing excellent soft tissue contrast. (5) A quantitative relationship between 3-fluorsorbitol production from 3-fluorodeoxyglucose and aldose reductase activity was established in the isolated lens of the eye, suggesting that this approach is a viable method of monitoring aldose reductase activity in vivo. Using optical spectroscopy the following advancements were made: (1)The effects of inner filters on the optical behavior of calcium indicating dyes and NAD(P)H fluorescence in the intact heart were defined and methods developed to correct for these serious artifacts. (2) Methods were further developed for the monitoring of tissue oxygenation in vivo using optical spectroscopy in the 500 to 650 nm range."}
{"text": "The overall objective of this work is to analyze the genetic relationships of the various populations of the Common Hose Mosquito present in the Boston area. By comparing the electrophoretic mobility of selected isozymes, we shall determine (1) whether the autogenous, anautogenous and rural forms may be characterized, 2) whether these populations may be genetically isolated, 3) if so, whether isolation is absolute, and 4) whether one population may possess more genetic variability than others. We shall study the blood-feeding habits of each population and determine whether man-biting may be associated with certain populations or combinations of populations. A manual will be prepared describing procedures for applying the polyacetate strip method of electrophoresis to populations of mosquitoes."}
{"text": "The proposed research-will use the technique of single-unit recording to examine the characteristics of neurons within the postsubiculum and limbic thalamic nuclei. Specifically, the properties of \"Head Direction\" cells will be examined. These cells respond only when an animal orients it's head in a specific direction (e.g., a cell may only fire when an animal points its head to the northeast) regardless of the animals absolute position in an apparatus. It is hypothesized that these cells play a significant role in spatial abilities. The experiments in this proposal will examine two basic issues with regard to head-direction cells: First, what type of navigational information determines head- direction cell activity? Second, do head-direction cells exhibit additional behavioral correlates (e.g., responses to stimulus presentation, goal approach, or reward) when examined in various behavioral paradigms? The experiments designed to answer these questions will provide a further understanding specifically of the determinants of head-direction cells activity, and in general, of neural bases for spatial abilities."}
{"text": "Studies on the cause of congenital cataracts in the progeny of female rats fed a vegetable protein diet during pregnancy and lactation have shown that supplements of either tryptophan or Vitamin E were effective in prevention of the opacities. An interaction was confirmed by preparation of a semi-purified diet using L-amino acids. With this diet, low levels of tryptophan (0.075 mg%) or Vitamin E (0.1 mg%) could be tolerated singly but caused 30% incidence of F1 cataract when fed simultaneously. Current studies are aimed at elucidation of the biochemical mechanism of this effect."}
{"text": "The antimetabolites have been used for the treatment of childhood cancer for more than 40 years. This class of agent continues to play a critical role in the treatment of childhood leukemia. The primary objective of this project is to study the clinical pharmacology of antimetabolites and other anti-leukemic agents to determine if they are being utilized optimally, and to develop rational, pharmacological- based alternative therapeutic approaches to the treatment of childhood leukemia. In our current lower risk ALL trial, we are applying observations made in our preclinical studies showing that thioguanine (TG) is 10-fold more potent than mercaptopurine (MP) against leukemic cell lines and lymphoblasts from patients with ALL. Pharmacodynamic correlation between plasma TG and RBC TG-nucleotide levels, and measures of drug toxicity and disease outcome, are being investigated. Results of this trial and the clinical pharmacologic assessment of TG has guided the design of the current CCG phase 3 randomized trial comparing TG to MP in the lower risk ALL population. We are also exploring a new class of antimetabolite specific for thymidylate synthetase (TS). ZD1694, a classical antifol which is a potent inhibitor of TS, is currently being evaluated in a pediatric phase 1 trial and pharmacokinetic study. A novel and sensitive enzyme inhibition assay for TS inhibitors has been developed and is being evaluated. Methotrexate is an antimetabolite widely used in pediatric cancer, and when administered in high doses must be followed by the rescue agent leucovorin. We are studying carboxypeptidase-G2, (CPDG2) a recombinant enzyme capable of hydrolyzing MTX into inactive metabolites. CPDG2 may offer distinct advantages over leucovorin in certain clinical situations. Based on our pre-clinical investigations, nationwide protocols have been developed to treat patients with severe renal dysfunction following HDMTX with CPDG2. To date, more than 70 patients with renal failure have been treated, with the enzyme producing a > 98% decrease in MTX concentrations within 15 minutes of administration. We have prepared the data for presentation to the FDA as part of an NDA for CPDG2 - Leukemia, Antimetabolites, Methotrexate, Thioguanine, - Human Subjects: Minor under 18 Years Old"}
{"text": "To maintain genomic integrity, eukaryotic cells duplicate their DNA precisely once before each cell division. In the G1 phase, pre-replication complexes (pre-RCs) are assembled at origins via the sequential recruitment of ORC, Cdc6, Cdt1, and MCM2-7. In S phase, when DNA replication initiates, pre-RCs are disassembled, and new pre-RC assembly is strictly prohibited. As a result, only one initiation event occurs at each origin, and DNA replication is limited to a single round. In vertebrates, pre-RC assembly is blocked in S phase due to Geminin and ubiquitin-mediated proteolysis of Cdt1. Using a cell-free system derived from Xenopus egg extracts, this laboratory made the surprising discovery that the destruction of Cdt1 in S phase is intimately coupled to DNA replication. Cdt1 is ubiquitylated on chromatin by the E3 ubiquitin ligase Cul4-Ddb1Cdt2, and this ubiquitylation event requires the interaction of Cdt1 with the processivity factor PCNA at the DNA replication fork. Studies from other laboratories suggest that PCNA-dependent Cdt1 destruction is conserved in humans, flies, worms, and possibly fission yeast. This pathway represents a new paradigm in which temporal control of proteolysis involves activation of a substrate (Cdt1) via its docking onto a cell-cycle regulated structure (chromatin-bound PCNA). This proposal contains experiments that will elucidate the mechanism by which PCNA-dependent Cdt1 destruction is temporally controlled. In Specific Aim 1, we address how Cdt1 interacts selectively with chromatin-bound PCNA to insure that Cdt1 is normally never destroyed in G1. In Specific Aim 2, we characterize the degron motif of Cdt1 and examine how it mediates binding to PCNA and Cul4-Ddb1Cdt2. In Specific Aim 3, we reconstitute Cdt1 ubiquitylation in a purified system so that we may distinguish between different mechanisms for how PCNA stimulates this process. Finally, in Specific Aim 4, we use real-time fluorescence microscopy to address how rapidly Cdt1 is destroyed at S phase onset, and we test a new model for local control of proteolysis by the PCNA/Cul4-Ddb1Cdt2 pathway. Together, these studies will illuminate the molecular mechanism underlying a novel proteolysis pathway that maintains genome stability. Narrative To maintain the integrity of our genomes and prevent diseases such as cancer, it is crucial that cells make one precise copy of their DNA before each cell division. This laboratory has discovered a new mechanism, which insures the accuracy of genome duplication in all higher organisms, and it involves the destruction of a DNA replication factor called Cdt1 after the first round of chromosome duplication has been initiated. The work is highly relevant for human health because failure to destroy Cdt1 is correlated with human cancer, and has been shown to cause cancer in mice. [unreadable] [unreadable] [unreadable]"}
{"text": "When individuals with Tourette syndrome (TS) are treated with medications to suppress tics, some improve, others have little or no response, and several may develop significant side effects. In order to explain this variability, pharmacogenetic research has focused on two major areas, drug metabolism and drug site of action. In this study, genetic variations of metabolizing enzymes (CYP2D6), dopamine & serotonin transporters (DAT, 5-HTT) dopamine receptors (D2, D3, D4) and serotonin receptors (5HT2a and 5HT2c) will be determined with the goal of predicting a TS patients response to treatment and a diagnostic category (TS or control). Fifteen established polymorphisms will be investigated in individuals with the diagnosis of TS using DNA obtained by buccal swabs. The first major objectives is to determine whether the presence of specific patterns of neurotransmitter polymorphisms predict the tic-suppressing pharmacologic effect of the atypical neuroleptic, risperidone. Patient response to pharmacotherapy will be based on a prospective evaluation of tic reduction, as measured by the Total Tic score of the Yale Global Tic Severity Scale, in 200 individuals with TS. This approach is similar to one that has been successfully used in schizophrenia to predict, with nearly 80% success, the response to the atypical neuroleptic, clozapine. Our second goal is to determine whether there are associations between genetic variations in dopamine or serotonin receptors and the etiology of TS. This question will be addressed in a cohort of 500 individuals with TS & compared with an equal number of age and sex-matched controls. This application, defines a study (to my knowledge, the first of its kind in TS), which will use DNA obtained from buccal mucosa, to establish a predictive therapeutic index in TS patients. The identification of genetic polymorphisms that are associated with clinical disease or treatment success, has obvious benefits."}
{"text": "In the developing mammalian brain, directed cell migrations establish the neuronal layers of the cerebellum and the neocortex. While a number of receptor systems have been studied in vertebrate neuronal migrations, an important avenue of discovery of genes that potentially regulate CNS migrations is to search for homologues of genes that function in neuroblast migration in C. elegans and Drosophila. During the prior funding period of this grant, we have begun studies with the laboratory of Dr. Cynthia Kenyon to explore the role of homologues of C. elegans neuroblast migration genes in mammalian brain development. Toward that end, we have cloned homologues of the C. elegans gene mig-13 and initiated studies on the role of Mmig13 in brain development. The overall goal of this Research Plan is to define the function of murine homologues of the C. elegans gene mig-13 in the formation of murine cerebellar and neocortical neural laminae. Our preliminary studies suggest that Mmig13 functions in the formation of the EGL in the embryonic cerebellar anlagen, prior to migration of postmitotic precursors along the Bergmann glial fibers, and in the formation of the cortical plate, the region where young neurons in the neocortex halt their movements along radial glial fibers and assemble into specific layers. The proposed research is aimed at testing and refining this general hypothesis by providing more detailed studies on the timing of expression of Mmig13 in these two brain structures, by imaging living neurons in cells and tissue from Mmig13 BAC transgenic mice, where the Mmig13 gene is marked by EGFP, and by evaluating the effects of either gain of function or loss of function on granule cell migration and/or cortical neuron migration. To better understand the mechanism of action of the gene(s), we will define their extracellular ligands and their intracellular signaling pathways. This information should broaden our view of the mechanisms that control CNS migrations and of the downstream events important to neuronal differentiation within the neural layers."}
{"text": "This proposal studies the behavioral pharmacology of oral cocaine self-administration. The optimal conditions for the induction of cocaine overindulgence in rats will be established by varying drug concentration and the food-schedule induction parameters which are known to produce excessive behaviors. The effect commonly- used and abuse licit agents (nicotine and caffeine) might have in increasing schedule-induced cocaine intake will be determined, as well as possible attenuating effects of promising therapeutic (desipramine) and blocking agents (e.g., chlorpromazine and pimozide). A number of commodities and activities will be tested as behavioral alternatives to available cocaine under the excessive-behavior-generating conditions. Extensive exposure to prior experiences with non-drug, highly-preferred substances such as saccharin (which are then made unavailable) under conditions that should induce cocaine abuse may block its development, as may pre-adaptation to the inducing conditions. The possibility that prior overindulgence of nicotine or caffeine may predispose to cocaine abuse development will be explored. The effect of parenteral cocaine injection on a fine motor control task will be determined, as well as the interactive effects of caffeine and of nicotine with chronic cocaine on this kind of performance. Serum cocaine and metabolite levels will be correlated with behavioral effects."}
{"text": "Project Summary/Abstract Prostate cancer is listed as a primary diagnosis for approximately 16% of older male VHA users, and contributes significantly to morbidity and mortality rates in these individuals. The majority of VA patients with metastatic prostate cancer, similar to most non-VA patients in the United States, receives hormonal treatment which is initially effective, but frequently fail indicative of the development of castration resistant prostate cancer (CRPC). The treatment options for patients who fail hormonal therapy are limited, despite numerous clinical trials testing multiple drugs. Hence, our laboratory has undertaken the innovative overall strategy of identifying therapeutic targets that can prevent prostate cancer progression to castration resistance, rather than trying to cure it after it has already developed. Studies performed during the previous funding period, which resulted in 14 publications in related topics, contributed to understanding the importance of the cell survival regulator Akt in prostate cancer development and progression. While a number of Akt inhibitors have been developed commercially, their utility has been compromised by high levels of toxicity when used in human patients, caused by the induction of apoptosis in normal tissue including non-tumor prostate regions. As a result, a goal of the project was to identify natural antagonists of Akt that selectively counteract the effects of the increase in Akt phosphorylation in the tumor without affecting basal levels of Akt activation in other cells. Two of our recent publications demonstrate that Filamin A (FlnA) may be one such protein. In vitro studies showed that FlnA localization to the nucleus resulted in castration sensitive growth of prostate cancer cells which was mediated by antagonism of Akt phosphorylation and its downstream effectors. FlnA nuclear localization is regulated by its phosphorylation, which in turn is regulated by PKA activity. The effects of FlnA nuclear localization are likely caused by its interaction with the androgen receptor (AR), a nuclear steroid receptor mediating the effects of the androgens on cell mechanisms. Therefore, in the current application, we will investigate the role nuclear FlnA plays in regulating castration sensitive cell behavior. We hypothesize that FlnA counters the effects of Akt phosphorylation and its downstream effectors, and that its localization is in turn regulated by its phosphorylation. The following aims will test this hypothesis. Specific Aim 1: To test the hypothesis that FlnA 16-24 nuclear localization sensitizes prostate cancer cells to androgen withdrawal by preventing the effects of Akt on AR activity and cell survival. We will determine whether FlnA16-24 induces sensitivity to androgen blockade in CRPC cells by antagonizing an Akt- dependent signaling pathway and preventing AR/TIF2 interactions. We will also investigate whether nuclear localization of FlnA 16-24 induces apoptosis by repressing NF-kB activation and stimulating AR-dependent p21Cip1 transcription. Specific Aim 2: To test the hypothesis that FlnA phosphorylation is a key factor responsible for preventing FlnA nuclear localization in castrate resistant prostate cancer. We will examine whether FlnA phosphorylation prevents FlnA nuclear localization and causes castration resistance in prostate cancer cell lines expressing a functional AR. We will also determine whether inhibition of FlnA phosphorylation prevent the development of castration resistance in a mouse model of recurrent prostate cancer. Specific Aim 3: To test the hypothesis that increased FlnA phosphorylation and decreased FlnA nuclear localization predict time to treatment failure in VA patients with metastatic prostate cancer on androgen withdrawal therapy who did not undergo radical prostatectomy. We have identified patients within the VA Northern California System who presented with metastatic prostate cancer. We will determine in biopsies of the prostates of these patients the localization of Filamin A, FlnA phosphorylation, AR levels and Akt phosphorylation and determine whether they respond to androgen withdrawal therapy."}
{"text": "Local BBB changes in drug-induced seizures and in acute hypertension were correlated in the same animals with rCBF changes. Our findings indicate that BBB changes are associated with marked regional elevations of the CBF. However, this relationship is relative and it does not indicate existence of any absolute threshold for rCBF elevation to be associated with BBB breakdown."}
{"text": "This project investigates the roles of the family in facilitating disease management among Navajo individuals with type 2 diabetes, specifically: 1) the support given to diabetics by their families; and 2) how family support and Navajo philosophical concepts interact with two current diabetes interventions conducted by the Indian Health Service. In the previous MBRS research project, family support emerged as a critical factor in determining the extent to which Navajos with diabetes adhere to prescribed self-care behaviors such as diet change, weight control, and exercise. Certain traditional Navajo philosophical concepts also were associated with use of self-management techniques. The project will involve two distinct groups of respondents. The \"Family Study\" will involve ethnographic interviews and focus groups with members of 12 families (both diabetics and non-diabetics) who have one or more diabetic relatives to provide a depth of understanding of the dynamics of family support and use of traditional philosophical concepts in assisting diabetic family members. The \"Intervention\" will follow three groups of clinic patients with regard to change in their health status either following participation in one of two ongoing diabetes interventions, or after a similar period of time with non intervention. The interventions vary in the degree to which family involvement and tenets of Navajo philosophy are incorporated. In both parts of the study, the outcomes considered will include objective measures of diabetes management taken from the medical record, as well as self-management behaviors as reported by the Navajo diabetics. Hypotheses are: 1) that individuals whose families offer consistent, active and varied support will manage their diabetes better than those lacking such family support; 2) that families expressing traditional Navajo philosophical concepts such as those described above will be more likely to provide support for their diabetic relative; and 3) that interventions which target family-wide change, and involve active learning will have a greater impact on management behaviors and ultimately blood glucose than interventions which target individual change and stress didactic presentation. The findings in each of these areas of study will have significant ramifications for improving disease management and health care interventions for Navajos with diabetes."}
{"text": "The mechanism of lysosomal localization and function of cholesteryl ester hydrolase will be studied in cultured vascular smooth muscle and endothelial cells, and in fibroblasts. The concepts of the secretion-recapture model for lysosome formation (Neufeld, Sando, Garvin and Rome), which were developed from studies of mucopolysaccharide-metabolizing enzymes in cultured fibroblasts, will be applied in these studies. Hormonal and nutritional factors thought to be associated with vascular disease will be studied for effects on the secretion and internalization of the hydrolase. The relationship of secretion and internalization of cholesteryl ester hydrolase to the regulation of cholesteryl ester metabolism will be compared in the above cell types. The relationship of low density lipoprotein uptake, internalization and subsequent regulation of sterol metabolism to lysosomal localization and function of the hydrolase will be examined."}
{"text": "The objective of this study is to gain operational and mechanistic understanding of mechanical and electrical effects on bone and cartilage growth and remodeling. Cells from chick epiphyseal cartilage, from rat embryo calvaria and from rat osteosarcoma are exposed to controlled mechanical or electrical perturbations in culture. The effect of the perturbation on cyclic AMP accumulation, ornithine decarboxylase induction and DNA synthesis are measured. Hypotheses on the mechanism of action of the stimuli are tested through manipulation of the composition of the medium. Results obtained so far show that mechanical and electrical stimuli modulate DNA synthesis via effects on cell membranes involving changes in cyclic nucleotides and Ca ion and Na ion fluxes. The response is cell specific and depends on the state of cellular differentiation."}
{"text": "In this application, we propose to test two new hypotheses concerning the MBP gene. These hypotheses are based upon data derived from the identification of two classes of unusual myelin basic protein (MBP)-related transcripts isolated from screens of mouse brain and human fetal spinal cord cDNA libraries. The first hypothesis is that the structure of the MBP gene is presently incomplete and contains at least one more exon upstream of what is currently believed to be exon 1 and an additional promoter. The second hypothesis is that portions of the MBP gene (including parts of at least one exon and one intron) are transcribed into mRNAs encoding proteins other than the MBP variants characterized thus far. The location of any additional exon sequences will be determined by conventional procedures of mapping and sequencing genomic clones using an MBP cDNA that contains sequence corresponding to the additional exon region. Sites of transcription initiation at this new upstream exon will be determined by S-1 nuclease and primer extension analysis. Experiments will be performed to determine if initiation of transcription at this start site and the one previously identified in the MBP gene varies during development or in different regions of the CNS. Experiments will also be performed to determine if all MBP variant forms are produced when transcription begins at this new transcription site. Full length cDNAs corresponding to the second class of MBP-gene related transcripts will be isolated, sequenced, and characterized. The structural relationship between this second class of transcripts and the MBP gene will be determined. The intron/exon arrangement of the gene coding for these MBP-related cDNAs will be elucidated and it will be determined if all the exons of this gene are encoded by genomic segments completely included within the MBP gene or if some exons lie outside the MBP gene. To determine if these transcripts are expressed in tissues other than brain or in cell types other than oligodendrocytes, Northern blot analyses of mRNA from a variety of tissues and cultured cells will be performed. The developmental appearance of the transcripts in the brains of normal mice and dysmyelinating mutants will be compared with the expression of the MBP mRNAs. In situ hybridization studies will be performed to determine the cellular and regional localization of the transcripts."}
{"text": "The population in the U.S. is aging. Data are sparse on cardiovascular disease in older adults, especially the oldest old, who are expected to number 18 million by 2050. The age at first myocardial infarction (MI) is increasing substantially, now to about age 71, and most of the cardiovascular disease (CVD) deaths in the U.S. occur in older individuals. CVD is the single most important cost of medical care in the US. In the early 1990s, the Cardiovascular Health Study (CHS), an NHLBI-supported cohort study of risk factors for coronary heart disease (CHD) and stroke in adults 65 years or older, recruited 5888 participants, who underwent extensive examinations at baseline and annually until 1999. Examinations included traditional risk factors as well as measures of subclinical disease. Since 1990, CHS has made major contributions to the understanding of CVD in older individuals, including the risks associated with subclinical disease, inflammation, diabetes, hypertension and renal disease. In 2008, CHS has the unique opportunity of evaluating clinical CHD in the oldest old-- MI, angina, heart failure (HF), atrial fibrillation-- and their contributions to both morbidity and mortality. In this competing continuation, the primary aim is to evaluate the incidence and determinants of cardiovascular disease and health in 1964 surviving participants aged 80 or older. The incidence of CVD in the oldest old will be related to demographic variables;measures of disability, physical functioning, and cognitive function;measures of subclinical disease;and traditional and novel clinical risk factors as well as their change over time. The data collection proposed in this application will increase the number of events in those 80 years and older by about 50% to 75% for stroke, CHD and HF. Power for assessing associations with stroke, CHD and HF is increased by 15 to 30%. The continued assessment of cardiovascular events is essential to identify determinants of successful cardiovascular aging among the oldest old. PUBLIC HEALTH RELEVANCE: Additional knowledge about the determinants of cardiovascular health and disease in older adults will help physicians and their patients make diagnostic, prognostic, and therapeutic decisions. PROJECT/"}
{"text": "Non-insulin dependent diabetes mellitus (NIDDM) affects people of Latino origin more often and at an earlier age than whites. The Latino culture influences dietary behaviors which affect diabetes. Although nutrition intervention studies have demonstrated reductions in weight and glucose, culturally sensitive programs for a specific population of Latinos with diabetes have not been developed or validated. Boston, MA is home to a large population of Caribbean Latinos who originate predominantly from Puerto Rico and the Dominican Republic. An NIH planning grant has been used by the investigators to create a multidisciplinary team of practitioners and researchers and to identify and characterize a population of urban Caribbean Latinos with diabetes mellitus. Preliminary data show that these Latinos consume traditional Latino foods (especially rice and beans) primarily at lunch and dinner, use oil and other high fat foods extensively and rarely eat outside of the home. There is a strong need for programs that are culturally sensitive and target specific behaviors. We propose to design, implement, and evaluate a culturally-sensitive program that reduces dietary lipid intake. Using the information gained during the planning grant year, we will develop a program that is oriented towards Caribbean Latino culture, utilizes principles of behavior modification, and consists of proper nutritional guidelines. The intervention will focus on recipe modification, meal planning, informed shopping and group nutrition counseling. After pilot testing and modifying the program, diabetic Caribbean Latinos will be recruited from Boston City Hospital and the South End Community Health Center and randomized to either an intervention nutrition (IN) group or a usual care (UC) group. A separate UC group of Caribbean Latinos from Whittier St. Neighborhood Health Center will be followed to determine whether crossover occurs. The IN group will meet for twelve consecutive weeks, and then at weeks 14,16 18, 20 and 24. The UC group will be followed at 6, 12 and 24 weeks. Follow-up for both groups will be done at 9 and 12 months. Parameters to be measured in both groups include dietary intake, weight, lipid profile, HgbA1C, WHR, and blood pressure. Metabolic studies will include body fat distribution using dual energy x-ray absorptiometry (DEXA) and indices of insulin sensitivity and resistance as measured by IV glucose tolerance test. By designing culturally sensitive programs that target specific behaviors, we believe that dietary fat intake can be reduced in urban Caribbean Latinos with NIDDM."}
{"text": "Recent evidence suggests that injection drug use is increasing among suburban residents. Little research has focused on this trend, and the economic, social, and medical consequences have not been considered. To close this gap, we are proposing to identify, recruit, and study a valid sample of injectors who reside in Connecticut suburbs using respondent driven sampling (RDS). RDS was selected because of its strengths in identifying individuals from hidden populations and allowing statistical analysis of the sample. We are submitting this application in response to PAR-06-114, \"Research on Pathways Linking Environments, Behaviors and HIV/AIDS\". Not only will be focusing on an environment - the suburbs - that has been neglected, but we will further focus on suburban injectors who inject in two distinct environments. Preliminary studies revealed that there are those who inject predominantly in the suburban town in which they reside and those who inject in urban areas where they obtain their drugs. We are building our study around the hypothesis that the two environments result in differences in (1) injection drug use patterns; (2) knowledge of, risk for, and prevalence of HIV, hepatitis B, and hepatitis C infections; (3) sociodemographic stability; and (4) the use of and barriers to services that reduce the negative consequences of injection. Our study will recruit a sample of 600 active injection drug users who reside in the suburbs. The sample will be studied through structured interviews, serology, and in-depth life histories. The sample will be followed longitudinally to ascertain whether the pattern of suburban or urban injection is stable and to identify factors associated with changes in injection environments and in risk for syringe-borne viral infection. Our study is designed able to answer questions including (1) how representative the sample is of the underlying population, (2) whether two groups of suburban injectors who differ based on where they inject differ in terms of disease knowledge, risk, and prevalences, (3) how much of the prevalence of HIV, HBV, and HCV can be predicted by individual, group, and environmental level variables, (4) how suburban injectors are geographically clustered and whether this affects disease prevalence, (5) how interaction with the criminal justice system influences drug use and risky injection practices, (6) which factors are associated with arrest and incarceration and (7) how injectors use drug treatment and disease prevention programs and what they perceive as the barriers to larger use of these services. To meet the objectives of this study, we have assembled an experienced, multidisciplinary team that can collect and analyze the quantitative, qualitative, and serological data and disseminate research findings to the scientific community through scholarly and public health channels and to the service community through our Center for Interdisciplinary Research on AIDS. [unreadable] [unreadable] [unreadable]"}
{"text": "Crohn's disease (CD) is a chronic and debilitating inflammatory bowel disease with unknown etiology;however, it is clear that there are both genetic components as well as environmental factors that are required for its development. Tremendous advances have been made recently in understanding the genetics of CD. Although several chromosomal loci have been associated with IBD susceptibility, until last year, only one gene had been conclusively identified as a CD susceptibility gene. This gene is NOD2 (nucleotide-binding, oligomerization domain 2), which encodes an intracellular sensor of bacteria in the innate immune system. Recent genome-wide association scans have identified multiple, novel CD susceptibility genes. Many of these new CD susceptibility genes also modulate immune responses, suggesting that dysregulation of an immune pathway rather than a single gene may be important in CD pathogenesis. One of these processes is autophagy. Autophagy (literally \"self-eating\") is best known as a cell survival mechanism in response to starvation, where organelles are broken down and recycled to provide nutrients. This mechanism also plays a crucial role in the clearance of intracellular bacteria by the innate immune system. Bacteria and bacterial components induce autophagy, linking innate immune microbial sensors to autophagy. One of these microbial sensors is Nod2, which detects a specific component of the bacterial cell wall called muramyl dipeptide (MDP). Our preliminary data demonstrates that stimulation of cells with MDP not only activates autophagy, but suggests autophagy may also be involved in activation of Nod2 by MDP. These results provide a link between two separate classes of molecules mutated in CD and lead us to propose the following central hypothesis: CD- associated mutations in the autophagy genes, ATG16L1 (autophagy related 16-like protein 1) and IRGM (immunity-related GTPase family, M), and NOD2 impair the same critical innate immune pathway and impairment of this pathway contributes to CD pathogenesis. We hypothesize autophagy and Nod2 function together in two non-exclusive ways: (1) MDP stimulation of Nod2 activates autophagy as an essential anti-microbial response, and (2) autophagy is involved in MDP internalization and Nod2 activation. This hypothesis will be tested by the following three specific aims using both primary cells and cell lines: 1) Determine the role of Nod2 in activation of autophagy - is autophagy a component of Nod2 stimulated anti- microbial responses?, 2) Characterize the mechanism of MDP internalization - is it autophagy?, 3) Analyze alterations in autophagy in phagocytic cells of CD patients. The results of our proposed studies may benefit individuals with CD by identifying a crucial processes altered in CD, leading to the design and use of more specific CD therapies targeting this pathway. PUBLIC HEALTH RELEVANCE: Recent advances in the genetics of Crohn's disease (CD) indicate that alterations in an immune pathway rather than a single gene may be important in CD pathogenesis. We propose to study the interactions of three genes associated with CD susceptibility and define if they are a part of the same innate immune system pathway. The results of our proposed studies may benefit individuals with CD by identifying a crucial process altered in the disease, leading to the design and use of more specific therapies targeting this pathway."}
{"text": "Estrogen is a fascinating hormone with seemingly paradoxical effects in the cardiovascular system. On the one hand, estrogen appears to be protective to the cardiovascular system of pre-menopausal women. Prior to the menopause have a lower incidence of cardiovascular disease than men. After the menopause, the incidence increases rapidly to reach and even exceed that of men. It has been widely assumed, form anecdotal evidence, as ell as several early studies, that replacement of estrogen would return the cardiovascular system of the post-menopausal woman to its pre-menopausal protected state. However, the early data from the Women's Health Initiative does not support the assumption; rather, these data suggest an increase in cardiovascular events in the first two years of hormone replacement therapy with estrogen. Estrogen remains the most likely cardiovascular protective factor for pre-menopausal women. Thus we propose that the failure to observe a protective effect of estrogen on cardiovascular function is the result of administering an estrogen that is an incomplete agonist in cardiovascular tissues, or that is administered in inappropriate mode. This project is designed to test the hypothesis that the dichotomous effects of estrogen on the cardiovascular system are due to differential Gene expression responses to different estrogen formulations and modes of administration. Two specific aims have been developed to test this hypothesis. Specific aim #1: To develop a profile of genes expressed in response to estradiol 17beta in vascular tissues and to examine the effect of estrogen formulation on the vascular reactivity of the mesenteric arteries."}
{"text": "A major goal of this laboratory is to understand the molecular mechanisms by which the nuclear hormone receptor peroxisome proliferator-activated receptor ? (PPAR?) controls adipogenesis and mediates antidiabetic effects of thiazolidinedione (TZD) drugs that improve insulin sensitivity but cause unwanted side effects. We hypothesize that target gene- and tissue- selective modulation of gene expression by PPAR? are dictated by different modes of PPAR? binding and synergy between PPAR? and cooperating transcription factors. Specific Aim 1 is to determine the direct transcriptional effect of TZDs in adipocytes and their relation to PPAR? binding on a genome-wide scale. We hypothesize that the direct effects of TZDs on transcription cannot be predicted from transcriptomic analysis. Preliminary Global Run-On sequencing (GRO-seq) results demonstrate the feasibility of measuring nascent transcripts in adipocytes on a genome-wide scale. Specific Aim 2 is to delineate the adipocyte epigenome and corepressor cistromes, and their relation to PPAR? binding and the effects of TZD treatment on a genome-wide scale. We hypothesize that TZD treatment of adipocytes alters coregulator recruitment to PPAR? at a subset of target genes, leading to epigenomic changes that alter gene transcription. This will be tested by ChIP-seq for coregulators and epigenomic marks in adipocytes treated with TZDs and non-TZD ligands with potentially fewer side effects. Specific Aim 3 is to understand the mechanisms of cell-type specific genomic binding and regulation by PPAR?. We hypothesize that PPAR? functions as a pioneer factor on some binding sites, particularly in adipocytes, whereas macrophage binding sites are more influenced by cell- specific transcription factors and epigenomic marks. This will be tested using cistromic approaches, and the ability of PPAR? and cell type-specific factors such as PU.1 to shape each other's genomic binding, as well as the adipocyte and macrophage epigenomes, will be explored using gain and loss of function studies. Our innovative approach will elucidate mechanisms underlying target gene- and tissue-specificity of PPAR?, which can be translated to the design of more selective insulin sensitizers to combat the epidemic of metabolic disorders."}
{"text": "Chronic administration of the beta-adrenergic agonist isoproterenol induces parotid gland hypertrophy and hyperplasia in the rat. This serves as a useful model for studying the physiological and biochemical alterations that occur in cell proliferation. An important event noted in the isoproterenol induced parotid acinar cell hyperplasia is the increase in cell surface 4 betagalactosyltransferase (Gal Tase). Numerous studies have shown that alterations in cell surface Gal Tase may be involved in cell-cell interaction during embryogenesis, differentiation and tumorigenesis. The elevated surface Gal Tase in parotid acinar cells then appears to interact with the EGF-R in a manner similar to growth factor associated 'receptor-ligand' interaction. It is believed that the EGF-R undergoes conformational changes in its carbohydrate moieties so as to serve as a substrate for cell surface-localized Gal Tase. However, it is not clear how the signal produced by such an interaction reaches the nucleus to modulate the processes such as DNA replication and gene expression. I therefore propose to examine the involvement of second messengers, and receptor crosstalk produced during the regulation of acinar cell growth. To accomplish this, phosphoinositol metabolism (along with their specific metabolic kinases), and adenylate cyclase G activating protein (GAP activity), will be studied in both the control and ISO stimulated parotid glands. Parallel studies with EGF and Gal Tase stimulation will be conducted to determine the extent of Gal Tase's ability to mimic EGF in regulating this specific receptor response. In addition, the characterization of the carbohydrate structure of EGF-R in the control and ISO-treated rat parotid gland will be made to confirm the unique Gal Tase - EGF-R interaction which appears to be a primary event in beta-agonist mediated signal transduction for proliferation. The injection of antibody to either Gal Tase or the EGF-R, along with ISO, have been shown to prevent acinar cell proliferation. The carbohydrate changes of the receptor may provide insights into the regulation and re-expression of fetal carbohydrate structures during parotid acinar cell hyperplasia. While not neoplastic, the expression of fetal carbohydrate antigens by these cells may reflect a common hallmark of transformed cells; namely a renewed synthesis of fetal developmental carbohydrate markers on the cell surface. These findings on the regulation of acinar cell signal transduction pathways in the isoproterenol induced parotid gland hyperplasia may be helpful in evaluating potential new treatments for the control of cell proliferation in human pathologies in general and those in particular of the oral cavity and soft tissue."}
{"text": "The broad, long-term objective of this research proposal is to improve the health and well being of the elderly via decreased risk for type 2 diabetes. The specific aim of the project is to examine the influence of an ad libitum 12- week low-fat/simple-carbohydrate diet, with and without aerobic exercise training, on body mass, energy balance, and insulin sensitivity in older subjects with impaired glucose tolerance. Preliminary data indicate that consumption of a low-fat/complex-carbohydrate diet, with no attempt at energy restriction, results in loss of body weight and improvement in insulin sensitivity. We hypothesize that replacement of dietary fat with simple rather than complex-carbohydrates will result in similar improvements in health. Thirty-six overweight men and women aged 55-80 years with impaired glucose tolerance will be randomized into one of three groups. Group 1 will receive a control diet (similar to baseline intake), group 2 will receive a low-fat/simple-carbohydrate diet (17% fat, 16% protein, 67% carbohydrate, 9g fiber/1000kcal), and group 3 will receive an identical low-fat diet and be placed in an aerobic exercise training program (4d/week, 80% VO2ma,). Body composition will be measured using air displacement plethysmography, energy and macronutrient metabolism will be measured using whole room calorimetry and de novo lipogenesis, and insulin sensitivity will be measured using a euglycemic-hyperinsulinemic clamp. Given the increasing prevalence of type 2 diabetes in the elderly U.S. population, completion of this project will more clearly define the specific dietary and exercise interventions that have the greatest potential positive impact on health in older individuals."}
{"text": "This competitive renewal proposal is to develop pulse saturation-recovery (SR) electron paramagnetic resonance (EPR) for application to spin labels in a context of site directed spin labeling (SDSL). The applicant's laboratory has recently made a breakthrough in SR detection based on a novel digital sub-sampling strategy, which has been termed time locked sub-sampling, and a new pulse spectrometer is under construction. Thorough engineering evaluation and design optimization including signal processing software are proposed for the next funding period. Feasibility for a new X-band bimodal loop gap sample resonator has also been demonstrated and further innovation is proposed. Engineering-based development follows naturally from the work of the previous funding period. SR can measure the rate of bimolecular collisions of spin labels with oxygen, and is a primary methodology for studying oxygen transport. Spin label oximetry is an important tool in identification of structural motifs such as alpha-helices or beta-sheets in proteins through SDSL methods. Three model systems are used here in each of three application-based specific aims. The model systems, in order of increasing biological relevance, are maleimide spin label in super-cooled sec-butylbenzene, hemoglobin labeled at beta93 in glycerol-water mixtures, and the ferric enterobactin receptor, Fep-A. The SR method is validated and compared with more conventional methodology. A novel SR method to separate overlapped spectra, termed discrimination by oxygen transport, is used to characterize multiple motional components of FepA. SR apparatus acquires intense free induction decay (FID) signals that are normally suppressed. Instead, this investigator proposes to use FID signals to measure site-to-site distances in double-labeled FepA. Frequency swept pulse electron-electron double resonance is introduced to characterize slow restricted angle motions in Fep-A in a situation where overall motion of the protein is negligible. The detection method, the bimodal resonator, use of FID signals for distance determination, use of SR to separate overlapped spectra, and measurement for the first time of restricted-angle motions are thought to be highly innovative. SDSL is a new and rapidly expanding methodology in structural biology. Addition of saturation recovery to SDSL methodology will, we hypothesize, revolutionize the field. The proposal lies at the foundation of modern biomedical research: development of tools that enhance one's ability to determine protein structure, dynamics and function. The investigator asserts that only on such a foundation can a rational approach to improved human health be based."}
{"text": "For risk assessment purposes it is important to establish the relationship between the applied or exposure dose and tissue specific concentrations and the relationship of this dose to toxicological or carcinogenic outcomes. It is therefore necessary to analyze tissue specific concentrations of 2,3,7,8-tetrachlorndibenzo-p-dioxin (TCDD) and other related chlorinated dibenzo-p-dioxins and furans in treated animals. We are therefore establishing a contract for analysis of TCDD and other related compounds in animal tissues. We will require high resolution gas chromatography/mass spectroscopy to quantitate these chemicals in these samples."}
{"text": "Fasting causes competition for nutrients between mother and fetus. The rate of maternal protein degradation may be lower in pregnancy in order to protect maternal stores while supplying the fetus. This rate will be measured by constant maternal infusion of I-13C leucine with application of steady state isotope dilution methods & will be compared in pregnancy and post-partum. Disturbance of the control of protein degradation may be responsible for altered fetal growth rate in gestational diabetes and preeclampsia."}
{"text": "Lysosomal storage diseases (LSDs) are a group of inherited metabolic conditions caused by defects in proteins critical for lysosomal function. While individually rare, their collective incidence is just over 1/7,000 live births, which makes the LSDs an important health problem. The majority of these diseases have a progressive neurodegenerative course, resulting in severe debilitation. Symptoms are not manifested unless there is more than 90% reduction in the residual activity of the deficient enzyme. Thus, an enzyme activity level above the critical threshold will prevent lysosomal storage and dysfunction, and can arrest, or reverse the disease process. Current therapies available for LSDs, such as enzyme replacement therapy (ERT) are only available to treatment of a few LSDs, and then only the non-neurological symptoms. Additionally, the approximate cost of over US$250,000/year/patient limits accessibility to ERT. For these reasons, therapies based on small molecules, which could cross the blood-brain barrier and be less expensive;appear to be an attractive approach. Small molecules can function as enzyme enhancement agents by binding, and stabilizing mutant misfolded proteins in the endoplasmic reticulum (ER), allowing them to evade the ER associated degradation (ERAD), and reaches the lysosome. Previously, we screened a small compound library of 1,040-FDA approved compounds and were able to identify and characterize two drugs that function as enzyme enhancement agents for two lysosomal enzymes: hexosaminidase A and glucocerebrosidase. My hypothesis is that small molecules may enhance residual activity found in patients with LSDs by direct interactions with the mutant enzyme, or by an indirect effect on different cellular pathways resulting in increased levels of the mutant and partially functional protein in the lysosomal compartment. I hypothesize that I can identify molecular probes that would restore arylsulfatase A (ASA), lysosomal enzyme deficient in metachromatic leukodystrophy (MLD), a neurodegenerative LSD for which a specific treatment is not available. To test this hypothesis, I aim to: (i) develop a robust and reproducible cell-based high throughput screening (HTS) assay to identify molecular probes that may function as enzyme enhancement agents for ASA;(ii) to configure a quantitative HTS assay for ASA, and carry out validation strategies for the \"active\" small molecules generated by the primary screening. I anticipate the submission of the screening project upon completion to the NIH Chemical Genomics Center for implementation. The candidate molecular probes generated by this screening will then be validated using different assays based on my experience previously acquired on the identification and characterization of enzyme enhancement agents for other lysosomal enzymes. The validated small molecules will be potential drug candidates to treat patients with MLD, and may also be used as tools to uncover novel biological pathways involved on the folding, maturation and trafficking of lysosomal proteins. PUBLIC HEALTH RELEVANCE: The development of high throughput screening assay (HTS) for arylsulfatase A (ASA) will allow the identification of molecular probes that, once characterized and validated, may be potential drug candidates to treat patients with metachromatic leukodystrophy, a lysosomal storage disease caused by deficiency of ASA. In addition, these compounds may be used as research tools to examine biological pathways involved on the folding, maturation and trafficking of lysosomal proteins."}
{"text": "This research project is aimed at developing a novel DNA sequencing, mapping, or diagnostic technique that requires no DNA labeling. The proposed innovative technique would even allow genomic DNA to be used for diagnostics, thus significantly reducing preparation cost (no polymerase chain reaction would be necessary). During Phase l we experimentally evaluated the most promising detection techniques and demonstrated the feasibility of the procedure. In Phase Il we plan to systematically determine the best parameters for probe immobilization and hybridization and determine the sensitivity of these procedures to variations in the parameters. We will also develop a less expensive prototype instrument that could be purchased by major facilities. Because of the potential cost effectiveness, accuracy and speed of this innovative technique, commercial benefits could be tremendous. PROPOSED COMMERCIAL APPLICATIONS: Commercialization could begin by supplying analytical services to researchers at national and private laboratories. Instrument sales would be first targeted to major research and clinical laboratories, but would grow to areas outside the biomedical field due to the wide applicability of the technique."}
{"text": "Candida albicans is both a ubiquitous part of the mammalian commensal flora and the most common systemic fungal pathogen of humans. Systemic disease is the fourth most common nosocomial infection and is associated with a ~40% mortality, due both to the severity of the infection and current inadequacies in diagnosis and treatment. Systemic, or disseminated, candidiasis mostly develops in patients whose innate immune system has been compromised by disease, chemotherapy, or medical intervention (surgery or implanted devices such as catheters). Thus, there is a fine line between Candida as a commensal and Candida as a pathogen and our long-term goal is to understand how this balance is maintained or disrupted to promote one state or the other. It is our premise that strengthening the immune system or weakening the fungus, even slightly, may tip this balance in favor of the patient, thus we have studied the interaction between C. albicans and cells of the innate immune system. From these studies, we have evidence that C. albicans secretes an immunomodulatory compound(s) that inhibits the release of nitric oxide (NO), a key antimicrobial and immunomodulatory compound, from macrophages. We have begun to characterize this inhibitor and have found that it is small, hydrophilic, heat-stable, and is not carbohydrate-based;one aim of this proposal is to identify this compound. In doing so, we have been aided by a series of genomic experiments that have defined the extensive and complex transcriptional response of C. albicans to phagocytosis by macrophages. One of the most surprising findings is an induction of the arginine biosynthesis pathway in its entirety. No other nucleotide or amino acid pathway is induced, making this a specific and unique response. In addition to being an essential amino acid, arginine is also the substrate for production of NO by the inducible Nitric Oxide Synthase (NOS2 or iNOS). Analogs of arginine inhibit iNOS;one such analogue has been shown to greatly reduce the anti-Candida activity of neutrophils. These arginine analogs fit our preliminary chemical characterization of the C. albicans-derived inhibitor. Thus, our central hypothesis is that C. albicans has co- opted the arginine biosynthesis pathway to produce an iNOS inhibitor, related to arginine, that promotes survival and pathogenesis of this organism. The experiments proposed here will test this hypothesis and identify the inhibitory compound. Serious fungal infections, caused mostly by Candida species, are increasingly common and severe. These affect mostly patients already debilitated by other medical treatments or illnesses and suggest that strengthening these patients'immune system would help fight these infections. We present data here that indicates that the fungus itself may be actively impairing the immune system and propose to characterize how this process occurs with the hope of eventually counteracting this ability."}
{"text": "1. It has been shown that the hypervariable region of flagellin of strains of Salmonella (potentially useful as typhoid vaccines) is a suitable locus for insertion of DNA coding for epitopes which are of interest as potentially effective immunogens for diseases other than salmonellosis. A nonapeptide which represents aa163-171 of Il-1 beta has been shown to be immunoenhancing. We shall test whether the expression of this nonapeptide enhances immune response to determinants which are juxtaposed to, near or spatially removed from this nonapeptide when it is inserted in the hypervariable region of vaccine strains of Salmonella. 2. Many polysaccharide antigens are considered to bc T independent in the sense that response to them does not require T cell help; anamnestic responses are not elicited and the immunoglobulin isotype response is restricted with such antigens. We propose to test a method to convert oligosaccharide determinants of such antigens to T dependence enabling both the elicitation of memory and unrestricted class switching. Furthermore, the process would make it possible to have such antigens expressed as peptide surrogates as piggy-back antigens in the hypervariable region of flagellin as in 1. A monoclonal anti Salmonella Vi will be used to screen a random peptide library for adventitious cross-reactive peptides which will be back-translated to DNA, and inserted for expression in the hypervariable region of flagellin. The essential hypothesis is that cross-reactivity as recognized in reaction will bc matched by cross-reactivity in induction of immune response. Specifically this might greatly enhance the utility of the typhoid vaccines being tested since they do not at present elicit anti-Vi."}
{"text": "Deficiencies in choroidal blood flow (ChBF) have long been suggested to underlie age-related retinal decline, and cause the outer retinal abnormalities that set the stage for AMD, given the necessary risk-conferring genes. Nonetheless, experimental evidence that this is the case has been lacking. We have been investigating the parasympathetic control of ChBF. We have found that the vasodilatory parasympathetic facial nerve input to choroid from the pterygopalatine ganglion appears to maintain high ChBF during low systemic blood pressure. We have found that this adaptive reflex, which we term ChBF baroregulation, is impaired by age, and when impaired by experimental manipulations in animal models leads to ChBF insufficiency and declines in retinal function. The studies proposed here will build on these findings and experimentally determine if impaired ChBF baroregulation leads to the types of outer retinal pathologies that characterize aging and/or early (i.e. dry) AMD. Three Aims will be pursued, all in rats. In the first Aim, we will determine the basic neural mechanisms by which ChBF baroregulation is mediated. These studies will provide basis for determining the defects that cause dysfunction of ChBF baroregulation, and identify points of intervention that might allow restoration of normal ChBF control. Our particular goal is to determine how blood pressure signals from the thoracic aorta via the aortic depressor nerve (ADN) regulate ChBF by their input to the nucleus of the solitary tract (NTS) and its output to the parasympathetic part of the facial nucleus (the superior salivatory nucleus, SSN), which project to the vasodilatory neurons of the pterygopalatine ganglion, which themselves innervate choroidal blood vessels. To this end, we will use imaging of circuit connectivity and pharmacological approaches to determine if SSN- projecting NTS neurons are excitatory and receive input from inhibitory ADN-receptive NTS neurons. In our second Aim, we will characterize by functional and morphological means the outer retinal abnormalities caused by long-term disturbance in ChBF baroregulation. We will be particularly interested in determining if the outer retina pathologies include those observed in aging, such as sub-RPE basal laminar deposits and photoreceptor loss, and/or those seen early in AMD, such as sub-RPE basal linear deposits, RPE and photoreceptor loss, and outer retinal waste accumulation (cholesterol, and proteins with advanced glycation end-product or peroxidized lipid adducts). Since light history is an AMD risk factor, we will also determine if impaired ChBF baroregulation exacerbates photoreceptor light damage. In the third Aim, we will determine if age-related choroidal baroregulatory impairments are associated with outer retinal dysfunction and pathology. Our studies may suggest choroidal baroregulation impairment as underlying age-related retinal decline, and as an AMD risk factor. This would suggest testing of choroidal baroregulation to assess AMD risk, and recommend drugs that boost ChBF as therapies. PUBLIC HEALTH RELEVANCE: Our studies will determine how the brain uses low blood pressure signals to maintain high blood flow in the choroidal layer of the eye, so the outer retina receives the nutrients it needs to remain healthy and function properly. We will also determine if impairment of this function underlies the retinal decline that occurs with aging, and explains why aging is the major risk factor for AMD. Our studies may lead to use testing of blood pressure regulation of choroidal blood flow as an AMD risk assessment, as well as lead to use of drugs that normalize neural control of choroidal blood flow to slow AMD onset and progression."}
{"text": "[unreadable] [unreadable] This proposal describes a 5 year training program for the development of an academic career in [unreadable] Cardiovascular Disease. The principal investigator (PI) has completed structured fellowship training in [unreadable] Cardiology at the University of Pittsburgh and will expand upon his scientific skills through interdepartmental collaboration. This plan emphasizes training in cardiac physiology, pathology, endocrinology, and biochemistry at the gross and molecular levels through investigation of the importance of leptin signaling in the heart. Dr. Charles McTiernan is an Associate Professor in the Cardiovascular Institute (CVI) and a recognized leader in the field of heart failure (HF) and cytokine research. Dr. Christopher O'Donnell, Associate Professor in the Pulmonary Division, is an expert on the study of leptin and murine physiology in heart failure. Together, both Drs. McTiernan and O'Donnell have trained numerous students and fellows in the past and will mentor the Pi's scientific development. To enhance training and provide scientific and career advise, an interdepartmental advisory committee will be formed. Dr. Michael Mathier, Assistant Professor in the CVI, will provide training on the functional assessment of heart failure in mice. Dr. Robert O'Doherty, Assistant Professor in Biochemistry, will provide guidance in the areas of leptin physiology, signal transduction, and cre-lox technology. Dr. Jorge Sepulveda, Assistant Professor of Pathology, is an expert in the field of cardiac pathology and will provide training in techniques related to leptin histopathology. The research proposed is based on preliminary data that shows benefit to leptin on cardiac function and survival after myocardial infarction (Ml). Therefore, we hypothesize that leptin is protective after Ml. Specific aims designed to test this over-riding hypothesis and provide mechanistic understanding of leptin's benefit [unreadable] after Ml include using mice to: 1) Examine of the effect of leptin-deficiency and systemic leptin replacement on survival and the development of HF after Ml. 2) Compare systemic versus central effects of leptin administration in ob/ob mice on survival and the development of HF after Ml. 3) Examine the effect of cardiac specific deletion of the leptin receptor on survival and the development of HF after Ml. Overall, the CVI at UPMC provides an ideal environment for training physician scientists by encouraging inter-disciplinary collaboration and sharing of resources necessary for productive scientific discovery. [unreadable] [unreadable] (End of Abstract) [unreadable] [unreadable] [unreadable]"}
{"text": "Breast cancer is the second most prevalent cancer in women, with a mortality rate of 40,000 per year in the US. However, if detected and treated early, more than 95% of breast cancer patients will survive. Current method for breast cancer detection relies heavily on X-ray mammography, which produces many false positive findings. These false positive readings cause substantial mental anguish in patients and in many cases, costly and painful biopsies. A full 80% of all biopies uncover only benign masses and calcifications at an estimated annual cost of 2.5 billion dollars. To improve breast cancer detection, we propose a highly innovative design for a dedicated Positron Emission Mammography (PEM) camera utilizing long, linear, lead-walled straw (LWS) detectors that provide high spatial resolution and rigorously accurate depth of interaction determination. Thus detectors with ample field of view to contain the entire breast without translation can be employed and very close detector pair spacing can be used without resolution degradation. Our proposed camera offers markedly increased sensitivity and resolution with substantially reduced cost compared to crystal-based PEM cameras under development. Phase I of the project will achieve proof of concept by constructing and testing two substantial submodules of a full scale camera. In Phase II, a full scale high sensitivity prototype camera will be constructed and tested."}
{"text": "By utilizing various strains of rats, Rattus norvegicus, a paradigm of human culture is being explored. This paradigm is based on the following theoretical considerations: The evolution of species of mammals, including human beings, has resulted in optimum group sizes of 12 adults. Since each group occupies a fixed territory, eventual occupation of all available space by such groups culminates in a stable adult populations of species. However, if conditions permits elaboration of social roles (particularly cooperative ones) and development of complex communication networks, then more individuals can survive in a given area without a resulting stressful increase in rate of interaction among members of the population that would otherwise trigger limitation of population size. This process which has permitted successive doubling of the human population is being experimentally simulated by providing computer controlled contingencies for rats living in a complex physical environment that fosters development of cooperative social roles and more complex communication networks."}
{"text": "The Eighth Conference on the Neurobiology of Learning and Memory is the eighth in a series of conferences organized by the Center for the Neurobiology of Learning and Memory at the University of California, Irvine. The theme of the conference is: Memory and Brain: Basic Mechanisms and Clinical Implications. It will be held in Irvine, California on March 11-14, 2006. The overall goals of the conference are to communicate and discuss recent research findings in five currently important topics in the neurobiology of learning and memory, to stimulate discussion about how basic findings might be translated into therapeutic or other applications, and to educate and inform researchers in the field. The 2006 meeting will include five sessions: 1) Mechanisms for acquisition and maintenance of reinforcement related behavior: Relation to addiction/compulsion, 2) Sensory and perceptual learning, 3) Memory and normal and abnormal aging, 4) Modulation of neuroplasticity and long-term memory, and 5) Molecular and cellular mechanisms of memory. To date, fifteen speakers have agreed to make presentations; we are planning a total of twenty. These speakers are among the world's leading investigators in the topics listed. The conference format emphasizes discussion by all participants. Chairs will be chosen for each session, to lead the interchange of ideas. The general sessions will take place at the Hyatt Regency Hotel in Irvine; a poster session (approximately 100 posters) will be held in the Herklotz Research Facility on the DC Irvine campus. We anticipate an attendance of 350-400 people, including faculty, postdoctoral researchers and graduate students from the U.S. and many foreign countries."}
{"text": "Further studies on the subunit structure of triton X-100 and papain solubilized receptors will be undertaken to get an insight into the molecular weight of the anchor piece which inserts the receptor to the membrane. The subunits will also be characterized with respect to IF-cbl and Ca ion binding. Membrane lipids or synthetic lipids will be used to make lipososmes with the receptor to study the uptake of 125I IF-(57 CO)cbl by the liposomes. Using suckling rats the membrane bound and soluble alkaline phosphatases will be further characterized about their lipid binding abilities in order to make liposomes."}
{"text": "Exposure to stressful events during withdrawal from psychostimulants, such as cocaine or amphetamine, induces drug craving and drug relapse in both animal models and human individuals. The effects of stress on drug-seeking behavior persist for months following psychostimulant abstinence, and play a large part in chronic relapses associated with psychostimulant addiction. It is the chronic nature of psychostimulant addiction and relapse that produces significant social, health and economic impact. Identifying the neurological alterations that occur during psychostimulant withdrawal will have significant impact on the development of pharmacological treatments designed to prevent drug relapse. The studies outlined by this proposal will determine whether psychostimulant abuse results in increased sensitivity of stress-related neural systems. These studies will also establish the duration of these effects during psychostimulant withdrawal. This will be achieved by using laboratory rat models of psychostimulant withdrawal. Changes in stress behavior, and alterations to the activity of neurotransmitter systems that regulate stress behavior, will be determined during acute and protracted withdrawal from amphetamine. Furthermore, studies will determine whether alterations to neural stress systems and stress behavior following psychostimulant withdrawal can be reversed by chronic treatment of the antidepressant, fluoxetine. Together, the proposed experiments will elucidate the neural substrates of stress sensitivity during psychostimulant withdrawal, and will determine the feasibility of antidepressant treatment to reduce stress behaviors and thus psychostimulant relapse. [unreadable] [unreadable]"}
{"text": "The objective of this proposal is to study the molecular basis for the decreased ability of the immune system in many elderly individuals, to produce high affinity antibodies and thus mount an effective immune response to vaccines or infections. To pursue this objective, the principal investigator will study the age-associated changes in the relationship between structure and function of immunoglobulin VH and VL genes which produce the natural anti-Gal antibody. Anti-Gal constitutes 1% of circulating IgG in humans and interacts specifically with the carbohydrate structure Gala1-3GalB1-4GlcNAc-R (termed alpha-galactosyl epitope). As many as 1% of circulating B lymphocytes can produce anti- Gal. The applicant has found that most of the sequenced VH genes in anti-Gal producing lymphoid clones, cluster within a distinct region of the VH3 immunoglobulin gene family, and that these genes undergo somatic mutations. Analysis of age-associated changes in activity of anti-Gal have demonstrated loss of affinity of this antibody in many elderly individuals. The principal investigator proposes to study two possible mechanisms which may cause the age-associated impairment in anti-Gal affinity in humans: 1) different utilization of VH and VL genes repertoire for anti-Gal synthesis; and 2) impaired affinity maturation in anti-Gal genes because of age-associated changes in the somatic mutations process. For this purpose he will construct combinatorial phage display libraries in young and in elderly individuals and isolate the anti-Gal displaying phage. Comparative analysis of the anti-Gal VH and VL gene repertoire in the young and elderly, and determination of the resulting antibody affinities, will enable the applicant to establish whether the age-associated decrease in antibody affinity correlates with the gene repertoire used for anti-Gal synthesis. Comparison of the V gene sequences with the corresponding germline genes will allow the investigators to establish whether the age-associated changes reflect the excess or paucity of somatic mutations in anti-Gal producing genes. The applicant will further determine whether elderly individuals with low affinity serum anti-Gal retain some high affinity clones, by selecting high affinity anti-Gal phage from the libraries of these individuals. Lastly, by correlating anti-Gal affinity with the immune response to an influenza virus vaccine, the principal investigator will determine whether anti-Gal can predict the competence of the immune system in elderly individuals to produce high affinity antibodies. Thus, by exploiting the anti-Gal system, the applicant aims to understand the molecular basis of the well-documented age-associated impairment of humoral immune responses in humans."}
{"text": "The goal of this research effort is to understand how various types of white blood cells recognize and respond to the presence of a microorganism or cancer cell in the body, or inappropriately recognize a normal component of the body (an auto-antigen). Our experiments are designed to provide a detailed understanding of how the substances (antigens) making up these microorganisms, cancer cells, or normal self-components, are made visible to the defending cells of the innate (anti-unspecific) or adaptive (antigen-specific) limbs of the host defense system cells and how recognition in infections or malignant cells is linked through complex cell-cell interactions to the induction of protective or self-destruction effector responses. Our previous work has described the events within a cell that bring together the antigen and MHC molecule and the cellular distribution of antigenic complexes within the body (antigen processing and presentation). We are now conducting studies primarily at the cell and tissue level to relate the physiology of antigen recognition to the development of effector function, immune memory, or tolerance and to add to this picture the activities of other cell types such as non-hematopoietic cells like fibroblastic reticular cells (FRCs). By understanding these events, we will be able to determine how best to deliver antigenic substances to stimulate an effective immune response or to interfere with autoimmune reactions and to understand how innate and adaptive components of the immune system participate in host defense under normal and pathological conditions. We previously reported a new method for the direct confocal microscopic visualization in real time of the interactions of T cells and antigen presenting cells in intact lymphoid tissue. These studies showed that individual T cells bind stably to antigen-bearing dendritic cells for several hours before activation-associated events lead to dissociation of the cell pairs and rapid migration of the T cells in the lymphoid tissue. Immunological synapses between T cells and a critical antigen presenting cell (the dendritic cell) were visualized in vivo as was clonal T cell division. This experimental system has now been improved by moving to multiphoton rather than confocal imaging and true intravital observation methods have been developed. We have worked out methods for imaging the bowel, which have allowed us to directly image the interaction of microorganisms with gut epithelium and dendritic cells in the mucosal immune system. Our studies in the small bowel have revealed the dynamic extension of lamina propria dendritic cell processes across the villus epithelium and the capture of lumenal bacteria by these processes. Other studies revealed the movements of B cells in lymphoid tissues and have provided evidence that dendritic cells outside of the follicle are involved in presentation of antigen not only to T cells but also to B cells. Using these new imaging approaches in concert with classical cellular immunological tools such a flow cytometry, we have recently shown that the small adapter protein SAP is essential for sustained interaction of antigen-specific T and B-cells that is needed to produce robust B cell clonal expansion and the development of germinal centers. Surprisingly, the same molecule is unnecessary for effective T cell interaction with dendritic cells, revealing an unexpected dichotomy in the molecular control of antigen-dependent cell-cell interactions involving T cells. New transgenic mice with cells ubiquitously expressing fluorescent proteins are being generated to provide new tools for long-term tracking of cells and genetically engineered strains of mice that have fluorescent reporters under the control of cytokine gene regulatory elements are being employed to relate the physical behavior of immune cells to their function."}
{"text": "We propose to hold an international conference involving approximately 350 scientists and 50 students from the U.S. and abroad. A major objective for the conference, Genetic Engineering of Animals: An Agricultural Perspective, is to bring together scientists working in the area of molecular genetics to discuss methodologies, philosophy, and potential applications as it relates to improvement of domestic animal agriculture and domestic animal research. To address the topic, we envision a tentative program that will be presented by approximately 24 of the leading scientists in genetic engineering of animals. The program will open with a conceptual session which will serve to meet a major objective of educating people who may have potential application of genetic engineering to increase the efficiency of their research efforts or to aid them in addressing important new research problems related to their research interests and the improvement of domestic animal production. A section on applications in laboratory and domestic animals will include discussions on sources of genetic material and examples of applications, i.e. influencing physiological, immunological, and developmental processes. A final section will address philosophical issues such as future application, moral, and legal implications, and where does it lead to. Proceedings of the conference will be published in an edited book within six months of the conference and the book should serve as a resource for the current state of our knowledge about genetic engineering as it relates to domestic animal agriculture."}
{"text": "The long-term goal of this Challenge Grant project is develop on a fast-track several existing new drugs which are already known to have activity in animal models of tuberculosis (TB), but which will not be developed by the respective companies in which they were discovered. Sequella, Inc. is in licensing negotiations for the rights to at least two, and possibly a third promising drug lead, selected with an emphasis on the following criteria: 1. Evidence of efficacy in a mouse model of tuberculosis 2. Simple chemical composition and synthesis, for ease of production and pricing 3. Representation of different drug classes 4. Evidence that they do not share cross-resistance with existing TB drugs. Sequella, Inc. will cost share with the NIAID to support the necessary remaining preclinical studies for each drug lead, according to the compound's present state of development. Preclinical studies include medicinal chemistry to enhance solubility and bioavailability, pharmacodynamic studies in animals, toxicological tests in animals and/or in vitro systems, and purity/stability testing in bulk drug prepared under GMP guidelines. Based upon the data obtained from the preclinical studies, Sequella, Inc. will apply for Investigational New Drug (IND) approvals for all compounds that show a duration of pharmacodynamic effects consistent with dosing intervals of no greater than once a day, and that appear likely to have an acceptable therapeutic ratio. The decision to proceed with Phase I/II trials of individual drug leads will be made cooperatively with NIAID staff. For each drug to be tested further, Phase I trials to determine tolerability will be carried out in volunteers at the NIH Clinical Research Center (if approved) or under contract. Phase II studies of each individual drug to test for efficacy in human TB patients will be carried out using a clinical trial design intended to reduce the risk of drug-resistant strains arising or spreading. The aims of the two-month Phase I time period would be to complete licensing agreements on targeted drugs (PA-824), identify promising TB drug leads in addition to those of which we are already aware, to meet with investigators who have worked with these compounds, to begin the negotiating process with Universities or companies to secure right-to-license agreements, and to outline clinical development plans for each TB drug candidate."}
{"text": "This application is in response to a Letter of Invitation (LOI-HD-05-111) to conduct community-linked studies to investigate the role of prenatal alcohol exposure in the risk for SIDS, stillbirth and FAS, and to determine how these different outcomes are inter-related. The proposed research will be conducted by the investigators the Prenatal Alcohol, SIDS, and Stillbirth (PASS) Research Network in a cooperative agreement with NICHD and NIAAA. This research involves the collaboration of: 1) two comprehensive clinical sites serving populations that are high risk for prenatal alcohol exposure, SIDS, and stillbirth, i.e. the American Indians in the Northern Plains and the Cape Coloured in Cape Town, South Africa;2) a central Developmental Biology and Pathology Center (DBPC);3) a central Data Coordinating and Analysis Center (DCAC);4) a central Physiology Assessment Center (PAC);and 5) program scientists and officers at the NICHD and NIAAA. This particular application pertains to the Tygerberg Comprehensive Clinical Site (CCS). The experimental design involves a prospective study of 7,000 pregnancies, and two retrospective, autopsybased studies of SIDS and stillbirth. The long-term goals of the SAFE PASSAGE STUDY are to decrease fetal and infant mortality and improve child health in communities at high risk for prenatal maternal alcohol consumption. The Specific Aims of the Network are as follows: 1) to determine the association between prenatal alcohol exposure and the risk for SIDS and stillbirth;2) to determine the role of the timing, pattern, and amount of prenatal alcohol exposure and other environmental factors in the risk for morbidity and mortality in early human life;3) to determine the role of specific genes in modifying the risk for morbidity and mortality in early life that is associated with prenatal alcohol exposure;4) to determine the role of alcohol exposure during pregnancy, and interactions among alcohol exposure and environmental and genetic modifiers, in altering profiles of autonomic activity of the fetus and infant, and neurobehavioral outcomes in the infant;5) to determine the role of maternal alcohol exposure, as influenced by specific environmental and genetic factors, in the impairment of placental function, and thereby the increased risk for fetal and/or infant morbidity and mortality;and 6. To determine abnormalities in key neurotransmitter systems in the brains of fetuses and/or infants that convey risk for sudden death, and to determine the role of prenatal alcohol exposure, as influenced by specific environmental and genetic factors, in their pathogenesis. The mission of the CCS is to ensure the proper identification of the perinatal risks of the use of alcohol during pregnancy, early detection of these risks and to perform basic research related to alcohol-induced injury in the human brain and placenta. This multidiscipline international study will determine whether the exposure of the fetus to alcohol during pregnancy leads to SIDS stillbirths and other complications. Knowledge gained from the study will help health care workers to provide better care for pregnant women."}
{"text": "The purpose of this study is to assess the efficacy and safety of 3,4-diaminopyridine in patients with myasthenia gravis and the Lambert-Eaton myasthenic syndrome."}
{"text": "The effect of prolonged space flight on the size and function of the heart is unknown. Using ultrasound, we measured the wall thicknesses, internal dimensions and the performance during blood ejection of the hearts of the Skylab 4 astronauts. Studies immediately following splashdown and subsequently over 68 days post-flight revealed no impairment of heart performance after 85 days in space."}
{"text": "Specific Aims 1. To facilitate the implementation of patient oriented HIV/AIDS related research at UCLA 2. To assist investigators with the regulatory aspects of biomedical and behavioral patient-oriented research 3. To establish and maintain a clinical database that will interface with the CFAR/CNICS program. The primary responsibility of the Clinical Research Facilitation Core is to facilitate the process of IRB submissions for faculty, fellows, new researchers and translational researchers who do not have a great deal of experience with human subjects research. While funding to facilities and equipment are crucial to the conduct of research, regulatory delays in the conduct of research are often overlooked. This core in itself is innovative as it provides investigators with resources not previously available to reduce delays. By its very nature, this core will support new initiatives. It will encourage research among new faculty, young faculty and fellows. The core is fully linked into the CFAR seed grant process and allows recipients to become productive in their research far earlier than if they did not have the Core to assist them. The Core has created both a website and a blog to share the resources learned while submitting applications. Additionally, the Core provides seminars to investigators and staff to increase knowledge of regulatory pitfalls and procedures. In reducing regulatory delays in. the conduct of patient-oriented research, this Core helps to maintain UCLA investigators on the cutting edge of research in HIV. PERFORMANCE SITE(S) (organization, city, state) University of California, Los Angeles Center for Clinical AIDS Research and Education 9911 W. Pico Blvd. Suite 980 Los Angeles, CA 90035 PHS 398 (Rev. 04/06) 614"}
{"text": "The principal objective of this multi-center study is to evaluate and compare safety and growth velocity effectiveness of graded Norditropin dose levels after administration to growth hormone deficient children for 2 years. The secondary objective is to determine safety and efficacy with chronic Norditropin administration and follow-up of patients until they reach adult height. There are 100 children enrolled nationally, randomly assigned at 3 dosage levels, 0.025, 0.050, and 0.1 mg/kg/day. The study is divided into 2 sequential segments: Segment I 24 months, Segment II lasts from end of Segment I until they reahc adult height. We have enrolled 6 children on this protocol 4 males, 2 females. Doses are as follows: 1 male on 0.1 mg/kg/day, 1 male on 0.05 mg/kg/day, 2 males and 2 females on 0.025 mg/kg/day. All children are growing well. We have lost one child for compliance reasons. No child has yet reached final height."}
{"text": "The melanoma stem cell can be identified by a few markers such as TIE-1, CD133, nestin and ABCG2. Recently, a novel marker, ABCB5 was identified by Schatton et al. This protein is a transporter protein responsible for the efflux of chemotherapeutic drugs (doxorubicin specifically) from melanoma cells. Schatton et al demonstrated that ABCB5 was expressed in CD133 + cells, and that ABCB5+ cells were capable of self-renewal, and could be serially transplanted in animal xenograft models. ABCB5 was expressed in both primary and metastatic melanomas, and constituted about 2-20% of the tumor cell population. Given recent data that suggested that the Wnt protein, Wnt5A, can affect the proliferation and maintenance of hemaopoietic stem cells, we are trying to determine what the role, if any, of Wnt5A in the maintenance and profileration of the melanoma stem cell might be, using ABCB5+ as a marker of stemness. We have recently shown that Wnt5A can cause the loss of expression of melanoma differentiation antigens (MDAs), while promoting metastasis. Melanoma stem cells give rise to differentiated tumor cells, and so we are currently exploring the link between ABCB5, MDAs and Wnt5A. We have found thus far that cells which express high levels of MDAs tenc to also express high levels of ABCB5. A very interesting finding is that PCR anlaysis indicates, in our less differentiated, more metastatic melanoma cells, ABCB5 is alternatively spliced. We are currently sequencing this gene to determine if this is due to mutation. If it is, we propose to analyze a series of patient tumors for the ABCB5 mutation, and then perform a functional anlaysis to uncover the significance of this mutation, using both in vitro and in vivo methodology.[unreadable] Another family of proteins involved in melanoma stem cell development, and melanoma metastasis is the Notch family of proteins. Current data from our laboratory indicates that Wnt5A and Notch proteins may interact to promote wound-healing, and melanoma metastasis. We are exploring this link further, and trying to better understand the interactions between these two families of proteins not just in a cancer setting, but also in the context of normal skin, and skin aging."}
{"text": "The goal of our proposed research is to improve diagnosis of patients presenting to the Emergency Department [ED] with dizziness, some of whom are misdiagnosed with potentiallygrave medical consequences. The prevailing diagnostic paradigm for the evaluation of the dizzy patient is based upon a 'pathophysiologic' approach. This approach begins a search for etiology with the assumption that the quality of symptoms (vertigo, presyncope, imbalance, or non-specific dizziness) reflects the underlying pathophysiologic mechanism (vertigo = vestibular, presyncope = cardiovascular, imbalance = neurologic, and non-specific = psychiatric). Although this assumption often holds true, the 'pathophysiologic' approach mandates a thorough etiologic search in each organ system, not only the one suggested by symptom quality. This strategy is well suited to the referral clinic setting where it was developed, but poorly suited to the time-pressured environment of the ED, where the high index of illness severity demands effective triage rather than diagnostic certainty. We hypothesize that (a) potentially serious misdiagnoses of dizzy patients are uncommon but not rare events in the ED and may result from an over-reliance on the diagnostic importance on symptom quality; (b) a novel 'triage' approach to diagnosis would reduce misdiagnoses and improve outcomes in an 'in vitro' computer model of the diagnostic approach to dizziness; and (c) a clinical decision-support system based on this approach would reduce misdiagnoses under simulated patient conditions. To test our hypotheses, we have designed three specific aims to: (1) measure the frequency, potential severity, and possible cause of misdiagnosis of dizzy patients in the ED (by gathering extensive case data on each ED dizzy patient and referencing ED physician [EP] diagnoses against those of a multidisciplinary expert panel); (2) design a computerized decision model to test a new 'triage' approach to diagnosis (by comparing 'in vitro' simulations of the two diagnostic approaches using hypothetical case scenarios); and (3) 'pilot' a web-based decision support system to reduce misdiagnosis of simulated ED dizzy patients (by comparing EP performance on a video-case-based examination with or without the use of the decision support system, using a randomized trial design). Results of this study will form the foundation for subsequent research into the effectiveness of error-reduction interventions among dizzy patients. The research career award candidate has devoted himself to acquiring the clinical and research skills required to complete this project and launch a successful career as an independent investigator. He has garnered the support and enthusiasm of both his clinical department and a large, multidisciplinary team that will enable him to complete the stated objectives. This research project and the research paradigms derived from it will form the nucleus of a career devoted to research in medical decision-making, causes of diagnostic errors, and methods to preventthem."}
{"text": "Accumulating evidence from many different types of scientific studies indicates that inadequate intake of the B vitamin, folate, increases the risk of colorectal cancer. Among the observations supporting this concept are those which indicate that a common polymorphism in the folate-dependent enzyme, methylenetetrahydrofolate reductase (MTHFR), is associated with modulation of colorectal cancer risk. The biochemical and molecular mechanisms by which the effect of folate is mediated remain ill-defined, although the prevailing concept focuses on folate's central role in DNA synthesis and repair, and biological methylation. Although less compelling, there exists both scientific rationale and empiric evidence that other B-vitamins involved in one-carbon metabolism can interact with folate status in determining the risk of neoplastic transformation in the colorectum. An understanding of the pathways by which folate modulates colorectal carcinogenesis would be greatly facilitated by defining the mechanisms by which vitamins B2, B6, B12, as well the common MTHFR polymorphism, interact with folate in determining cancer risk. These factors can therefore serve as mechanistic probes to further our understanding of the processes involved. The proposed experiments will be conducted in established mouse models of B-vitamin depletion and intestinal carcinogenesis, as well as in some novel mouse models that are under development. Experiments are planned to examine the means by which interactions of the abovementioned dietary and genetic factors alter the biochemical, molecular and cell cycle mileu of the intestinal epithelium and thereby modulate the risk of carcinogenesis. Although several genetic pathways will be examined proactively, identification of novel, previously unsuspected, pathways towards cancer will be elucidated by use of DNA microarray technology. Elucidating the mechanisms responsible for the effect of folate on carcinogenesis will greatly enhance our ability to translate this field of scientific investigation into public health initiatives that effectively reduce the risk of cancer."}
{"text": "Description (adapted from applicant): The goal of the COEP is to provide environmental health information and developing educational programs in response to identified community needs. Reflecting the Center?s research theme on environmental agents as modulators of disease processes, Center faculty are actively involved in COEP delivering presentations at community forums and teacher workshops, assisting in curriculum development, or participating in the Community Advisory Board. Outreach efforts span the entire range of the community, from children to older adults, from rural to urban residents. In addition to the Community Advisory Board, the COEP Community Outreach Initiatives include a Lead Poisoning Prevention Program, Occupational and Health Care Provider Outreach, Community Lectures, Newsletters, and Web Site. The COEP Science Education Outreach Initiatives include three main programs: ?My Environment, My Health, My Choices?, an Environmental Health Curriculum for middle and high school students; Project Begin which involves the ethical, legal, and social implications of genetic research in environmental health sciences; and the Life Sciences Learning Center, itself comprised of five programs?Laboratory Investigations, Lab Skills Development Program, Saturday Morning Science, Summer Science Camps, and Teacher Workshops. Many of these COEP programs are new initiatives within the Center, developed since the 1999 NIEHS Center grant renewal."}
{"text": "Toxic oxygen species are important mediators of lung injury. An array of antioxidant defenses have evolved to protect cells against reactive oxygen species. An important component of this defensive system is glutathione. The specific aims in this study are to identify and characterize genetic factors which can influence the intracellular concentration of glutathione in mouse embryonic stem cells, and to generate transgenic mice from these cells. We hypothesize that selected mouse embryonic stems cells with alterations in glutathione content will display either increased or decreased resistance to reactive oxygen metabolites. Transgenic mice formed from such stem cells should also show altered sensitivity to free-radical damage. During the study period we will 1) isolate retrovirus-induced mutants of mouse embryonic stem cells with altered GSH content, by using a fluorescent dye specific for GSH; 2) isolate and characterize the mutated DNA that resulted in altered GSH metabolism; 3) analyse the effects of the altered GSH on cell sensitivity to free-radical damage; and 4) establish colonies of transgenic mice bearing the GSH mutations of interest. Creation of transgenic mice with genetically altered GSH content will be especially useful in future studies of free radical-induced lung injury."}
{"text": "Summary: Infections due to Neisseria gonorrhoeae represent a significant cause of morbidity and mortality in women and children throughout the world. The pathogenesis of gonococcal infections is not well understood, particularly with respect to the effect of patient hormonal status on the infectious disease process. Currently, there are no licensed vaccines for prevention of gonorrhea. This project is investigating the effect of reproductive hormones, estradiol and progesterone, and oral contraceptives, on the expression of gonococcal outer membrane antigens, and whether or not these hormones affect attachment and/or invasion to human cervical and endometrial tissue culture cells. The goal is to identify potential new antigens that may be particularly relevant in gonococcal infections in women and to determine if hormonal status may be a factor to be considered in the evaluation of models of infection. The specific aims of this project are: 1. to assess the effect of estradiol and progesterone on expression of gonococcal outer membrane antigens; and 2. to determine whether estradiol and progesterone alter gonococcal adherence and/or invasion to human cells. Progress to date on specific aims: 1. We have determined the effects of reproductive hormones on the cultured cell line Hec1B: estradiol was not inhibitory to the growth of Hec1B cells in culture over a range of concentration from 1x 10-6 to 1 x 10 -12 M/L, and the cell line grew optimally at concentrations of 1x 10 -8 and -9 M/L. These concentrations are similar to the physiologic levels measured in serum taken from women during the early proliferative stage of the menstrual cycle. Progesterone and combined progesterone and estradiol are slightly inhibitory to cell proliferation. Phenol red has a weak estrogenic-like activity and we have identified this common component of cell culture media as a possible confounder in gonococcal adherence assays. 2. Outer membrane proteins from HEC1B cells grown with and with out estradiol were analyzed and at least two proteins at 80kDa and 180 kDa demonstrated increased expression when exposed to estradiol. These proteins will be excised from gels and sequenced using Liquid chromatography mass spectroscopy/mass spectroscopy. 3. Gonococcal adherence assays were conducted and an increase in gonococcal adherence in the presence of 1 x 10-9 M/L estradiol was observed. Our laboratory has recently developed an assay that will more accurately quantitate the number of bacteria bound to cultured cells. 4. Outer membrane proteins isolated from gonococci bound to Hec1B cells in the presence or absence of estradiol revealed an altered protein profile by SDS-PAGE. Several proteins were down-regulated and one protein was up-regulated. These proteins were excised from gels and sequenced using Liquid chromatography mass spectroscopy/mass spectroscopy. Sequenced proteins were used to search the FA1090 Neisseria genome at the University of Oklahoma and the NCBI-Blast database. The down regulated genes were identified as groEL, dnaK, L23, and a gene encoding a putative lipoprotein. The up regulated protein was identified as neisserial catalase. The gene for each identified protein has been cloned from MS11mkC genomic DNA. Promoterless b-galactosidase (lacZ) and GFP reporters were constructed which will be used to evaluate differences in gene transcription of identified genes in the presence and absence of hormones. Our laboratory is in the process of identifying other proteins that differed in their expression when in the presence of estradiol. High precentage SDS-PAGE is being used to isolate lower molecular weight proteins."}
{"text": "Project 3: Dissecting p53 Action in a Mouse Lymphoma Model Scott Lowe, Ph.D. p53 is a potent tumor suppressor that limits cancer development and contributes to the anti-tumor effects of certain cytotoxic agents. Accordingly, p53 mutations are associated with aggressive cancers, poor patient prognosis and, in some cases, therapy resistance. Our project studies the p53 network using the Ep-myc transgenic mouse, a well characterized model of B cell lymphoma in which p53 profoundly impacts tumorigenesis and treatment outcome. We previously used the E?mu?-myc system to study how p53 loss promotes tumor development and drug resistance, and to explore the relationship between cancer genotype, tumor progression, tumor maintenance, and treatment sensitivity. These studies produced new insights into p53 regulation and effector mechanisms in diverse contexts where it limits proliferation. They also established that p53 loss is required for tumor maintenance and that translational control of cell survival is a druggable mediator of oncogenesis. To facilitate our goals, we developed methods to genetically manipulate E?mu?-myc lymphomas, enabling the rapid and cost-effective production of mice harboring tumors with diverse genotypes. We also incorporated small animal imaging methods to track lymphoma progression and response, and stable and inducible RNAi technology to fine tune and study loss of function phenotypes. Moving forward, we seek to further explore the interplay between cancer genetics and cancer therapy in the E?mu?-myc system in order to produce a more complete picture of the p53 network and its action in vivo. We also wish to understand in detail how p53 mutations impact tumor behavior, and identify new therapeutic targets based on the vulnerabilities they create. We will perform focused RNAi screens to identify new modulators of p53 action relevant to suppressing tumorigenesis and/or modulating therapy response, test the hypothesis that the deletions on chromosome 17p influence cancer biology beyond simply inactivating p53, and identify and characterize drug targets that interact with the p53 tumor suppressor network. Our approach uses genomic data from human lymphoma to inform function studies in mice, and implements a suite of new animal modeling and genetic tools that are time and cost effective."}
{"text": "The purpose of this project will be to evaluate a simple blood test and an interview process proposed as methods to screen for the presence and control of diabetes. If accurate these tests may be clinically useful for dentists interested in treating geriatric patients and needing to asssess the presence and degree of control of diabetes in this population prior to initiating dental treatment. Methods: 100 subjects of which at least 30 will be diabetic, will be recruited from the patient population of two extramural geriatric dentistry clinics run by the U.S.C. School of Dentistry. A medical history and relevant symptom report will be taken from each subject using an interview procedure. In addition, the diabetic subjects will be asked to respond to questions regarding the history and control of their diabetes. All subjects will be asked to fast from midnight, and will then have three tests run: a venous blood glucose, a hemoglobin A 1c, and a capillary blood glucose test. Those subjects who have never been diagnosed as being diabetic will then be given a 75 gram carbonated drink for glucose testing. Two hours later venous and capillary blood will be drawn. The data received will be tabulated and coded and subjected to descriptive statistical analysis. Significance: It has been well-reported that the prevalence of diabetes increases with age. With the increasing numbers of elderly patients seeking dental care, and the significant complications that can occur in treating an undiagnosed or uncontrolled diabetic, and in-office technique to uncover undiagnosed diabetics or to assess the level of control of diagnosed diabetes is needed. Proper understanding of the role of diabetes in the production and maintenance of oral disease will not only benefit the patient community, but it can be of great significance to dental clinicians who often treat oral diseases as isolated phenomenon with little or no knowledge of the interrelationship between unsuccessfully managed or undiagnosed diabetes, and the oral problem they may be seeing."}
{"text": "Mucosal organs such as the intestine are highly vascular organs with extensive metabolic demands. Epithelial cells which line the intestine function to orchestrate a multitude of mucosal responses, and given their anatomic location, are primary targets for diminished blood flow and resultant tissue hypoxia. Our previous studies have explored the response of intestinal epithelial cells to hypoxia and these studies defined a transcriptional signaling pathway mediated by hypoxia-inducible factor (HIF). Activation of HIF results in the coordinated induction of a cluster of apically-localized, barrier protective gene products. Such induction parallels the accumulation of polymorphonuclear leukocytes (PMN, neutrophils). In this proposal, we will test the hypothesis that HIF coordinates protective epithelial responses to hypoxia. Three specific aims are proposed to test this hypothesis. First, we will elucidate the role of PMN to inflammatory hypoxia using in vitro and in vivo models of intestinal inflammation. Second, we will build on recent findings to further explore the role of HIF signaling to intestinal inflammation. In particular, we will define the contribution of HIF-1 and HIF-2 to protection afforded by inflammatory hypoxia. Third, we will extend our recent findings with pharmacological approaches that activate HIF (prolyl-hydroxylase inhibitors) to define specific targets and mechanisms of protection in both chemically- and genetically-induced murine models of intestinal inflammation. The overall aim of this proposal is to elucidate the how hypoxia and inflammation coordinately influence disease outcomes at the mucosal interface. PUBLIC HEALTH RELEVANCE: These studies are proposed to better understand basic mechanisms of inflammation in the intestine. Specifically, these studies will define how metabolic shifts present during episodes of inflammation might be harnessed to develop novel therapies for mucosal diseases. It is our hope that extensions of this work in human patients might impact inflammatory disease outcomes."}
{"text": "The overall direction of the Molecular Mechanisms of Tumor Promotion Section is to understand the regulation of the signaling pathways downstream from the lipophilic second messenger diacylglycerol, to elucidate the basis for heterogeneity of response to different ligands which function through this pathway, and to exploit this understanding for developing novel ligands with unique behavior that function through this pathway. A complementary direction is to understand the regulation and structure activity relations for the vanilloid receptor. The vanilloid receptor is a downstream target of the diacylglycerol signaling pathway, shares partial homology in its ligands to this pathway, and shares with the diacylglycerol signaling pathway an important role in inflammation. Both directions impact both our understanding of biological regulation and the potential development of therapeutic agents. Protein kinase C, the best studied downstream target for diacylglycerol, represents the classic system for tumor promotion and is a therapeutic target for cancer chemotherapy. The vanilloid receptor represents a promising therapeutic target for cancer pain, among other indications, and thus represents an important direction in palliative care for cancer patients. DAG-lactones represent a synthetically accessible platform for probing the structure activity relations of protein kinase C and the other targets downstream of the second messenger diacylglycerol. We have continued to evaluate DAG-lactones designed to have selectivity for the RasGRP subclass of diacylglycerol targets. This class is of particular importance because it functions as an activator of Ras, which is many tumors shows enhanced activity without being mutated. The atypical protein kinase C isoforms zeta and iota play important role in cellular polarity and cellular invasion. We have previously identified the specific structural differences between their DAG recognition domain and that of typical DAG responsive signaling proteins such as the classical and novel PKC isoforms. We have described a first generation of DAG lactones with selectivity for the specific differences in the recognition domains of PKC zeta and iota. We have evaluated the role of aliphatic side chains on the DAG lactone for providing selectivity for protein kinase C epsilon, a pro-tumorigenic protein kinase C isoform. Bryostatin is an agent in clinical trials with a unique mechanism of action. It binds to protein kinase C with high affinity and activates the enzyme but paradoxically antagonizes many protein kinase C mediated responses. We find, using microarray analysis of gene expression along with detailed examination of the time and dose dependence of genes representative of the differences in expression revealed by the microarray analysis, that transient duration is the predominant difference in the mode of action of bryostatin as compared to typical protein kinase C activators such as the phorbol esters. In collaboration with the groups of Gary Keck and Michael Krische, we seek to define the critical structural elements in bryostatin conferring its unique pattern of activity, with the goal of developing the next generation of bryostatin analogues. One recent insight is our identification of the B ring structure as critical for the bryostatin like activity. Protein kinase C is subject to post-translational modification, in particular phosphorylation. In collaboration with the CCR Collaborative Protein Technology Resource, we have shown that the extent of protein kinase C modification is much more extensive than had been recognized. Moreover, the pattern of modification was different for different ligands such as phorbol ester or bryostatin. We suggest that such modification signatures may be of particular value for the structure activity evaluation of ligands with complex effects where, as in the case of protein kinase C, ligand binding causes both activation and change in subcellular localization. For protein kinase C or other targets of diacylglycerol, ligand binding drives membrane association. We show that the charge on the DAG recognition domain plays a critical role in the selectivity for membrane phospholipids. Further, this selectivity also depends on the specific ligand, with more hydrophilic ligands showing greater sensitivity to membrane composition. On our TRPV1 project, we have made considerable progress in defining the optimal structures for binding of antagonists to human TRPV1, as part of our effort which seeks to advance the development of drug candidates for this target. We further are continuing our efforts to use photoaffinity labeling to confirm directly the ligand binding site on TRPV1 as well as to use mutational analysis to refine computer modeling of the putative binding site."}
{"text": "Centralized Omics REsource (CORE) Technologies is a multiple award Indefinite Delivery Indefinite Quantity (IDIQ) begun in 2015 and provides IDIQ contract awards for whole genome sequencing and other omics measures, including DNA methylation, RNA expression patterns, and metabolic profiling. CORE, in conjunction with NHLBI's overarching TOPMed Program, seeks to discover factors and variants that influence disease risk, identify subtypes of disease, and develop more targeted and personalized treatments. Studies and proposed study designs and omics are selected via peer review and are approved samples supplied to one of eleven selected omics centers; the resulting raw and processed data is processed using TOPMed approved and designed pipelines and shared with originating studies, TOPMed PIs working in TOPMed domain working groups, and are also made available to NHLBI directed data repositories for sharing with approved investigators. TOPMed studies include multiple non-European (underrepresented populations) as well as studies that include or are entirely female."}
{"text": "Icosahedral capsid assembly is a highly coordinated process involving sequential addition of multiple proteins, ultimately leading to an infectious virion of proper size and morphology. The long-term goal for this project is to achieve a mechanistic understanding of the protein: protein interactions involved in capsid assembly. The development of new anti-viral drugs is impeded by a lack of understanding of how viral capsid proteins are programmed to adopt the correct conformations to produce the correct assembly product. Capsid assembly will be investigated using bacteriophage P22, a model dsDNA virus. In phage P22, herpesvirus and many other dsDNA viruses, the capsid is formed from a coat protein having the ubiquitous HK97 fold. The initial assembly product is a procapsid (PC). Scaffolding protein (SP) directs proper assembly of coat protein (CP) to form PCs. SP also directs the incorporation of the portal protein complex, which is essential for genome encapsidation. Phage P22 provides an excellent model assembly system because complex in vivo processes are easily mimicked in vitro. The simple genetics and well-established biochemistry of phage P22 offers significant advantages as an assembly model over complex mammalian dsDNA viruses. Our central hypothesis is viral capsid assembly is driven by specific weak protein: protein interactions, which control nucleation and elongation to form the proper assembly products. In this granting period we will test our central hypothesis with the following aims. Aim 1. Define how communication between domains of coat protein affects capsid morphology. Our data suggest that proper capsid morphology is controlled by communication between domains of CP, thereby affecting the curvature of the subunit. We will test this hypothesis by generating and characterizing site- directed mutants in different domains of P22 CP. Aim 2. Determine the structure and function of the I-domain from distantly related phages. We defined important roles for an inserted domain in the folding and assembly of P22 coat protein. Phages Sf6 and CUS-3 have low coat protein sequence identity but have an identifiable inserted domain. We will determine the NMR solution structure, and the function of the inserted domain in the CP folding and assembly from these related phages. Aim 3. Elucidate the protein conformational changes occurring during assembly. Protein conformational changes regulate proper capsid assembly. We will use single molecule FRET experiments to understand how individual capsid proteins in a population change conformations between monomeric and assembled states. Aim 4. Investigate the assembly of the portal protein complex during PC assembly. Portal protein is essential for genome packaging, yet how this complex is assembled into procapsids at a single vertex is not understood. Incorporation of portal protein will be characterized using RNA atpamers identified by SELEX."}
{"text": "Studies of the effects of environmental agents on human health are a vital part of the mission of the institute. As such studies are almost always observational rather than experimental, interpretation of the data is complex. Good statistical collaboration in all phases, including conception, design, analysis, and interpretation, is essential to the success of this work. During the current year, we were active collaborators on many projects. Included were studies evaluating biomarkers, examining health effects of various environmental or occupational exposures and drugs, and describing biology of several conditions."}
{"text": "Cervical spine injuries, degenertion (spondylosis), nerve root compression (radiculopathy) and spinal cord compression (myelopathy) are the major causes of pain, deformity and neurological disfunction in the cervical region. Studies of the spinal column alone, while important, provide limited information about the cord and nerve roots. The purpose of the present proposal is to study in a comprehensive manner the mechanical basis of local, radicular and myelopathic symptomatology in the cervical spine. Such information is not available presently. This comprehensive study has three basic parts. Firstly, to determine ligamentous strains and intervertebral foramenal changes due to physiological movements of the cervical spine. Secondly, to measure physiological movements of, and strains in,, the nerve roots and spinal cord. Finally, to quantify 3-dimensional anatomy of the cervical spine in various physiological postures using cryomicrotome sectioning, X-rays and CT-scans. Significance of this work will be in several different areas. The comprehensive set of biomechanical data about the functioning relationships between the various components of the cervical spine system, namely, vertebrae, ligaments, spinal cord and nerve roots will be available for the first time. It will provide the basis for understanding the functioning of the cervical spine system. Thereby making it possible to correlate precisely the anatomic derangements with clinical presentation of symptoms in a patient. It will also be helpful in the design of new and more efficient diagonostic and treatment modalities for the various problems of the cervical spine."}
{"text": "The objective of this project is to develop and implement sophisticated point-of-care EHR-based clinical decision support that (a) identifies and (b) prioritizes all available evidence-based treatment options to reduce a given patient's cardiovascular risk (CVR). After developing the EHR-based decision support intervention, we will test its impact on CVR, the components of CVR, in a group randomized trial that includes 18 primary care clinics, 60 primary care physicians, and 18,000 adults with moderate or high CVR. This approach, if successful, will (a) improve chronic disease outcomes and reduce CVR for about 35% of the U.S. adult population, (b) maximize the clinical return on the massive investments that are increasingly being made in sophisticated outpatient EHR systems, and (c) provide a model for how to use EHR technology support to deliver \"personalized medicine\" in primary care settings. PUBLIC HEALTH RELEVANCE: The objective of this project is to develop and implement sophisticated point-of-care EHR-based clinical decision support that (a) identifies and (b) prioritizes all available evidence-based treatment options to reduce a given patient's cardiovascular risk (CVR). The prioritized list of treatment options is provided in different formats to both the primary care physician (PCP) and patient at the time of each office visit made by a patient with moderate to high CVR and sub-optimally controlled and potentially reversible CVR factors. Available evidence-based treatment options are prioritized based on the magnitude of potential CVR reduction of each treatment option. This intervention strategy, referred to as Prioritized Clinical Decision Support (PCS), is specifically designed for widespread use in primary care settings and has the potential to substantially augment current efforts to control CVR in the 35% of American adults with 10-year Framingham CVR of 10% or higher. To assess the ability of the PCS intervention to reduce CVR in adults, we will randomize 18 primary care clinics with 60 primary care physicians (PCPs) and approximately 18,000 eligible adults with baseline Framingham 10-year risk of a major CV event (either heart attack or stroke) of 10% or more into one of two experimental conditions: Group 1 includes 9 clinics (with 30 PCPs and 9,000 patients) that will receive prioritized clinical decision support (PCS) to reduce CVR at the time of each clinical encounter made by an eligible adult. Group 2 includes 9 clinics (with 30 PCPs and 9,000 patients) that receive no study intervention and constitute a usual care control group. The study will formally test the hypothesis that after control for baseline CVR, post- intervention 10-year Framingham CVR will be better in Group 1 than Group 2 at 12 and 24 months after start of the intervention. In addition, impact of the intervention on specific components of CVR (BP, lipids, glucose, aspirin use, and smoking) will be assessed, and the cost-effectiveness of the intervention will be quantified. This innovative project builds upon 10 years of prior work by our research team, and extends prior successful EHR clinical decision support interventions by introducing prioritization, by providing decision support to both patients and PCPs at the time of the office visit, and by extending the decision support across the broad and critically important clinical terrain of CVR reduction. The results of this project, whether positive or negative, will extend our understanding of how to maximize the clinical return on massive public and private sector investments now being made in sophisticated outpatient EHR systems. If successful, this decision support tool could be broadly used to both standardize and personalize care delivered by case managers, pharmacists, and other providers in a wide range of care delivery configurations."}
{"text": "Bcr/abl gene is derived from the t(9;22) reciprocal translocation and is present in most of chronic myelogenous leukemia (CML) and a cohort of acute lymphoblastic leukemia (ALL) patients. BCR/ABL oncogenic tyrosine kinase modulates response to DNA damage inducing resistance to genotoxic therapies. In addition BCR/ABL stimulates genomic instability, which may lead to mutations in BCR/ABL kinase causing resistance to imatinib mesylate. We hypothesize that: BCR/ABL elevates the levels of reactive oxygen species (ROS) which induce spontaneous DNA lesions (for example uracil residues), whose unfaithful repair introduces amino acid substitutions in the BCR/ABL kinase domain causing resistance to IM. The role of ROS in generation of oxidative DNA damage leading to mutagenesis and resistance to imatinib mesylate will be studied in CML hematopoietic stem cells (HSC), common myeloid progenitor cells (CMP), and granulocyte/macrophage progenitor cells (GMP) using anti-oxidant approaches and in vitro and in vivo models of BCR/ABL leukemogenesis. The efficiency and fidelity of the mechanisms processing ROS-dependent oxidative DNA damage in BCR/ABL leukemia cells will be determined by studying base excision repair (BER), focusing on UDG glycosylase removing uracil residues. These reactions will be examined using well-defined reporter/substrate systems and a combination of different approaches including immunofluorescence, phosphorylation-less and interaction- deprived mutants, transgenic mice, and sequencing. BCR/ABL-UDG functional interaction will be investigated by mutagenesis and targeted by aptamers to inhibit resistance to imatinib mesylate."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. It is becoming clear that regulation of protein function is often acheived via shifts in the populations of active and inactive conformers. X-ray crystallographic analysis gives only snapshots of possible configurations populated in solution. Taking advantage of our expertises in theoretical and experimental analysis of protein folding and assembly, we seek to use a combined SAXS and theoretical approach to define the structural ensembles present in the functional landscapes of multidomain proteins. This approach extends beyond current simulation analysis of SAXS data to include local and global folding/unfolding transitions that are important in function. Our initial choice in developing this methodology is the enzyme Csk as it is not only an important therapeutic target but also is a system we have characterized in terms of the optimal enzymatic and folding conditions. There is growing evidence that biases in the structural ensemble may play an important role in the regulation of Csk. The methodology developed here is broadly applicable to biological macromolecules and will provide useful information about what ensembles of conformations are consistent with the experimental data as well as the the ubiquitous dynamic reversible folding/assembly processes inherent in biology."}
{"text": "In this proposal we aim to investigate a role of the Activation-inducible isoform of Hypoxia-inducible Factor- 1alpha (aiHIF) in regulation of anti-bacterial response of T cells during sepsis. Our hypothesis indicate aiHIF as a potential target for improvement of anti-bacterial clearance in septic patients. This assumption is based on our findings that aiHIF plays inhibitory role in TCR-activated T cells, and we found that HIF-1alpha prevents T cells from fully contribute into anti-bacterial response during sepsis. Our preliminary studies support the hypothesis that aiHIF plays major role in the negative regulation of T cells during sepsis. Using our recently created aiHIF-deficient mice we will test whether the total or T-cell-specific deficiency of aiHIF enhances the pathogen destruction and survival in murine sepsis models. Using in vivo models of live bacterial sepsis in mice we will test whether aiHIF is a major negative regulator of activated T cells during sepsis. In addition, we will determine the mechanism of T-cell inhibition by aiHIF. The aiHIF-deficiency is expected to de-inhibit T cells by rendering them resistant to inhibition in hypoxic inflamed areas, and to allow T cells to fully participate in orchestrating the overall anti-pathogen response, which will improve sepsis survival. These studies will identify aiHIF as a novel molecular target and provide proof of a principle for a feasible strategy to improve therapy of sepsis, especially when combined with our recent discovery of previously unknown aiHIF isoform in human T cells that will allow our mouse sepsis studies of aiHIF to translate into humans. PUBLIC HEALTH RELEVANCE This proposal aims to provide proof of principle for a novel strategy of improving the therapy of sepsis by prevention of T cells inhibition by activation-inducible HIF-1alpha."}
{"text": "Various storage and retrieval algorithms have been studied. The development of flexible and efficient storage and retrieval algorithms is very useful, because such algorithms are used in almost all computer programs. Thus, biomedical computation in particular can benefit from improved storage and retrieval methods. In FY85, work continued on the analysis of trie data structures and the development of algorithms and analysis for a new type of doubly-chained trie capable of holding prefixes. This work is of particular import for text storage and retrieval, including dictionaries, indices to document collections, and various special thesari. The proposed data structure provides a general mechanism for storing arbitrary words and/or phrases without restriction on content. The storage scheme is fast and parsimonious, and the retrieval scheme is also fast. Prior to discovering how to handle prefix items, tries could not be as easily employed for arbitrary collections of words."}
{"text": "Thymocyte maturation and thymic architecture is dependent upon precise intrathymic cell migration. The orchestration of thymocyte trafficking, however, is not well understood at the molecular level. Recently, we described the highly-regulated plexinD1 expression on CD4+CD8+ double positive (DP) thymocytes which is further modified by T cell receptor/co-receptor engagement. Activation of plexinD1 via its ligand, semaphorin 3E (sema3E), represses CXCR4/CXCL12 signaling in pre-selection DP thymocytes and CCR9/CCL25 signaling in CD69+ post-selection DP thymocytes. Using Plxnd1-deficient fetal liver cell-transplanted mice, loss of plexinD1 causes CD69+ thymocytes to remain in the cortex, maturing in situ to form ectopic single positive (SP) thymocyte clusters. As a consequence, the boundary between DP and SP thymocytes at corticomedullary junctions is disrupted and medullary structures form under the thymic capsule. These results demonstrate the importance of plexinD1 in directing migration of maturing thymocytes via modulation of biological responses to chemokine gradients. The dysmorphic corticomedullary structures observed in Plxnd1-/- and Sema3e-/- knockout mice are further consistent with this view. To address the cellular interactions and functional consequences of Plxnd1 deficiency in thymic development, the present proposal will pursue three major aims. First, we will focus on the role of plexinD1 in regulation of CCR9/CCL25 and CXCR4/CXCL12 pathways and the directed migration of DP thymocytes using Ccr9-/- and Cxcr4 conditional knockout mice. Specifically, the links between these chemokine signaling pathways and Plxnd1 deficiency as well as the associated retarded thymocyte migration towards the medulla of pre-selected (CD69-) and post-selected (CD69+) DP thymocytes will be assessed. Second, we will analyze by multi-photon excitation microscopy the dynamics of GFP- expressing (green) Plxnd1-/- thymocytes in fetal liver-transplanted mice interacting with tdTomato (red) cortical thymic epithelial cells within renal subcapsular fetal thymic implants, using CFP-expressing (blue) wild type cells as an internal control. Third, we will analyze the impact of Plxnd1 deficiency on negative selection during T cell development using OT-I/OT-II TCR and RIP-OVA transgenic mice and fetal liver cell transplantation, in particular screening mice transplanted with Plxnd1-/- fetal liver cells for evidence of autoimmune phenomena. Collectively, our studies will offer insight into the molecular basis of plexinD1-mediated regulation of DP thymocyte movements within the thymus with consequential effects upon thymocyte differentiation and selection that may lead to autoimmune pathology in the mature immune system. PUBLIC HEALTH RELEVANCE: T cell function is critical for mammalian host protection against a wide variety of infectious disorders and cancers. T cells emanate from the thymus. Hence, understanding of the processes required for normal thymic architecture, differentiation and maturation will contribute to generation of more effective immunity."}
{"text": "The overarching goal of the BioRepository Center (BRC) is to facilitate translational research. Tight coordination between surgical staff, pathologists, and BRC enables collection of specimens to fulfill investigators' needs. Our protocols for rapid acquisition of tissue samples allows for preparation of high quality analytes. Patients' rights and privacy by our participation in obtaining and confirming informed consent for tissue collection; guidance with regard to preparation and submission of IRB documents is offered to help ensure compliance. The BRC also promotes collaborations between clinical and basic scientists, and can aid in the design and execution of prospective, hypothesis-driven studies to examine the impact of genetics or treatment on disease progression. By promoting investigators access to appropriate biospecimens, the BRC enhances the ability of NYULMC Cancer Institute members to perform meaningful translational research."}
{"text": "Traumatic brain injury (TBI) is a significant problem in the pediatric population. Ninety per cent of pediatric TBIs present to an emergency department, but only 8% are hospitalized. Since the majority of pediatric patients with mild TBI are therefore discharged home with the diagnosis of concussion, accurate assessment of the severity if concussion and consequent outpatient management and instructions are critical for ensuring safe recovery from injury. Without state-of-the-art knowledge and clinical tools, mild TBI (mTBI) may go undiagnosed and untreated, leaving individuals who have sustained a mTBI with an increased risk for functional problems. The ACE and ACE care plan were developed as part of the CDC's \"Heads Up: Brain Injury in your Practice\" toolkit for physicians to manage mTBI. Adapting the ACE for the ED and implementing a standardized clinical protocol by ED physicians systematically should improve management by ensuring accurate diagnosis and improving patient education and adherence with discharge recommendations. The goal of this research is to demonstrate the capacity to improve diagnosis and management of mTBI presenting to the Emergency Department (ED) by the feasible application of systematic procedures in the form of the ACE and the ACE Care Plan. This study will be conducted collaboratively by Children's National Medical Center and UPMC/ Children's Hospital of Pittsburgh with the specific aims to: (1) evaluate the feasibility of the ACE and ACE Care Plan for standardized implementation in the ED setting (2) determine if the ACE and ACE-ED Care Plan can be implemented by the ED staff and disseminated to the Primary Care Providers and (3) determine if routine use of the ACE-ED and ACE-ED Care Plan will improve patient/family follow-up behavior and patient recovery. We have designed the study to progress in two stages: Stage 1 proposes to develop expert consensus agreement regarding the importance and feasibility of using the ACE and ACE Care Plan in the ED setting, including an understanding of current concussion management care pathways. An outcome of Stage 1 will be the consensus-based adaptation of the ACE and ACE Care Plan for the ED, referred to as the ACE-ED and the ACE-ED Care Plan. Stage 2 applies these revised tools via a pilot implementation study for patients age 5-22 years old presenting with mTBI. The primary outcome will be patient/family follow-up behavior with the primary care/specialist. As secondary outcomes, we will examine clinician adherence to use of the ACE-ED and ACE-ED Care Plan, and its dissemination to primary care providers. Feasibility of implementation will be further evaluated by identifying the actual facilitative and barrier conditions to ACE-ED/ Care Plan use within the ED setting. We will also develop estimates of effect of this implementation on patient recovery. Upon study completion, key data will be available to support the development of"}
{"text": "The p53 protein plays a critical role in tumor suppression, and represents a pivotal mediator of cellular response to physiological stress. One of the major regulators of p53 activity and abundance is the MDM2 oncoprotein. The long-term goals of this research are to better understand the complex regulation of the p53 tumor suppressor and the MDM2 oncogene, specifically regarding interacting factors that modulate p53 function. In normal cells, p53 has a short half-life and its abundance is low. In response to cellular stress, p53 becomes stabilized and activated as a transcription factor, and the expression levels of p53-target genes increase of decrease. The physiological outcome of such p53 activation is growth arrest or cell death (apoptosis). Thus, elucidation of the parameters that control p53 abundance and function in a cell remains an important area of study in cancer biology. They have identified two proteins that can regulate the activity and stability of p53 when bound. One of these factors, the co-repressor mSin3A (Sin3), was previously determined by them to be a critical mediator of p53-dependent trans-repression and apoptosis. Thus, p53 transcriptional repression activity and stabilization may be functionally linked. The other factor, MDMX, is a homologue of the MDM2 oncoprotein. While MDMX stabilizes p53 by interfering with MDM2-mediated degradation of p53, Sin3 appears to stabilize p53 by a novel mechanism, not dependent on MDM2. The broad aims of this proposal are to elucidate the mechanisms whereby interaction with MDMX and Sin3 influence p53 regulation. Significantly, they have found that Sin3 can bind the p53 homologue p73 as well. This indicates that transcriptional repression and stabilization by interaction with Sin3 may be conserved properties of the family of p53-related transcription factors, with implications for understanding pathways of apoptosis. Therefore, as a natural corollary to this work, this hypothesis will also be tested."}
{"text": "Our aim is to test the common assumption that aging in mammals involves a nonrandom decrease in neuron number and to consider the hypothesis that the decrease is preceded by changes in connections made and received by those neurons. The hypothesis derives from well-established data that at critical periods early in life, the number of neurons is adjusted downward by about 50% in many regions of the nervous system as a normal programmed event, and that axonal innervation of target cells also may be adjusted downward. Do such adjustments occur late in life and what controls them? Our analysis uses standard mice of the inbred C57BL/6 inbred strains, at ages from early adulthood to beyond 30 months of age, which is unusually old for mice. Complete serial section series through the whole brain are being prepared by a new embedding and sectioning technique. We have developed computer-assisted quantitative methods applicable at light and electron microscopic levels for scoring the numbers and positions of neurons of defined classes, counting and classifying axons with statistical reliability in central tracts and peripheral nerves, and measuring volumes and surface areas of brain regions and of individual cells. With the support of this grant, we are among the first to be able to measure fiber number and sizes automatically, directly from the electron microscope image, without photographic mediation. Further efforts will focus on the difficult micro-alignment and other problems inherent in three-dimensional quantitative reconstruction of synaptic organization at high magnification. We are concentrating overall on 3) numbers of cerebellar neurons in the major classes as a function of age, 2) the relation of retinal ganglion cell size, number, and distribution to the corresponding parameters in optic nerve, and 1) sampling and statistical methods for motor and sensory components in mixed peripheral nerves. Our objectives is to establish methods and a normative baseline against which to test experimentally in the future, whether long-term neuronal survival is influenced by quantitative synaptic relationships at an earlier period of life."}
{"text": "Experimental evidence is emerging that the gasotransmitter hydrogen sulfide serves an important role in the cardiovascular system especially during tissue ischemia. However, specific cellular sources, targets, and mechanisms of hydrogen sulfide within the vasculature remain largely unknown. Work from our lab and others demonstrate that hydrogen sulfide is protective against chronic tissue ischemia, which involves increased vascular remodeling responses such as angiogenesis. Importantly, very little mechanistic information exists regarding how hydrogen sulfide modulates ischemic vascular remodeling in vivo or in vitro. Data in this application reveals a novel finding that hydrogen sulfide emanating from cystathionine !-lyase (CSE) selectively augments ischemic tissue nitrite reduction to NO that mediates increased angiogenic activity involving HIF-1 activity and VEGF expression. Experiments in this proposal will determine molecular mechanisms for these novel results through the hypothesis that endothelial cell CSE dependent hydrogen sulfide generation augments ischemic nitrite reduction to NO that increases ischemic vascular remodeling. The hypothesis will be examined through the pursuit of three specific aims including: 1) determine how endothelial cell CSE expression regulates ischemic vascular remodeling responses, 2) determine the mechanisms by which hydrogen sulfide increases NO generation in ischemic tissue and how this regulates HIF-1 activity and VEGF expression, and 3) determine the effect of endothelial cell CSE dependent hydrogen sulfide on diabetic ischemic vascular remodeling responses. Successful completion of this project will significantly advance the fields understanding of hydrogen sulfide biology in the vascular system, provide clear mechanistic information on hydrogen sulfide-NO pathway interactions, and identify novel approaches for ischemic vascular disease therapy."}
{"text": "Indole-3-carbinol (I-3-C), a normal dietary constituent, has been shown to decrease chemical carcinogenesis in experimental animals. N-nitrosodimethylamine (NDMA) requires metabolic oxidation in order to express its hepatotoxic, mutagenic and carcinogenic potential. Our results show that treatment of mice by gavage with I-3-C decreases the covalent binding of (14C)NDMA metabolites to DNA or protein up to 90% with no change in the rate of NDMA demethylation of cytochrome P-450 levels. We found that ethanol or methylene chloride extracts of livers from mice treated with I-3-C decreased the covalent binding of NDMA metabolites to DNA and protein in liver cell fractions from untreated mice. In order to explain these observations, we developed the hypothesis that treatment of mice with I-3-C gives rise to lipophilic nucleophiles that protect tissue macromolecules from electrophilic attack by a scavenging mechanism. To test this hypothesis, we developed a procedure to assign a relative value of nucleophilicity to chemicals or chemical mixtures, such as tissue extracts. The nucleophilic index value (NIV) for liver tissue extracts is inversely proportional to the rate of covalent binding of MDMA metabolites to DNA and protein, and independent of the rate of NDMA demethylation. The Specific Aims of this project are: a) to find the I-3-C dose regimen to maximize tissue NIV; b) to correlate NIV with indices of hepatotoxicity and DNA damage; c) to fractionate solvent extracts of livers from I-3-C treated mice in order to purify and identify the active nucleophilic material(s), using the HPLC separation techniques in parallel with liquid scintillation counting and high resolution mass spectrometry compound identification procedures; d0 to establish the mechanism of protection by I-3-C. The objectives of this research plan are a) to establish and validate a model system which uses the NIV of the liver to predict biochemical and functional susceptibility of the liver to damage from the hepatotoxic and caracinogenic chemical NDMA; b) to identify the chemical species and the mechanisms involved in the I-3-C protection from molecular and cellular damage. This project offers a predictive model for tissue susceptibility to, and a molecular basis for dietary protection from certain environmental toxic and mutagenic/carcinogenic insults."}
{"text": "Recent evidence indicates that the ingestion of increased amounts of the trace mineral selenium (Se) during the developmental period of tooth formation increases dental caries in man and experimental animals. The long term goal of the research proposed in this application is to provide a biochemical understanding of how selenium causes an increased susceptibility to dental caries. Utilizing C14 labeled radioactive precursors, we plan to study the integrity of metabolic pathways for: (1) the oxidation of amino acids, glucose, and fatty acids; (2) the synthesis of proteins, glycogen and lipids; and (3) the synthesis of matrix proteins in developing teeth, in rats receiving drinking water containing inorganic and organic selenium. Because of the dental, as well as metabolic significance, of selenium- induced depressions in food intake, results will be compared to control animals receiving diet provided by a pellet dispensing device in the same pattern of food intake as the selenium animals."}
{"text": "Present cage assignments for primates are based solely on the animals' weight. Variation in shape between species of primates of the same weight indicate that the current weight-based standard may be inappropriate. A large number (410) of primates of four different species have been measured (arms, legs, chest, tail, crown to rump, crown to heel) in order to determine association of and variations in weight as functions of shape measurements. The results of this study provide for the assignment of cages based not only on animal weight, but also on allometric measurements and accounting for species differences. This project has been completed."}
{"text": "Type I diabetes is characterized by the permanent loss of pancreatic insulin-producing beta cells. Our goal is to understand the mechanisms determining pancreatic beta cell mass, as a first step toward the development of regenerative therapies for diabetes. In collaboration with the BCBC Coordinating Center and members of the consortium, we will use transgenic mouse technology to examine the hypothesis that beta cells have a significant regenerative capacity, which is modulated by specific signals. The specific aims are to: 1. Develop a system for regulated ablation of pancreatic beta cells. Our preliminary studies indicate that mice can recover from a pulse of beta cell ablation. We will characterize in detail the physiological and histological aspects of ablation and recovery, in order to understand the mechanisims of beta cell regeneration. 2. Determine the cellular origins of regenerating beta cells. We will employ genetic lineage tracing to definitely determine the contributions of stem cells and pre-existing beta cells to beta cell regeneration. 3. Characterize the signals that regulate beta cell mass. The relative importance of blood-borne signals will be determined, with emphasis on the role of insulin and glucose metabolism. In addition, we will assess the significance of beta cell dedifferentiation in vivo and in vitro. Insights from these studies will be applied to primary beta cell cultures, with the aim of improving yield, preventing dedifferentiation and minimizing cell death"}
{"text": "Preliminary studies in our laboratory are consistent with a relationship between hyperglycosylation (additional antennae and fucose residues on N-linked oligosaccharides) and nicking or cleavage of hCG. Other preliminary studies indicate the structural instability and diminished biological activity of hyperglycosylated and of nicked hCG molecules, and how they may be the source of free Beta-subunit in the circulation and Beta-core fragment in urine samples. A large number of papers have been published showing raised hCG levels, and raised proportions of free Beta-subunit and Beta-core fragment levels in serum or urine samples from patients with Down syndrome pregnancies, preeclampsia, trophoblast disease, or hyperemesis gravidarum. It is our hypothesis, that all of these raised levels arise from the hyperglycosylation of hCG, through a pathway involving nicking of hyperglycosylated hCG, ineffective autocrine control of hCG production, and rapid dissociation to generate nicked free Beta-subunit, and degradation to Beta-core fragment. Five sets of experiments are proposed to test this hypothesis and investigate the clinical ramification of the observations in normal and abnormal pregnancies. Studies are proposed to confirm and compare the levels of hCG, free Beta-subunit and Beta-core fragment in 720 sets of parallel serum and urine samples from normal and abnormal pregnancies (Aim 1A); to purify hCG from normal first, second, and third trimester pregnancy urine samples and trophoblast disease, preeclampsia, hyperemesis gravidarum and Down syndrome pregnancy urine samples (3 each) and compare the extents and sites of nicking, and the type, amounts and locations of hyperglycosylated N-linked oligosaccharides (Aim 2); to investigate the hyperglycosylation-nicking relationship by comparing normal and hyperglycosylated hCG as substrates for nicking enzymes (Aim 3); to examine the biological activities of normal and abnormal pregnancy hCG and abilities to feedback through to the placenta and control hCG synthesis (Aim 4); to investigate the and compare the stabilities of hCG from normal and abnormal pregnancy, and determine whether dissociation could explain the raised proportion of free Beta- subunit (Aim 5); and to use the parallel serum and urine samples to investigate the clinical utility of new immunoassays specifically measuring nicked or hyperglycosylated hCG molecules (Aim 1B)."}
{"text": "The over-all objective of this program is to test a theoretical model which attempts to account for the frequently-encountered phenomenon of increase longevity of irradiated insects. The major tenet of the model is that radiation damage to DNA induces higher levels of various DNA-repair enzymes which would otherwise decline in differentiated postmitotic tissues. It is generally accepted that many enzymes which repair DNA damaged by radiation also play a role in repair of DNA lesions produced by chemicals, including, presumably, some metabolic products. Thus, the model predicts that DNA-damaging chemicals would also increase longevity in insects (and other organisms whose somatic tissues are predominantly or entirely postmitotic); that nonrepairable damage, such as that produced by high LET radiations, would be effective, in the manner of gratuitous inducers; that there would by age-related declines in resistance to two types of deleterious agents: those which act by damaging DNA in differentiated tissues, and those whose effects must be overcome by transcribing the genetic information encoded in such tissues. The model also predicts that effects on adult longevity would not be correlated with decreased fecundity or reproductive activity."}
{"text": "HIV-positive adult male and female study subjects will be recruited from Moore Clinic patients who have positive HCV antibodies and positive HCV RNA on stable antiretroviral regimens (>4week). Thirty (30) subjects will be selected for study participation according to inclusion and exclusion criteria in the clinical protocol."}
{"text": "More than 29 million children have a dental restoration. The majority of restorations now placed are composites. Most resin-based composites placed in children contain a derivative of Bisphenol A (BPA) called BPA-glycidyl methacrylate (BisGMA). When BisGMA was developed in 1962, no studies were required by the FDA to evaluate biological toxicity. BPA is a widespread xenoestrogen that may affect brain, endocrine, and reproductive development. National dental organizations maintain the amount of BPA from BisGMA-based dental materials is too small to be relevant to human health. There is scant scientific evidence for or against this assertion. Contrary to this claim, our pilot data and data from the only other prospective study in children show urinary BPA (uBPA) concentrations are a magnitude of 2 to 6-fold higher post-treatment. Studies show exposure to medical products increase BPA concentrations, yet there are no studies in children that quantify BPA exposure from medical products used in anesthesia (tubes, syringes) for dental treatment. We need to determine the contribution of dental-related BPA exposure to overall BPA load, and distinguish between different sources of dental-related BPA exposure. The aims of this application are to: (1) Quantify the magnitude and duration of BPA exposure resulting from dental treatment; (2) Determine associations between number of BisGMA-based treated surfaces and BPA, overall and by type of material (composites, sealants); and (3) Determine the association between type of anesthesia and BPA. We will measure uBPA concentrations in 210 children 4 to 8 years old receiving BisGMA-based dental materials with different types of sedation at the University of Washington Center for Pediatric Dentistry 2 times before and 4 times after treatment from 24 hours to 16 weeks. To ensure we have sufficient numbers of highly exposed children we will employ stratified sampling. We will recruit children who are treated with <4 surfaces (n=105) and =4 surfaces (n=105) with BisGMA-based dental materials. Within each of these two groups, we will recruit 35 patients in three groups who receive: (1) no sedation, (2) nitrous oxide; or (3) general anesthesia. We will administer surveys to collect demographic data and data on food security and other sources of BPA. We will measure the height and weight of the child, and collect detailed treatment records. We will conduct the first large study in children to comprehensively examine multiple sources of dental-related BPA exposure. Distinguishing BPA from dental materials and medical products used in anesthesia may enable us to develop interventions to reduce dental-related BPA exposure. By oversampling children receiving treatment on =4 surfaces with BisGMA-based dental materials and children receiving GA, we will include high-risk children likely to have high baseline BPA who may receive among the largest amounts of dental-related BPA exposure from materials or anesthesia, and who may be most likely to experience adverse health effects from BPA."}
{"text": "Agents that inhibit the reaction of activated GPIIb/IIIa (alphallb/betaS integrin) with fibrinogen and other ligands are a promising family of anti-thrombotics now widely used to prevent adverse events following coronary angioplasty. Acute, severe thrombocytopenia, often occurring within hours of first exposure to one of these agents, is a recognized side effect of all drugs in this class. In the previous period of support, we obtained evidence that drug-specific antibodies, which can be naturally occurring (or at least pre-existing), are the major cause of this complication, characterized clinical and serologic aspects of this group of disorders and obtained evidence that the responsible antibodies recognize ligand (drug)-induced structural conformers of GPIIb/IIIa. We now propose to extend these observations with the following specific aims: 1) Characterize epitopes on GPIIb/IIIa recognized by antibodies causing thrombocytopenia in patients treated with GPIIb/IIIa inhibitors. Epitopes on ligand-occupied GPIIb/IIIa for which this apparently unique class of immunoglobulins is specific will be defined at the molecular level. 2) Develop new methods for identification of clinically significant antibodies not detected in conventional immunoassays. We hypothesize that platelet destruction in a significant subset of patients is caused by low affinity antibodies not detected in most immunoassays and propose to improve diagnostic yield with assays that a) detect antibody binding in real time and/or b) increase the Ka by preserving the structural integrity of the target. 3) Characterize the incidence, clinical significance and genetic origin of pre-existing (\"naturally occurring\") immunoglobulins (NA) that recognize GPIIb/IIIa-inhibitor complexes. The incidence of potentially \"dangerous\" NA specific for ligand-occupied GPIIb/IIIa in the general population, their genetic origin and their relationship to antibodies causing thrombocytopenia will be defined and their implications for platelet physiology and transfusion therapy and for the design of \"safe\" GPIIb/IIIa inhibitors will be explored. Findings made are expected to elucidate a previously unrecognized mechanism of disease resulting from the immune response to conformational changes induced in an integrin by its ligand or a ligand-mimetic drug and to advance understanding of the role of \"natural\" antibodies in health and disease."}
{"text": "1) Detailed laboratory procedures for isotachophoresis, \"quantitative\" PAGE and electrofocusing on polyacrylamide gel were written; 2) An apparatus for packing of multiple 6 and 18 mm Sephadex electrofocusing gel tubes was designed and constructed; 3) a zwitterionic monomer was synthesized, polymerized to a cross-linked gel and tested for its capacity of bridging conductance gaps in EF and its effect on protein mobilities; 4) Restacking of unstacked protein zones, postulated by the Jovin theory of multiphasic buffer systems, was demonstrated for the first time and applied to the simultaneous isolation of several hCG isohormones; 5) Purify of protein isolation by Steady-State Stacking was greatly improved by replacing polyacrylamide with agarose in the extraction and concentration step."}
{"text": "Project Abstract: Aging is associated with enhanced inflammation. An altered ratio between angiotensin receptors AT1R and AT2R results in induction of inflammation in animal models. The effects of aging on the expression of AT1R and AT2R in humans and the contribution of changes in AT1R and AT2R to increased inflammation in the older have not been previously studied. Our preliminary evidence suggests that frail older adults have up-regulation of AT1R, down-regulation of AT2R expression and implicate for IL6 in this imbalance. We hypothesize that human frail aging is associated with up-regulation of AT1R and down-regulation of AT2R expression and function in immune system cells. We hypothesize that these receptor changes contribute to decreased immune system cells phagocytic capacity and to the increased production of inflammatory cytokines in older individuals which will further heighten the divergence in AT1R and AT2R expression. In order to test these hypotheses, we propose a comprehensive study of AT1R and AT2R using immune system cells (lymphocytes and monocytes) from young, healthy adults (age 20-30) and four comparison groups that consist of (1) robust, older adults (age 70-90), (2) robust, older adults (age 70-90) treated with AT1R blockers, (3) frail, older adults (age 70-90), (4) frail, older adults (age 70-90) treated with AT1R blockers. From these subjects we will collect lymphocytes and monocytes that will be utilized for the following proposed studies: 1. Measure changes in gene expression, protein synthesis, and signaling pathways of AT1R and AT2R in immune system cells from young control (20-30Y) and the four comparison groups (N=33 in each group) using Q-PCR, western blot, confocal microscopy, flow cytometry and Bio-Plex Phosphoprotein Cellular Signaling Assays. 2. Evaluate age-related difference and contribution of Angiotensin receptors blockade to monocytes phagocytic function by incubating immune system cells from the same individuals with specific AT1R and/or AT2R blockers and measuring changes in monocytes phagocytic function with Phagocytosis Assay at baseline and in response to treatment(s). 3. Evaluate contribution of AT1R and AT2R to cytokine production in the older individuals by incubating immune system cells from the same individuals with specific AT1R and/or AT2R blockers and measuring cytokines with ELISA and Bio-Plex cytokine assays at baseline and in response to treatment(s). 4. Assess the feedback of inflammation on AT1R and AT2R expression and function by incubating immune system cells from the same subjects with IL-6. Q-PCR and western blot will be used to quantify the change in expression of AT1R and AT2R in response to IL-6 treatment."}
{"text": "This study is designed to examine the immune regulatory functions of monocytes and lymphocytes as a function of age of otherwise healthy subjects and to examine co-factors in the development of tuberculosis particularly as regards the immune status."}
{"text": "The research is designed to evaluate the relative roles of the cell mediated and humoral arms of the immune system in experimental candidiasis. Unmodified (normal) mice and mice immunosuppressed following adult thymectomy or treatment with cyclophosphamide will be studied from various perspectives. First, the development of cell-mediated immunity in such animals will be studied in vivo by footpad testing and in vitro by assays for lymphocyte transformation and migration inhibition factor. Three extracts of Candida albicans will be studied in the in vitro assays, but one extract, designated ppt-HEX, will be studied in more detail both in vivo and in vitro. Secondly, since normal mice develop resistance to reinfection following cutaneous infection, attempts will be made to passively transfer such resistance to naive normal and immunosuppressed mice with cells or serum from normal immune mice. If cells transfer the resistance, attempts will be made to abrogate the transfer by preincubation with anti-theta serum. Thirdly, cutaneous lesions developing in normal and immunosuppressed mice in response to one or two inoculations of viable C. albicans yeasts will be analyzed at selected intervals following inoculation for the number of colony forming units, and by immunofluorescence techniques for the infiltration of T and B lymphocytes into tissue surrounding the lesions. Finally, if time permits, additional studies are planned to assay for cell-mediated immunity in animals infected intravenously, to investigate the specificity of ppt-HEX, and to attempt to induce resistance to reinfection by artificial immunization with subcellular fractions of Candida."}
{"text": "Volunteers will have their sway measured on two separate outpatient visits using the Biomechanics platform."}
{"text": "An integrated approach has been taken to elucidate important factors contributing to the pathogenesis of microangiopathy. Dynamic studies measuring mean retinal circulation time by means of fluorescein retinography will be continued to be performed in populations of normal subjects, prediabetics, mild diabetics and severe diabetics. A simplified, but highly standardized system for obtaining consistent fluorescein retinograms including an automatic scanning microdensitometry system and a computerized format for the computation of circulation times has been perfected. A particular heavy focus will be placed on prospective studies of retinal blood flow in all of the above named populations. Relationships between the circulation time and metabolic factors such as the degree of carbohydrate intolerance, the magnitude of serum insulin and growth hormone responses during a variety of stimulation tests and plasma lipids, such as triglyceride and cholesterol will be sought in the milder diabetic patients. In the more severe diabetic patients the duration of the severity of diabetes and the degree of control will be placed in juxtaposition to the circulation time parameters."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have reported on experiments in vitro showing that in response to live Borrelia burgdorferi (Bb) and its purified outer surface lipoprotein A (L-OspA), IL-10 dampens the prototypic IL-6, IL-12, TNF-alpha and IL-1beta inflammatory responses in mouse J774 macrophages. We also showed that mouse macrophages incubated with IL-10 together with either live Bb or L-OspA additively augmented the expression of the suppressor of cytokine signaling (SOCS) 3 in these cells. In this study we first used a chemokine/cytokine multiplex approach to determine the extent of the IL-10-mediated inhibition of inflammatory mediators in macrophages in response to live Bb and L- OspA. RNA interference was then used to silence socs3 gene expression in macrophages to determine the effect of silencing on IL-10 inhibition of inflammatory mediators in response to live Bb and L-OspA. Multiplex analysis revealed that IL-10 significantly (P0.05) down-regulated the production of several cytokines (TNF-alpha, IL-12, IL-6, IL-1alpha, IL-1beta, IL-9) and chemokines (G-CSF, CXCL1, CXCL5, CXCL10 and CCL5) in macrophages stimulated with live Bb or L-OspA. In all instances live Bb induced higher levels of cytokines and chemokines than did L-OspA. All mediators induced by both stimulants in macrophages were down-modulated by IL-10 in a similar fashion and correlated with enhanced SOCS3 expression in these cells. Silencing of SOCS3 expression in macrophages abrogated the IL-10-anti-inflammatory actions against IL-6 production in response to live Bb and L-OspA. This study demonstrates that SOCS3 in part mediates the IL-10 anti-inflammatory effect in macrophages stimulated with live B. burgdorferi spirochetes and lipoproteins. Comparisons of macrophage genome-wide transcriptomics data before and after the silencing of SOCS3 will ultimately identify the extent of the SOCS3-mediated anti-IL-10 inhibitory effect in response to B. burgdorferi."}
{"text": "Experiments are proposed which will determine mechanisms governing synthesis and assembly of acetylcholine receptor. The nicotinic ACh-receptor is an integral membrane glycoprotein of muscle which binds ACh release the nerve. The mechanism by which ACh binding results in a change in membrane permeability culminating in muscle contraction is under intensive study in a number of laboratories. Mechanisms governing receptor synthesis and destruction also deserve intensive study because these are important to the overall function of AChR during development, disease, and regeneration. The synthesis of ACh-receptor as well as other specific proteins is regulated by nerve induced muscle activity. My goal is to study the regulation of synthesis and assembly of the ACh-receptor at the molecular and cellular level. Studies will include the development of immunochemical methods for detection of nascent receptor polypeptides and receptor synthesis in cell free protein syntheiszing systems. Combination of these techniques will provide a means for direct study of receptor synthesis at the translational and transcriptional level. Studies of ACh-receptor regulation should serve as a model for the study of other receptors. Control of receptor synthesis may be involved in the regulation of synapse formation, and thereby in the mechanism of establishment of synaptic specificity. Regulation of ACh-receptor content is perturbed in the experimental autoimmune animal model for Myasthenia Gravis. Studies of this experimental model may lead to improved therapeutic treatment of the human disease. They may also serve in understanding other receptor diseases."}
{"text": "Heart failure is the scourge of the elderly accounting for nearly $35 billion dollars and causing substantial morbidity and mortality; ~50% of all aged patients with CHF have heart failure with a preserved ejection fraction (HFpEF), a multi-factorial condition associated with several abnormalities in diastolic function. Stimulated by previous work from this research program the global objective of the next 5 years is to test novel strategies to prevent and reverse the impaired diastolic function associated with sedentary aging and HFpEF. These include: A) novel exercise training strategies implemented early enough in life while CV plasticity still exists; and B) pharmacologic probes of underlying pathophysiology targeted to improve relaxation in the elderly and HFpEF patients, and restore functional capacity. The aims of the program are: Specific Aim 1: to test the hypothesis that exercise training implemented 4-5 times/week for 2 yrs in sedentary middle aged men and women (45-64yr) will improve cardiac and vascular compliance to a degree equivalent to life-long exercisers (and sedentary young). We will perform invasive and non-invasive assessment of cardiovascular structure and function before and after an exercise program involving high intensity aerobic intervals, lower intensity endurance and strength training or yoga control. Specific Aim 2: to test the hypothesis that the stiff, slowly relaxing heart of patients with HFpEF causes a marked elevation in pulmonary capillary pressure during exercise which leads to premature fatigue prior to achieving maximal HR, thus causing apparent chronotropic incompetence. We further hypothesize that both sedentary aged and HFpEF patients have slowed relaxation due to down regulation of SERCA2a activity, the putative cellular mechanism underlying age related impaired lusitropic function. We plan to perform 2 sets of experiments for this aim: 1) to measure the HR response to two separate interventions: a) maximal activation of central cardiovascular pathways (central command) from static handgrip at 40% maximal voluntary contraction to fatigue; b) incremental doses of isoproterenol in the face of ganglionic blockade to isolate ? adrenergic responsiveness without reflex compensation; and 2) to perform invasive and non-invasive assessment of cardiac function at rest and during exercise in patients with HFpEF compared to age matched and young controls: a) after infusion of istaroxime an experimental drug with prominent lusitropic properties due to its ability to upregulate SERCA2a activity; and b) after Na+/K+ ATPase inhibition control (digoxin). After these aims are accomplished, we will have established a novel, practical exercise training strategy designed to prevent the cardiovascular stiffening with aging, and ultimately to prevent HFpEF. Such a determination would have enormous public health significance since this condition is quite difficult to treat once established. In addition, we will use novel physiological and pharmacological probes to determine the mechanism of the apparent chronotropic incompetence and impaired exercise capacity in patients with HFpEF which has the potential to open up new therapeutic options for this challenging syndrome."}
{"text": "The general purpose of the proposed research is to gain an understanding of the true dynamic picture of proteins and membranes rather than the more common static picture. The off-resonance rotating frame spin-lattice relaxation technique we have just developed will permit the investigation of the rotational motion of proteins and internal motions of different parts of proteins. The influence of substrates, inhibitors, and allosteric effectors on those motions will be explored. We have also demonstrated that spin-lattice relaxation in the rotating frame can be used to determine the lateral diffusion coefficient of a phospholipid in a model membrane bilayer. This technique will be employed to determine the lateral diffusion coefficient of lipids in model membranes and biological membranes as a quantitative parameter characterizing membrane \"fluidity\" which is an important factor for various membrane functions. In particular, the effect of the composition of the phospholipid, temperature, phase, and presence of other membrane constituents such as other phospholipids, cholesterol, and membrane proteins will be examined. The effect of general and local anesthetics on the lateral diffusion coefficient of the phospholipid in the bilayer will also be investigated."}
{"text": "Compare the safety and virologic efficacy of nelfinavir and DMP-266 when used separately or in combination with nucleoside-containing regimens for patients previously in ACTG 302 or 303."}
{"text": "We plan to continue our studies on the mechanisms of action of antitumor antibiotics. In particular, we are studying the nature and mechanism of the damage induced in DNA by the protein antibiotic, neocarzinostatin. Such damage can be induced in HeLa cells in culture or in vitro with isolated DNA. We are exploring the possible relationship between the two observations."}
{"text": "The overall objective of our study is to understand the manner in which the genetic information of mammalian cells is expressed and regulated and to determine how these processes are altered in cancerous cells. Our immediate goal is to describe the steps involved in the synthesis of mRNA in the nucleus, any modifications that it may undergo before being expressed in the cytoplasm, and finally its fate during and after translation. In addition we wish to characterize the early events associated with globin mRNA synthesis in erythroleukemia cells. These studies are in extension of our earlier work concerned with the identification, isolation and characterization of the rabbit and mouse globin mRNAs. During this time we have successfully translated these mRNAs in a variety of cell-free systems, described their translation efficiency, purification, poly(A) content, molecular weight, methylated base content, presence in ribonucleoprotein particles and their distribution between membrane bound and free polysomes. We have initiated studies concerning the synthesis of globin mRNA in the nucleus and described a specific poly(A) shortening mechanism. Our immediate aims are: (1) to continue our studies on the shortening of the poly(A) sequence of mouse globin mRNA, (2) to describe the location of the methylated nucleosides in globin mRNA, (3) to isolate and characterize the globin mRNA precursor, (4) to measure the synthesis and turnover of globin mRNA following the committed division in erythroleukemia cells and (5) to ascertain if post-transcriptional regulation occurs during differentiation of these cells. The ability to isolate molecules containing globin mRNA sequences using mRNA specific affinity column prepared by attaching globin cDNA to cellulose is an important technical advance which will aid these studies. We have prepared such a column and found it successfully isolates globin mRNA. The column will now be used to prepare precursors to globin mRNA."}
{"text": "The University of California, Berkeley School of Optometry (UCBSO) seeks 5 years of support to train clinician-scientists in accordance with the guidelines of the Institutional Clinical Scientist Development Program (K12). The Berkeley Clinical Scientist Development Program will provide interdisciplinary training to enable well-established clinicians to carry out high-level, independent, clinical (patient-based) vision research. The University of California, Berkeley (UCB) K12 program will draw upon the rich resources of world-renowned departments and groups on the Berkeley campus, including Vision Science, Optometry, Public Health, Neuroscience, Infectious Disease, Bioengineering, Health and Medical Science, and Molecular &Cell Biology. The Berkeley K12 Program will feature two unique elements: (1) The recruitment of established clinicians who wish to make a career shift from clinical practice and teaching to one centered on the combination of teaching and independent, federally funded, patient-based vision research. (2) The multidisciplinary composition of a distinguished and diverse mentoring group composed of faculty with expertise in vision research, plus faculty whose primary expertise is not in vision, but who have extensive experience in clinical research. The program will provide the awardees with focused, cutting-edge didactic and mentored clinical research training tailored specifically to the individual's career plan. The curriculum will consist of course work, seminars, research in campus-wide laboratory facilities and clinics, and closely mentored supervision from a group of nationally recognized scientists and clinicians. Trainees will conduct research in one of seven clinical research areas: Retinal Disease, Corneal Disease, Strabismus, Amblyopia, Visual Processing, Low Vision &Blindness Rehabilitation, and Lens &Cataract - all of which are consistent with the Programs and Research Priorities of the National Eye Institute. The Berkeley program is designed so that upon completion, the trainees will be able to compete successfully for research resources and maintain a productive and independent career in patient-based vision research. Several strategies will be used to recruit established clinicians (e.g., OD, MD, DVM, DDS, or equivalent professional degree) whose clinical expertise is either within or outside the ophthalmic community. Four trainees would be recruited during the first year, and then one additional trainee admitted in each of the following four years (total admitted by 5th year = 8)."}
{"text": "It is proposed to investigate the use of somatic cell gene therapy for the correction of genetic diseases using domestic animals as test systems. A large number of models of human genetic disease have been discovered in domestic animals and characterized in detail at the Univ. of Pennsylvania School of Veterinary Medicine. Among these diseases are the mucopolysaccaridoses (MPS) which are recessive and result from defects in single genes encoding enzymes involved in the degradation of glycosaminoglycans (GAGs). Breeding colonies of MPS cats and dogs have been established. Thus, diseased animals are immediately available as test subjects. MPS cells can endocytose exogenous normal enzyme and degrade GAGs. Therefore, the experimental strategy will be to construct retrovirus vectors containing cDNA copies of normal genes, introduce the vectors by infection into bone marrow cells, and autologously transplant the cells to repopulate the donor/patient with cells producing normal enzyme. The sponsors, Drs. D. Patterson and M. Haskins, are members of the University-wide NIH Human Genetics Center and have extensive interactions with scientists working on genetic diseases in man. However, there is no one at the Veterinary School who has the expertise in molecular genetics who can concentrate on constructing and testing the vectors that are needed to perform the proposed experiments. Therefore, it is proposed that the candidate, Dr. John Wolfe, receive the necessary further training in molecular genetics to enable him to conduct these experiments. Therefore, it is proposed that the candidate, Dr. John Wolfe, receive the necessary further training in molecular genetics to enable him to conduct these experiments. He is a graduate of the NIH Veterinary Medical Scientist Training Program where he received training in immunogenetics, retrovirology and protein chemistry and is now an Exxon Fellow in Molecular Genetics at Sloan-Kettering Institute. Thus, he has training in most of the techniques needed to do the proposed experiments. A leading researcher on retroviral vectors, Dr. E. Gilboa of Princeton Univ., has agreed to collaborate with him on the construction and evaluation of the vectors. Dr. R. Schmikel of the Medical School who is the principle investigator of the Genetics Center and Dr. R. Brinster of the Veterinary School who is a leading investigator in the technology of gene transfer into embryo cells have agreed to participate in Dr. Wolfe's training. It is anticipated that the proposed training in recombinant DNA technology will enable him to establish a laboratory at the Veterinary School that will concentrate on the molecular aspects of genetic diseases and gene therapy using the domestic animal models at test systems."}
{"text": "This second revision of an R29 application seeks to advance our understanding of the molecular mechanisms by which the movement of water across the human cervical epithelial barrier. The applicant will use cultured human epithelial cell models developed in his laboratory to explore three hypotheses: 1) that estrogen increases transcervical permeability by means of an effect on cervical cell tight junctions, 2) that estrogen increases calcium mobilization in the cells, which in turn increases the permeability of the intercellular space, and 3) that the mechanism of Ca++ mobilization in the human cervical cell is a Ca++-dependent stimulation of cellular KCl transport, which results in a reduction in cell volume. It is proposed that the results of these studies will increase both our understanding of the basic biology of cervical fluid homeostasis, but also a better understanding of clinically relevant settings in which the cervical fluid status is abnormal."}
{"text": "The generally held view that the immobilization of ciliated cells by specific immune serum results from antibodies cross-linking adjacent cilia is only superficially correct. Our current study has shown that the ciliary agglutination occurs only in living cells and is characterized in its later stages by a fusion into a continuous unit membrane of those ciliary membranes that come into contact with one another. In a number of key aspects, this agglutination process resembles the \"capping\" observed in other systems more than it resembles microorganisms or cell suspensions treated with specific antiserum. Globular material is seen to accumulate at the ciliary tips by phase contrast and fluorescence microscopy. When cells are examined by SEM, the fused ciliary tips are seen to be distended, discoidal membranes. TEM often reveals several ciliary axonemes within a single, enlarged membrane that is oriented with the ferritin-labelled second antibody directed against the i-antigen antibody on the outer surface only. Fixed cells or living cells treated with immune Fab do not show membrane changes, but do bind antibody. Membrane fusion occurs only if cells are alive and the i-antigen is cross-linked by intact immune IgG. In addition, the redistribution of the immune complex appears uneffected in the presence of colchicine. In conjunction with these studies on the immobilization of cells, the surface i-antigen designated C was purified and characterized."}
{"text": "Early clinical reports of severe morbidity and mortality in infants born passively addicted to opiates are being reinterpreted in light of recent reports of an improved prognosis in children born to women on methadone maintenance programs who avail themselves of antenatal and well-baby clinics, and in whom illicit polydrug abuse and poor nutrition are kept to a minimum. Most of the preclinical animal literature dealing with effects of methadone during development would suggest that methadone should never have been released for clinical use by people of childbearing age or who are pregnant. However, we believe that there are many flaws in the design and interpretation of these experiments and have completed or are in the process of extending a series of experiments with the opiate 1-alpha-acetylmethadol (LAAM) or its active metabolites in which we can duplicate many effects reported for methadone, but which appear to be preventable or nonexistent, if careful attention is paid to animal modeling and potential epiphenomena. We propose to continue these studies as well as incorporate several new studies to determine the relevance (i.e. generality or specificity) of our observations with LAAM, to study the consequences of hypoxia and hypercapnia equivalent to that produced by (acutely toxic) doses of methadone typically used by other laboratories for such studies. Very little is known about the effects of cocaine on the developing child, when exposed in utero. The dearth of data is more recently being added to by anecdotal, small group or clinical case reports of neonates born to cocaine abusers who also invariably are polydrug abusers. This makes it difficult or impossible to separate direct from indirect effects, as well as effects due solely or primarily to cocaine exposure, except in those instances where clearcut acute toxic manifestations are responsible (e.g. subdural hematoma or other evidence of CVA or seizures). Even then, combinations of multiple drug exposure, poor nutritional or health status (i.e. infectious hepatitis due to i.v. drug self-administration) may be more responsible for morbidity/mortality of cocaine-exposed neonates. We propose to study the effects of cocaine, administered at different doses, to pregnant rats (prior to and throughout pregnancy or at different stages of pregnancy) or to breeder male rats in order to assess the neurobehavioral consequences in offspring of such exposure. Rats will be tested in adulthood, after exposure prior to fertilization (gametes) and during in utero development. Treatment of neonates with cocaine and drugs known to attenuate or block opiate withdrawal in rats and/or humans will be used to determine if cocaine-exposed neonates show morbidity or mortality as a result of residual cocaine or if toxicity and/or a withdrawal syndrome is treatable during the neonatal period."}
{"text": "PNU-142731 is a pyrrolopyrimidine, non-corticosteroid anti-inflammatory agent developed by the Pharmacia & Upjohn Pharmaceutical Company to be used in asthma patients. It is an oral, once daily agent proposed for use in the chronic treatment of asthma. Animal studies have shown that PNU-142731 can ameliorate both the early-phase and late-phase asthmatic response. Three human trials have shown that PNU-142731 is tolerated well by humans. The proposed study is a double-blind, randomized, placebo-conrolled trial of two doses of PNU-142731 to evaluate the efficacy and safety of PNU-142731 in subjects with moderate persistent asthma. The end-points of the study include direct measures of asthma including lung function, daily symptom logs, and beta-agonist use as well as indirect measures of asthma such as assessment of inflammatory markers in induced sputum and serum. A secondary purpose of this study is to validate the use fo asthma quality-of-life (QOL) questionnaires in clinical trials.\""}
{"text": "Plasma high density lipoprotein (HDL) elicit their anti-atherogenic effect by transporting cholesterol from arterial macrophages to the liver for degradation, a process known as reverse cholesterol transport (RCT). Current HDL therapies are inadequate and new interventions for increasing plasma HDL levels and RCT are needed. HDL contains exchangeable apolipoproteins (apos) that are potential targets for HDL therapy. These include apo A-l, the major protein, and apos A-ll and E, which contain cysteines that are the sites of apo homo/heterodimerization. This project will fill gaps in our knowledge about HDL assembly, structure, and properties by testing new hypotheses about a] the mechanistic coordination of HDL phospholipidation and the dimerization of apos E3 and A-ll, b] the configurations of apos on HDL subfractions sorted by size and apo composition, and c] the hepatic production and phospholipidation of HDL-apos. These hypotheses will be addressed by the three specific aims: Aim 1: To use chemical kinetics to identify physico-chemical determinants of apo dimerization. This aim addresses a fundamental question about intracellular oxidative folding that is applicable to other cysteine-containing lipophilic proteins, including apo B. Aim 2: To use peptide mapping with and without chemical cross-linking and mass spectrometry to determine the apo configurations on native and reconstituted HDL. The configurations of apos on various HDL species determine HDL interactions with cell surface lipid transporters and with hepatic HDL receptors. Aim 3: To identify the itinerary of nascent HDL speciation in hepatocytes with respect to apo phospholipidation and in the case of cysteine-containing apos, the homo/heterodimerization of apos A-ll and E, by steady state and pulse chase labeling methods. Fibrates, which increase apo A-ll synthesis, produce modest elevations of HDL-C, an effect that might occur through altered HDL biogenesis. Distinct mechanisms for assembly of each apo into HDL could provide distinct therapeutic targets for increasing plasma HDL-C and RCT. Obesity-linked diabetes, a serious and growing public health problem in the United States, is associated with a cluster of lipid risk factors that include low plasma HDL-C. Although niacin and the statin class of hypolipidemic drugs can increase HDL-C, their effects are modest and better HDL therapies are needed. The information derived from our research would permit a rational approach to the development of new interventions that could operate at the level of the three main components of RCT-hepatic HDL production, remodeling in the plasma compartment, and hepatic HDL-cholesterol uptake."}
{"text": "The study will investigate the pathophysiologic effects of both acute and chronic treatment of rats with various amount of cadmium. The principal focus wwll be on cardiovascular changes, and will include effects on venous and arterial pressure, on cardiac activity and upon neural control of these functions. Histologic examination of tissues will be correlated with altered functional activity observed during life. Different pharmacological agents, including other metallic radicals, will be used to modify or alter the primary responses with a view to gaining further sight into the mode of cadmium's action. The responses will be correlated with sex, size, and strain, and with concurrent changes in body and urinary electrolytes and hematology. There are circumstances under which cadmium treatment may induce changes in carbohydrate metabolism, either hypoglycemia or hyperglycemia. These will be explored in depth."}
{"text": "This project aims to investigate the unique regulation of ornithine decarboxylase (ODC), the key rate limiting enzyme of polyamine biosynthesis, involving a unique protein termed antizyme. Antizyme binds to ODC, inhibits ODC activity and targets the degradation of ODC by the 26S proteasome without poly-ubiquitination. In our current studies we have been studying the stoichiometry and characterization of this interaction. For this purpose we have purified both yeast ornithine decarboxylase and yeast antizyme to homogeneity both for attempts at crystallization and for biophysical studies on the interactions of the two proteins. Recently we have developed methods to purify the ODC:antizyme complex and studied their interactions by various biophysical and biochemical techniques (Gel filtration, size exclusion-static-light scattering, dynamic light scattering, equilibrium ultracentrifugation, CD spectra). The binding affinity for yeast AZ to yeast ODC is 5X10-11 M. Using purified His-tagged AZ as a binding partner, we have purified the AZ:ODC heterodimer, which has no ODC activity. The native molecular weight of the complex is 90 kDa, which suggests a 1:1 stoichiometric binding of AZ and ODC in vitro. Circular dichroism (CD) spectra of the two individual proteins and the ODC:AZ complex show no significant change in the secondary structure of either ODC or AZ after binding to each other. These results indicate that yeast antizyme and ornithine decarboxylase form a heterodimer, consisting of each monomer and that there is no change in secondary structure in the AZ:ODC complex."}
{"text": "We propose to investigate the role of circadian rhythms in photoperiodic time measurement. One of the goals is to establish the relation between biological clocks, photoperiodism and seasonal reproductive activity in representative mammals. The participation of the nervous and endocrine systems in these phenomena will be detailed. The overall goal is to describe the ways in which temporal integration of the animals' internal milieu is affected and the fashion in which behavior is synchronized with cyclical fluctuations in the external environment."}
{"text": "The goal of this R21 application is to refine an experimental design to examine the impact of commonly used pain medications on sleep and next-day fatigue. However, before such studies can be conducted in patients with acute or chronic pain associated with cancer or other severe chronic illness, crucial information is needed to optimize the study design. This proposal seeks to answer certain experimental methodological design questions that can be safely, quickly, and inexpensively addressed in healthy volunteers. This R21 application will generate data guiding the research design and power estimates for subsequent RO1 applications intended to address the impact of pain medication on sleep and fatigue in patients with acute or chronic pain. There are 5 specific aims: 1. Characterize the effect of a typical nighttime dose of MS-Contin and methadone on cytokines, polysomnographic sleep and on rest/activity patterns in healthy volunteers: 2. Characterize daytime fatigue the day after drug administration with self-reported measure of fatigue and mood 3. Characterize neuropsychological performance on the day after drug administration. 4. Estimate the covariance structure of sleep measures to assess the comparative advantages of a crossover or parallel groups design. 5. Evaluate the necessity of repeated first nights and whether this is the optimum design for follow-up studies. Over a two-year period, 50 healthy volunteer subjects will be examined with polysomnography, actigraphy, and neuropsychological tests. Data will be collected on self-reported mood and fatigue, as well as proinflammatory cytokine levels (IL-6, IL-1beta, and TNF-alpha. The study design involves a double-blind, placebo-controlled crossover. Each individual will be admitted to the UCSD GCRC on 3 pairs of nights. Each admission will feature an acclimation night in the sleep laboratory followed by a night providing placebo, MS-Contin (15 rag) or methadone (5 mg). On the morning after each dosing subjects will complete fatigue and mood ratings and will perform a brief neuropsychological test battery. They will also undergo actigraphy monitoring for 3 continuous days to examine whether there are subtle lingering effects of the drugs. Blood levels ofproinflammatory cytokines will be obtained at various points before and after drug dosing and the next morning to test the hypothesis that opioid-induced changes in these cytokines are associated with sleep disruption and increased fatigue."}
{"text": "The long term objective of this proposal is to increase the sensitivity of DNA concentration measurements by 1000X over the state of the art. In addition, the microfluidic platform we will develop to accomplish this will also enable, for the fist time, the targeted recovery of genome-size DNA fragments out of a heterogeneous sample. Nucleic acid analysis through technologies like microarrays and next-generation sequencing are transforming the way that many biological questions are addressed. The power of these approaches stems from their ability to obtain system-wide information with single molecule detail. However, a major technical barrier that often prevents their effective application is that many biological systems are too complex to be straightforwardly sequenced: Even with bioinformatic algorithms, the billions of short, sequence dreads are often too complex to piece together into useful information. To address this challenge, we will develop a technology that allows nucleic acids to be quantitated and sorted in a heterogeneous sample. Importantly, this method will utilize specific multiplexedTaqMan PCR assays performed in giant-unilamellarvesicles (GUVs); this will make it much more specific and targetable than methods that sort DNA based on size or staining properties. Moreover, by assaying individual molecules or cells in GUVs, we will be able to perform billions of PCR assays in parallel, enabling massive, heterogeneous samples to be screened to identify and recover extremely rare targets. This basic technology will be broadly useful throughout biological research and has immediate human health impacts, including for detecting and sequencing cancer DNA in the blood early in the disease, analyzing genetic heterogeneity in tumor cells, and identifying immune cells latently infected with HIV. The aims are: Specific Aim 1: Demonstrate microfluidic generation and FACS sorting of thermostable femtoliterGiant-Unilamellar Vesicles. We will develop the microfluidic hardware and processes for generating billions of monodisperse femtoliter GUVs. We will also optimize processes to FACS GUVs, both into positive and negative pools and, individually, into wells on a microliter plate. Specific Aim 2: Optimize and characterize GUV-PCR and benchmark against aqueous droplets. We will explore different PCR reagents to optimize the GUV-PCRs and benchmark the efficiency of these reactions against ones performed in aqueous-in-oil droplets generated with our own microfluidics and with the Bio-rad QX100 digital PCR machine. We will measure efficiency using endpoint fluorescence, fraction of positive reactors, and yield of DNA recovered out of the reactors. We will also optimize protocols for rupturing GUVs to access their contents. Specific Aim 3: Demonstrate 1000X greater sensitivity than competing platforms by performing over 1 billion digital GUV-PCRs, and recovery of positive molecules with FACS. We will demonstrate the superiority of GUVs for digital PCR by performing 1000X more reactions than is possible with existing droplet technologies. We will also demonstrate the ability to recover rare DNA molecules by FACS sorting the positive GUVs."}
{"text": "1. In vivo anti-tumor activity of CD3-AK cells. (A). CD3-AK cells were induced by activation of mouse splenocytes with alphaCD3-AK antibody. The conventional LAK cells were induced by activation of mouse splenocytes with purified human recombinant IL-2. We found that different tumors showed different degrees of susceptibility to the in vivo killing by CD3-AK or LAK cells and the results also correlated with the in vivo effect of the killer cells on these tumors. A plasmacytoma P815 was much-more susceptible to the killing by CD3-AK than by LAK cells, and the in vivo growth of P815 cells was inhibited by simultaneous inoculation of CD3-AK and was not affected by LAK cells. In contrast, a melanoma JBMS was susceptible to the killing by LAK but was resistant to - CD3-AK mediated killing, and the in vivo growth of JBMS was retarded by LAK cells and was not affected by CD3-AK. Due to the ability of CD3-AK to expand rapidly in vitro, CD3-AK may be an attractive candidate for immunotherapy of susceptible tumors. 2. Mechanisms for lymphocyte-mediated cytolysis. (B). Two subsets of killer cells were induced by alphaCD3. The killer cells that were maintained in IL2 and alpha-CD3 (CD3-AK+) induced fast lysis, whereas the killer cells that were maintained in IL-2 alone induced slow lysis. Protein kinase C (PKC) was not required in fast lysis but was required in slow lysis. Cytolytic granules might be involved in fast lysis but had very little effect in slow lysis. On the other hand, cytokines such as IL-2, IL-4 and TNF were involved in slow lysis but had no effect in fast lysis."}
{"text": "Our objective is to define structural events at the cellular level in intestinal antigen processing. Our investigations are focused on Peyer's patches where M cells in the epithelium take up soluble and particulate antigen, then transport it to lymphocytes. Giardia which are too large to be transported by the M cell penetrate the intestinal epithelium over Peyer's patches and are taken up by macrophages. We will determine by morphometric analysis whether lysosomal volume within the cytoplasm of M cells is reduced compared to adjacent columnar cells. This may explain how M cells can transport luminal material without degrading it. We will determine whether lymphocytes within M cell interstices are B or T cells using peroxidase labeled antibodies. The pathway of radiolabeled ferritin from Peyer's patch through the circulation to other peripheral lymphoid organs will be determined by autoradiography and scintillation counting. Traffic of lymphocytes to lymphoid follicles will be studied using scanning electron microscopy of fractured post capillary venules. The specificity of Giardia trophozoite uptake by macrophages will be studied in vitro to determine the effect of host and parasite strain and the influence of immune or non-immune serum on phagocytosis."}
{"text": "The aim is to gain information leading to a deeper and more coherent understanding of enzymatically catalyzed glycosylation, the process whereby complex saccharides of living organisms are synthesized from (and degraded to) their sugar components. A unique experimental approach, developed in our laboratory, is being used and has proved highly effective in providing fresh insight into the catalytic capabilities and mechanisms of a variety of enzymes considered to be well-characterized by traditional studies. This approach consists of the study of glycosylation reactions that occur without glycosidic bond cleavage. We have, for example, recently demonstrated that exo-alpha-glucanases are able to attack both alpha- and beta-forms of appropriate glycosyl fluorides, and that these enzymes, long considered strict hydrolases, have a second (nonhydrolytic) mode of action that is stereocomplementary to the well known (hydrolytic) mode. Studies in progress show, moreover, that exo-glucanases as well as alpha- and beta-glucosidases are able to create specific anomeric configuration anew from enolic substrates that lack alpha- or beta-configuration. From the results obtained with these several systems, a new mechanistic principle has emerged which may well be applicable to carbohydrases in general; namely, that the catalytic groups have functional flexibility beyond that expected from the principle of microscopic reversibility. A potential consequence of such functional flexibility is that a carbohydrase may not be limited to a single reaction mechanism with all substrates. This is already apparent with most of the enzymes being studied, and will be further investigated. We also plan to study the extent to which classic types of endo-glucanases (alpha-amylases, pullulanase, cellulases) can catalyze polymerative glycosylation reactions in which water is kept out of a succession of steps, using appropriate glycosyl fluorides as substrates."}
{"text": "The Dana-Farber Cancer Institute is an independent, federally dcsignated, Comprehensive Cancer Center affiliated with the Harvard Medical School. The aims of the Institute can be summarized as follows: (a) to develop and maintain clinical and laboratory research programs of high quality related to the biology, cause and prevention, diagnosis and treatment of cancer; (b) to provide total care for cancer patients on an interdisciplinary basis and in association with neighboring institutions; c) to serve as the major cancer center in the Harvard-Longwood medical area and to play a leadership role for cancer research and patient care in New England providing expertise in cancer diagnosis and treatment; (d) to add to the total oncology resources of the medical community in terms of available staff, facilities and expertise; (e) to participate in the teaching and training of medical students, house staff, fellows and graduate students and in the continuing education of practicing physicians and other health professionals, and to provide post-doctoral research training. To maintain these activities, the present application requests renewal of the Institute's Cancer Core Support Grant."}
{"text": "Many neurons of the cerebellum are spontaneously active, firing 10 to 100 action potentials per second even in the absence of synaptic input. This high basal activity correlates with information coding mechanisms that differ from those of cells in circuits that are generally quiescent until excited synaptically. For example, in the cerebellar nuclei, long-term changes in the strength of excitatory synaptic inputs are not generated by classical Hebbian rules of coincident synaptic excitation and postsynaptic firing. Instead, synaptic currents are potentiated by patterns of stimulation that combine inhibition and excitation, in a manner that resembles the activity of (inhibitory) Purkinje afferents and (excitatory) mossy fiber afferents predicted to occur during cerebellar associative learning tasks. Such results support the idea that cerebellar circuits have rules for information transfer and storage that distinguish them from other well studied brain regions. The present proposal is motivated by the question of how spontaneous firing sets the stage for plasticity that is independent of spike timing. In the proposed research, experiments will be performed on neurons of the cerebellar nuclei in cerebellar slices of mice. Voltage-clamp and current-clamp recordings of synaptic responses, ionic currents, and action potentials, as well as imaging of Ca signals in nuclear cell dendrites, will be directed toward identifying the mechanisms of potentiation of excitatory synaptic responses to mossy fiber input, as well as toward examining the influence of spontaneous activity in Purkinje afferents and nuclear cells on plasticity. The resulting data will provide general information about the fundamental properties of signal encoding across brain regions, as well as specific information about the ionic mechanisms underlying cerebellar synaptic plasticity under normal and pathophysiological conditions. PUBLIC HEALTH RELEVANCE: In the present work, we are studying cells in the cerebellum, a part of the brain that controls the learning and execution of coordinated muscle movements, and whose electrical and chemical signaling patterns are disrupted in ataxia, dystonia, dyslexia, and autism. Because signaling patterns by brain cells depend on specialized proteins called ion channels, we are studying the properties of these ion channels, with the goal of understanding how their activity leads to long-lasting changes in cerebellar signals that are important for motor learning. These data can be used to make comparisons to disrupted signals in the cerebella of animals that have ataxia as a result of genetic mutations of ion channels, with the goal of understanding what goes wrong under pathophysiological conditions and whether ion channels can serve as a target for therapeutic interventions."}
{"text": "Work under this contract includes: The establishment and maintenance of cancer reporting system in Pureto Rico (The system will utilize the standard abstracts of records of all patients with active disease seen in any of the Puerto Rican Hospitals.); The follow-up of patients to determine their vital status and end-results of cancer treatment; Providing National Cancer Institute with computer tapes containing cancer incidence and end results data; and Setting up a working relationship with other registries in Latin American countries regarding ad hoc studies and epidemiological investigations."}
{"text": "ACTG 303 - Influence of Risk Status for Disease Progression on the Response to Antiretroviral Interventions: A Follow-up Study to ACTG 175. The hypothesis of this trial was that HIV-infected patients who had received prolonged combination therapy with zidovudine (ZDV) plus didanosine (ddl) or ADV plus zalcitabine (ddC) would show virologic and immunologic improvement upon addition of lamivudine (3TC) or after changing to ADV plus 3TC. The virologic and immunologic responses of these patients was expected to be influenced by virus load, CD4+ cell count, biologic phenotype of the HIV-1 isolate (ability of HIV-1 to induce syncytium formation in vitro), and presence or absence of symptoms of HIV disease at the time the new treatment regimens are instituted. The primary goals outlined in this trial were to carefully characterize subjects who have a well defined nucleoside exposure history with respect to the symptomatic state, their CD4 count trajectory, and their virologic profile, and to relate this to the response to new antiretroviral interventions in HIV infected patients who had been treated either with monotherapy (ZDV or ddl) or dual combination therapy (ZDV/ddl or ZDV/ddC) while participating in ACTG 175. Results of this trial are not yet available."}
{"text": "Electron paramagnetic resonance (EPR) spectroscopy is being evaluated as a potential technique for the early detection of cancers and is being used to elucidate the nature of free radicals and macromolecular conformational changes involved in cancer. Detailed EPR measurements on ceruloplasmin obtained from blood samples of large numbers of patients with diagnosed cancer are being compared with same kinds of measurements on blood samples from selected groups of control volunteers to determine whether statistically reliable differences exist which might be used in screening patients for the early detection of cancers. The effects of surgery, chemotherapy, and radiation therapy on the EPR spectral propertis of plasma ceruloplasmin and certain spin labelled blood components are being determined for a large number of patients to evaluate statistically the use of these properties as reliable indicators of the success or failure of the therapy. A variety of techniques, including ENDOR and electron spin echo, are being employed to identify and characterize free radical changes and changes in ceruloplasmin molecules associated with cancer. The information provided by this study may verify that EPR can be successful as a screening technique in the early diagnosis of cancer and in the monitoring of cancer therapy."}
{"text": "The virtual slide scanner and quantitative software is absolutely essential in allowing pathology truth to define imaging truth. We are funded through NIH grants to undertake this important task for lung and prostate cancer in the human. The proposed instrumentation, the Olympus VS120 slide-scanning microscope with VisioMorph newCAST software is critical for the completion of several very important NIH funded research projects and NIH funded educational grants at the University of Iowa. Some of the immediate applications include distinguishing malignant from benign lung nodules, 3D reconstruction imaging of mouse, ferret and pig lungs as a baseline for animal models in cystic fibrosis animals, defining the development of cystic fibrosis phenotype in the pancreas. The successful completion of Dr. Henry's research will lead to identification of patients at risk for lethal prostte cancer at stages early enough for effective treatment. Dr. Hammond seeks clarification of the role of inflammation in chronic pain leading to enhanced therapies for a higher quality of life for chronic pain patients. Dr. Dunnwald and Dr. Murray are analyzing the role of the protein Irf6 in immune cells associated with wound healing. Dr. Bonthius will test the efficacy of gene therapy against Alexander's disease, a disease with devastating and lethal affects on infants and children. The virtual slide scanner will also support the microscopy component of a current National Library of Medicine funded educational research and resource proposal that is aimed at increasing the efficiency and cost effectiveness of laboratory test ordering in clinical medicin. Updating and expanding the Virtual Slidebox for medical, dental and graduate students. The Virtual Slidebox increases accessibility, student satisfaction and efficiency of learning. This database is also used to assess the diagnostic competency of resident physicians. Enhanced medical student and resident education will continue to improve the lives and health of countless patients."}
{"text": "Noninvasive prenatal genetic testing, which utilizes cell-free fetal DNA and advances in sequencing technology, is revolutionizing the practice of obstetrics. While currently used as a screen for a limited number of aneuploidies and genetic conditions, noninvasive testing is anticipated to employ whole fetal exome and genome sequencing to identify not only monogenic disorders but also microduplications, microdeletions, and variants of uncertain clinical significance. This will give rise to a vast and complex body of information that expectant parents and healthcare providers must be prepared to interpret. Given the impact of fetal genetic information on reproductive choices, it is critical that effectiv informed consent practices are in place to ensure pregnant women and their partners make informed and value-reflective decisions about incorporating genomic technologies into their prenatal care. However, there is a critical lack of knowledge about how to structure an informed consent process that effectively meets the needs and preferences of expectant parents and is responsive to the challenges posed by noninvasive prenatal genomic testing and the clinical practice of medicine. The primary goal of this study is to ensure patient-centered counseling and effective informed consent practices are in place for noninvasive fetal genomic testing. Our central hypothesis is that the current absence of data concerning the informed consent process for such tests will not only have serious clinical and ethical implications for the delivery of prenatal care but also interfere with 1) patients' informed access to emerging applications noninvasive testing and 2) the translational process of other new prenatal technologies from the bench to the bedside. This project has two aims: 1) to describe the components of an effective informed consent process for noninvasive fetal genomic testing from the perspectives of pregnant women and partners and 2) to determine obstetric and genetic healthcare providers' perspectives regarding approaches and barriers to an effective informed consent process for noninvasive fetal genomic testing. We will conduct this work in collaboration with a multidisciplinary team of leading experts in the field of obstetrics, genetics, ethics, law, and medical decision-making and use a combination of qualitative and quantitative methods. This project is innovative because it will provide a new framework for the informed consent process and bring in the perspectives of partners in decisions about the use of noninvasive fetal testing. This work is significant because it is the first step in a line of research expected to develop and implement clinically relevant strategies to support expectant parents' informed consent about the use of next generation sequencing technologies. These results are expected to have an important positive impact on public health, as informed consent is not only foundational to the ethical practice of medicine but also a key component of healthcare quality, access, and outcomes. We anticipate that this study's findings will ultimately contribute to empowering pregnant women and their partners to make informed choices that reflect their needs and preferences as individuals and parents."}
{"text": "The term pseudohypoparathyroidism (PHP) is used for three distinct disorders, PHP type Ia (PHP-Ia), pseudo-PHP (pPHP), and PHP type Ib (PHP-Ib). PHP-Ia is characterized by PTH-resistant hypocalcemia and hyperphosphatemia, impaired function of several other endocrine systems, and a variety of developmental/skeletal defects (Albright's hereditary osteodystrophy (AHO)), including short stature, obesity, impaired intelligence, and skeletal abnormalities. Patients with pPHP are also affected by AHO, but show no resistance toward PTH or other hormones. PHP-Ia and pPHP occur within the same family, and individuals affected by either form show, as a result of inactivating mutations, reduced activity of the stimulatory G protein (Gsa); the Gsa gene is located on chromosome 20q13.3. Recent findings provided strong evidence for paternal imprinting in PHP-Ia/pPHP (i.e. patients with AHO), and showed that the abnormal control of calcium homeostasis, occurs only if the disease is inherited from an obligate female carrier. PHP-Ib patients show only resistance toward PTH, but no AHO phenotype. Since PTH-resistance is the only discernible abnormality in PHP-Ib, the disorder was first thought to be caused by inactivating PTH/PTHrP receptor mutations, but these were excluded by us and others. To continue our search for the molecular defect in PHP-Ib without prior knowledge of its primary structure, we collected several large PHP-Ib kindreds, conducted a genome-wide search with greater than 350 polymorphic microsatellite markers, and obtained linkage with a maximal combined LOD score of 6.48 to chromosome 20q13.3 (the Gsa gene is in same region). Furthermore, we showed that PHP-Ib is paternally imprinted, as is PHP-Ia/pPHP. We now propose to further reduce the linked interval and to confirm the 20q13.3 locus with additional PHP-Ib kindreds (Aim 1), to determine whether Gsa mutations cause the disease, and, if this candidate gene can be excluded, to search for the PHP-Ib gene (Aim 2), to explore the role of the PHP-Ib gene with regard to PTH/PTHrP receptor function (Aim 3), and to explore the importance of the stimulatory G protein in endochondral bone formation using heterozygous Gsa gene ablated mice (Aim 4)."}
{"text": "In the proposed study we intend to focus on the long term effects of increased cortisol activity in depressed patients. The major aims of the research are: 1) to Confirm that patients with psychotic major depression (PMD) exhibit greater cognitive impairment as measured by neuropsychological testing than nonpsychotic major depressed (NPMD) patients at baseline and to test the hypothesis that cognitive impairment at baseline correlates with plasma and urinary measures of cortisol in both PMD and NPMD patients; 2) to test the hypothesis that in comparison to NPHD patients, PMD patients have poorer outcome on measures of functioning, but not necessarily on measures of symptoms, at 15 months after the index episode; 3) to test the hypothesis that baseline measures of plasma and urinary cortisol do not predict outcome on measures of symptoms and functioning in PMD and NPMD patients at 4, 9, and 15 months after the index episode; 4) to test the hypothesis that at each of the three follow-up time points (4, 9, and 15 months), higher cortisol levels are associated with poorer outcome on measures of functioning, independent of depressive symptoms; 5) to test the hypothesis that at 15 month follow-up, plasma and urinary measures of cortisol in depressed patients correlate with cognitive impairment; and, 6) to explore whether in healthy control subjects there exists a relationship among urinary and plasma measures of cortisol, neuropsychological testing scores, and measures of social and occupational functioning. Eighty-one patients with diagnoses of unipolar major depression (42 psychotic and 39 nonpsychotic will be evaluated at baseline on measured of symptoms, social and occupational functioning, neuropsychological tenting, and plasma/urinary cortisol. Psychopharmacologic treatment will then be given according to a standardized protocol. Patients will be reevaluated at 4, 9, and 15 months on the same measures as at baseline except for the neuropsychological testing which will be repeated only at 9 and 15 months. Further understanding of the relationship between cortisol and long-term functioning in depression should help clinicians and researchers make more knowledgeable decisions regarding pharmacologic and psychological treatments during the first 15 months after the index episode."}
{"text": "Aggression is a complex behavior expressed in many different social contexts. Often abused, yet essential for the survival of organisms, little is known of the biological roots of this behavior. While model systems may not duplicate aggression in all of its many human manifestations, they can be highly informative about: the relative importance of genes, hormones and experience in laying down patterns of aggression in nervous systems;detailing the neural circuitry involved;and addressing questions of how animals choose among the varied complex behaviors available within their behavioral repertoires. It is the last two matters that are the major focus of the present application. In recent years, the Kravitz laboratory has established a fruit fly model of aggression making this important behavior available for experimental analysis in an organism that is highly suitable for genetic analysis. With fruit flies aggression is complex and is shown by both males and females, the animals engage in many other inter-related behaviors, and the behaviors can be quantitatively analyzed. In addition, however, the genome has been sequenced and incredibly powerful methods are available that allow experimental manipulations to be performed with fruit flies that cannot yet even be approximated in most other animal model systems. Recently, it has been found that the same gene that determines who flies court also determines whether flies fight like males or females. The present application extends these studies and has the following Specific Aims: I. To explore decision-making between courtship and aggression, two sexually dimorphic patterns of behavior in flies, by mapping and manipulating the fruitless/doublesex-associated circuitry within the subesophageal ganglion (SOG), the taste center in fruit fly brains;II. To map the distribution of receptors for amines and peptides associated with the SOG circuitry;and III. To introduce genes into neurons that can alter the function of these nerve cells in behaving animals and to begin to address the question of how the same neurons differ in male and female animals. These studies use a wide variety of state-of-the-art genetic, behavioral and anatomical methods in exploring these Aims. For example, in collaborative studies with the Chiang laboratory in Taiwan, neuronal maps will be made that will allow examination in great detail of the possible connections between individual neurons involved in courtship and aggression. The focus will be to define the brain circuitry important in aggression and courtship at much higher levels of resolution than are now available, and then to ask how that circuitry gets established in fly brains and how animals choose between behavioral responses when conflict situations arise. PUBLIC HEALTH RELEVANCE: All organisms, including humans, must be capable of rapidly evaluating social situations and selecting proper responses from what may be a wide variety of possible behavioral choices. Such selections must be made correctly in order to allow the survival of organisms both as individuals and as species. How organisms make such choices and how patterns of innate behavior of great complexity get established in nervous systems are not well understood, and are the theme of this application."}
{"text": "Basic science investigations have identified an increasingly large number of genetic factors important in cancer. By communicating this genetic information to high risk individuals, we can facilitate informed decision- making about participation in cancer prevention and early detection programs. The purpose of the proposed program is to improve adherence to cancer control regimens among persons with a genetic susceptibility to develop cancer. Subjects for the primary project will be 750 women aged 40-65 who have a positive family history of breast cancer in at least one first-degree relative. A randomized factorial design will be used to evaluate the impact of Breast Cancer Risk Counseling (BCRC), a form of genetic counseling for women at high risk for breast cancer. In addition, two different styles of presenting cancer control recommendations in BCRC will be tested. The BCRC intervention will be based on the traditional medical genetics counseling model, but will concentrate on breast cancer surveillance, rather than on reproductive decisions. Telephone interviews will be conducted prior to the interventions to collect familial and medical data to calculate individualized breast cancer risks and to assess baseline risk perceptions, psychosocial functioning, and breast screening patterns. Post-intervention and follow-up surveys will be used to assess the short- and long-term impact of the interventions on these outcomes. Also, subgroups of women who are most and least likely to benefit from the different BCRC interventions will be identified, based on their precounseling psychosocial characteristics and demographic backgrounds. The information obtained from this primary project will lay the foundation for other studies to improve adherence to cancer control practices in diverse high-risk populations. The interventions proposed for these studies are replicable, and, if effective, can be administered by nurses and physicians in medical practice."}
{"text": "Cell migration is essential for cellular morphogenesis and tissue repair, as well as a number of other important biological outcomes, and has a critical involvement in various developmental disorders and disease states. Of particular interest have been the regulatory events underlying cell migration as they relate to cell invasiveness, metastasis, and cancer progression. Tissue transglutaminase (tTG), a GTP- binding protein which also possesses an enzymatic transamidation activity that allows it to cross-link proteins, has been shown to play an important role in cell migration and invasion. As a signaling protein downstream of the epidermal growth factor (EGF) receptor, tTG becomes activated and is recruited to the leading edges of cells in response to EGF. In addition to its role in cell migratin and invasion, tTG has been shown to be important for Ras-driven transformation. The primary focus of this application is to learn more about how the activation and localization of tTG to the leading edges of migrating cells participates in the enhanced cell migration and invasive activity exhibited by human cancer cells and contributes to Ras- mediated cellular transformation. The following lines of investigation will be carried out to achieve this goal. 1) Determine the signalig mechanisms responsible for localizing tTG to the leading edges of actively migrating cells. EGF receptor signaling through Ras to JNK is important for tTG activation and localization. This aim will involve identifying the substrates of JNK that mediate these effects. In particular, I will determine whether tTG or Hsp70 can serve as a substrate for JNK or whether other known substrates (i.e. paxillin) are important for regulating tTG activation and localization. 2) Determie how the chaperonin function of heat shock protein 70 (Hsp70) helps target tTG to the leading edges of cells. This line of study stems from our recent findings that Hsp70 and tTG interact and co-localize along the leading edges of cells and this localization is blocked in the presence of inhibitors of the ATP hydrolytic activity of Hsp70, myricetin, methylene blue, and VER 155008. This aim will involve identifying the client proteins of Hsp70 which help target tTG to the leading edges of cells. 3) Determine how tTG works with Ras to promote cellular transformation. EGF signaling through Ras results in activation and localization of tTG to leading edges and Ras needs tTG for its transforming potential. In this aim, I will determine whether tTG- promoted Ras transformation occurs as a result of tTG's ability to enhance Ras activity or the activation of one of its downstream effectors, or whether tTG activates a Ras-independent signaling event that synergizes with Ras signaling to drive malignant transformation. These studies should provide insight into the significance of coupling tTG activation and localization to the leading edges of cells and how tTG is contributing to cell migration and invasion as well as cellular transformation."}
{"text": "A necessary operation for language comprehension and production is coreference, which involves keeping track of concepts in memory, updating them, and forming correct links in the mind between different coreferring forms, often over long distances. The process of coreference in text comprehension has been well studied, while the contribution of discourse and prosodic cues relevant for the speech and gesture modalities has received considerably less attention. This proposal seeks funding to investigate the process of coreference in speech-gesture comprehension and production in order to model the organization and mechanisms of the adult, end-state, language system more completely. Four hypotheses will be tested. (1) Prosodic stress accesses and makes use of the same underlying memory mechanism(s) and organizing principles as found for text comprehension. (2) Gesture can be used to mark antecedents and this coreferent gesture is co-expressive with speech. (3) Coreferring gestures also make use of the same underlying memory mechanism(s) and organizing principles as prosodic stress and text comprehension. (4) Gesture codes antecedent representations in a motor-kinetic format."}
{"text": "According to the Guide to Clinical Preventive Services, the common form of screening for thyroid abnormalities, neck palpation, lacks the sensitivity (33%) for detecting thyroid cancer, and ultrasound has too great a false positive rate to avoid invasive tests such as biopsy, to rule out cancer. It is the aim of this work to provide sensitive ultrasound tests to non invasively diagnose cancer in the thyroid earlier and more accurately than current imaging techniques. Elastography, which images the stiffness properties of tissues, and parametric backscatter imaging that displays the frequency dependence of ultrasound reflections offer novel signal processing approaches that provide new information about tissues. These advanced ultrasound techniques will be incorporated into a new, software-controlled research ultrasound platform. Phase I efforts will tune the platform so that it provides data and software control needed to fully develop and evaluate these novel imaging methods. Further work will evaluate algorithms for producing elastograms using raw radio frequency echo data available on the research scanner. It also will develop acoustic parametric images that are based on the frequency dependence of scattering and on attenuation, offering modes that will compliment conventional amplitude mode processing. A software development kit (SDK) that accesses fast digital signal processing (DSP) boards on the scanner will also be developed. Quantitative images of thyroids will be derived to demonstrate the new modes. Phase II will implement elastograms in real time and provide other parametric images during image freeze. Real time elastography will be achieved through careful evaluation of different algorithms for use on the DSP circuit. Algorithms will be assessed for the quality of elastograms produced and the computational overhead incurred in their generation. Phantom and preclinical tests will be done to select the most effective for thyroid imaging. Parametric images based on attenuation and the frequency dependence of scattering, characteristic of the scatterer size will be developed. Novel features will be to incorporate reference phantom data for calibration directly into the scanner memory to facilitate rapid processing, and to apply compound scanning for reducing statistical fluctuations in quantitative images. The system will be evaluated by testing in patients with thyroid nodules and in patients with proven breast masses."}
{"text": "Alcohol abuse can have devastating impacts on the brain and behavior and is a big burden on public health. However, the molecular underpinnings of alcohol induced neural cell injury are not fully understood. Recent advances in functional genomics suggest that long non-coding RNAs (lncRNAs) may play critical roles in alcohol- induced neural cell death. The recent work from our laboratory support this hypothesis. Therefore, a comprehensive screening system of lncRNAs related to alcohol-induced neural cell death is of significant benefits for identifying potential therapeutic targets. Genetic editing tools such as Zinc Finger Nuclease (ZFN) and Transcription Activation-Like Element Nuclease (TALEN) have great potential for the functional study of genes or the application of gene therapy through knockout or knockin techniques. A new type of genetic editing tool based on bacterial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR?Associated System [Cas]) has been successfully used in various organisms from bacteria and yeast to mammals. Relative to ZFN and TALEN, CRISPR/Cas is advantageous because it only requires changing the sequence of the guide RNA (gRNA) and it can also be directly delivered into embryos to generate gene-modified organisms. Furthermore, the multiplexing capability of CRISPR/Cas makes it possible to target multiple genes simultaneously. In this study we will use our recently developed dual guide CRISPR/Cas approach to generate an lncRNA knockout (gRNA) library in SH-SY5Y cell model. We will then perform a genome-wide screening for lncRNAs involved in alcohol-induced cell death pathways. Our preliminary data indicated that alcohol increased the expression of a nuclear paraspeckle lncRNA in SH-SY5Y cells. Our previous work have demonstrated that alcohol activates the oxidative stress-apoptotic cell death pathway in a dose dependent manner to reduce neuronal cell viability and increase cellular oxidative stress. We expect that the application of the high throughput screening platform in alcohol-induced neuronal death system will allow for the accurate identification of all other specific lncRNAs as well as their potential roles in mediating alcohol-induced neurodegeneration. The generated lncRNA gRNA library in this study can also be available for the characterization of cell toxicity induced by other neurotoxins or other substance abuse. Thus this research has a potential to reduce the health impacts of alcohol abuse and also has benefits for other substances abuse related public heath burdens."}
{"text": "DESCRIPTION: (Applicant's Abstract) Opioids have been demonstrated to alter immune functions. The natural opioid peptide methionine enkephalin (Met-ENK) has been reported to boost T-lymphocyte numbers and function in a cytokine-like manner. Thus, this peptide has been suggested as an adjunct to antiviral chemotherapy in the treatment of acquired immunodeficiency syndrome (AIDS). The combination of antiviral and immunostimulatory substances has been under serious evaluation for effectiveness in treating AIDS for several years. However, this approach has been limited by the need to be conservative in designing clinical protocols. The current proposal uses a murine retrovirus-induced immunodeficiency model to study various combinations of the anti-retroviral drug azidothymidine and Met-ENK. Initial studies indicate that Met-ENK can significantly enhance protection against the mouse retrovirus infection, as measured by reduced splenomegaly and increased survival. The use of the murine model, which uses various numbers of subjects treated by drug combinations, is advantageous in allowing studies that fully evaluate the interaction between these agents. These studies will focus on determining: 1) the optimal drug combination in dosing, time, and route of administration; 2) whether or not the Met-ENK effects are via opioid receptors; 3) if the effects of Met-ENK result in a cytokine pattern (type I cytokines) that favors resistance to disease; and 4) whether therapy reduces viral load temporarily or long term, and whether viral infection is actually cleared from the mice. This model has excellent potential for establishing whether the therapeutic combination of antiviral and immunostimulatory agents may be applied in the future to human AIDS or other retroviral infections."}
{"text": "Our proposed research is aimed at providing a systematic approach and an eventual mechanistic understanding of the kinetic factors associated with cholesterol gallstone dissolution in bile from the physical chemical viewpoint. It involves the experimental and theoretical evaluation of the influences of the principal constituents of human bile by conducting dissolution rate studies of cholesterol gallstones and cholesterol monohydrate \"model gallstones\" in chemically defined media and comparing these results with those obtained with human gallbladder bile. Emphasis is being directed toward the assessment of the importance and the understanding of the interfacial resistance factor which rate limits stone dissolution in vitro and, probably, in many in vivo situations corresponding to certain bile compositions. Results of current studies will be used to examine in vivo dissolution data from patients being treated for stones with orally administered chenodeoxycholic acid. Also the present results are being used to provide an explanation of why certain formulations for T-tube infusion treatment of retained common duct stones appear to be more clinically effective than others. Efforts are being put into seeking out and studying conditions, procedures, and chemical agents that may, from a kinetic viewpoint, significantly accelerate cholesterol gallstone dissolution. An animal model will be studied for testing in in vitro findings with regard to effectiveness."}
{"text": "Approximately 5-20% of psychiatric patients hospitalized in public facilities receive long-term, high-dose antipsychotic treatment. Most of these patients &e prescribed the high-dose treatment because they have severe symptoms of schizophrenia. This treatment, associated with higher risk for side effects, was not demonstrated to be therapeutically more effective than the (lower) standard dosage regimens. Plasma level of 10 ng/ml of haloperidol (approximately 18 mg/day by mouth) was demonstrated to provide sufficient antipsychotic effects in schizophrenic patients. The main goals of the current proposal are to identify schizophrenic inpatients receiving long-term high dose haloperidol treatment; to reduce their plasma levels gradually and under controlled conditions to 10 ng/ml, and to determine the effects of that reduction. The subjects will be 290 schizophrenic inpatients whose treating psychiatrists prescribed oral doses of haloperidol yielding plasma levels greater than 15 ng/ml. Patients will be randomly assigned to one of two groups: level reduction or level continuation. The patients in the first group will have their doses reduced over a period of 12 weeks; the target plasma level will be 10 ng/ml. They will be maintained on that level for the subsequent 16 weeks. The patients in the continuation group will be maintained on their level for 28 weeks. Any relapsing patients will have their medication adjusted. Comparison of these groups outcomes will show whether the high doses were needed. A subgroup of patients who need the high-dose treatment may be identified and characterized. Proposed 5-year study will provide a rational basis for the dosage regimens of typical neuroleptics in the treatment of severe forms of schizophrenia."}
{"text": "The proposed research involves the collection of quantitative and qualitative data on past reproductive behavior and the prospective follow-up of the demographic dynamics of a population of about 13,000 people in rural Cambodia. The first aim of this study is to provide the accurate and timely demographic indicators much needed for development planning in Cambodia. To date, the 1998 census and recent surveys have yielded conflicting results on current levels of fertility and child mortality. The data collection proposed here and extant techniques of demographic analysis will provide several indicators of the quality of the existing census and survey data. The population follow-up will provide reliable and timely indicators of current demographic levels and trends. The second aim is to document how the dramatic events of the 1970s have shaped the survivors' reproductive system, both at the community and the family level. A study of post-war reproductive behavior will combine analyses of our individual interviews and focus group discussions with males and females at different stages of their reproductive careers, with recent census and nationally representative survey data. The unusual features of recent Cambodian demography can provide unique insights on the long-standing theoretical debate on the impact of mortality changes on marriage and reproduction. The third aim of this study is to lay the foundations for longitudinal studies of social change in this population and, most immediately, of the demographic and social impact of the HIV epidemic. The study of the impact of HIV will be initiated during the two years of the follow-up but the bulk of the impact is likely to be felt in the later part of this decade. During the initial two-year period, however, we will be able to test a light structure for demographic surveillance that could be maintained indefinitely at low cost. The existence of this long-term observatory will complement and enhance future substantive research in this population."}
{"text": "We have initiated the development of assay techniques that will allow us to understand the sulfation code in chondroitin glycosaminoglycan (GAG) chains. These assay techniques take advantage of specific chromatography techniques (ion exchange, hydrophilicity) to separate the different disaccharides and monosaccharides that comprise the GAG chain. This is the only technique capable of doing this. We have also initiated mass specrtrophotometric analysis of these separated GAG chains to begin to determine the sequence of sulfations on the different parts of the GAG chain. We have continued experiments to modify the biological activity of GAG chains by altering the level or activity of enzymes that either synthesize or degrade sulfated chondroitin. In particular, we are developing methods to specifically alter the sulfation of GAG chains in particular positions in order to evaluate if they alter the biological activity of the GAG chain. We showed that a specific reduction in 4-sulfation abolishes the biological activity of CSPGs, thus suggesting that sulfation on the 4 position of GalNAc is essential for biological activity. We have started experiments to evaluate novel intracellular signaling mechanisms in response to GAG chains. Growth cones turn in response to bound GAG chains. We have used this property of growth cone turning to assay the activity of drugs and other compounds. In particular, we have determined that the activity of particular intracellular molecular motors and other components of the cytoskeleton are essential for this growth cone turning. Modification of their activity by drugs may ultimately prove to be a useful therapeutic approach for treatment of brain injury. Because many of these mechanisms are also found in injury to heart and blood vessels, these approaches may have a more general applicability."}
{"text": "Virginia Commonwealth University (VCD) and the University of Chile will jointly offer translational research training in reproductive biology to junior scientists from throughout Latin America. Potential trainees will submit applications to the University of Chile, which will be responsible for screening, interviewing, and selecting program participants. Eligibility will be based on completion of doctoral degree requirements (plus clinical training for MD candidates) within the past 5 years; area of research interest (relevance to public health priorities in reproductive medicine in their home country); commitment to return to home country to establish research program; and letters of reference. Trainees will complete 1-2 years of coursework and laboratory training at the University of Chile before coming to VCU to conduct a 1-3 year intensive mentored research project. Each year, 2 trainees will be supported in Chile and 1 in the US. While at VCU, trainees will complete an NIH grant-writing workshop tailored to the needs of international researchers that will be open to all Fogarty population science training program participants; this workshop will be converted to an online distance education course. Additional distance education offerings led by presenters from VCU and Chile will cover research areas covered by training mentors, including polycystic ovary syndrome and insulin resistance, preterm birth, pre-eclampsia, fetal membrane rupture, cell signaling, gonorrhea vaccine development, steroidogenesis, and promotion of healthy pregnancy. Training in the responsible conduct of research will be provided at both the University of Chile and VCU, and practical scientific management skills (Making the Right Moves) will be taught so trainees can understand how best to partner with researchers at US institutions and how they can apply these skills in their home setting. Other enrichment activities include Endocrine Grand Rounds, Journal Club, Clinical Conference, and lab brainstorming on a weekly basis; a monthly Visiting Professor series; and annual seminars on basic endocrinology research and women's health research. A detailed evaluation plan with measurable outcome indicators will be used to track the success of the program, of trainees who participate, and of research mentors involved. Trainees who complete their mentored research at VCU will be eligible for a re-entry grant of up to $5000 per year for up to 3 years (continued funding contingent on completion of productive research in their home country)."}
{"text": "The overall objectives of the proposed research are: 1) Prediction of safe exposure to noxious vapors and gases, with respect to exposure schedule, physical status of the exposed subject, physico-chemical properties of inhaled substance, and the effect of other chemical agents; and 2) design of an effective system for monitoring biological samples for evaluation of occupational exposure. Mathematical modeling will be used as a tool to achieve these objectives. Compartmental models will be used, because they afford insight into the effect of physiological parameters and physico-chemical properties of the substance on uptake, distribution, and elimination of inhaled vapors. These insights are crucial to the prediction of bio-equivalent exposures and to the monitoring and evaluating of exposure from biological samples. In our attempt to explore mathematical modeling to generate information applicable to inhalation toxicology (with special emphasis on evaluation of occupational exposure), our research is taking four courses: 1) Refinement of animal models to generate basic data, and to corroborate experimentally the predictions made by the mathematical model; 2) refinement of the existing mathematical models to accomodate the complexity of occupational exposure, and use of the model to evaluate the effect of individual factors on uptake, distribution, and elimination of inhaled vapors; and 3) simplification of the mathematical model and programming of the mathematical solution of the model for a programmable pocket calculator or microcomputer to make modeling accessible to toxicologists and hygienists for design, monitoring, and evaluation of occupational exposure; and 4) obtainment of insight on kinetics of inhalation of mixtures of vapors."}
{"text": "The effect and mechanisms through which lipoproteins regulate MVA metabolism and act as competence factors for cultured cells will be analyzed using as models normal diploid cells (capillary endothelial cells (CE), vascular smooth muscle cells) and an established cell line (MDCK cells). The ability of very low density lipoproteins (VLDL) which are LDL precursors to support cell shape and proliferation will be determined using cultures exposed or not to compactin. Their effects will be compared to that of HDL. As a specific VLDL and HDL metabolic response, their effect on HMG-CoA reductase will be measured and considered as a reflection of substrate flux into sterols and nonsterol metabolites. We will determine which key nonsterol products resulting from MVA metabolism would be relevant to the control of cell proliferation and cell shape and would explain the ability of lipoproteins to act as competence factors. Our attention will be focused on the synthesis of terpenes which modify specific polypeptides post translationally. The effect of HDL, VLDL and isolated components (phospholipid liposomes, apolipoproteins) on terpene synthesis and their incorporation into polypeptides as well as the effect of b FGF ( a progression factor for CE cells) on such a process will be examined using as models sparse CE cells exposed or not to compactin."}
{"text": "(A) Study pathogenesis and pathophysiology of high biocontainment viral pathogens utilizing molecular technologies including reverse genetics systems: Bunyaviruses: We are currently in the process of establishing the first nonhuman primate model for Crimean-Congo hemorrhagic fever virus (CCHF) utilizing Cynomolgus macaques. The model will be extremely valuable for pathogenesis studies and countermeasure development. (studies ongoing) Filoviruses: The Syrian hamster model for Ebola virus infection displays almost all hallmark features of human disease. Using this model, we have investigated the mechanisms in which hamsters are protected from wild-type Ebola virus infection and determined that a CD4+ T cell response, facilitating a neutralizing antibody response, is responsible for natural immunity. (studies ongoing) We continued to utilize the Collaborative Cross (CC), a panel of reproducible, recombinant inbred strains spanning the genetic breadth of three murine subspecies, to identify host factors important for Ebola virus disease progression. Transcriptomics revealed that the host responses dependent on genetic background determine susceptibility to infection independent of virus replication. (studies ongoing) We have studied the virulence of several Ebola-Makona isolates from the West African epidemic in macaque models and could show that these isolates are slightly attenuated compared to the Ebola-Mayinga prototype strain. More interesting, these new isolates did not cause lethal infection in the IFNR-/- mouse model, an established rodent model for non-rodent-adapted Ebola isolates. We have started to utilize humanized mice to study Ebola virus pathogenesis and immune responses. (studies ongoing) (B) Study immune responses to infection and vaccination of high containment viral pathogens and develop new vaccine candidates: Arenaviruses: We continued a project focused on the elimination of Lassa virus from the reservoir species, Mastomys natalensis. For this, we used two vaccine platforms, a recombinant attenuated strain of Salmonella and a recombinant cytomegalovirus vector, to immunize the peri-domestic reservoir with the hope to block Lassa virus transmission among rodents and to humans. (studies ongoing) Bunyaviruses: We have continued to characterize and optimize the adenovirus based vaccine for CCHF. Ongoing transfer and depletion studies using the adenovirus-based vaccines expressing the CCHF nucleoprotein or glycoproteins indicated that antibodies are the mechanism of protection. We also developed VSV-based vaccine vectors. Efficacy testing in the mouse model has shown that the glycoprotein itself is insufficient to protect animals. Filoviruses: The VSV vaccine efforts are reported under the 'Trivalent Filovirus Vaccine' project. In the meantime, we have proceeded with the development of alternative vaccine candidates for filoviruses. One approach is targeted towards wildlife vaccination, in particular the great apes, using the concept of a disseminating vaccine vector. The idea is the introduction of a recombinant CMV expressing the Ebola virus glycoprotein. Proof-of-concept studies have been successful in the mouse and rhesus macaque model using a murine and rhesus CMV vector, respectively. WE have also continued to investigate a vaccine based on Modified Vaccinia Ankara (MVA) expressing filovirus-like particles; initial rodent and nonhuman primate studies have revealed very promising results. (C) Study vector/reservoir transmission of high containment viral pathogens using appropriate animal models: Arenaviruses: We have studied infection kinetics of different Lassa virus in the Mastomys reservoir utilizing a unique colony established here at RML. The animals support virus replication and shedding for several weeks before Lassa virus gets cleared. The model will allow for important transmission studies. (studies ongoing) (D) Utilize in vitro and in vivo systems to study the interactions between viral pathogen or viral components and host cells and develop new antiviral strategies: Arenaviruses: Previously we had tested the efficacy of two antivirals, T-705 and ribavirin, in the guinea pig model of Lassa virus infection. In particular, T-705 showed promise as a treatment for Lassa virus with protection even when treatment was started post-disease onset. More recently, we were able to demonstrate efficacy of T-705 against Lassa infection in the nonhuman primate model. (studies ongoing) Filoviruses: Ribavirin can effectively extend the time-to-death of hamsters infected with Ebola virus, but resistance will rapidly develop. Surprisingly, T-705 protected hamsters against Ebola virus infection when animals were treated for two weeks beginning the day after infection. This promising treatment option is currently being followed up. We have also identified a few promising broad spectrum antivirals against Ebola virus, which are in development as treatment options for other RNA viruses. Further confirmation is underway. In contrast, treatment with anti-malaria drugs, including chloroquine, failed in Ebola rodent models and should not be further considered. (studies ongoing) (E) Study the epidemiology and ecology of high biocontainment pathogens utilizing newly developed rapid, sensitive and specific diagnostic test systems including those that can be applied under field conditions: Filoviruses: From August 2014 through May 2015 we have operated a diagnostic laboratory in Monrovia, Liberia to support the international response to the West African Ebola epidemic. We have started to analyze data for scientific purposes under approved protocols. So far we have demonstrated the importance of performing on-line clinical chemistry and malaria differential diagnostics. Most importantly, we have identified the interesting observation that plasmodium co-infection increases the survival rate of Ebola-infected patients. Future studies will be designed to test this observation in animal models and to understand the underlying mechanism."}
{"text": "Although much information has become available concerning the biochemical changes taking place during the chronic administration of morphine, it still is not clear which of these changes are related to the development of tolerance and addiction to the narcotic agents. A different approach to these studies may be useful at this time. Animals made thiamine deficient by feeding diets deficient in the vitamin will be used to study the development of morphine tolerance and various associated biochemical changes. The effect of thiamine deficiency on catecholamine and serotonin turnover with morphine will be studied by monitoring the incorporation of C14-tyrosine or C14-5- hydroxytryptophan into the amines, since synthesis is decreased in the deficiency state. The mechanism of decreased motor activity in deficient animals has been investigated using agonists, antagonists and precursors of the catecholamines. The effect of morphine on the various thiamine-dependent systems will be examined in tolerant and non-tolerant animals, as well as in vitro. Studies will be conducted to determine whether morphine will cause the release of thiamine from nerve membrane preparations and whether it will either inhibit or enhance the release of thiamine induced by other agents."}
{"text": "There has been a huge increase in demand for comprehensive quantitative analysis of neurovascular imaging data produced in the clinical setting for diseases such as multiple sclerosis, traumatic brain injury, stroke and dementia. Our objective in this project is to design and develop advanced image processing software that can rapidly and accurately analyze such data. To achieve this objective, we propose a range of novel algorithms to process data from the following MR imaging sequences widely used in the aforementioned applications: time resolved 3D contrast enhanced MR angiography (CE-MRA) for the assessment of vascular anatomy, time resolved 2D phase contrast flow imaging (PC-MRI) for the evaluation of vascular hemodynamics, susceptibility weighted imaging (SWI) for quantifying iron deposition in the brain, and fluid attenuated inversion recovery (FLAIR) imaging for the detection of white matter hyperintensities (WMH) and lesions. A variety of tools will be designed and implemented to tackle these problems including: tissue similarity mapping and active shape models to segment the vasculature in both CE-MRA and PC-MRI images; automatic tissue segmentation in the basal ganglia and thalamus for a two-region of interest analysis for iron quantification with SWI; and finally adaptive approaches incorporating fuzzy C-means, shape factor analysis, compactness and fractional anisotropy to quantify lesions and WMHs. To exploit the advantages provided by different imaging sequences, co-registration algorithms will be used to improve segmentation of vessels between CE-MRA and PC-MRI, and between 3D T1 weighted imaging and SWI. Upon finishing this project, we expect a multi-fold increase in processing efficiency and a significant increase in accuracy will be achieved. The resulting software will not only help the growth of our company, but also improve the diagnosis and treatment of neurovascular diseases. PUBLIC HEALTH RELEVANCE: The huge increase in demand for a more comprehensive and accurate analysis of the vast amount of clinical MR imaging data for neurovascular diseases such as multiple sclerosis, traumatic brain injury, stroke and dementia is the driving force for th development of more advanced image processing software in our company. In this project, we propose an integrated approach to develop a set of processing software for imaging sequences that target the assessment of both anatomy and function of the neurovasculature system. The results will lead to a better access to quantitative data about the brain's vasculature, flow, hemodynamics and iron content present in neurovascular diseases. The completion of this project will not only help the growth of our company by increasing processing throughput and accuracy, but also improve the diagnosis and treatment of patients with neurovascular disease."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To convert human pluripotent stem and somatic cells into hematopoietic stem cells (HSCs) and self renewing cardiac progenitor cells. The Midwest Progenitor Cell Consortium is a collaboration between the UW-Madison and the University of Minnesota, which will serve as a research hub within the NHLBI's Progenitor Cell Biology Consortium. The goal is to develop new strategies to convert human pluripotent stem and somatic cells into hematopoietic stem cells (HSCs) and self renewing cardiac progenitor cells through identification of genetic program leading to formation of pre-HSCs and cardiac progenitors and activation of self-renewal program in lineage restricted cells. Dr. Slukvin's involvement in the consortium relies on WNPRC Stem Cell Resources."}
{"text": "Nocturnal cyclic intermittent hypoxia, such as experienced by patients with obstructive sleep apnea (OSA) is thought to alter vascular tone and function leading to peripheral vasoconstriction and consequent arterial hypertension. Studies in patients indicate that sleep apnea results in diminished reactivity to endogenous vasodilators, and altered sensitivity to some endogenous vasoconstrictors. Furthermore, OSA patients demonstrate an augmented pressor response when exposed to hypoxia and fail to decrease forearm vascular resistance (FVR) when exposed acutely to progressive isocapnic hypoxia as do non-apneic volunteers. Our own preliminary data, collected in normal volunteers and OSA patients, suggests, however, that intermittent hypoxic exposure may lead to vasodilation rather than vasoconstriction. Normal volunteers exposed to intermittent hypoxia for 14 nights have no change in vascular resistance despite increased sympathetic activity and also fail to vasodilate when acutely exposed to isocapnic hypoxia after the repetitive exposure. Based on these and other observations we propose three hypotheses. First, we hypothesize that hypoxic exposure causes either sustained vasodilator release (e.g., epinephrine, NO) that persists during normoxia or altered sympathetic transduction with diminished vasoconstriction (e.g., altered receptor density or transmitter release), or both. Second, we speculate that an acute re-exposure to hypoxia after a prior intermittent exposure results in an abrupt increase in sympathetic nervous system activity but little further increase in vasodilator release, thus resulting in impaired vasodilation. Finally, we hypothesize that maximum vasodilator release declines over time with continued hypoxic exposure, resulting in a gradual increase in arterial pressure. To test these hypotheses we plan a series of investigations in normal volunteers before and after an exposure to cyclic nocturnal hypoxia for 28 nights, and in OSA patients before and after 30 days of monitored therapy with nasal CPAP. These studies will use selective intra-arterial infusions of specific pharmacological agonists and antagonists to assess the roles of endogenous vasodilators and vasoconstrictors in altering vascular tone following an exposure to hypoxia. We anticipate that these studies will significantly enhance our understanding of how intermittent hypoxia and obstructive sleep apnea influence vascular tone and function."}
{"text": "Two genetic loci, one dominant, Ir-Z1, the other recessive, ir-Z2, controlling immune responsiveness to aqueous solutions of Beta-galactosidase have recently been found. It is proposed to characterize the function of these genes. First, the location and the number of genes involved will be investigated in hybrid and congenic strains. Subsequently, cell-to-cell interactions will be studied by adoptive transfer experiments and by in vitro synthesis of antibodies. The methodology developed for studying immune responsiveness to Beta-galactosidase is such that activating antibody can be detected at the single molecule level and antigen-binding cells can be easily enumerated. The objective is to establish the type of cell in which the product of the Ir-Z genes are expressed and ultimately, to isolate and characterize the gene products."}
{"text": "Human health depends strongly on adequate nutrition. Symbiotic fixation of atmospheric dinitrogen by legume rhizobia is the major source of nitrogen in the human diet, as well as the major factor limiting legume crop yield. This project aims to characterize the alterations entailed in differentiation of the alfalfa symbiont Rhizobium meliloti from free-lving bacteria, which do not fix nitrogen, to the nitrogen-fixing bacteroid form. Three interrelated studies will be carried out. (1) Fix genes, required for differentiation, will be identified by transposition mutagenesis with a Tn5-lac promoter probe, which will allow characterization of regulatory circuitry. (2) Changes in the outer and inner bacterial membranes will be characterized as a function of bacterioid differentiation. (3) The heat shock response, which depends on a specific RNA polymerase sigma subunit, will be characterized in R. meliloti, as a possible genetic approach to identification of their sigma subunits suspected to be involved in differentiation. These studies should begin to define the molecular changes required for differentiation of the bacteria to a form that can fix nitrogen. Such information will ultimately be necessary for genetic engineering of nitrogen fixation, with benefits to human health and agronomy."}
{"text": "This is the firs application from a new investigator with the overall objective to investigate the regulation of caudal-related homeobox (CDX) proteins in normal colon and colorectal cancer. CDX1 and CDX2 are transcription factors that are involved in regulation of proliferation and differentiation of the intestinal epithelial cell. Additionally, expression of replacement of CDX gene expression in colon carcinoma cell lines dramatically inhibits cellular proliferation by cell cycle arrest in G1. Thus, altered regulation of CDX genes in colonic epithelial cells is involved in the development of the neoplastic phenotype. Therefore, this proposal is based on the hypothesis that an understanding of the regulation of CDX genes in both normal and neoplastic cells will elucidate pathways that control the phenotype in cancer cells. The goal of this project is to elucidate the normal regulation of CDX1 and CDX2 and to determine changes that occur in the colonic neoplasia. Preliminary are presented that demonstrate variable of CDX genes in colorectal cancer cell lines and that this differential expression is regulated the level of gene transcription. To accomplish this goal, four specific aims are proposed: 1) To elucidate the transcriptional regulation of the human CDX1 gene in normal and neoplastic colonocytes using DNase I hypersensitivity. mapping and transfection and DNA-protein interaction studies. Preliminary data shows that there is a specific hypersensitive site in a cell line that expresses CDX1 that is absent in non-intestinal cell lines and intestinal cell lines and intestinal cell lines that do not express CDX1. The function of this site will be explored and the remainder of the gene will be studied for additional sites. 2) To elucidate the transcriptional regulation of CDX2 in normal and neoplastic tissue. Similar to CDX1, we have mapped and characterized and characterized the CDX2 gene. Transfection studies suggest that elements for directing intestine-specific expression are outside the immediate upstream region of the gene. DNase I hypersensitive site analysis will also be employed to identify potential regulatory regions. 3) To study the regulation of CDX genes in transgenic mice. These studies will validate the function of regulatory elements identified in Aims 1 and 2 in the whole animals. 4) To examine cellular signaling pathways that regulate CDX genes in colon cancer cells. Once regulatory elements are defined, the cellular pathways that regulate expression in normal and neoplastic cells will be studied. Completion of these studies will provide a detailed picture of the transcriptional regulation of CDX genes in normal and neoplastic cells. This will provide the basis for understanding the network of cellular pathways that control these important cell specific transcription factors and how they are altered in neoplasia."}
{"text": "Lactase non-persistence (adult-type hypolactasia) results in digestive system malabsorption of lactose, the carbohydrate macronutrient present in milk. Human DNA sequence variants associated with intestinal lactase persistence and non-persistence have recently been identified. This research project focuses on characterization of the genetic determinants of intestinal lactase persistence in humans. We have previously characterized molecular mechanisms regulating intestine-specific expression of the rat lactase gene in cell culture and in living animals. We will now direct attention to characterization of the molecular mechanisms mediating human lactase gene persistence and non- persistence and the temporal decline of lactase in rodents. Preliminary studies have shown that DNA sequences in the region of lactase gene polymorphisms can bind nuclear proteins and enhance lactase promoter activity. We hypothesize that DNA polymorphisms associated with lactase persistence function to regulate lactase gene transcription in adult human enterocytes via differential interaction with specific nuclear protein transcription factors. Research objectives are therefore aimed at defining the mechanistic roles of genetic determinants of human lactase persistence. In Aim 1, we will characterize a functional role for DNA polymorphisms associated with persistence of lactase gene transcription in humans. Functional characterization of lactase gene polymorphisms will be investigated in cell culture and in vivo in transgenic mice. In Aim 2, we will define the molecular mechanisms regulating human lactase persistence gene transcription. The goals of this aim will be to identify and characterize nuclear proteins capable of interacting with DNA sequences in the region of lactase gene polymorphisms associated with lactase persistence and to characterize whether the nuclear proteins function to regulate lactase transcription. In Aim 3, to complement our investigation of the mechanism of lactase persistence in humans, we will further elucidate mechanisms regulating the temporal decline of lactase in rodents. Knowledge of mechanisms regulating human lactase gene expression may provide future novel diagnostic, therapeutic or prognostic strategies for gastrointestinal disorders that result in loss or inappropriate gain of epithelial cell digestive function. Our combination of in vitro and in vivo experimental approaches will elucidate roles for the genetic determinants associated with lactase persistence and non-persistence. PUBLIC HEALTH RELEVANCE: Adult-onset hypolactasia, lactase non-persistence, renders much of the world's adult human population intolerant of excessive consumption of milk and other dairy products. In some adults, however, high levels of lactase enzyme activity persist in adulthood presumably due to inheritance of a genetic mutation that prevents the normal maturational decline in lactase expression. Knowledge of mechanisms regulating expression of the digestive lactase gene may provide novel strategies for enhancing expression of digestive enzymes in intestinal diseases and will have broad implications for understanding regulation of other genes with aging."}
{"text": "Approximately two thirds of patients with intrahepatic cancer present with unresectable disease. Our previous studies show that high dose conformal radiation combined with chemotherapy appears to prolong the survival of patients with unresectable intrahepatic cancers. However, attempts to increase radiation dose still further have been limited by the development of radiation-induced liver disease (RILD). The pathology of RILD is veno-occlusive disease, which is characterized by thrombosis within the central veins of the liver producing post hepatic congestion. In the past, efforts to develop models to estimate the likelihood of developing RILD have been based primarily on the planned radiation dose distribution for the normal liver. These analyses have demonstrated that increasing mean liver dose correlates with the likelihood of developing RILD. While these models have permitted the safe delivery of far higher doses of radiation than have previously been possible, they also suggest that there is a broad range of individual patient sensitivity that is not reflected by predictions made solely based on the physical dose distribution or general clinical features. As the basic pathophysiology of RILD is venous occlusion, we develop the hypothesis that early monitoring of portal venous perfusion and hepatobiliary function would have the potential to predict liver function after irradiation, thereby permitting to safely deliver the higher dose to the tumor without an increase of complications. Our preliminary data provide evidence to support this hypothesis. In this R01 application, we propose to develop a model to predict liver function after the completion of radiation therapy based upon the radiation treatment plan, and the values of portal venous perfusion and hepatobiliary function assessed by DCE MRI and SPECT prior to and during radiation therapy. Also, we assess individual and regional radiation sensitivity during radiation therapy using the liver dose response function that we have developed, for prediction of liver function after irradiation. Our proposed approach is highly innovative and represents a new paradigm to investigate radiation toxicity in the liver. It has potential to be a tool for individualized therapy."}
{"text": "Tuberculosis (TB) continues to be an important public health problem, with an estimated 9.4 million cases and 1.7 million deaths in 2009. Multidrug resistant TB (MDR-TB, defined as resistance to at least isoniazid and rifampicin), and HIV-associated TB pose important threats to TB control. The high case fatality rate of HIV- associated MDR-TB has major implications for sub-Saharan Africa. The lack of laboratory capacity for Mycobacterium tuberculosis culture and drug susceptibility testing (DST) in resource limited settings poses important challenges to the fight against MDR-TB. In 2008, only 7% of the estimated 440,000 MDR-TB cases worldwide were detected. Xpert MTB/RIF, a novel rapid diagnostic that simultaneously detects M. tuberculosis and rifampicin resistance within two hours, was recently (Dec. 2010) recommended by the WHO as the initial test in those suspected of MDR-TB or HIV-associated TB. The assay detects rifampicin resistance with 95.1% sensitivity and 98.4% specificity, high negative predictive value (99%) but relatively low positive predictive in people with a low pre-test probability of MDR-TB. While rapid diagnosis of rifampicin resistance could revolutionize the fight against MDR-TB, this technological advance in rapid diagnosis will only result in improved patient outcomes if the rapid diagnosis is linked to rapid access to optimal treatment. In 2008, only 11% of MDR-TB cases were appropriately treated. To ensure optimal treatment outcomes and avoid amplification of resistance, knowledge of the complete resistance profile is required. Using Xpert MTB/RIF, clinicians will need to make a treatment decision based on the knowledge of rifampin resistance only, and thus risk the initiation of a suboptimal regimen. To guide the initial standardized management of rifampicin resistance detected by Xpert MTB/RIF, we will comprehensively characterize the phenotypic and genotypic resistance profiles in a large (n=474) number of patients with rifampicin resistant TB. To evaluate the impact of Xpert MTB/RIF on patient outcomes, we will perform implementation research nested within the phased implementation of Xpert MTB/RIF by the South African Department of Health (DOH). We will document treatment decisions made in a cohort of rifampicin resistant TB cases, and compare outcomes (time to culture conversion, amplification of resistance, drug toxicity, and survival rates) during the first 6 months of treatment between 237 patients diagnosed with rifampicin resistance on Xpert MTB/RIF and 237 patients with rifampicin resistance detected on culture based-DST (patients without routine access to Xpert MTB/RIF). The proposed implementation research aims to change the current paradigm for screening, diagnosis and treatment of MDR- TB, by building a robust knowledge base on the resistance profiles of patients diagnosed with rifampicin resistant TB on Xpert MTB/RIF, by quantifying the impact of the assay on amplification of resistance and treatment outcomes in patients with drug resistant TB. The evidence generated will contribute to the successful implementation of this novel assay into routine practice in resource limited, high TB/HIV burden countries."}
{"text": "DESCRIPTION: (Applicant's Description) The broad long-term goal of this revised research program is to produce an educational program to train cancer registrars (CR) to code co-morbidity information for inclusion into cancer registries. There are three specific aims: 1) to teach CR to code co-morbidity information with the use of a valid co-morbidity instrument; 2) to assess accuracy, consistency, and efficiency of co-morbidity coding, and 3) to develop an educational program consisting of written materials and a videotape to train cancer registrars at other hospitals. Cancer patients often have other non-neoplastic diseases or co-morbidity. These other diseases can be so severe as to impact on prognosis, treatment selection, and outcomes. At present, data collected by cancer registries include demographic descriptions of the patient and morphologic descriptions of the tumor. However, no information about co-morbidity is presently collected. Several valid measures of co-morbidity exist and their inclusion in cancer registries would improve the scientific accuracy of cancer statistics and evaluation of treatment. This project will be a two-year, prospective, longitudinal study with three distinct phases. Phase I will include education of CR on the importance of co-morbidity to the pretreatment evaluation and classification of cancer patients. Phase II will include coding the medical records of 400 newly diagnosed cancer patients and assessing the accuracy, consistency, and efficiency of coding. To test the generalizability of the instrument, one CR from three of the BJC-affiliated hospitals, will be asked to use the instrument to code co-morbidity for 100 consecutive cancer patient records. Phase III will include the preparation of the educational material. This work is significant because if cancer registrars are shown to be able to code co-morbidity accurately then this information can be included into standard oncology data collection efforts. Improved accuracy of cancer statistics and evaluation of treatment effectiveness will decrease mortality and improve quality of life for cancer patients."}
{"text": "Examination of the potential therapeutic effect of 4-ASA on patients with ulcerative colitis. This is an FDA \"Orphan Drug\" sponsored double blind randomized 6 week controlled trial that includes state of the art assessmen of the severity of the disease."}
{"text": "We have examined various aspects of hormonal control of adipocyte metabolism with isolated rat epididymal adipocytes as a model system. (A) In 32-Pi-loaded cells, the phosphorylation of a 65 KDa protein, found associated with the lipid fraction of adipocyte homogenates, was examined under conditions of unchanging, steady- state cAMP-dependent protein kinase (A-kinase) activity. Steady- state phosphorylation was achieved following a pulse, or overshoot, of phosphate incorporation, indicating that increases in A-kinase are accompanied by increased phosphate activity, and that the concerted action of both kinase and phosphatase provide the cell with a means to produce graded responses to graded increases in cellular cAMP. In a manner independent of A-kinase activity. Insulin also leads to the removal of phosphate from this protein. Insulin stimulates the phosphorylation of an abundant 62 KDa protein, also located exclusively in the lipid fraction: phosphorylation of this protein is abolished by increased A-kinase activity. Such data reveal a tight interplay, or crosstalk, between adenylate cyclase-linked receptors and the insulin receptor. (B) A high affinity antibody against hormone-sensitive lipase (HSL) was raised and used to probe a fat cell cDNA library. Also, a probe for the HSL gene has been produced from a peptide sequence derived from HSL purified in this laboratory. (C) Analysis of fat cell G proteins showed that fat cells contain 3 different subspecies of Gi and 2 different Gs proteins, all located primarily on plasma membranes. However, intracellular membranes contain a large \"Gs-like\" protein of 55 KDa that is ADP-ribosylated by cholera toxin, both in vivo and in vitro, and recognized by affinity purified antibodies against Gs."}
{"text": "Proteomics is a rapidly expanding field but current methodologies remain inadequate for achieving its full potential. The most basic enzymological tool for characterizing proteins is the protease. Proteases are already an essential part of proteomic analysis but more sophisticated tools are needed to identify low abundance proteins in highly complex samples. We have engineered prototype restriction proteases which are active in denaturing conditions and which cut specifically at well-defined cognate sequence motifs. Our basic innovation would be the ability to parse a proteome into sequence-edited slices and to detect these edited portions with high resolution and high sensitivity. In Phase I we tested the prototype restriction protease for suitability in proteomic analysis and implemented a novel directed evolution methodology for the selection of proteases that cut new sequence motifs. The Phase II objective is to develop a sophisticated set of protease tools to facilitate proteomic analysis in the way restriction endonucleases have facilitated genomic analysis. PUBLIC HEALTH RELEVANCE: The complex and dynamic nature of proteomes make them rich with useful information but difficult to characterize. Our long-range objective is to develop a sophisticated set of protease tools to facilitate proteomic analysis in the way restriction endonucleases have facilitated genomic analysis. Better proteomic tools will lead to earlier detection of disease states, better treatments, better predictability of the effects of various treatments, and the development of individualized therapies."}
{"text": "APPLICANT'S ABSTRACT: Chronic alcoholism in humans or chronic experimental alcohol treatment in rats results in a multitude of endocrinologic, metabolic and physiologic abnormalities such as sexual and reproductive failure and hepatic dysfunction. Although there is a wealth of data available on alcohol's effects on some aspects of drug metabolism, our overall understanding of the mechanisms underlying alcohol-related hepatic changes is superficial. The overall goal of the current proposal is to distinguish the direct effects of ethanol on liver metabolism from those that are secondary to or synergistic with changes in endocrine or nutritional systems. There is much evidence to indicate that part of the induction of P-450j (IIE1) by ethanol is a direct post-tranlational stabilization of the protein. However, the nutritional deficiencies seen in alcoholic men may also play a synergistic role in the induction of this and other cytochrome P-450 isozymes after chronic ethanol abuse. In addition, ethanol has inhibitory effects on the hormones of the hypothalamic-pituitary-gonadal axis, resulting in hypoandrogenization and hyperestrogenization in men and rats. Since hormones appear to have an important regulatory role in the expression of liver cytochrome P-450 isozymes, the interaction between ethanol abuse, endocrine function and drug metabolism requires investigation. The focus of this proposal is on 5 principal areas: 1) the direct effects of ethanol on the hepatic microsomal monooxygenase system (HMO); 2) the indirect hepatic effects of ethanol secondary to alterations in nutritional or endocrine status; 3) the in vitro/in vivo correlation of ethanol induced changes in drug metabolism and activation; 4) the interaction of chronic ethanol with other inducers of the microsomal monooxygenase system; and 5) the characterization of a new ethanol induced cytochrome P-450 isozyme. Many alcohol abusers as well as social alcohol users, are taking multiple medications, and are exposed to a wide range of xenobiotics in the diet and in the environment. Although many compounds are known to induce HMO in addition to alcohol, few studies have addressed the question of how ethanol consumption might alter induction of cytochrome P-450 forms other than P-450j. Results from our studies should provide valuable molecular and hormonal information regarding the mechanisms or ethanol induced changes in liver metabolism and provide a rational clinical basis for; 1) reducing toxic drug interactions in alcohol users, and 2) optimizing nutritional support during recovery from alcoholism."}
{"text": "PROJECT SUMMARY/ABSTRACT The Drug Discovery, Delivery and Translational Therapeutics (DT) Program at the Markey Cancer Center (MCC) is scientifically focused on identifying novel targets and biomarkers and discovering and developing new drugs targeting these biomarkers. The MCC catchment area population has both a high cancer risk related to excessive carcinogen exposure and lack of access to cutting-edge clinical trials due to geographical isolation and poor socioeconomic status. The DT program vision is to understand the unique molecular and phenotypic markers of cancer in Kentucky as well as barriers to accessing care and integrate that knowledge to inform drug discovery, development, and delivery of early phase clinical trial efforts for a hard-to-reach Appalachian Kentucky population. MCC investigators are international leaders in biomarker discovery (Theme 1) with ongoing translational studies including more than 600 participants, evaluating the role of environmental carcinogens and identifying biomarkers of lung cancer. DT pharmaceutical scientists work to discover and develop new anticancer agents targeting identified mutations and phenotypes (Theme 2), partnering with Cancer Cell Biology and Signaling (CS) and Genomic Instability, Epigenetics, and Metabolism (GEM) program members. For example, a novel modulator of 4E-BP1 phosphorylation, a validated colon cancer target, was identified from the Appalachian natural products repository. DT investigators lead clinical trials focusing on cancers relevant to the catchment area (Theme 3) and have enrolled more than 500 patients to lung, colon and ovarian interventional treatment and diagnostic trials. They regularly partner with Cancer Prevention and Control (CP), CS and GEM program members to inform and advance MCC basic science, for example, translating early identification of the anticancer activity of PAR-4 in CS to clinical trials focused on a PAR-4 secratagogue. DT is a cross-disciplinary program of 47 investigators from 6 colleges and 18 departments who work together to develop novel anticancer therapies and translate these therapies into the clinic. This productive program has total annual external cancer-related funding of $8.5M ($5.9M annual direct costs, of which 28% is from the NCI). Members published 366 publications over the current funding period, 99 (27%) of which are inter-programmatic, 84 (23%) are intra-programmatic, and 189 (52%) are inter-institutional. The DT program Co-leaders, Drs. Jill Kolesar and Jon Thorson, have a long-standing collaboration and bring complementary expertise in biomarker discovery, drug development, and early clinical trials. Both direct key resources supporting the DT program, the MCC Precision Medicine Center and the UK Center for Pharmaceutical Innovation, respectively. Each leader brings critical strengths including local, national and international collaborations, entrepreneurial relationships, and active participation in NCI initiatives. Taken together, the DT program has a cohesive and collaborative team that translates novel biomarkers into targets for effective anticancer treatments."}
{"text": "This is an application for a K01 Mentored Research Scientist Award for Dr. Scott Beeman, a postdoctoral research associate in the Mallinckrodt Institute of Radiology at the Washington University School of Medicine. He has a strong background in developing magnetic resonance techniques for non-invasive measurement of diabetes-related physiologic changes. Herein, he seeks to expand his scientific foundation to include specific expertise in the pathophysiology of diabetes and clinical investigation. A primary goal of Dr. Beeman is to become an independent investigator and a leader in diabetes-related imaging research, thus he proposes a career development plan in which he will train with world's experts in metabolism and diabetes (Drs. Abumrad and Klein) and magnetic resonance and biophysics (Drs. Ackerman and Garbow). From Drs. Abumrad and Klein he will learn: (i) the molecular and cellular mechanisms which relate to type 2 diabetes and its pathogenesis, (ii) husbandry, genotyping, and diet-modification techniques used to produce mouse models of type 2 diabetes, (iii) the gold-standard histological and assay techniques used in type 2 diabetes research, (iv) the ethical and legal issues involved in clinical and translational research, (v) clinical study design and subject recruitment, and (vi) clinical techniques required to study obesity and type 2 diabetes in human subjects. From Drs. Ackerman and Garbow he will learn: (i) a deep theoretical understanding of the biophysics which underpin magnetic resonance signals, (ii) magnetic resonance pulse sequence design, and (iii) magnetic resonance hardware development, and (iv) the nuances of managing a major research laboratory. Insulin resistance is a defining feature of type 2 diabetes - an obesity-related disease with severe morbidity and mortality. Recent studies have suggested that adipose tissue hypoxia is a key step in the cascade that leads to systemic insulin resistance. Still, the longitudinal adipose oxygen partial pressure (pO2) profile during pathogenesis of insulin resistance has not yet been resolved. Characterization of adipose pO2 profile during the pathogenesis of insulin resistance would be a major advance in understanding the hypoxia-driven insulin resistance mechanism. The goals of this work are to: (i) develop methodology to non-invasively quantify in vivo adipose oxygen partial pressure (pO2) with magnetic resonance, (ii) characterize adipose pO2 longitudinally during pathogenic expansion of adipose in mice, (iii) demonstrate the hypoxia-driven insulin resistance mechanism in humans, and (iv) show that exercise intervention restores hypoxic adipose normoxia in humans. The larger goal of this proposal is to develop Dr. Beeman into a productive independent investigator. The knowledge obtained during the proposed career development and research plans will provide him the tools to secure R01 funding and thrive as an independent investigator."}
{"text": "Chemosensory responses are critical components in the control of several essential behaviors of insects that are vectors for pathogens responsible for many important human diseases. In particular, olfaction plays a major role in host seeking and oviposition selection behaviors of blood-feeding female mosquitoes and, as such, constitutes a critical component of the mosquito's ability to transmit diseases such as malaria, dengue, yellow fever and West Nile virus encephalitis. Within this context, and together with our colleagues, we have undertaken a molecular and cellular examination of several elements of the olfactory signal transduction cascade in the principal African malaria vector mosquito Anopheles gambiae sensu stricto and the arbovirus vector Aedes aegypti. An increased understanding of olfactory mechanisms and their underlying chemical cues in these systems may provide insight into the processes of insect behavioral responses in general and disease transmission by vectors in particular and would likely be instrumental in the development of novel mosquito control strategies. We have made significant progress in the characterization of several aspects of olfactory process in both mosquito systems leading to, among other things, the identification and initial characterizations of a family of odorant receptor proteins in An. gambiae (AgORs) and Ae. aegypti (AaORs) that lie at the heart of the olfactory signaling pathway. Building on those advances, this proposal for competitive renewal focuses on extending several elements of our ongoing program including characterization of: (1) structure/function relationships between AgORs and AaORs, (2) the role of AgORs and cognate odorant binding proteins (AgOBPs) in the context of larval olfaction and odor coding in vivo and (3) the molecular events underlying oviposition site selection by gravid adult females. PUBLIC HEALTH RELEVANCE: We have undertaken a molecular and cellular examination of elements of the sense of smell (olfaction) that largely drive several important behaviors in malaria and arbovirus vector mosquitoes. An increased understanding of olfactory mechanisms and their underlying chemical cues in these systems may provide insight into the processes of mosquito behavioral responses and disease transmission by vectors. This information would likely be instrumental in the development of novel mosquito control strategies. Thus far, we have made significant progress and now propose to extend, our characterization of several aspects of olfactory process in both mosquito systems that focus on the role and interactions of a large family of odorant receptor proteins in An. gambiae (AgORs) and Ae. aegypti (AaORs) that lie at the heart of the olfactory signaling pathway."}
{"text": "Cellular immunity (CI) to tumor-antigens (TA) was evaluated and monitored using microassays of direct and indirect migration inhibition (MI), isotopic footpad and Winn neutralization tests as a function of growth of mouse mammary tumor-virus (MuMTV)-induced tumors, SV-40 virus-induced sarcomas, chemically induced sarcomas and plasmacytomas, and guinea pig hepatocarcinoma. CI to TA and antigens of MuMTV was observed in tumor bearers. CI decreased as magnitude of tumor-burden increased. Immunological \"eclipse\" with plasmacytomas was associated with Sephadex G10 adherent cells and the effect was reversed by levamisole therapy. Direct capillary tube and indirect MI microassays demonstrated production of leukocyte inhibitory factor (LIF) of human breast and lung carcinoma, Ewing's sarcoma and melanoma patients in response to tissue culture tumor extracts, and of breast cancer patients to MuMTV. High titered LIF supernatants were generated with small numbers of mononuclear cells by PPD and lung and breast cancer extracts and MuMTV. Physicochemical separation of supernatants was successful in partially purifying LIF. Evaluation of indicator cells for indirect human and mouse MI demonstrated that some humans and mouse strains were insensitive to lymphokine. In mice this was due to suppressor cells."}
{"text": "Studies of the mechanism(s) by which anthracyclines are toxic to isolated heart cells have resulted in the isolation by gel electrophoresis of a protein that may be related to the phenomenon. The protein is present in mitochondrial fractions, has a molecular weight over 120,000, and covalently binds doxorubicin in a time-dependent fashion which correlates with the drug's toxicity to these cells. Efforts are underway to identify the high affinity protein, and to determine if it is involved in the development of the unusual, delayed cardiotoxicity of anthracyclines in vivo."}
{"text": "Existing two-phase solvent systems for high-speed countercurrent chromatography cover the separation of hydrophobic to moderately polar solvent systems, but often fails to provides a suitable partition coefficient values for highly polar compounds such as sulfonic dyes, catecholamines and twitter ions. The present paper introduces a new solvent series which can be applied for separation of these polar compounds by spiral tube countercurrent chromatography. It is composed of 1-butanol, ethanol, saturated ammonium sulfate and water at various volume ratios. The system consists of 10 steps which are arranged according to the polarity of the solvent system so that the two-phase solvent system with suitable K values for the target compound(s)can be found in few steps of search. Each solvent system gives proper volume ratio and high density difference between the two phases, and provides a satisfactory level of retention of the stationary phase in the spiral column assembly. The method is validated by partition coefficient measurement of four typical polar compounds including methyl green (basic dye), tartazine (sulfonic dye), tyrosine (twitter ion) and epinephrine (catecholamine), all of which show low partition coefficient values in the polar 1-butanol-water system. Recently, the above ultra polar solvent systems (Organic/high ionic strength aqueous solvent system) has been improved by increasing the solvent composition in 20 steps (1 to 10 and 11 to 20). Using this new system, a graphic determination of two-phase systems can be done in two steps, i.e., first the sample is partitioned in system 10. If the K value is over 1, the second K determination is performed at system 1 whereas if the K value in system 1 is below unity, the second K determination is performed at system 20. By drawing the line between these two points, the optimum K value of near unity can be easily obtained from the crossing point with the K = 1 line in each case. The method was successfully applied to separation of tartrazine (K=0.77), tryptophan (K=1.00), methyl green (K= 0.93), tyrosine (0.81), metanephrine (K=0.89), tyramine (K=0.98), and normetanephrine (K=0.96). Three sulfonic acid components in D&C Green No. 8 were separated by HSCCC using the graphic selection of the two-phase solvent system This new method was also applied for separation of ultra polar samples polar compounds including nucleobases, nucleosides and nucleotides."}
{"text": "All organisms, from bacteria to humans, need to recognize where they are, and respond accordingly. A pathogen must sense its location in the infectious cycle, then produce factors necessary for that site while repressing synthesis of inappropriate factors. Thus, disruption of key regulatory pathways can inhibit a pathogen's ability to infect, indicating useful targets for developing novel antibiotics. In our studies of th Lyme disease bacterium, Borrelia burgdorferi, we discovered that regulation of a new DNA/RNA-binding protein named BpuR is required for mammalian infection. B. burgdorferi regulates production of BpuR, synthesizing it at low levels during mammalian infection processes, and at high levels during colonization of vector ticks. We genetically engineered B. burgdorferi to constitutively produce high levels of BpuR during murine infection, and found the mutant to be significantly impaired in its ability to colonize mice. Transcriptomic analyses (RNA-Seq) demonstrated that BpuR significantly affects expression of approximately 5% of B. burgdorferi operons, disturbing cellular levels of several known virulence factors and metabolic enzymes. We hypothesize that disruption of the processes controlling production of BpuR will significantly inhibit B. burgdorferi infection. The planned studies are designed to elucidate the mechanism by which B. burgdorferi controls expression of this critical regulatory factor, and determine how disrupting that mechanism impacts upon infection processes."}
{"text": "1. X-linked retinoschisis (XLRS) gene therapy clinical trial. XLRS is a genetic disease caused by mutations in the retinoschisin (RS1) gene. An absent or mutated RS1 protein in the retina of patients with XLRS leads to abnormalities in the normal laminar structure of the retina, resulting in impaired visual acuity and increased propensity to retinal detachment. In collaboration with Dr. Paul Sieving's group, we have developed and optimized a self-complementary AAV vector that is capable of mediating stable and retinal specific expression of human RS1 protein. To meet the FDA requirements for gene therapy clinical trials, we have completed a preclinical efficacy study in a retinoschisin knockout mouse model, and a vector toxicology study in both mouse and rabbit models. The investigative new drug application has been approved by FDA, and a Phase I/II clinical trial has started. A total of nine patients with XLRS will receive (or have received) the vector administration in year 2015. 2. Preclinical gene therapy studies for retinitis pigmentosa due to RPGR or RP2 mutations. X-linked forms of retinitis pigmentosa (XLRP) are relatively severe blinding disorders, resulting from progressive photoreceptor dysfunction primarily caused by mutations in RPGR or RP2 gene. Gene therapy for RPGR-XLRP has been hampered by the relatively slow disease progression in mouse models and by difficulties in cloning the RPGR-ORF15 cDNA that includes a purine-rich 3-coding region. We managed to overcome these problems and have generated AAV vectors carrying full-length mouse and human RPGR ORF15 coding sequences. We have also developed a self-complementary AAV vector carrying human RP2 expression cassette. We have completed long-term (18-24 months) dose efficacy/toxicity studies in mouse models with RPGR or RP2 deficiency. Our results demonstrate that administration of the RPGR AAV vectors at appropriate doses can significantly preserve the retinal function and delay the photoreceptor loss in the mice with RPGR deficiency. Additionally, administration of the RP2 vector with a broad dose range can remarkable maintain the function and viability of cone photoreceptors in the mice with RP2 deficiency. In order to bring the therapies to the clinic, we are currently optimizing the vectors and testing them in mice with different background/mutations. 3. Gene therapy for Leber congenital amaurosis due to CEP290 mutations. Leber congenital amaurosis (LCA) is one of the most common causes of blindness in children. People with this disease typically have severe visual impairment beginning in infancy. Mutations in the CEP290 gene account for 20-25 percent of LCA, afflicting an estimated 20,000 people worldwide. Since the size of CEP290 coding sequence (7.4 kb) exceeds the packaging limit of AAV vector (5kb), we are seeking several approaches including the use of dual-vector and functional subunit of CEP290 to deliver the therapeutic genes into the retina. We have made a series of AAV vectors and have tested them on a mouse model with Cep290 mutation. We recently identified one vector that is capable of preserving the retinal function and structure in the mouse model. A detailed study is being conducted. 4. Application of CRISPR for treating retinal degenerative diseases. We have recently initiated a new project involving the use of CRISPR technology to knockdown dominant gene mutations in the retina. We are hoping that we can develop the CRISPR technique into a treatment for autosomal dominant retinal diseases."}
{"text": "Trafficking of mRNAs from the nucleus to the cytoplasm is essential for gene expression and is highly regulated by pathogens and signaling pathways. To exit the nucleus, most mRNAs interact with the receptors NXF1-p15 (TAP-p15) via adaptor proteins, such as E1B-AP5. Another mRNA export factor is Rae1, which aids in docking mRNAs onto nuclear pore complex proteins, such as the nucleoporin Nup98. The mRNA export complex is then translocated through the nuclear pore complex to the cytoplasm. We have reported the interaction of viral proteins with constituents of the mRNA export pathway, which induced inhibition of Mrna export and provided insights on regulatory mechanisms of mRNA export. We showed that the vesicular stomatitis virus (VSV) matrix (M) protein binds Rae1 and that the nonstructural protein 1 (NS1) of influenza virus interacts with NXF1-p15, Rae1, and E1B-1AP5. NS1 is a major virulence factor of influenza A virus that is essential for pathogenesis. These viral-host interactions inhibit expression of antiviral proteins. However, Nup98 and Rae1 are up-regulated by interferons (IFN), which constitute a mechanism of antiviral response that can revert the mRNA export block mediated by these viral proteins. Another nucleoporin involved in antiviral response is Nup96, which is regulated by IFN and in turn preferentially facilitates expression of IFN-regulated mRNAs. Using viral proteins as tools, we propose to uncover novel molecular mechanisms of mRNA export. Our specific aims are: 1. To investigate the mechanisms through which viral proteins disrupt the mRNA export machinery. We have evidence that NS1 and VSV M proteins interact with different forms of the mRNA export complex. We have also identified novel constituents of the complex. Biochemical approaches will be used to assemble these mRNA export complexes in vitro. Functional studies will be carried out using knockdown, overexpression, and mutagenesis strategies in combination with mRNA export assays. 2. To study regulation of mRNA export by antagonists of viral-mediated mRNA export block. We have identified novel chemical inhibitors of the NS1 protein of influenza virus, some of which target constituents of the mRNA export machinery. Biochemical analyses of interactions between these inhibitors and the mRNA export machinery will be performed to investigate key regulatory mechanisms. Functional assays to assess the effect of these inhibitors on mRNA export will also be performed in vitro and in vivo. 3. To determine the role of mRNA export in antiviral response. We have reported that Nup96 is up-regulated by IFN and is involved in antiviral response to facilitate mRNA export of IFN-regulated mRNAs. Nup96 interacts with Sec13 and Seh1. To study the relationship between these Nups in nuclear transport and Nup96-mediated regulation of mRNA export and IFN response, we have generated novel hypomorphic mice and cells that allow gradual down-regulation of Sec13 and Seh1 alone or in combination with Nup96. Altogether, these studies will generate new information on basic mechanisms of mRNA export and on how viruses and host regulate this machinery to their own advantage. PUBLIC HEALTH RELEVANCE: Trafficking of molecules between the two major compartments in the cell, the nucleus and the cytoplasm, involve processes that are targeted by viruses, as they are important for antiviral defense. Our laboratory studies the mechanisms and key players of these trafficking processes. By understanding these mechanisms, we were able to design inhibitors or drugs that work against viral toxicity."}
{"text": "Breast cancer susceptibility gene, BRCA1, has been implicated in the maintenance of genomic integrity. However, the molecular mechanism(s) by which BRCA1 plays a role in maintenance of genomic fidelity remains to be defined. Interestingly, recent studies in our group and others have demonstrated that BRCA1 can regulate the GADD45, a p53-regulated stress inducible gene that plays an important role in cellular response to DNA damage. These results have evidently linked GADD45 to BRCA1 and raised the possibility that GADD45 might be a BRCA1-downstream effector and mediate BRCA1's role in maintenance of genomic stability. Therefore, this proposal seeks to define the biochemical mechanism(s) by which BRCA1 transactivates the GADD45 gene, and to define the role of the BRCA1-GADD45 pathway in the induction of apoptosis following genotoxic stress. The long-term objective described in this application will specifically focus on three key issues: (1). To characterize the GADD45 gene as a BRCA1's downstream effector. We will define the BRCA1-regulatory elements in the GADD45 promoter and identify the proteins that modulate the BRCA1 transactivation of the GADD45 promoter. (2). To define whether the GADD45 is an essential player in the BRCA1-induced apoptosis. We will analyze the induction of apoptosis following expression of GADD45 and BRCA1. We will also examine the alterations of the BRCA1-activated apoptosis and BRCA1-induced growth suppression in GADD45- deficient cells. (3). Our most recent studies demonstrated that BRCA1 is cleaved by caspase-3 during apoptosis induced by DNA damage. This DNA damage-activated cleavage results in an accumulated 90-kDa band of the BRCA1 C-terminus. Therefore, we will first determine whether the BRCA1 cleavage is required for BRCA1-activated apoptosis following DNA damaging agents. We will also determine the functional role of the cleaved 90-kDa band of the BRCA1 C-terminus in activation of apoptosis. Finally, we will analyze the role of this cleaved product in the BRCA1 transactivation of the GADD45 promoter. The studies proposed in this application would define a novel pathway (BRCA1-GADD45) controlling apoptosis following DNA damage and provide information regarding the biochemical mechanism by which BRCA1 regulates its targeted genes. Since apoptosis is closely associated with the therapeutic sensitivity, the perspective outcome will also provide insight into the development of therapeutic agents."}
{"text": "In the United States, human papillomaviruses (HPVs) cause an estimated 6 million new infections annually. Some HPVs cause the most prevalent benign viral sexually transmitted hyperproliferative disease, genital warts, and are referred to as low risk (LR) HPVs (e.g., HPV 6). Others cause cervical cancer and some head and neck cancers and are, therefore, referred to as high risk (HR) HPVs (e.g., HPV 16). All HPVs are maintained as low copy number extrachromosomal genomes in the undifferentiated layer and amplify their genomes and complete their life cycles in the differentiated compartment of the epithelium where cells normally have exited the cell cycle. All HPVs have to create an S-phase environment in this latter compartment to complete their life cycle. While the HR HPVs have been extensively studied because of their ability to cause human cancer, it is imperative that we understand the mechanism of pathogenicity of the LR HPVs, clinically significant pathogens in their own right. We have been focusing on the E7 proteins encoded by LR and HR HPVs because of their pivotal role in both the life cycle and oncogenesis. This application builds on our novel observation that both LR and HR HPVs can target the pRb family member, p130/pRB2, for degradation while only HR HPVs can target other members of the pRb family. Previously, it was thought that only HR HPV E7 could target proteins for degradation. Building on our paradigm-shifting observation, the Specific Aims of this exploratory R21 application are: 1) to test the hypothesis that LR and HR HPV E7 target other (non-pRb family member) proteins for degradation and 2) to test the hypothesis that LR HPV E7 binds members of the ubiquitin ligase complex. We will use mass spectrometry to detect endogenous proteins that are differentially ubiquitylated in the presence of LR HPV 6 and/or HR HPV 16 E7. We will identify proteins that form a complex with LR HPV 6 E7 by tandem affinity purification coupled with LC MS/MS. Results will be appropriately validated. By generating the first global comparison of the substrates ubiquitylated in response to LR and/or HR E7 expression, a profile of proteins that contribute to the viral life cycle versus those involved in oncogenesis will be developed. The analysis of HPV 6 E7 binding partners, by comparison to HPV 16 E7 binding partners, will identify ubiquitin ligases that potentially mediate LR HPV 6 E7 and/or HR HPV 16 E7 targeted degradation, providing possible novel therapeutic targets for directed inhibition of such degradation. By identifying shared E7 activities in Specific Aims 1 and 2 we will gain new insight into potential biomarkers of and therapeutic targets pertinent to the life cycle of all HPVs. Subtracting out these shared activities will identify biomarkers and therapeutic targets specific to the oncogenic activity of the HR HPVs. The feasibility of these experiments is high based on data in the literature and the expertise of the two laboratories involved in their execution. PUBLIC HEALTH RELEVANCE: Human papillomaviruses, as causative agents of cervical cancer and some head and neck cancers, have a major public health impact. Although the FDA-approved HPV vaccine holds promise of preventing infection, it cannot cure existing infections. This application offers the potential of identifying new biomarkers for diagnosis and monitoring of disease status as well as potential therapeutic targets, and thereby the translation of the results from the bench to the bedside. [unreadable] [unreadable] [unreadable]"}
{"text": "Hemagglutinin molecule is a viral attachment protein. Another antigenic protein, the neuraminidase, is also on the surface of the virus particle, in conjunction with the hemagglutinin. The examination of influenza virus with high-resolution cryo-SEM would be extremely useful to reveal the conformational change of hemagglutinin."}
{"text": "Compaction of the myelin membrane into lamellae is essential for its function as an insulator in axons of the peripheral nervous system (PNS). P(O), the most abundant glycoprotein in the PNS, is believed to hold these lamellae together. The long term goals of this study are to clarify in molecular terms the role of P(O) (as well as other proteins) in myelination in the hope of contributing to our understanding of the mechanisms of demyelinating diseases such as Guillain-Barre syndrome (GBS) and in neuronal regeneration in the PNS. The extracellular domains of P(O) undergo hemophilic P(O)P(O) interactions in which the carbohydrate moieties play a major role. Heterophilic interactions of P(O) with neurons are believed to promote neurite outgrowth. The overall goals of this proposal are to determine what roles various regions of P(O) play in the synthesis and compaction of myelin. Our adhesion assay specifically developed to identify extracellular regions of P(O) participating in interactions will be used to pursue the following aims. First, to determine by mutation whether a 5-amino acid sequenced in immunoglobulin-like domain of P(O) is crucial to adhesion. This sequence is also found in a flu virus vaccine which elicited GBS, therefore the results may also bear on the mechanisms of demyelination in this disease. Second, to clarify the role of the intracellular domain of P(O) in the adhesive properties of its extracellular domain and in the sorting of P(O) to myelin. Sorting will be assessed by expressing shark P(O) in rat Schwann cells, allowing myelination to occur, and determining by means of species-specific antibodies whether truncated forms of P(O) reach myelin. Third, to elucidate the interactions of P(O) with myelin associated glycoprotein, possibly in the initial stages of myelination, by mixed adhesion assays and by determining the adhesive properties of cells co-expressing both proteins. Fourth, to map the regions of P(O) that may be involved in heterophilic interactions with neurons by comparing the ability of cells expressing unaltered and mutated P(O) to promote neurite outgrowth. Accomplishment of these aims should substantially advance the long term understanding of the mechanism of myelin organization."}
{"text": "Objectives: The Developmental Core will play a prominent role in the revitalization of CSDA as it reorganizes in line with current budget realities. Though important to all CSDA Associates and Affiliates, the core will be instrumental in seeking to identify and incorporate promising young population researchers as they join the faculty."}
{"text": "The Cancer Control & Population Sciences Program (CCPS) organizes all of the cancer prevention-related research activities of the Winship Cancer Institute (WCI), which span the cancer prevention continuum from primary to secondary to tertiary prevention at the individual, select population, and societal levels. The overall goals of the Program are to reduce cancer risk, incidence, morbidity, and mortality in Georgia through describing and tracking cancer incidence and mortality trends of all cancers; conducting etiologic research in humans; conducting chemoprevention trials in Georgia and in national collaborations; applying theories of human behavior to prevent cancer through early detection; conducting multi-disciplinary research that describes, interprets, and predicts the impact of interventions and other factors on cancer outcomes important to decision makers; characterizing the scope of behavioral co-morbidities that occur in patients with cancer; and understanding relevant mechanisms and treatments of these behavioral co-morbidities. To promote the development of the different areas of research associated with cancer control and population sciences across the entire prevention continuum, and to do so in a coordinated manner, the program is organized into four general research areas, each headed by a senior investigator: 1) Epidemiology, Biomarkers & Chemoprevention (EBC); 2) Health Behavior Research (HBR); 3) Outcomes & Quality (OQ); and 4) Symptom Management & Control (SMC). The CCPS Program includes members from 17 departments across all three schools in the health sciences at Emory UniversityPublic Health, Nursing, and Medicineand the Emory College. Beginning with few members and little funding at the start of the P20, through recruitment and internal development, the Program now has 62 members, of whom 41 are Core, eight Research, eight Clinical, and five Coalition, and a portfolio of 52 NIH and other national, peerreviewed, cancer-related grants headed by 25 Core members, with a combined funding for this year of $9,931,319 in Program direct costs (out of a total of 84 externally-funded cancer research grants with $12,185,701 in Program total costs). The number of peer-reviewed cancer publications by members of the Program during the last grant period was 455 of which 22% were intra-programmatic and 7.5% were interprogrammaticproportions that are rapidly escalating in this young program."}
{"text": "The use of thermometric methods for analyzing serum components is being investigated. Currently three activities are in progress. First, the study of enzyme calorimetry, including creatinine phosphokinase and various transaminases. Second, the use of enzyme inhibition to monitor low levels of heavy metal ions and third, the development of a flow calorimeter for rapid analysis."}
{"text": "The Hematopoietic Stem Cell Facility is part of the NCI Case Western Reserve University Ireland Cancer Center. This protocol is established for obtaining blood and bone marrow samples from normal donors and patients under a wide variety of research programs at CWRU including, for the most part, the CWRU Cancer Center members and their related protocols. The bone marrow aspirations done on normal donors are conducted on the GCRC by the CAP. This facility was just refunded by the NCI."}
{"text": "[unreadable] [unreadable] Epilepsy affects over 2.5 million people in the US, and over 180,000 new cases of the disease are diagnosed each year. Nearly half of the people suffering from epilepsy are not effectively treated. Moreover, currently used anticonvulsants can cause significant side effects, which often interfere with compliance. Clearly, there is a need for new, safer drugs to treat epilepsy. Glutamate, by stimulating NMDA receptors, has been implicated in the neuropathology and clinical symptoms of epilepsy, and NMDA receptor antagonists are potent anticonvulsants. Antagonists at the GlyB co-agonist site inhibit NMDA receptor function and are also anticonvulsant. Importantly, GlyB antagonists have fewer side effects than classic NMDA receptor antagonists and other antiepileptic agents, making them a safer alternative to available anticonvulsant medications. 7-Chlorokynurenic acid (7-C1-KYNA) is one of the most potent and specific GlyB antagonists currently known and is a powerful anticonvulsant when injected into the brain. However, like almost all GlyB antagonists developed so far, 7-C1-KYNA crosses the blood-brain barrier very poorly and is therefore ineffective following peripheral administration. Its pro-drug, L-4-chlorokynurenine (4-C1-KYN), on the other hand, readily gains access to the brain. Following systemic administration, 4-C1-KYN is efficiently converted to 7-C1-KYNA and prevents seizures in animal models of epilepsy. 7-C1-KYNA formation occurs preferentially in brain areas that suffer seizure-related brain injury, thereby reducing the risk for side effects with chronic use. Furthermore, 4-C1-KYN forms a second metabolite, which blocks the synthesis of the proconvulsant quinolinic acid in brain. These unique properties make 4-C1-KYN a highly innovative candidate for the treatment of epilepsy. The project proposed here by Vistagen will advance the preclinical development of 4-C1-KYN as a treatment for adult and childhood epilepsy. We will conduct extensive pharmacokinetic and ADME analyses to further delineate the oral bioavailability of the drug observed in pilot studies, will subject 4-C1-KYN to a battery of safety and toxicology studies and will test anticonvulsant efficacy of the compound in two chronic animal models of epilepsy, CLE and kindling. Finally, we will begin to develop the protocols needed to submit an IND for FDA approval to conduct clinical testing of the drug in humans as a new and improved anti-epileptic agent. [unreadable] [unreadable] [unreadable]"}
{"text": "Abstract for Coordinating Center Core (CCC) The UPCI-CMCR-CCC will provide three critical services to NIAID and the other components of the 5 ? 7 CMCR Programs. The CCC will cooperatively develop a program for archiving and distributing data from 5 ? 7 CMCR sites and will maintain electronic data storage and develop security passcodes and validations for retrieval of data. The CCC will develop a program for timely submission of data and data analysis at NIAID/NIH and maintain rapid availability of electronic exchange of records to CMCR Programs as well as the general scientific community. The CCC will coordinate administrative activities for the scientists organizing national meetings, coordinating conference calls, establishing three levels of firewalls to distribute information at levels of CMCR Centers locally, CMCR Programmatic Centers for all 5 ? 7 programs, and to the general scientific community. Finally, the CCC will administer funds for the development of web-based training programs in four key areas: 1) Principles of Radiobiology, 2) Use of assays, methods, reagents, and animal models, 3) Technologies to study radiobiology and develop new product regulatory process, and, finally, 4) A course on triaging radiation casualties in the event of a radiation terrorist event."}
{"text": "Diadenosine tetraphosphate (AP4A), two adenosine moieties bridged by 4 phosphates, is an endogenous purinergic ligand found in brain. Previous studies have shown that AP4A reduced neurodegeneration caused by dopaminergic neurotoxin 6-hydroxydopamine in rat striatum and substantia nigra. The purpose of this study was to determine whether AP4A is protective against methamphetamine (MA) mediated toxicity in dopaminergic neurons. Primary neuronal cultures were prepared from rat embryonic (E15) ventral mesencephalic tissue. On DIV8, cultures were treated with 2 mM MA for 48 hours. Application of MA increased LDH levels, decreased TH immunoreactivity, and increased TUNEL. All these changes were reduced by pretreatment with AP4A. The protective effect of AP4A was further examined in vivo. Adult Sprague Dawley rats were injected with AP4A or vehicle intracerebroventricularly followed by 4 doses of MA (5mg/kg), given subcutaneously every two hours. Administration of MA increased caspase-3 immunoreactivity in striatum and cortex. Pretreatment with AP4A significantly reduced the density of caspase-3 cells. Using microdialysis, dopamine (DA) release was monitored in dorsal striatum in freely moving rats. AP4A did not acutely alter MA-evoked DA release, suggesting that AP4A -induced protection is not directly mediated through a change in DA overflow. Taken together, these data show that AP4A has protective effects against MA-mediated neuronal injury both in vitro and in vivo. The mechanism of action may involve suppression of MA -induced apoptosis.[unreadable] [unreadable] Astaxanthin (ATX) is a carotenoid antioxidant found in crustaceans. Other carotenoids, such as beta-carotene, lutein and lycopene have been shown to have beneficial effects against oxidative stress. The purpose of this study was to examine whether ATX has protective effects against ischemic brain injury. Adult male Sprague-Dawley rats were anesthetized with chloral hydrate. ATX or vehicle was administered intracerebroventricularly 10-20 minutes prior to a 60-min middle cerebral artery ligation. At 2 days after MCA occlusion (MCAo), rats that received ATX had an increase in locomotor activity. ATX significantly reduced cerebral infarction and TUNEL labeling in ischemic brain. At 8-hour after MCAo, ATX antagonized ischemia/reperfusion induced loss of aconitase activity, an indirect marker for the production of reactive oxygen species, and translocation of cytochrome C from mitochondria. Ischemia or free radical -induced glutamate release was examined using microdialysis and HPLC. Pretreatment with ATX reduced ischemia or H2O2 mediated glutamate release in cerebral cortex. ATX did not alter blood gases, blood pH, blood pressure, body temperature, brain temperature and cerebral blood flow. Collectively, our data suggest that astaxanthin has protective effects against free radical toxicity and ischemia-related injury in vivo through the inhibition of oxidative stress, inhibition of glutamate release, and anti-apoptosis."}
{"text": "It is proposed to study the binding affinities and behavioral effects of several classes of hallucinogenic (psychotomimetic) agents, i.e. tryptamines, phenethylamines, phenylisopropylamines. The serotonin receptor binding affinities will be determined using the isolated rat fundus preparation, as we have previously described. The behavioral studies will be performed by investigating the discriminative stimulus properties of these agents, and their ability to generalize with a 5-methoxy-N,N-dimethyltryptamine (5-OMe DMT) stimulus. In this manner, information can be obtained regarding mechanism of action, structure-activity relationships, structural requirements of the serotonin receptor, etc... Agents which produce similar interoceptive cues (in the generalization studies) and which possess similar binding affinity characteristics will lend support to the hypothesis that certain of these agents may produce their effects via a common mechanism. Using rats, cannulae will be implanted in various brain regions so that 5-OMe DMT can be administered directly into the brain. Employing the discriminative stimulus paradigm, generalization between centrally and peripherally administered 5-OMe DMT will yield information on the brain site of action. Dopaminergic, as well as serotonergic sites will be investigated. Bufotenine esters and other novel compounds which possess, within the same molecule, features related to more than one class of hallucinogens, are proposed for synthesis. The behavioral effects and receptor affinities of such compounds should aid in determining whether various hallucinogens act via a common mechanism."}
{"text": "Abstract Hedgehog signals are the key regulators of embryonic patterning and adult tissue homeostasis. Consequently, defects in Hedgehog signaling can cause developmental diseases, congenital heart disease, and cancers such as basal cell carcinoma. There is an urgent need to understand the molecular mechanisms that underlie the modulation of Hedgehog signaling pathway for its potential preventative and therapeutic value. It is known ver- tebrate Hedgehog signaling relies on the ciliary trafficking of Hedgehog signaling receptors, among which smoothened (SMO) is the central positive mediator of Hedgehog signaling. If mutations occur in either intrafla- gellar transporters (IFTs), or in the ciliary transition zone, SMO activities can be severely disrupted. However, the molecular mechanism of how IFT particles and transition zone regulate trafficking of SMO is currently un- known. Determination of the molecular regulation mechanisms is the objective of this application. Our prelimi- nary data acquired by Stochastic Optical Reconstruction Microscopy (STORM) showed the colocalization of transition zone proteins with SMO and IFT88, suggesting that transition zone proteins and IFT particles interact with SMO. Based on previous studies and our own primary data, my central hypothesis is that the transition zone serves as a checkpoint for Hedgehog signaling receptors, and IFTs help Hedgehog signaling receptors cross the transition zone. This transition zone checkpoint model represents a novel mechanism for the control of cilium trafficking and the cross-interaction between different ciliary cargos. It could potentially allow new ap- proaches to manipulate Hedgehog signals, and underlie the foundation for treatments of diseases caused by defects in Hedgehog signaling. To approach to the project, I plan to map SMO molecules and IFT particles in the transition zone using multicolor 3D STORM. It will reveal the spatial relationship between SMO molecules and these ciliary components at a resolution of ~15 nm. Algorithms will be developed to reduce the uncertainty of the spatial easements caused by structural heterogeneity and immunostaining, providing a ~ 5nm precision of the distance between investigated proteins, indicating protein-protein interaction. Equally important as the static structural study, I also plan to detect the interactions among SMO molecules, IFT particles, and transition zone proteins dynamically using single-particle tracking and photoconversion imaging. The proposed project will not only offer new insights into the molecular mechanisms of Hedgehog signaling regulation, but also ad- vance a suite of microscopy-based technologies and algorithms that can be broadly applied to the fields of cell signaling and structural biology. Furthermore, the results are expected to have broad impact, because the reg- ulatory components to be identified by this project will provide new mechanisms and new drug screen for pre- ventive and therapeutic interventions. 1"}
{"text": "This proposal addresses a fundamental topic relative to Complementary and Alternative Medicine (CAM) treatments: How do we measure the context of an encounter? Encounters are defined as what patients experience between when they arrive at a CAM practice site and when they leave. The encounter can be divided into two parts: (a) the experience of the main treatment intervention (the intervention) and (b) all other experiences before, during, and after the intervention itself (the context). Contextual effects play two critical roles in assessing the efficacy and effectiveness of CAM interventions. First, contextual effects may mediate how well a treatment works (context as mediator). Second, context effects may actually contribute to outcomes directly (context-as-intervention). In a classic RCT, investigators typically want to control for context-as-mediator effects and measure context-as-intervention effects to disentangle what portion of the results are due to the intervention and what are due to the context. In CER, investigators are less concerned about controlling for context-as-mediator effects, but they would like to understand what part of the encounter accounts for any positive results. In either case, investigators MUST know how to measure the context. Without such measures, it is impossible to assess either type of context effect. This proposal has 3 specific aims: 1. to understand what kinds of contextual factors patients are exposed to during CAM encounters? 2. To determine how to measure such contextual factors reliably via observation and/or patient and provider recall; and 3. To assess the degree to which contextual factors might vary within and across (a) CAM modalities (i.e., chiropractic versus osteopath); (b) practice sites, (c) providers, and (d) individual patient encounters This study will use the chiropractic encounter as an exemplar for CAM and develop a rapid ethnographic observation method to study the context of the encounter. The objective of this project is to develop a reliable, valid, and rigorous methodology for collecting data about the context. The proposed study addresses a fundamental issue of all outcome studies in health: how to measure the effect of the context on the health outcomes."}
{"text": "Activated TGF-beta family factors signal via dual Type II (TbetaRII) and dual Type I (TbetaRI/Activin-like kinase 5/ALK5) transmembrane serine/threonine kinases receptors and effector Smad transcription factors. While TGF-beta signaling is critical during endocrine pancreas development, whether TGF-beta plays a role in maintaining adult beta-cell function is unclear. Interestingly, TGF-beta levels are elevated in diabetes, diabetes-associated complications, and obesity. [unreadable] [unreadable] Using primary islets and established beta-cell lines, we are examining the role of various TGF-beta isoforms and their respective receptors on the regulation of key pancreatic specific genes. Smad proteins are the established intracellular effectors of TGF-beta signaling and upon TGF-beta ligand binding, the activated TbetaRI/ALK-5 receptors recruit and phosphorylate receptor-Smads, i.e., Smad2 and Smad3. We are examining the roles of various Smad proteins, in the regulation of gene expression specific to the pancreas. [unreadable] [unreadable] TGF-beta signalling is known to suppress proliferation and promote growth arrest, differentiation/apoptosis of cells of the epithelial lineage. However, the role of this pathway in the growth, proliferation, differentiation and death of constituent pancreatic epithelial cells is unknowm. Using primary islets and established pancreatic ductal and beta cell lines, we are examining the role of TGF-beta signaling in these processes. [unreadable] [unreadable] Of particular interest will be the contribution of TGF-beta signaling in regulation of beta cell mass amd beta cell function. We are using primary islets, and established beta cell lines to examine the effects of TGF-beta isoforms, its receptor activation/inactivation, on beta cell mass and beta cell fucntion. In addition the role of downstream Smad proteins is also being investigated. Several parallel approaches will be taken to explore this issue, which will hopefully lead to an improved understanding of the role of this complex signaling pathway in pancreatic development and specifically in regulation of beta cell mass and function. The information gained will be useful to delineate the role of this pathway in diseases of the pancreas, primarily diabetes, and hold promise to allow design and development of novel rational therapeutics for these diseases."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The purpose of this study is to understand the effects of body fat, particularly fat locked within the abdomen, on metabolism. Patients who are scheduled to undergo surgery, will be consented to donate a sample of their abdominal fat (omental adipose tissue), a small amount of abdominal wall muscle, and some of the fluid in their belly. We are interested in ways in which abdominal fat can mimic the immune system in causing inflammation."}
{"text": "The objective of the proposed Drug Abuse Research Center is to determine the extent and magnitude of the effects of narcotics on brain biochemistry and ultrastructure during the development of tolerance and physical dependence. The specific goal of the proposed research is to lay a foundation of basic information on the neurochemical and morphological effects of the narcotics from which studies of the actual \"mechanisms\" involved in tolerance and dependence can logically progress. We shall use a variety of micro-analytic chemical and immunochemical techniques, radioassays and quantitative-histochemistry to determine the effects of narcotics on: 1) The biogenic amines; 2) Brain protein synthesis; 3) Brain metabolism; 4) Representative cerebral enzymes; 5) Brain morphology and ultrastructure; and 6) Additional studies will be conducted to examine the neuroanatomical correlates of tolerance to and dependence on narcotics. Our approach will be highly integrated and interdisciplinary and we shall correlate, whenever possible, pharmacologic, biochemical, and morphological observations within a given region of brain during various phases of morphine treatment and subsequent withdrawal. All of these determinations will be made in histologically well-defined regions of brain, including the major nuclei of the hypothalamus, thalamus, caudate, midbrain, medulla, cerebellum and cerebral cortex."}
{"text": "PROJECT SUMMARY/ABSTRACT The cerebral cortex mediates all of human and animal cognition, encompassing a diverse set of abilities including sensation, perception, decision making, and motor planning. Dysfunctions of the cerebral cortex are thought to underlie numerous neurological and psychiatric disorders. A major obstacle both to understanding normal behaving and to treating pathology is the high degree of complexity of cortical circuitry, which has remained largely enigmatic. The conventional view of neocortex has been that sensory processing begins in layer 4 (L4), which was identified a century ago as the principal target of thalamic axons carrying information from our sensory organs. Sensory transforms are widely believed to occur as excitation spreads serially along the densest axonal pathways (thalamus?L4?L2/3?L5/6). Recently we discovered that the cerebral cortex, rather than being a monolithic structure, may contain two entirely separate processing systems, activated by the same signals arising from the thalamus. L4 is thus not an obligatory distribution hub for cortical activity, and thalamus activates two distinct ?strata? of cortex in parallel. This proposal's goal is to identify the behavioral and computational roles of the upper (L2-4) and lower strata (L5/6) as well as the interactions between them. We will investigate the behavioral roles of these layers in the mouse whisker system. Specific layers will be optogenetically disrupted in a series of tactile behavioral tasks, in which task complexity is progressively increased. Interlaminar interactions will also be studied by recording electrophysiologically from specific layers during behavior and using novel machine learning techniques designed to identify the type of computation performed in different levels of ?deep networks?. The dimensionality of the representation in a layer will be estimated under normal behaviors and when specific layers are inactivated. Identifying fundamental functions of upper versus cortical layers will likely pave the way for future studies in other neocortical systems and in higher-order species. Moreover, as the different layers contain molecularly and biophysically distinct cell types and project to distinct downstream targets, specific neurological disorders may involve dysfunction of specific pathways, cell types, and layers. Establishing the behavioral and computational roles of these elements may contribute to development of targeted therapies."}
{"text": "This proposal describes an investigation of the effects of aluminum and other trivalent cations on cholinergic activity and other metabolic pathways in rat brain. These findings are crucial because aluminum toxicity has been proposed to play a major role in the development of senile dementia of the Alzheimer type (SDAT) and selective impairmant of the cholinergic system has been established in patients with SDAT. Both in vitro and in vivo effects of aluminum will be sought. Brain slices will be incubated with (U-14C) glucose to test the effects of aluminum on carbohydrate utilization, lipid synthesis and acetylcholine (ACh) synthesis. Synaptosomes will be incubated with deuterium labelled choline to test the effects of aluminum on high affinity choline transport, ACh synthesis, spontaneous and K+ induced ACh release and the release of choline from lipids. (3H)-Choline will be used to measure the rate of incorporation of choline into lipids in synaptosomes in the presence and absence of aluminun. The effects of several other trivalent cations on these processes will be compared to those of aluminum to establish their relative potencies and to obtain information about the mechanism of the toxic effects of aluminum. Rats will be treated in vivo with aluminum followed by determination of the rate of turnover of ACh in rat brain regions using pulse labelling techniques with deuteriumzlabelled choline. Endogenous and deuterium labelled choline and ACh will be measured using combined gas chromatography mass spectrometry. In other rats treated with aluminum we will (i) measure the activity of ChAT, (ii) prepare synaptosomes and compare the rates of Ch transport and ACh synthesis and release to those observed in synaptosomes from control rats, and (iii) measure the rate of utilization of (U-14C) glucose in brain slices. This investigation will establish whether or not aluminum causes impairment of cholinergic activity or several other metabolic processes, will indicate how specific for aluminum these toxic effects are and will allow a comparison of the biochemical effects of aluminum toxicity and SDAT."}
{"text": "This research proposal tests the hypothesis that identification of the requirements for in situ activation of tumor-infiltrating lymphocytes (TIL) will provide the biological basis for immunotherapy of human solid tumors without infusion of ex vivo- activated lymphocytes. Specific aims are as follows: 1. Characterization of TIL from melanoma, renal carcinoma and sarcoma. There are at least six different TIL subsets as follows: CD4-CD8+ T cells with (i) autologous tumor-specific or (ii) MHC- nonrestricted (LAK) cytotoxicity; CD4+CD8- T cells with (iii) autologous tumor-specific The activity or (iv) LAK activity, (v) CD3-CD16+ NK cells with LAK activity and (vi) CD4-CD8- T cells. We will (i) purify each of these subsets before and after incubation with rIL2; (ii) establish cloned cells; (iii) investigate their functions; (iv) elucidate requirements for activation of each TIL subset (IL-2,other cytokines, autologous tumor cells or autologous macrophages), and (v) investigate molecules involved in the TIL activation (TCR, CD3, CD2, CD4, CD8, CD16 and LFA antigens on TIL). 2. MHC-restriction of antitumor activity. We will investigate if autologous tumor-specific CTL or The activity in melanoma TIL is restricted by MHC-Class I or Class II antigen expression on tumor targets respectively by determining MHC-haplotypes of tumors. 3 and 4. Determination of requirements for CD4 CD8- TIL to produce IL-2, and differentiation of resting blood lymphocytes into TIL. Role of macrophages. Requirements tested include: IL-l, IL-2, IL2R-inducing factor, anti-CD3 mAb., hybridomas expressing anti-CD3 mAb, autologous monocytes, tumor cells, CD4+CD8- or CD4-CD8+ cloned TIL. 5. Antitumor activity of lymphocytes from LN with melanoma metastasis. LN-lymphocyte subsets will be incubated in the presence of IL-2, anti-CD3 mAb, IL-l and/or autologous monocytes, followed by testing their proli- feration and antitumor activity. 6. Identification and purification of IL-2 receptor- inducing factor (IL2R-IF). Culture supernatants from the established CD4+ TIL line (TILh-Ll) and PBMC stimulated with PHA and PMA will be used for this study. Knowledge of immunological properties of TIL and understanding the mechanisms involved in TIL activation and the biology of host-tumor relationships will provide important findings for development of in situ lymphocyte activation and new strategies in the immunotherapy of human solid cancers."}
{"text": "Vibrational Optical Activity studies consisting of vibrational circular dichroism (VCD) and Raman circular intensity differential (CID) will be applied to several biochemical systems. These techniques are exceptionally sensitive to structural features of the molecules being studied and offer distinct advantages over currently used spectroscopic techniques. The data obtained will be used to give new insight into the secondary structure of polypeptides and nucleic acids, into the conformations of cyclic peptides, and into the stereochemistry of phosphorylation reactions. In particular, the sensitivity of VCD to coupling of strong electric dipole moment transitions will be exploited to study amide linkages in poly- and cyclic peptides, base stacking arrangements in oligo- and poly-nucleotides, drug induced conformation changes in poly-nucleotides, phosphate coupling in DNA and RNA, and deoxy-ribose ring conformations. These interactions and their resultant VCD are straight-forward to model with the coupled oscillator and fixed partial charge theories that we have previously tested on small molecules."}
{"text": "Hypothetical examples of dental clinical trial data were developed to demonstrate the utility of a statistical technique of blocking on baseline DMFS. In the six trials studied, crude means ranged from one extreme of highly significant caries reduction in the fluoride group for one trial to the opposite extreme of highly significant caries reduction in the control group for another trial. Blocking on baseline DMFS, however, revealed that the trials were essentially identical."}
{"text": "PROJECT SUMMARY Direct cell reprogramming has the potential to facilitate the development of safer and more effective patient- specific cell-based therapies. However, current methodologies that induce direct reprogramming face major translational hurdles, including heavy reliance on viral transfection. While the results are promising, biosafety concerns, capsid size constraints and/or the stochastic nature of conventional methods (viral and non-viral) pose significant limitations. We developed and novel 3D nanochannel electroporation (3D NEP) platform technology that overcomes these barriers by enabling deterministic transduction of reprogramming factors with single-cell resolution, and without the need for viral vectors. This nanotechnology-based approach promotes remarkably fast and efficient direct cellular reprogramming, as demonstrated with well-established and newly-developed models of induced neurons and endothelium, respectively. Non-viral direct derivation of induced endothelial cells, in particular, could find applications in the treatment of a number of disorders, including critical limb ischemia and stroke. Ischemic strokes, for example, result in significant cellular deficiencies (e.g., vascular, neuronal) that could lead to death or major morbidity. Nevertheless, currently no study has looked into developing methods for virus-free direct reprograming of endothelial cells. Moreover, the regenerative potential of these cells has not been investigated within the context of stroke. Here we are proposing to develop an optimized method for direct derivation of endothelial cells by 3D NEP, and to study the extent to which these cells induce functional recovery in a middle cerebral artery occlusion (MCAO) mouse stroke model. In the US, strokes occur every ~40 s and have a death rate of ~42%. With an estimated cost of >70 billion dollars, strokes represent a substantial burden to the health care system. 1"}
{"text": "The proposed NRSA Short-Term Training Grant at the University of Michigan (UM) builds upon a strong foundation and its long-standing commitment to support medical student research in aging. The UM has served as a training site for 90 medical students who participated in its Hartford Foundation supported program since 1989. Its research and training environment provides an ideal institutional setting to support medical student research in aging. A partnership with Wayne State University Medical School and Institute of Gerontology will expand opportunities for medical student and faculty participation. Fifty-five qualified faculty mentors with active aging-related research programs have been identified as potential mentors for medical student trainees. The overall goal of the proposed program is to provide quality experiences in aging-related research for up to 18 medical students each year. The program's specific objectives are: 1. To provide medical students exposure to and participation in aging-related research; 2. To give students the opportunity to learn research concepts and methodology from experienced investigators, and assess whether they wish to pursue careers in aging-related research; 3. To expand the scholarly and research experiences of medical students as part of an overall career development strategy aimed to increase the number choosing aging-related research careers in basic science, health services or clinical research; 4. To increase the number of underrepresented minority medical students who participate in a research experience, and ultimately choose careers as physician scientists; 5. To promote research experiences that can develop into longer-term student-faculty mentoring relationships; 6. To provide trainees with experience in scientific presentation and publication of research; 7. To provide students with a high quality didactic program in research with an emphasis on unique aspects of aging-related research and on the ethical and responsible conduct of research; and, 8. To provide medical students with an introduction to clinical geriatric medicine and career options in geriatrics through structured clinical \"shadowing\" opportunities."}
{"text": "By utilizing an in vitro cell line established from Drosophila melanogaster, we intend to expolit the genetic, cytogenetic and molecular biological technology of this organism toward the goal of understanding the structure and function of genes. First, we intend to define mRNA from Drosophila cells with respect to their size, poly A content, sequence relatedness, and to localize at least those redundant transcripts as to their genetic sites by means of in situ hybridization. Second, we intend to explore the nature of what appears to be an endosymbiotic double-stranded RNA virus that exists in one of our cell lines."}
{"text": "During the prior year we demonstrated that there are distinct forms of Drd1a-mediated activation of ERK (receptor coupled kinase) signaling in the dorsal striatum and the nucleus accumbens (Gerfen et al., 2008). Using Drd1a- and DARPP32-deficient transgenic mice we show that in the dopamine intact accumbens, psychostimulants activate ERK1/2 via a Drd1a- and DARPP32-dependent mechanism. This mechanism is activated in the dorsal striatum only following dopamine depletion as occurs in Parkinson's Disease. Work done during the past year is a continuation of studies to determine the affects of Drd1a-mediated activation of ERK1/2 in the striatum. To this end we have developed a transgenic mouse model in which ERK1 and ERK2 are deleted in the different subpopulations of striatal neurons. Developing these mice has taken over 4 years of intensive work and we are now at the stage of having the mice available to use them in animal models of Parkinson's Disease with L-DOPA induced dyskinesia. Further work on the cellular mechanisms underlying neurologic and pscyhiatric disorders is limited by the fact that neurons throughout the brain utilize common mechanisms for critical functions such as synaptic plasticity, whereas specific neural systems are affected in different disorders. A large part of the Laboratory is now working with the Gene Expression Nervous System Atlas (GENSAT) project ( funded by NINDS and NIMH ) to produce transgenic mice with Cre-recombinase under the regulation of promoters to drive expression in specific neuron populations. Our part of this work is to characterize the expression of Cre in the brains of such transgenic lines and to provide such lines to the Mutant Mouse Regional Resource Center (MMRRC) for distribution to the research community. In the past year we characterized 50 Cre driver lines and are in the process of characterizing 75 lines for the current year ( Gong et al., 2007)"}
{"text": "The overall goal of this project is to test the hypothesis that Specific Language Impairment (SLI) is associated with deficits in acoustic processing. To this end, the mismatch negativity (MMN) of event-related potentials will be used to assess a variety of basic capacities associated with pre-attentive, automatic processing of acoustic events. These capacities include: 1. detection of changes in intensity, frequency and stimulus duration; 2. identifying features of acoustic stimuli that are constant while other features change; 3. determining that the order of two-tones pairs has changed; 4. determining that the serial order of three to five tones has changed; 5. forming an abstract rule as to the direction of change between the frequencies of two-tones pairs across a series of pairs (e.g., that they all rise in frequency from the first to the second tone), while the absolute frequencies of the tone pairs varies; 6. the duration of transient memory upon which the MMN relies. In addition, SLI and control subjects will receive training on the paradigm used in points 1-4 above which yields the poorest MMN to determine whether the MN will become larger in amplitude and/or shorter in latency."}
{"text": "Our studies on novel small molecules inhibitors of HIV integrase have led to the identification of new potent phenanthrene diketoacid (DKA) HIV integrase inhibitors that also inhibit HIV replication in cell culture, with a selectivity index up to 10. In light of the need for more effective and less toxic anti- HIV drugs, these results appear promising and warrant follow-up optimization and preclinical studies. However, since phenanthrenes are polyaromatic hydrocarbons (PAHs) with potential carcinogenicity we propose to investigate these novel phenanthrene diketoacids for carcinogenicity potential. Our hypothesis going forward is that appropriate substitution with electron withdrawing groups can minimize mutagenicity and produce useful anti-HIV agents. This is supported by the example of the new anti-malarial drug halofantrine, a substituted phenanthrene that is effective against multidrug resistant malaria, and is neither mutagenic nor teratogenic. We thus believe that appropriate substitution of our HIV integrase inhibitory phenanthrene DKAs will make them non-mutagenic and non-carcinogenic. Thus the specific aims of this proposal are: 1) to synthesize and test the IN inhibitory activity of new phenanthrene diketoacids with multiple electron withdrawing substituents and 2) to determine the carcinogenicity potential of the phenanthrene DKAs synthesized in Specific Aim 1. We will use the Ames test for in vitro carcinogenicity testing. The success of this project will provide vital information to decide whether or not to go ahead with optimization of the potent phenanthrene DKA HIV integrase inhibitors that we discovered recently and shown to selectively inhibit HIV replication in human peripheral blood mononuclear cells. If this approach to reducing carcinogenicity is successful it will not only provide a means of eliminating carcinogenicity of the phenanthrene DKAs, but may also provide a general method for reducing the carcinogenicity of PAH containing drugs. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: This project is aimed at investigating whether or not a novel class of potent phenanthrene diketo acid HIV integrase inhibitors that have been shown to suppress HIV viral replication in cell culture, are carcinogenic. The results of the research will determine whether to carry on with further work on optimizing this class of new anti-HIV agents towards AIDS therapeutics development. [unreadable] [unreadable] [unreadable] [unreadable]"}
{"text": "Erythropoietin is a cytokine required for red blood cell formation. Erythropoietin receptor expression is not restricted to hematopoietic cells and erythropoietin activity extends beyond blood and includes neural protection and cardiac protection demonstrated in select animal models. Using the erythropoietin receptor null mouse that develops a lethal embryonic anemia, we demonstrated that erythropoietin is required for normal endocardium and myocardium development. We also observed erythropoietin receptor expressed on skeletal muscle satellite cells. Erythropoietin stimulates myoblast proliferation and modifies expression of MyoD and myogenin transcription factors to delay cell differentiation and fusion to myotubes. Erythropoietin response is lost with down regulation of its receptor after skeletal muscle differentiation. We test the hypothesis that erythropoietin administration or up regulation of its receptor on myoblasts can contribute to muscle maintenance and/or repair. Specifically we examine erythropoietin protection in myocardial ischemia-reperfusion injury, in myoblast survival in skeletal muscle and in skeletal muscle wound healing. [unreadable] We found that in a mouse model for myocardial ischemia-reperfusion injury, erythropoietin administration significantly reduced infarct size. Erythropoietin receptor is expressed on endothelial and cardiac myocytes and an animal model was created with erythropoietin receptor restricted to hematopoietic and endothelial cells to examine cell specific response. Erythropoietin cardioprotection in this animal model is in progress. [unreadable] Myoblast transplantation for the treatment of myopathies has the potential to retard or stop muscle degeneration. We identified erythropoietin receptor expression on primitive/early myoblasts as well as more mature myoblasts isolated from mouse skeletal muscle. Erythropoietin stimulation of primary myoblasts decreased apoptosis associated with inflammatory cytokine treatment. Forced expression of erythropoietin receptor provides further protection with erythropoietin treatment. Transplantation of myoblasts into mice is being conducted to determine the ability of erythropoietin treatment to increase donor cell survival. [unreadable] In culture, we observed that erythropoietin protected myoblasts from hypoxic stress as well as inflammatory cytokines, suggesting a potential for erythropoietin activity in skeletal muscle repair. The potential of erythropoietin to influence wound healing of skeletal muscle or myoblast survival is being evaluated in a mouse model system of mechanical or toxic injury.[unreadable] These studies address the role of erythropoietin in muscle maintenance and repair. Experiments are designed to elucidate regulatory mechanisms that determine endogenous erythropoietin receptor expression in myogenic cells, to reveal the nature of erythropoietin myoprotection and to illustrate the potential utility of erythropoietin administration in myoblast transplantation. The ability of erythropoietin to stimulate muscle progenitor cells demonstrates its potential to activate proliferation and/or act as a survival factor to prevent apoptosis in multi-organ systems, an activity that is closely linked to the presence and level of receptor expression."}
{"text": "These studies are directed towards definition of the tumor-specific transplantation antigen(s) (TSTA) recognized by Lyt-l+ T effector (Te) and Lyt-2+ T suppressor (Ts) cells responsive to the 51509a (H-2[unreadable]a[unreadable]) methylcholanthrene-induced fibrosarcoma. T cells derived from S1509a-hyperimmune A/J (H-2[unreadable]a[unreadable]) hosts proliferate in vitro in response to soluble tumor-specific protein antigens present in S1509a ascites fluid, predominantly against a 60 to 70 kilodalton molecule with a buoyant density of 1.2-1.32 gm/cc CsCl. Proliferation is specific in that no response is observed with soluble YAC (H-2[unreadable]a[unreadable]) tumor antigens, but appropriate cross-reactivity is exhibited with related SAl (H-2[unreadable]a[unreadable]) soluble antigens. Tumor-specific Ts do not appear to bind the crude antigen preparation, supporting previous observations on the differential specificity of suppressor and effector T cells for S1509a and SAl tumor-associated antigens. A panel of syngeneic monoclonal antibodies (mAbs) reactive with soluble and/or cell-bound 51509a tumor antigens and exhibiting varying degrees of cross-reactivity with other ALJ tumors and with ALJ spleen cells is now being evaluated for recognition of the 51509a TSTA relevant to T-cell immunity. Our second interest is in the administration of anti-class II mAbs as an immunotherapeutic approach to regulating transplantation immunity. Treatment with minor-\\or class I-incompatible skin graft prolongs survival 2-\\to 3-fold and results in 50% indefinite survival of heterotopic heart allografts. Treatment is associated with delayed development of donor-specific DTH and CTL reactivity concomitant with the appearance of alloantigen-specific T[unreadable]s[unreadable]. Failure to enhance H-2-incompatible graft survival is to the differential recognition of class II versus class I or minor alloantigens by host T[unreadable]s[unreadable]. This therapy may prove beneficial in class II-matched organ transplants, since treatment can be discontinued once suppression has been established, leaving the host response to environmental pathogens intact. (LB)"}
{"text": "DESCRIPTION: (Applicant's Abstract) Hormones have been shown to influence the growth characteristics of epithelial ovarian carcinoma, but their mechanism of action is still unclear. In particular, low and high affinity LHRH receptors have been demonstrated on ovarian tumor cells. Also, LHRH agonists have been used successfully in phase II clinical studies of ovarian carcinoma. This project will investigate the antitumor activity of Cetrorelix, the first LHRH antagonist suitable for clinical use. A phase II study of Cetrorelix will be performed on patients with advanced recurrent ovarian cancer. Included in this study is a pharmacological investigation of different dose levels of Cetrorelix to determine the endocrine inhibitory effect of this drug in these ovariectomized patients. In order to understand the hormonal dependency of some ovarian carcinomas, we will evaluate the modulation of hormone and growth factor receptors on the membrane of ovarian tumor cells treated with Cetrorelix. Laboratory results obtained will be compared to clinical responses. The ultimate goal is to predict which tumor is sensitive to this LHRH antagonist by identifying relevant prognostic factors applicable to clinical use."}
{"text": "Actins are ubiquitous, highly conserved proteins that fall into classes: muscle actin (alpha-actin) and nonmuscle or cytoplasmic actins (beta- and gamma -actin). Because of its highly conserved nature, most antibodies to actin has an unusual specificity: in addition to reacting with the immunogen, this antiactin recognizes cytoplasmic vertebrate actin and not skeletal muscle actin. It is therefore an ideal probe for studying the distribution of cytoplasmic actin in skeletal muscle. Our previous immunocytochemical studies of human skeletal muscle showed that cytoplasmic actin is present and associated with membranes. Our more recent immunofluorescence studies of rat diaphragm muscle suggest that this actin may somehow stablize acetylcholine receptors in the postsynaptic membrane at the neuromuscular junction. To understand its function in muscle, an immunological approach will be used to localize cytoplasmic actin in sections of adult skeletal muscle and developing muscle cells at the level of the light and electron microscope. An antibody raised to squid tubulin also will be used to study the organization of microtubules in muscle. Lastly, dystrophic and myasthemic muscle will be examined to identify fine defects in the cytoskeletal structure of these tissues."}
{"text": "For the past three years, the Depression Unit and the Clinical Research Unit have collaborated in a study which surveys a population of individuals maintained on methadone to determine the prevalence of depression. The group studied included about 100 volunteers, and was generally representative of the entire clinic population in terms of such features as age, race and sex. In this research participants were evaluated based on several standardized rating scales measuring depression as characterized by verbal self-report, behavior, and symptoms. On the basis of the survey result, over 30 percent of these individuals were considered to be significantly depressed, including some patients with rather severe degrees of depressive symptoms. In most clinical settings, the degree of depression as shown by members of this group is considered of sufficient intensity to require antidepressant medication. We therefore propose a further study which assesses the efficacy of an antidepressant medication in treating depression in individuals maintained on methadone. The study will include 50 patients who exhibit significant depression at the time of admission to the Methadone Program. The selected individuals will be randomly assigned to receive dosages of the antidepressant Tofranil (imipramine) or a placebo on a daily basis for eight weeks. All medication will be dispensed on a double-blind basis and will be combined with daily methadone dosages. Throughout this drug trial, depression will be measured by self-reports, and by clinical assessment of behavior and symptoms. It is expected that the study will take two years, and will provide a sufficient sample to assess the efficacy of antidepressant medication in treating depression in this population."}
{"text": "Abstract: This cost center is fulfilling an essential service for the EDRN by receiving samples for our specimen reference sets, often aliquoting them to smaller sizes, maintaining these reference sets in freezers, and shipping sets out to investigators who obtain approval to test their biomarkers. These sets are used only for pre-validation or validation studies of biomarkers which is one the chief purposes of the EDRN. Without the maintenance of these reference sets the effectiveness of EDRN to test newly emerging biomarkers would be severely hampered. These reference sets differ than the extensive collections obtained through studies like PLCO or NLST in that the numbers of samples kept are much less being selectively chosen to address a particular clinical situation, include properly matched controls in that clinical context, and are powered statistically to permit adequate analysis of the biomarkers being tested."}
{"text": "Methods of enzymology, chemical and enzymatic kinetics, and synthetic, physical and analytical chemistry are being used to investigate the mechanisms of action of reverse transcriptase (RT) and protease enzymes of HIV-1, with the ultimate goal of developing specific inhibitors for these enzymes. Optical assays for retroviral proteases and P's, developed in this laboratory, are being employed in studies of the effects of reaction conditions and potential inhibitors on the activity of both RT and retroviral proteases. i) A continuous UV spectrophotometric assay described in a previous Report was used to study the elongation, catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I, of a partially self-complementary \"hairpin\" shaped oligonucleotide consisting of 35 mononucleotide residues. The sensitivity of the method permits the measurement of incorporation of a single base. ii) Concentrations of sodium chloride up to 4.5 M accelerated the hydrolysis of a chromogenic peptide substrate catalyzed by the protease of avian myeloblastosis virus. Similar effects have been observed by us for the cleavage of a peptide substrate catalyzed by HIV-1 protease, and have also been reported by others for cleavage of several peptides by this enzyme. These salt effects appear to be unrelated to the association of subunits that is required for the activity of retroviral aspartyl proteases, since very similar behavior is observed with the monomeric, mammalian enzyme pepsin. iii) We have identified two amino-acid repeat motifs in RT which are conserved in ten HIV-1 and eight HIV-2 isolates, and which occur in a region of the protein that may be involved in association of the p66 and p51 subunits of this enzyme. One motif is a modified \"leucine zipper\", whereas the other motif involves five tryptophans. Since the activity of HIV-1 RT has been suggested to require subunit association, an understanding of this process may lead to novel approaches to the inhibition of this enzyme."}
{"text": "The in vitro carcinogenesis model derived from organ culture of the whole mammary glands of BALB/c female mice will be used to study the transforming action of the environmental carcinogens, benzo[a]pyrene (B[a]P) and N-diethylnitrosamine (DENA). Appearance of the potentially neoplastic nodule-like alveolar lesions (NLAL) in the mammary glands in vitro after exposure to B[a]P or DENA will be used as a morphological marker for preneoplastic transformation. Carcinogenecity of the mammary epithelial cells transformed in vitro will be confirmed by the ability of cells to produce mammary carcinomas after transplantation into gland-free mammary fat pads of syngeneic virgin mice. The unique organ culture model of mammary cell transformation also will be used to study the mechanics of chemopreventive action of the dietary compounds, a new retinoid, Beta-carotene and selenium, during the neoplastic process induced by DMBA, B[a]P or DENA. Influence of the chemopreventive agents on the molecular events such as cellular accumulation of the electrophilic reactants, DNA alkylation and DNA repair activity initiated by the carcinogenic chemicals will be examined. Attempts will be made to ascertain whether influence of the chemopreventive agents on the molecular events of \"initiation\" may alter morphological expression of the transformed cells as NLAL in the glands in vitro and/or in the mammary tumors in the mammary fat pads in vivo. Studies also will include determination of the influence of the chemopreventive agents during hormone mediated \"promotion\" of the initiated (transformed) cells, both in vitro and in vivo. These studies will examine influence of chemopreventive agents at the specific stages of transformation between normal -- preneoplasia and preneoplasia -- neoplasia. The results should provide elucidation o the mechanics of chemopreventive action of these dietary compounds."}
{"text": "The nuclear enzyme DNA topoisomerase I relaxes supercoiled DNA and participates in DNA replication and transcription. Topoisomerase I localizes predominantly to nucleoli, and is the cytotoxic target for drugs of the camptothecin class. Several analogues of this class are currently in Phase I/II clinical trials, and early results of these traials have shown them to have activity against several types of \"refractory\" solid tumors. Intracellular factors reported to influence the cytotoxicity of the camptothecins include the amount of nuclear enzyme, the drug-induced increase in covalent topoisomerase I-DNA complexes, and the requirement for ongoing DNA synthesis. We suggest that additional, previously unidentified cellular events also significantly affect the cytotoxicity of the camptothecins. Therefore, we propose to test the hypotheses that: 1) Camptothecins limit their own cytotoxicity by altering the subcellular distribution of topoisomerase I and other nuclear proteins and by decreasing the amount of nuclear topoisomerase II, resulting in decreased topoisomerase I-DNA complexes and decreased DNA synthesis; 2) Intermittent, repeated exposures to the camptothecins that allow cells to maintain nuclear enzyme levels and nucleolar integrity, and consequently the ability to form topoisomerase-DNA complexes, will have the greatest cytotoxic effect; and 3) Based on biochemical observations, markers that predict the antitumor effect of topotecan in samples of primary tumors or bone marrow metatases of pediatric patients with neuroblastoma or medulloblastoma cal be identified."}
{"text": "Induced pluripotent stem cells (iPSCs) and their application to tissue engineering and disease modeling have great potential to change current medical practices. Current research is largely focused on devising efficient virus-free protocols to produce large numbers of iPSCs. Direct delivery of proteins obviates the risk of mutagenic insertion and enables more accurate control of the highly sensitive reprogramming process. However, cell-penetrating peptide methods currently provide reprogramming efficiencies that are too low for clinical use. The microfluidic delivery technology proposed has demonstrated its ability to deliver proteins at high efficiencies to human fibroblasts and it eliminates the need fo chemical modification or the use of exogenous compounds. Moreover, preliminary results indicate that the technique can be developed into a universal delivery method capable of delivering a range of macromolecules to different cell types underserved by current technologies. The current prototype is capable of delivering high throughput rates of 10,000-20,000 cells/s and can yield up to 1 million delivered cells per run. This combination of single-cell level control and macro-scale throughput places this device in a unique position relative to existing delivery methods. Aim 1: The mechanism of protein delivery and cell recovery will be investigated to better understand the system and direct its optimization. Preliminary results indicate macromolecular delivery occurs through a pore formation mechanism. To validate this hypothesis, model fluorescent macromolecules and proteins will be used in experiments designed to control against endocytosis and image membrane pores directly. Results will be used to develop a predictive model of the delivery system and conduct optimization studies to improve delivery efficiency, uniformity and cell viability. The design of future device generations will be guided by the gained mechanistic understanding and will aim to incorporate features such as coupling with electroporation. A streamlined version of the system will also be developed for use in collaborating laboratories. Aim 2: The intracellular delivery method will be optimized for protein-based reprogramming of fibroblasts to iPSCs. The robust delivery capabilities of the device will allow studies on the biological aspects of the reprogramming process itself, such as the optimal combination of transcription factors to produce maximum reprogramming efficiency and identification of the role of individual factor in the overall process Moreover, the device will be used to investigate potential improvements by combining other macromolecules, such as microRNA and mRNA, with protein-based reprogramming. In addition to reprogramming applications, such a high throughput microfluidic device platform capable of delivering a range of macromolecules with minimal cell death could enable unprecedented control over cellular function. Hence, in the future, it can be implemented in studies of disease mechanisms, identification of macromolecular therapeutic candidates, stem cell differentiation, and diagnostic applications with reporter cell lines."}
{"text": "This is a biobehavioral, longitudinal investigation of the role of emotion in the development of psychopathology in adolescence. The focus is on (a) the role of multiple components (experience, expression, regulation) of negative emotions (anger, anxiety, sadness) in the evolution of psychopathology, and (b) socialization experiences and biological processes that contribute to emotion dysregulation and disorder. The dysregulated experience and expression of emotion is implicated in both externalizing (antisocial patterns) and internalizing (anxiety, depressed mood) disorders. Adolescence is a critical juncture in the development of these disorders because of both the increased incidence of psychopathology during this time period and the increased differentiation of clinical problems. Four groups of youths are studied (N= 60 in each group): (1) comorbid externalizing and internalizing problems, (2) externalizing problems predominate, (3) internalizing problems predominate, (4) asymptomatic. Half the youth in each group are 11-13 years, the other half 14-16 years in order to conduct age cross-sectional analyses at each time point. Equal numbers of males and females participate, in order to examine sex differences in symptoms and emotion regulation. One theme pertains to the integration of emotions across systems, and how different patterns of emotion relate to psychopathology. A second theme pertains to the developmental changes in how disorders are manifested, e.g. increased differentiation of disorder along gender specific pathways. During the past year a primary focus has been on data collection, and both laboratory sessions and home visits are close to complete. This year will be spent (a) coding, analyzing, and preparing articles for publication based on Time 1 data, and (b) collecting the Time 2 data for longitudinal analyses. - emotions & psychopathology; adolescent clinical problems; co-morbidity; gender - Human Subjects & Human Subjects: Interview, Questionaires, or Surveys Only"}
{"text": "The sense of hearing is initialized in the inner ear by signal transduction; i.e., the transformation of sound to electrical signals. Normal cochlear function relies on the integrity and functionality of the hair cells that act as mechano-electric transducers. Cochlear transduction is vulnerable to structural and metabolic alterations in the inner ear that consequently lead to hearing loss; therefore, techniques that can assess cochlear transduction would be a desirable clinical tool to evaluate cochlear function. The long-term goal of the proposed research is to characterize cochlear transduction in cochlear disorders and, in turn, to develop a diagnostic tool that can no invasively distinguish cochlear hearing losses. The transduction processes in the hair cells produce sound or acoustic emissions that are measurable in the ear canal. Evoked by two-tone stimuli, the distortion product otoacoustic emissions (DPOAEs) are generated from the nonlinearity in cochlear transduction. Recently, we have developed a technique to derive a nonlinear transducer function of the inner ear from low-frequency modulation of DPOAEs. The modulation patterns of the DPOAEs provide physiologic indices of cochlear transduction. To continue our research towards the long-term goal, three studies, two in animals and one in the humans, are proposed. In the animal studies, the specific aim is to determine the sensitivity and specificity of the transducer function and the associated indices in detecting cochlear pathologies. The sensitivity of the transduction indices will be tested in animals with moderate temporary hearing loss induced by exposures to pure tones. The specificity will be evaluated by differences in the change of transducer functions obtained from animals with hearing losses produced by different ototoxic drugs. The specific aim of the human study is to obtain normative values, the variability and reliability of the cochlear transduction indices derived from the low-frequency modulation of DPOAEs. Successful completion of the proposed research will provide a solid base for future development of a clinical tool that is helpful in the early identification of cochlear disorders, a critical practice in otology and audiology for treatment and hearing preservation. [unreadable] [unreadable]"}
{"text": "The use of alcohol off and on the job by employed adults represents an important social policy issue because it can undermine employee health and productivity, and may further interfere with employers' ability to compete effectively in an increasingly competitive domestic and global economic environment. Therefore, it is imperative that we develop a better understanding of the workplace causes of employee alcohol use both off and on the job. The most frequently explored work-related cause of employee alcohol use is exposure to work stressors. Nonetheless, because past research on work stress and alcohol use has left many issues unresolved or unexplored, it is not surprising that the findings to date have been weak and inconsistent. The proposed study will systematically extend past research on work stress and alcohol use in a number of ways. First, a broad taxonomy of work stressors will be used to determine the general types of work stressors that may be related to employee alcohol use. Second, a broad set of alcohol measures will be used to determine if work stressors are more strongly related to certain dimensions of alcohol involvement. The taxonomy of alcohol use will include several dimensions of both context-free (i.e., overall) alcohol involvement and work-related context-specific (use before work, use during the workday, use after work, and use on off-work days) alcohol involvement. Finally, variables expected to moderate or mediate the relation between work stressors and alcohol use will be examined. Mediating variables include negative affect, inability to relax or unwind after work, physical and psychological fatigue, and the desire to use alcohol (cravings). Moderating variables include person characteristics (affect and performance regulation expectancies, avoidant coping, impulsivity, and stress-related rumination) and environmental characteristics (overall and workplace physical and social availability of alcohol). To explore these issues, a national telephone survey will be conducted using a representative sample of 3,500 employed individuals 18 to 65 years old. The proposed study will help shape future research on work stress and alcohol use by leading to a more comprehensive model of work stress and employee alcohol use. It will contribute to organizational policy and shape future intervention research on workforce and workplace alcohol use by identifying work conditions and vulnerable subgroups that can be the focus of intervention efforts and work redesign. [unreadable] [unreadable] [unreadable]"}
{"text": "This project involves the analysis of Missouri vital statistics data to determine if using a cohort method of analysis is biased in understanding the effect of prenatal care on pregnancy outcome. Cohort methods appear to underestimate the effect of prenatal care and suggest that late entry into prenatal care is more beneficial than early entry."}
{"text": "An attenuated monocyte-tropic clone of SIV (SIV/17E-C1) has properties which are unique to a live attenuated SIV vaccine. Infection with SIV/17E-C1 induces an early vigorous type-specific neutralizing antibody response which broadens during the first 7 months postinfection to include activity against SIV/DeltaB670, a heterologous primary isolate. The induction of protective immune responses coincides with the switch from type-specific to group-specific neutralizing antibody, and, at least in part, appears mediated by antibody since sera from protected monkeys passively protected 2 of 4 recipients. A nef-deleted variant of this monocyte-tropic clone, but not the lymphocyte-tropic parent SIVmac239deltanef, also induced detectable class I restricted CTL during the first 6 months postinfection. These unique properties are likely due to a unique gp120 conformation imposed by sequences which enable it to replicate in macrophages, and/or selective presentation to the immune system by the infected macrophage. We propose to further characterize the protective responses induced by SIV/17E-C1 infection, and to utilize this information to design a subunit vaccine that can induce the protective responses observed with the attenuated virus. The protection observed by intravenous challenge with cell-free virus of SIV/e-C1- infected monkeys will be extended to protection against infected cells, delivered both intravenously and at the intestinal mucosal surface. The role of antibody in passive protection will be confirmed by passive transfer of purified immunoglobulin. Antibodies binding to native versus denatured gp120 will be affinity purified, characterized in vitro and similarly analyzed to define the specificity of protective responses. Passive transfer studies will also be performed with monoclonal antibodies derived from SIV/17E-C1-infected monkeys to identify the specific epitope(s) required for protection. The contribution of cellular immunity will be determined by determining the kinetics of class I restricted CTL induction in infected monkeys, and correlating these responses to protection. The efficacy of a purified recombinant gp120 vaccine comprised of SIV/17E-C1 sequences will be evaluated to determine whether these sequences per se uniquely induce protective immunity. The ability of DNA vaccines comprised of SIV/17E sequences, alone or in combination with cytokine expression vectors, to elicit protective responses will be assessed using both direct injection of DNA and the particle delivery system developed by Agracetus. The optimal HIV vaccine strategy is one which is efficacious in both SIV (disease) and HIV (infection) model systems. Thus, the ability of SIV/HIV recombinant viruses to serve as live attenuated vaccines will be determined to extend the observations gained from SIV to HIV glycoproteins. A model for assessing effective SIV subunit vaccines, as well as heterologous (inter- clade) protection, will be established using SHIV constructs."}
{"text": "The Universidad Central del Caribe (UCC), through its Center for Addiction Studies and the Caribbean Basin and Hispanic Addiction Technology Transfer Center (CBHATTC), is requesting support to implement \"The Second National Conference on the Hispanic/Latino Family: The Role of the Hispanic/Latino Family as a System of Drug Use Treatment and Prevention,\" to be held August 18-20, 2004 at the San Juan Marriott in San Juan, Puerto Rico. The Conference is part of the Center for Addiction Studies and CBHATTC's educational commitment to continually pursue the goal of \"Blending Research/Science to Practice.\" The conference is designed both to build a knowledge base among participants regarding up-to-date research findings in each of the themes of the conference to provide an environment in which Hispanic drug treatment providers, others who serve Hispanic populations, and researchers can interact and develop relationships and coalitions that may result in conjoint research endeavors. The conference program is structured around the subtheme of \"From molecules/genes to community to culture,\" and is comprised of pre-conference workshops, plenary sessions, workshops, and poster sessions. Major topics to be covered include the neuroscience of addiction; family-centered approaches to prevention and treatment of substance use; community and cultural factors which may facilitate or impede the family's ability to address substance use among its members; and issues of trauma and violence, sexual identity, and co-occurring disorders as they relate to substance abuse and its treatment and prevention. A Conference Proceedings document is to be developed in Spanish and English, which will be available to all states through the SAMHSA/CSAT ATTC Network and the NIDA National Hispanic Science Network. The conference approach is guided by elements of Hispanic culture, and the theories of Diffusion of Innovation and Successful Technology Implementation."}
{"text": "Acquired immune deficiency syndrome (AIDS) occurs at a very high rate in Zaire and attacks females at approximately the same rate as males. It is clear that the epidemiology of the disease in Africa differs from that seen in the United States and studies of the disease in Zaire could lend valuable information in understanding and combatting the disease in this country. For this reason, a Collaborative AIDS Project in Zaire (CAPZ) has been established by the Centers for Disease Control and the NIAID to study epidemiologic and immunologic aspects of this disease in Zaire. The study is being set up, laboratory facilities prepared and equipment being shipped. Dr. Francis will be responsible for the laboratory aspects of the project and for some epidemiologic studies of special interest to the NIAID and Dr. Mann will be responsible for most epidemiologic study design and implementation. It is planned that a clinician will be added to the project in the near future. The project is estimated to take two years to complete."}
{"text": "PROJECT SUMMARY This application entitled ?Circadian Rhythms of Skin Barrier, Pruritus and Inflammation in Atopic Dermatitis,? is being submitted by Dr. Anna Fishbein at Northwestern University Feinberg School of Medicine/ Ann & Robert H. Lurie Children's Hospital of Chicago for a Mentored Patient-Oriented Research Career Development Award. Dr. Fishbein is a pediatrician, allergist and immunologist with a background in translational research. Eczema (atopic dermatitis) is the most common chronic skin disease of childhood, affecting 10% of US children. Nocturnal eczema is the itchy nighttime exacerbation of atopic dermatitis (AD) that afflicts more than 50% of children with AD, resulting in devastating scratch and sleep disturbance. Circadian rhythms are 24 hour biological cycles that appear to underlie the nocturnal worsening of atopic dermatitis. The objective of this project is to elucidate how circadian rhythm dysregulation is related to underlying AD pathophysiology. Dr. Fishbein's novel approach will apply state of the art tools to test her overarching hypothesis that the circadian system drives skin barrier disruption, pruritus and inflammation in nocturnal atopic dermatitis flare. She will test the specific hypotheses that 1) blunting of the normal skin barrier function rhythm in AD is associated with increased scratch and sleep disturbance and 2) melatonin increases release of TH2/TH22 cytokines that are known to disrupt skin barrier function and increase itch. Modified constant routine protocol (28 hours in a controlled-laboratory setting with dim light) will isolate the contribution of the circadian system to atopic dermatitis. The specific aims to test the hypotheses are: 1) To determine how changes in the skin barrier function rhythm exacerbate nocturnal atopic dermatitis flare; and 2) To determine the role of melatonin in atopic inflammation in AD. Through the proposed research, Dr. Fishbein will work towards her long-term goal of becoming an independent translational physician investigator by developing chronotherapy (timed based treatment) for atopic dermatitis. Her short-term goal is to apply circadian biology to study skin barrier, pruritus and inflammation to determine the mechanism of nocturnal AD flare. Her training goals for this award are to: 1) build a foundation in circadian research techniques and molecular assessment of rhythms to study inflammatory skin disease, 2) enhance her knowledge base in immunology with a focus on circadian rhythms in T-cell biology, 3) advance personalized medicine for children with AD and 4) become an independently funded investigator. Dr. Fishbein's clinical training and prior experience in patient-oriented research make her an ideal candidate to accomplish this multidisciplinary project. Ann & Robert H. Lurie Children's Hospital of Chicago/Northwestern University provides an optimal training environment with top-notch expertise. Primary mentor Dr. Phyllis Zee is an expert in circadian biology and has successfully trained many academicians. Co-mentors Dr. Amy Paller and Dr. Robert Schleimer are world-renowned researchers in atopic dermatitis and immunology. ."}
{"text": "DESCRIPTION: We intend to refine the affinity-reagent discovery process so that it is not only cheaper and faster than currently possible, but also so that we produce reagents that are more versatile and useful than current monoclonal antibodies. We propose to use in vitro combinatorial recombination of pre-defined synthetic complementarity determining regions (CDRs) of single chain variable fragment (scFv) antibodies to synthesize a novel type of highly diverse synthetic display library. We have termed this new library-type as a 'pre-defined CDR' (PDC) library. This novel library system will have properties that will also enable it to greatly facilitate both the downstream affinity-reagent selection and the maturation processes. Methods for testing this library using microfluidics and emulsion screening are discussed."}
{"text": "This proposal is to develop a revolutionary new type of angioscope. The new technology will achieve the following improvements compared to existing angioscopes: 1)a factor of two brighter image, 2)a 67% improvement in resolution, 3)a factor of five greater flexibility, and 4)the ability to make quantitative measurements such as the degree of vascular stenosis. The broad objectives of this project are to fabricate angioscopes of several sizes with this new technology. They will be designed to be plug-in compatible with the already installed base of electronic cameras, illumination systems, and monitors. The specific aims of Phase I are : 1)to fabricate image guides of various resolution, 2)characterize the optical and mechanical properties of these guides, and 3)design an advanced angioscope which incorporates the new technology and has the ability to perform quantification, and spectroscopy. Heart and vascular disease is the single largest killer in the USA. This new approach to angioscopy will provide access to 100% of the vascular tree with unprecedented image quality, and quantification. The proposed technology has never previously been used in endoscopy. The successful demonstration of the feasibility of this technology will have wide ranging implications for innovation in all aspects of medical and industrial imaging. PROPOSED COMMERCIAL APPLICATION: The world wide market for angioscopes is about $20M. The new technology being proposed will make a substantially better product at a lower cost and permit penetration of this market. Success in the small diameter endoscope market will be expanded into the rest of medical endoscopy and industrial imaging, which is a several hundred million dollar market."}
{"text": "The purpose of this project is twofold: 1) gain additional training in conducting research and primary data collection/analyses in developing countries, with a focus on issues related to early intervention for children with disabilities; and 2) to obtain more knowledge about the influence of psychosocial, resource and cultural factors on the needs of children with disabilities and their families in developing countries. The candidate wishes to pursue a career in international rehabilitation, with the ultimate goal of contributing to improved conditions for children with disabilities and their families in developing countries. Her immediate goal is to combine her backgrounds in cross-cultural and disability studies to address the global issue of the factors that influence the conditions of children with disabilities and families. The candidate's long-term goals include embarking on a career of independent research and teaching in international rehabilitation for children with disabilities and their families and conducting field research. This award will allow the candidate to benefit from a period of mentored research in international rehabilitation for children and gain necessary experience in conducting field research and primary collection in a developing country. The candidate plans to spend about five months per year for this five-year project primarily in Hanoi, Vietnam, and the remainder of the year at the New York State Institute for Basic Research in Developmental Disabilities in the U.S. under the guidance of Drs. Peter Vietze, Tran Trong Hai, Judith Ladinsky and Nguyen Viet Nhan. During this time, she will attend relevant seminars and courses for additional training in the field, including courses in Vietnamese language, advanced statistics, and longitudinal research methods at the City University of New York and New York University. The early intervention study will involve a comprehensive assessment of children with disabilities at the time the parent enters the treatment study and again at 3, 6, 12, 18, and 24 months. The primary aims of the study are: 1) to examine the level of child's and family variables over the course of the intervention program; 2) to explore potential factors that cause these changes including stressors and family patterns; and 3) to test whether changes in child adaptation, the hypothesized variables, and the relation between parent and child symptoms differ as a function of the type of services they received. It is expected that the study will represent a unique opportunity to further our understanding of the familial context for child disabilities that can serve as a guide for the development of intervention programs for young children with disabilities in developing countries."}
{"text": "Despite great scientific advances over the past decade, the translation of these discoveries to the benefit of patients has been hampered by a number of traditional barriers. The overall goal of the iWioyinit Siimai ImstitiLiiites of ClBrnical &Tiramslatioinial Sciences is to establish a new research paradigm at Mount Sinai, which will create an integrated multidisciplinary environment that will enable translation of cutting-edge research more quickly and easily from \"bench-to-bedside-to-implementation\" in the community;train and create an \"academic home\" for the clinical and translational investigators of tomorrow;and supply the governance, facilities, resources and opportunities needed to elevate the clinical research enterprise so that clinical investigators can take full advantage of Mount Sinai's Translational Research Institutes and potential collaborators in Mount Sinai Hospital and its affiliates, the School of Medicine and partnering institutions, and other CTSA programs around the country. This transformation will be achieved by fulfilling the following Specific Aims: (1) redesign the institutional research enterprise infrastructure and governance by integrating research functions across administrative, departmental and intellectual boundaries, enhancing and promoting interactions between basic scientists and clinical investigators, and streamlining administrative procedures to facilitate the start-up of clinical trials and the dissemination of results;(2) establish a Translational Discoveries Program to facilitate and expedite clinical research by providing consultation, oversight and facilities for clinical and translational research, engaging the surrounding community and our affiliates to rapidly translate health benefits to the general population, and developing new methodologies to improve clinical trial design and reduce participant burden;(3) train and support the translational investigators of tomorrow by overseeing a multi-disciplinary, doctoral degree-granting program in clinical and translational research for all health care professionals, developing a program to recruit, enhance career development and retain clinical and translational researchers, providing an academic home where new collaborations can be formulated;(4) establish an Experimental Therapeutics&Technologies Program to stimulate the development of new methodologies in clinical and translational research by developing an institution-wide mentored program to identify and develop novel clinical and translational research projects; and (5) establish an institution-wide biomedical information infrastructure that can connect basic researchers, clinical researchers, caregivers and laboratories with an integrated network of information. RELEVANCE (See instructions): The establishment of the ilfloynt Siinao Institutes of Clinical &Tiranslational Sciemces would create an effective, efficient and centralized research administrative structure, foster and reward interdisciplinary collaborations, educate and retain the clinical and translational investigators of the future, enable translation of basic scientific discoveries into clinical practice, and deliver to our diverse community new therapies and a standard of care that would have been unimaginable even five years ago."}
{"text": "Accumulating evidence suggests that cerebral amyloid beta (Abeta) deposition, which begins rapidly following traumatic brain injury (TBI), occurs in response to axonal and oxidative damage. Our long-term goal is to validate the use of positron emission tomography (PET) imaging with a novel molecular probe, [18F]FDDNP, for visualizing Abeta plaques, in vivo, in the setting of acute TBI. We hypothesize that the amount of [18F]FDDNP binding will correlate with both the concentration of diffusible Abeta in the cerebrospinal fluid and patient outcome. The Specific Aims are to 1) confirm the specificity of [18F]FDDNP for cerebral Abeta deposition [18F]FDDNP uptake following TBI using autoradiography and immunohistochemistry of brain specimens and, 2) determine the relationship between cerebral Abeta plaque deposition, assessed by [18F]FDDNP-PET imaging, and CSF Abeta concentration acutely following TBI. [18F]FDDNP-PET has been used successfully in imaging amyloid pathology in Alzheimer's disease patients, allowing early diagnosis and improved understanding of the disease. We believe that the ability to visualize amyloid deposition in vivo with PET, in a broad range of TBI injury severity, will allow us for the first time to assess the incidence, time course, and regional distribution. More importantly, it may also offer a noninvasive tool to monitor the efficacy of treatments aimed at decreasing amyloid deposition (including neuro-inflammation modulators). In brief, the research design entails studying a range of mild to severely head-injured patients acutely after injury (3-10 days) with FDDNP-PET. The results may have important public health implications in that improved understanding of the pathological consequences of cerebral amyloid deposition may lead to medical treatments that will improve outcome following TBI. [unreadable] [unreadable] [unreadable]"}
{"text": "This interdisciplinary study is primarily designed to assess the effect of a prototype intervention program designed to improve cardiovascular health of rural, American Indian, Hispanic, and Anglo fifth graders. The basic program will be compared with a control condition and a condition in which the program is supplemented by peer modeling. The program will involve instruction about the heart and cardiovascular system, obesity, nutrition, exercise, smoking and self-control techniques for changing behaviors related to cardiovascular health. A variety of instructional methods including parental and community involvement, lessons in food preparation and storage, and an emphasis on cultural tradition as well as traditional audiovisual and written materials will be used. A secondary purpose of the study will be to collect some descriptive data on health-related knowledge and attitudes in a population which has not previously been studied. Measures of short-term and long-term effects will include a) physical measures: height, weight, blood pressure, heart rate, and skinfolds, b) physical fitness: the one mile run/walk, c) knowledge measures about the heart and cardiovascular system, nutrition, obesity, smoking, exercise and behavior change, d) self-report measures of eating, exercise and smoking behaviors, e) measures of attitudes toward obesity and self-perception of their own weight and fitness, f) a posttest assessment of their feelings about the program, for those not in the control groups. It is hypothesized that subjects in both experimental conditions will show changes on all measures at the end of the semester in which the major part of the material is presented. Maintenance of the changes is expected to be superior for those in the peer modeling condition, who will have the opportunity to serve as peer models in subsequent years. The focus on incorporating behavioral changes into the life styles of the participants is hoped to lead to permanent reductions in risk factors related to cardiovascular disease."}
{"text": "PROJECT SUMMARY/ABSTRACT The Cancer Genetics and Epigenetics (CGE) Program of Winship Cancer Institute of Emory University is a laboratory-based basic science program that seeks to better understand how altered genetic and epigenetic components of the genome contribute to the initiation and progression of human cancers. Under the leadership of Paula Vertino, PhD (leader) and Jin-Tang Dong, PhD (co-leader) the CGE Program includes 27 core members drawn from 14 departments and three schools across Emory University, including the School of Medicine, Emory College, and Rollins School of Public Health. The goals of the CGE program are to better understand the mechanisms that govern the maintenance of genome stability and proper gene regulatory networks, how these become corrupted in cancer cells, and how their disruption contributes to the initiation and progression of cancer. The CGE Program seeks to promote the expanded application of genomic technologies in the molecular classification of cancer phenotypes and outcomes. The scientific focus of the program is organized around three inter-related and complimentary themes: (1) DNA Damage, Repair, and Cellular Responses to Stress, (2) Epigenetics and Gene Regulation, and (3) Cancer Genetics and Genomics. Over the last competitive cycle CGE Program members have published 256 cancer-relevant scientific articles. Of these, 55 (21%) were intra- and 105 (41%) were inter-programmatic collaborations, and 123 (48%) represented a collaboration with another cancer center or other academic organization. As of March 31, 2016, CGE held $9.1 million in annual total cancer-relevant research funding, of which approximately $3 million (33%) was awarded from the NCI. Strong scientific synergism has led to important discoveries. Key insights into the mechanism of DNA double strand break repair, how replication stress is resolved, and radiation mutagenesis are leading to important predictors of chemotherapy and radiation response. Landmark investigations into the structure and function of the TET dioxygenase enzymes have revealed oxidized methylcytosine residues to be more than a transient intermediate in the turnover of 5mC, but a distinct component of the epigenetic `code' governing gene expression programs. Significant strides have been taken towards the successful translation of the program's genetics/genomics efforts, including new insight into the contribution of key `driver' gene mutations and the unique cellular vulnerabilities that such alterations unmask, an RNA-based biomarker panel for the early detection of aggressive prostate cancer, a combined RNA-DNA test for risk stratification in oropharyngeal carcinomas, and the implementation of a clinical genomics workflow to guide decision making in several cancer types. Overall, the CGE Program promotes center-wide goals for improvements in cancer outcomes across the state of Georgia by identifying of key points of cancer cell vulnerability that can be exploited towards new therapeutic opportunities and the development of genomic and epigenomic signatures as predictors of clinical outcomes and population risk."}
{"text": "Project Summary Overwhelming evidence suggest that mitochondria plays a causative role in diabetic endothelial dysfunction. How mitochondria become dysfunctional remains enigmatic. Our exciting preliminary data indicate that diabetes instigates aberrant mitochondrial fission by suppressing autophagy-dependent degradation of the dynamin-related protein 1 (DRP1), a molecule essential for mitochondrial fission. The central hypothesis of this application that selective impairment autophagy-mediated DRP1 degradation in diabetes instigates vascular endothelial dysfunction and accelerates atherosclerosis. This hypothesis will be tested using gain-/loss-of-function strategies in both animal models and cultured cells. Aim 1 is to test the hypothesis that defective vascular autophagy increases mitochondrial fission in the development of endothelial dysfunction and atherosclerosis by characterizing the spatial and temporal dynamics of vascular autophagy, mitochondrial fission, and endothelial dysfunction in Akita mice (type 1 diabetes) and ob/ob mice (type 2 diabetes), determining whether inhibition of autophagy aggravates mitochondrial fission, endothelial dysfunction, and atherosclerosis in diabetic LDLr-/-/beclin1 heterozygous (beclin1+/-, with reduced autophagy) mice and their littermates, and examining whether enhanced endothelial autophagy lessens oxidative stress and improves endothelial function in diabetic Atg7 endothelium-specific transgenic mice (with enhanced autophagy). Aim 2 is to delineate the mechanisms by which diabetes-inhibited autophagy increases mitochondrial fission. This aim will test the hypothesis that diabetes induces mitochondrial fission by inhibiting autophagic degradation of DRP1. Proposed studies are significant as the completion of this proposal will provide the rationales for developing new therapeutics to prevent/treat cardiovascular complications in diabetes."}
{"text": "Rapid eye movement (REM) sleep is a behavioral state characterized by activation of the cortical and hippocampal EEG, rapid eye movements and muscle atonia. While some progress has been made in recent years in the effort to delineate 1) the locus of the pontine switching circuitry for REM sleep, 2) the neurotransmitters regulating REM phenomenon, i.e., muscle atonia, activation of the cortical and hippocampal EEG, and 3) how dysfunction of this circuitry may form the neuropathologic basis of REM sleep behavior disorder, major gaps remain in our knowledge. Recent work by our laboratory has revealed the presence of mutually inhibitory REM-off and REM-on areas in the mesopontine tegmentum that may form the neuroanatomical basis of the switching circuitry for REM sleep. These findings, which form the basis of the present research plan, posit a REM switching circuitry model that is analogous to an electronic 'flip-flop'switch. In this flip-flop switch arrangement, GABAergic REM-on neurons (located in the sublateraldorsal tegmental nucleus (SLD)) inhibit GABAergic REM-off neurons (located in the ventrolateral periaqueductal gray matter (vlPAG) and lateral pontine tegmentum (LPT)) and vice versa. Inside this pontine brainstem \"switch\" the REM-on area contains two populations of glutamatergic neurons, the first of which projects to the basal forebrain and regulates EEG components of REM sleep and the second which projects to the ventromedial medulla and spinal cord and regulates atonia during REM sleep. To demonstrate the critical role of glutamatergic SLD neurons in producing REM without atonia, we will selectively eliminate glutamatergic neurotransmission in the SLD by stereotaxically injecting an adeno-associated virus containing the gene for Cre recombinase (AAV-Cre) into the SLD of conditional knock-out mice with lox-P modified alleles of the vesicular glutamate transporter 2 (VGLUT2) genes. We will similarly eliminate GABAergic neurotransmission in the SLD and LPT by stereotaxically injecting AAV-Cre into mice with lox-P modified alleles of the vesicular GABA transporter (VGAT). Finally, we will examine the role of the ventromedial medulla in REM atonia by combining injections of orexin-saporin into rats and AAV-Cre injections into VGAT and VGLUT2 mice. Findings from the present proposal will provide a context for understanding the pathophysiologic mechanisms and etiological bases for a variety of sleep disorders, including REM sleep behavior disorder."}
{"text": "This research focuses on the central unsolved problem of psychiatric epidemiology, the probem of how to measure psychiatric disorders independently of treatment status in the general population. Its major aim is to test the sensitivity and specificity of the newly developed Psychiatric Epidemiology Research Interview (PERI) as a first-stage instrument to screen functional psychiatric disorders among adults in a demographically complex urban population. To this end, we will investigate PERI's relationship to other potential screening instruments on a sample of 600 adults from the general population and calibrate PERI on a sample of 500 psychiatric patients with DSM-III diagnoses of schizophrenia and the schizophreniform psychoses, affective disorders, various types of disorder under the heading of neurosis, anti-social personality, and other selected personality disorders. The research setting is Washington Heights, a section of Manhattan in New York City that surrounds Columbia Presbyterian Medical Center. It is a demographically complex urban area containing large percentages of blacks and Purerto Ricans as well as members of more advantaged ethnic groups. The results should provide a major step toward the development and testing of a two-stage procedure for case identification and classification. The findings should also be important for needs assessment and for the planning and evaluation of mental health services in Washington Heights."}
{"text": "Protein-protein interactions underlying molecular recognition are studied, utilizing monoclonal antibodies (Mabs) specific for the protein hen egg white lysozyme (HEL), a protein which has long served as a prototype for investigating the specificity of immune recognition. The X-ray structures of HyHEL-5 Fab complexed with 2 single site-directed mutants of HEL have been solved and refined: R68K which reduces the affinity of the complex by a factor of over 10 to the 3rd power, and R45K, which reduces affinity by only 10-fold. These results represent the first time structures have been obtained for 3 antigens, differing at only a single critical residue, complexed to the same antibody, and will provide valuable insight about the role of Arginine side chains in protein-protein interactions. We are beginning to define fundamental principles that will allow prediction of function from structure, principles that are critical to such applications as antibody design and vaccine development. We are also approaching the problem of vaccine development by investigating immunogenicity and protective epitopes in Shigella flexnerii. In addition, experiments with specific-pathogen- free and conventional BALB/c mice have established that plasmacytoma- refractory SPF BALB/cAnPt mice have naive T cell responses as well as associated restricted B cell responses. These results suggest a significant influence of antigenic exposure, particularly viral antigens, on development of the specificity repertoire and plasmacytomagenesis."}
{"text": "The feasibility of a device to enable precise and repeatable calibration of single-breath lung diffusion (DLCO) instruments will be investigated. The calibrator will enable the accuracy of DLCO instruments to be verified between pulmonary laboratories, allowing greater confidence in a diagnosis based on the single breath DLCO test. This study will verify the performance of a unique gas collection and mixing system which will allow generation of precise carbon monoxide (CO) and Helium (HE) gas fractions without using gas analyzers. This will allow production of a DLCO calibrator which will not require any calibration of its own."}
{"text": "Continued study of human fetal hemoglobin in normal and abnormal hematological conditions will be made to increase insight into the genetics and control of the linked beta, gamma, and delta structural genes. In particular, examination of cord bloods will be made to gain more information about a newly detected but poorly understood anomaly in the regulation of the gamma genes. These studies will be made in collaboration with Prof. T.H.J. Huisman of the Medical College of Georgia. It is hoped to complete the sequence studies on the catalases. If possible, studies of sequence in lactoperoxidase will be carried further."}
{"text": "We are requesting beam time to investigate how the presence of methane affects the structure of intermediate Q in M. tricosporium methane monooxygenase. We would like to determine when and how methane interacts with the diiron active site."}
{"text": "The major goal of this research project is to understand the molecular mechanisms by which steroid and peptide hormones regulate the expression of specific genes in differentiated cells. We will focus our attention on the chick transferrin and ovalbumin genes because these two egg white genes represent well-characterized examples of tissue-specific expression and hormonal regulation. The ovalbumin gene is expressed exclusively in the oviduct in response to estrogen and progesterone; however, tissue culture experiments have recently demonstrated that this steroid response is completely dependent on the additional presence of an insulin-related growth factor (IGF). The transferrin gene is expressed and regulated in at least three differentiated tissues: the oviduct, liver, and testis. Our specific objectives are to (1) study the IGF-mediated events which modulate the response of the ovalbumin gene to estrogen (2) identify the DNA sequences that interact with steroid receptors and regulate transcription of the transferrin and ovalbumin genes (3) explore the mechanisms which lead to gene commitment and tissue-specific expression of the transferrin gene. Our approach will utilize hormone-responsive oviduct cells in culture as an in vivo assay system for regulatory proteins and DNA sequences that can be introduced into the living cells by microinjection. The tissue-specific expression of the transferrin gene will be examined by introducing the chick transferrin gene into the mouse genome via oocyte microinjection. The expression and regulation of the chick gene in differentiated mouse tissues will then be examined. We expect these studies to further elucidate the basic mechanisms by which reproductive hormones control the developmental program of growth, differentiation, and specific gene expression in target tissues."}
{"text": "Project Summary Every year, a substantial number of children injure a growth plate, a cartilaginous region found at the end of all long bones in children that provides signals for the bones to lengthen. The growth plate is the most fragile structure in a child?s developing bones, making it prone to injury. Damaged cartilage within the growth plate is often replaced by unwanted bone, forming a ?bony bar?, which can lead to angular deformities or halt bone growth completely. Current surgical methods to correct bone growth defects are invasive, prone to infections and have low success rates. There is no current treatment that leads to complete repair of an injured growth plate. Innovative treatment strategies that prevent bony bar formation, restore functional growth plate cartilage, and permit normal longitudinal bone growth in affected individuals are greatly needed. This proposal seeks to develop an injectable hydrogel biomaterial system that could prevent bony bar formation and promote the formation of cartilage tissue, and which could ultimately be examined for its ability to heal growth plate injuries in children. It has been shown that mesenchymal stem cells (MSCs) infiltrate the injured growth plate and undergo osteogenic differentiation. Here, two important areas for inhibiting the osteogenesis of MSCs via a biomaterial system will be studied: (1) the role of biopolymer hydrogel substrate mechanics in preventing osteogenesis, and (2) the delivery of short interfering RNA (siRNA) from biopolymer hydrogels that can block osteogenic differentiation. We hypothesize that hydrogel systems that are less stiff and that provide sustained exposure of MSCs to p38 MAPK siRNA will inhibit osteogenesis. This proposal seeks to engineer hydrogel systems with these characteristics as a first step towards creating new technologies for helping to heal growth plate injuries in children. This will be accomplished as by two Aims: Aim 1 - To engineer a hydrogel system with mechanical cues that prevent osteogenic differentiation of MSCs. Aim 2 - To design a hydrogel that would provide sustained release of siRNA targeting p38 MAPK to prevent osteogenesis of MSCs. The p38 MAPK pathway has been linked to osteogenesis in various cell types, including MSCs. Local inhibition of this pathway by siRNA could prevent MSC osteogenesis from occurring after growth plate injury. For both Aims, these studies will be conducted in vitro and also in vivo in a rat growth plate injury model. This project is a first step towards successful development of a biomaterial system that can prevent MSC osteogenesis, which could ultimately aid in growth plate tissue repair."}
{"text": "The inhibitors of DNA or differentiation (Id proteins), members of the helix-loop-helix transcription factor family, act in a dominant negative fashion to control cell cycle and differentiation by dimerizing and inhibiting the function of the basic helix-loop helix (bHLH) transcription factors. We hypothesize that removal of 3 out of 4 copies of the Id1 and Id3 genes, and the resultant deficiency of Id, disrupts normal bHLH transcription factor regulation, thus allowing the formation of normally regulated or restricted protein-DNA complexes. The deregulation of bHLH transcription factors may result in the inhibited tumor-associated angiogenesis phenotype previously described in these mice. Therefore, the major goal of this study is to identify and characterize the protein-DNA complexes and the interactions that occur in spontaneous tumors as a result of the deficiency in Id and to determine changes in gene expression. To perform these studies, Id 3 out of 4 copies knocked out mice (Id1 +/ Id3-/-) will be crossbred with the p53-/- murine tumor model. The resulting mice will be a source of tissue that will be examined histologically and on a molecular and biochemical basis to identify novel interactions and gene expression that the absence of Id allows. These studies will help us understand the role of Ids in regulating tumor-associated angiogenesis and may identify new targets for use in blocking tumor vascularization and designing more specific anti-angiogenic drugs."}
{"text": "Hypertensive kidney disease commonly progresses. The primary objective of the AASK (African American Study of Kidney Disease and Hypertension) Cohort Study is to determine prospectively the course of kidney function and risk factors for kidney disease progression in African-Americans with hypertensive kidney disease who receive recommended antihypertensive therapy. A secondary objective is to determine the occurrence of cardiovascular disease and assess its risk factors. The AASK Cohort Study is a prospective, observational study that is an extension of the AASK trial. The AASK trial tested the effects on kidney function of 3 medications used as initial antihypertensive therapy (ramipril, metoprolol and amiodipine) and 2 levels of blood pressure control. Of the 1,094 trial participants, approximately 650 to 700 individuals who have not reached end stage renal disease (ESRD) will likely enroll in the Cohort Study. Risk factors to be studied include environmental, genetic, physiologic, and socio-economic variables. The primary renal outcome is a composite clinical outcome defined by doubling of serum creatinine, ESRD, or death. Medication treatment for hypertension, beginning with the angiotensin converting enzyme inhibitor ramipril, is offered to all participants. In this fashion, the study directly controls two of the major determinants of kidney disease progression (treatment of hypertension and use of reno-protective, antihypertensive medication). The minimum duration of follow-up in the Cohort Study is 5 years (total of 9 to 12 years, including the period of the AASK trial). Ultimately, data from the AASK Cohort Study should enhance our understanding of the determine the progression of kidney disease. Such results might eventually lead to new ESRD."}
{"text": "Prevalence of HIV-associated neurocognitive disorders (HAND) remains high despite the introduction of combined antiretroviral therapy. HAND is associated with elevation of pro-inflammatory factors in blood and subsets of activated infected monocytes, both shown to cause blood brain barrier (BBB) impairment that contributes to HAND. Therapeutic strategies that disrupt monocyte migration can slow progression of HIV infection and BBB injury, thereby ameliorating HAND. During the previous period of funding, we focused on studies of (1) molecular mechanisms of BBB injury and (2) anti-inflammatory and barrier protective properties of glycogen synthase kinase (GSK) 3? inhibition in neuroinflammation driven by HIV infection. We uncovered molecular mechanisms of BBB dysfunction [tight junction (TJ) phosphorylation, CD40/CD40 ligand interactions at BBB and signaling events behind the direct effects of HIV on brain endothelium]. We demonstrated barrier tightening following GSK3? suppression in brain endothelium due to TJ stabilization. We uncovered potent anti-inflammatory effects of GSK3? inhibition in brain endothelium (suppression of monocyte migration, diminution of inflammatory factor production, and BBB protection) in vitro and in vivo. GSK? inhibition in monocytes attenuated their adhesion/migration across the BBB, down regulated active integrin expression via suppression of the small GTPase, Rac1, and protected the BBB. Yet, BBB shielding properties or inhibition of monocyte migration/adhesion were not fully attained in vitro or in vivo, suggesting that additional pathways complimentary to GSK3? are necessary for the restitution of BBB function. In search of such molecules, we turned our attention to poly(ADP-ribose) polymerase-1 (PARP-1) and its inhibitors, recently recognized as anti-inflammatory/immune modulating agents with significant neuroprotective properties. Based upon preliminary data, we propose that PARP inhibition will attenuate BBB injury caused by HIV-1 via effects on monocytes, brain endothelium, activated microglia and HIV-1 infected macrophages. Indeed, preliminary data indicate that PARP suppression in primary human brain microvascular endothelial cells (BMVEC) improved BBB integrity and augmented expression of TJ proteins. PARP inhibitors prevented barrier disruption caused by inflammatory factors, diminished monocyte adhesion/migration across a BBB model, down regulated adhesion molecules/pro-inflammatory molecules and decreased activity of RhoA/Rac1. In monocytes, PARP inhibitors down regulated the active ?-integrin that paralleled RhoA/Rac1 suppression. PARP inhibitors decreased expression of pro-inflammatory molecules and diminished HIV replication in human macrophages. Although the modulatory effects of PARP inhibitors on immune cells have been studied to some extent, nothing is known about their effects in the setting of HIV CNS infection. PARP inhibitors have now reached the stage of clinical testing for cancer treatment, assuring quick translation to therapy of immune/inflammatory disorders."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project looks to expand the research that outlines the developmental trajectories of young adult and alcohol use and related outcomes. It also seeks to examine the effects of genetic and non-genetic factors on these trajectories."}
{"text": "Freeze-fracture electron microscopy has revealed organized sites of aggregated intramembranous particles in granular cell luminal membranes from toad urinary bladder after vasopressin (ADH) treatment. This response appears to be a primary hormonal effect which is reversible and quntitatively and specifically related to induced alterations in membrane water permeability. The proposed research is to investigate the aggregation response in terms of onset-offset mechanisms, to determine wether sites of aggregation are actual sites for water passage, and to determine the applicability of this to the mammalian kidney."}
{"text": "PROJECT SUMMARY Although genome-wide association studies (GWAS) have been extremely successful in identifying numerous germline variants associated to risk for schizophrenia and bipolar disorder, at the vast majority of these loci, the causal mechanism between genetic variation and disease risk remains unknown. This limits the development of novel drug targets and/or personalized treatments. Schizophrenia and bipolar disorder share many genetic risk loci thus motivating approaches to gain insights into the shared molecular basis of these two diseases. Post-GWAS studies are experiencing a ?big data? revolution driven by the exponentially decreasing costs of high-throughput genomic assays, including transcriptome levels, epigenetic modifications, and localization of tissue-specific regulatory sites, which are being collected in increasingly large cohorts of individuals. Here we propose a rigorous framework aimed at loci where shared or disease-specific risk for schizophrenia and bipolar disorder is mediated through alteration in gene expression levels, regulated via epigenetic control. To increase power for discovery while also facilitating validation of new findings, we will generate new disease-specific expression and chromatin variation data in subjects with known disease status. We propose to examine risk loci for schizophrenia and bipolar disorder to prioritize causal variants and genes and to validate them in functional assays."}
{"text": "Although posttranslational modifications of lens proteins play a causative role in biogenesis of human senile cataracts, surprisingly little is known concerning the identity of specific amino acids that are modified in these lenses. Nonenzymatic deamidation of asparagine and glutamine residues is probably the most prevalent modification occurring in the human lens, and studies from other tissues have suggested that resistance to deamidation is a general property of stable proteins. The central hypothesis of this proposal is that the aged human lens contains gamma/beta crystallins with very low rates of deamidation, and that if deamidation does occur, it could result in deleterious effects to the lens, such as the formation of high molecular weight aggregates, low molecular weight cleavage products, and eventual cataract. To identify and quantitate deamidation, we have developed a novel approach involving the use of synthetic peptide standards, HPLC, mass spectral analysis and N-terminal sequencing. This methodology will be used to identify and quantitate deamidation of specific residues from the high molecular weight aggregate and low molecular weight cleavage products, as well as total proteins from dissected nuclei of human cataractous and aged-matched normal lenses. Molecular modeling will be used to correlate known structural properties of crystallins, with their observed rates of deamidation. Finally, human gamma/beta crystallins containing specific residues found to be deamidated will be expressed in a recombinant system and characterized by various biochemical and biophysical methods. Together, this project will identify and quantitate deamidation of specific residues of gamma/beta crystallins in the aging normal and cataractous human lens, followed by determination of the consequences of these modifications upon the stability and structural properties of the expressed deamidated proteins."}
{"text": "The goal of this project is to show that sphincter prosthesis placement is an alternative treatment to external sphincterotomy in patients with spinal cord injury (SCI). The sphincter prosthesis consists of a surgical alloy wire mesh prosthesis inserted transurethrally under endoscopic control and is intended to hold the external sphincter open permanently. This project is designed to test the hypotheses that: 1) Sphincter prosthesis placement is as effective as external sphincterotomy for detrusor sphincter dyssynergia (DSD) based on urodynamic parameters of voiding pressure, residual urine volume, and bladder capacity. 2) Sphincter prosthesis placement has less complications than external sphincterotomy of bleeding, erectile dysfunction, autonomic dysreflexia, and reobstruction. 3) Sphincter prosthesis is associated with shorter length of surgery, shorter length of hospitalization, and reduced hospitalization costs than external sphincterotomy. Surgical alternatives to external sphincterotomy and improved treatment of DSD has not been examined in a randomized controlled manner. A total of sixty SCI patients with DSD will be entered into a prospective randomized study between stent placement and external sphincterotomy at three centers: 1) Regional Spinal Cord Injury Center of Delaware Valley (RSCICDV), Jefferson Medical College, Philadelphia, PA, 2) Ranch Los Amigos Medical Center, Downey, CA, and 3) Spinal Cord Injury Service, Department of Veterans Affairs, Palo Alto, CA. Urodynamic studies and testing will be done prior to surgery and at 1, 3, 6 and 12 months post-surgery. The protocol will provide a direct comparison of effectiveness between treatment of DSD patients with a sphincter prosthesis and transurethral sphincterotomy, the currently accepted surgical treatment. The short- term health related benefits of this project are to improve management of the dysfunctional bladder, reduce complications such as bleeding, impotence, urinary infection, multiple operations, and to reduce health care costs. The long-term benefits are to enhance the ability of subject population to more fully participate in regular life activities including school and work environment and to avoid long-term urological complications and death from renal failure and urosepsis."}
{"text": "To date, there is insufficient empirical evidence exploring the long term health consequences of childhood sexual abuse on adulthood reproductive health. Although negative health behaviors subsequent to the abuse and mental health correlates have been identified, the future health consequences are not as well understood. The purpose of this dissertation is to investigate the long term reproductive health outcomes associated with childhood exposures to physical, sexual, and emotional abuse. In order to examine this relationship, an observational dataset of a large cohort of female nurses, Nurses Health Study II, is being explored to see whether a relationship exists between childhood maltreatment and adult pregnancy outcomes, such as, preterm delivery and spontaneous abortion. Furthermore, the relationship of psychopathology to reproductive outcomes will be explored, by assessing the relationship between post-traumatic stress disorder (PTSD) symptoms with these pregnancy outcomes. Utilizing a life course model of health linking childhood exposures to adult health outcomes, this research will help explore the potential role of childhood abuse as an exposure with repercussions that last far beyond the immediate period of childhood and adolescence. There is a persistent and growing need for research to provide empirical evidence with regards to the possible long term health impacts related to childhood maltreatment. This research can be used to support the promotion of high quality care to meet the needs of children who have suffered maltreatment, as well as increasing awareness of the need for prevention services that will be of benefit not only to children, but may also serve to decrease the burden of disease across the life course. PUBLIC HEALTH RELEVANCE: This research will investigate whether childhood abuse increases risk for poor pregnancy outcomes, and the possible role of mental health and health behaviors in the development of these conditions. This research would be important to increasing awareness among researchers, clinicians, and policy makers as to the long term consequences of childhood abuse, as well as beginning to address how these experiences might lead to poor health."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In addition to the offered training of biomedical research investigators the Resource takes every opportunity to disseminate information regarding its function, capabilities and accomplishments. Major dissemination approaches range from the publication of scientific publications and our Newsletters, active involvement and participation in symposia and workshops, to regular seminars in areas relevant to the Resource. An up-to-date web page additionally has the objective to attract new users as well as inform prior users about new developments at the Resource. The training of medical research investigators in research, technology, use of laser instrumentation and data analysis related to spectroscopic experiments is another important goal of this Resource. By offering an integrated chemical-physics and biomedical environment, visitors, post-doctoral fellows and graduate students gain a wide range of expertise in the application of laser spectroscopy methods to biomedical research questions."}
{"text": "EVALUATION OF ELECTRODE MATERIALS"}
{"text": "The dramatic increase in the use of interventional spine procedures and the rising health care costs over the past decade have raised lots of questions about the utilization and outcomes of treatments for back and neck pain. Several articles have addressed some concerning scenarios related to spinal surgeries and catastrophic adverse events after epidural steroid injections have been reported, related to steroid use. The purpose of this study is to describe and characterize the treatment of back and neck pain, while examining temporal and geographical trends, physician specialty, and setting variations. We will characterize treatment trajectories in newly diagnosed back and neck pain patients, and identify efficient treatment paths and potential gateways to high cost and utilization. The characterization of patients treatment paths will include tracking of manual and percutaneous treatments, use of opioids, imaging utilization, surgical procedures, and safety events. We will examine the outcomes and adverse events following interventional spinal procedures by tracking patients for at least one year after their procedures. Identifying efficient and safe treatment paths for back pain will lead to better guidelines for back pain management. A characterization of the outcomes and side effects of interventional spinal procedures will help patients and physicians make more informed decisions about treatment options. Data This project currently focuses on Medicare administrative health records for patients diagnosed with back pain between 2000-2011. A contract to access this data has been established between NIH and CMS. Since the previous annual report, we have achieved the following: Renewed our seat for access to a CMS virtual research data center (VRDC) that allows the analysis of CMS data in SAS, on CMS servers. Produced preliminary results based on a subset of the data of interest and evaluated the feasibility of future work. Presented preliminary results at academic conferences and meetings. Submitted a data addendum request to CMS, who approved the proposal. Obtained access to CMS Carrier, Inpatient, Outpatient, and Part D files for the years 2000-2011 for almost 47 million patients with diagnoses of back pain. Started collaborations with academic partners. Identified codes of diagnoses, procedures, drugs, complications, and outcomes relevant to our projects. Additional data runs have been conducted on the updated dataset and cohort that focus on patient demographics and numbers of injections over the period under study. Differential complication rates by steroid type and injection approach are being examined. Preliminary results have been presented at Grand Rounds style conferences."}
{"text": "Abstract: Unexplained medical symptoms in functional medical illnesses present a challenging diagnostic dilemma for physicians. These conditions usually involve pain or pressure together with other symptoms which suggest a combination of biological and unexplained medical factors. Interstitial cystitis (1C), more recently labeled Painful Bladder Syndrome (PBS), is a prime example of this type of disorder. Recently, intense interest has focused on the psychological/psychiatric components of these disorders. In particular, our team has demonstrated that somatization disorder, when present, may influence the course and management of these functional medical syndromes. Patients with somatization disorder disproportionately consume clinicians'time and medical resources. Thus, understanding the role of somatization disorder in IC/PBS has the potential to yield clinical and economic benefits. In the present study, 120 patients with IC/PBS will be assessed for physical and psychological symptoms, medically unexplained symptoms, and somatization disorder, and they will also monitored over 12 months for temporal symptom patterns and outcomes. The study will demonstrate the proportion of patients with IC/PBS who also have somatization disorder and the degree to which these patients represent a significantly distinct IC/PBS subgroup with different illness presentation, progression, and outcome. Previous research by this team from similar studies of Irritable Bowel Syndrome (IBS) has generated highly useful information and will serve as a template for this research. The comparability of the findings of this study to previous work on IBS will help guide recommendations for modification of pharmacological and psychological interventions for this patient population. Recognition and appropriate management of somatization disorder in patients with IC/PBS has the potential to benefit a substantial proportion of patients with IC/PBS and inform future research. This proposed study truly investigates the interface of medicine and psychiatry in its approach to these disorders."}
{"text": "In the current project, genetic determinants of type 2 diabetes mellitus and obesity are being sought using techniques of genetic linkage and association analysis. Lymphoblast cell lines have been established from informative pedigrees. DNA is available from other families in nuclear pellets extracted from blood specimens obtained in the epidemiologic studies and is amplified by whole genome amplification when needed. An autosomal genome-wide linkage study identified strong evidence for a locus influencing diabetes and diabetes/obesity on chromosomes 1q and 11q. Efforts to identify the causative polymorphism or polymorphisms in both of these regions are currently underway using both a systematic analysis of linkage disequilibrium and analyses of candidate genes. Genome-wide association mapping methods are also being used and exhaustive association analyses are being conducted of regions identified by the genome-wide association studies and of other candidate genes. Whole genome sequencing studies are also being pursued. Genetic variants in the trehalase gene, which is in a region linked to diabeteson chromosome 11q, are strongly associated with plasma trehalase activity and one of these variants is also consistently associated with diabetes. Several candidate genes that have been associated with type 2 diabetes in other populations have been evaluated. The majority of genes seen in other populations appear to have consistent effects in Pimas, though only a few show statistical significance. Some variants identified in other populations (e.g. TCF7L2) appear to have little effect. Variants in established type 2 diabetes genes, MOB2, KLF14 and KCNQ1, are subject to parent-of-origin effects and these parent-of-origin effects replicate in Pimas. The effect of the KCNQ1 variants is particularly strong; with consideration of the parent-of-origin effect these variants account for 4% of liability in susceptibility to diabetes. Recently genome-wide association studies initially with 100,000 markers and later with 1,000,000 markers have identified several additional potential susceptibility genes for young-onset diabetes and for obesity. These include potential diabetes-susceptibility variants in a number of genes and potential obesity-susceptiblity variants in SIM1 and MAP2K3. Replication studies in larger sample sizes are planned, as are functional studies. Variants in candidate genes, such as LEPR, LPGAT1 and SIRT1 show nominally significant association with diabetes, obesity and related traits in Pimas; rare variants in MCR4 are also associated with obesity particularly in childhood. Currently fine-mapping studies with additional variants are being conducted to extract more of the genetic information in regions identified as potentially involved in diabetes susceptibility. Through collaborations, studies are being conducted to determine if any of the signals identified in the present mapping studies replicate in other populations. Variants reproducibly associated with type 2 diabetes from other populations are also being typed to determine their role susceptibilty to diabetes and obesity in the Pimas. Whole genome sequencing is also being conducted in a small number of participants. The data from these studies will initially be used to impute untyped variants from the genome-wide association data; data from these studies suggest that existing reference panels are not accurate for imputation in American Indians. Additional American Indian participants are being recruited for replication studies."}
{"text": "The detailed mechanisms underlying the fundamental membrane events involved in the visual process will be investigated at the sub-molecular level by x-ray and neutron diffraction and nuclear magnetic resonance and optical spectroscopy. These studies center initially on a determination of the dynamical structure at the sub-molecular level of the rhodopsin-containing retinal receptor outer segment disk membrane and the nerve axonal membrane. The dynamical structure of the natural and structurally modified membranes together with their various structurally modified functionally relevant model membranes will be studied \"at rest\" and during the time course of their natural or artificially perturbed visual function. \"Functionally relevant\" here refers to model membranes which exhibit selective light-modulated, chemical-modulated and electric field-modulated ion binding or permeability. The detailed mechanisms will primarily be determined through a correlation of systematic time-independent and time-dependent dynamical-structural variations of the membranes with their resulting modified functionality."}
{"text": "Since our neurodegerative disease research group received COBRE grant support we have administered 5 separate research pilot grant programs and provided financial support to investigators totaling in excess of $1,000,000. The purpose of the pilot grant program has been to increase the scope of research conducted at UND related to the theme of the COBRE grant and to increase the competitiveness of UND investigators for funding from extramural organizations. Resulting from this investment in research pilot grant support, the recipients brought into the institution over $5.2M. Thus, pilot grants have been an excellent investment in the on going success of our COBRE grant supported neurodegenerative disease research group. The following is a brief description of the research pilot grant program that we are proposing for the COBRE Phase III funding period. First, we will solicit proposals for our research pilot grant program. Second, the applications will be submitted electronically to the PI/PD. Third, the grants will be distributed to the grant review committee consisting of Dr. Geiger (PI/PD), Drs. Vaughan and Brown-Borg of our Internal Advisory Committee, and Dr. Fyffe of our External Advisory Board. Fourth, the reviewers will meet with the applicants to discuss the hypothesis proposed, the methodological and scientific approaches to be taken, the respective involvement of named investigators, and their timelines and benchmarks of success. We have found that this; site visit approach provides to the applicants a very high quality review and an outstanding opportunity for mentorship. Fifth, midway through the pilot grant year investigators will submit a progress report and then again meet with the grant review committee to evaluate progress, receive advice, and review plans for the remaining one-half of the pilot grant year. At the end of the pilot grant period, the investigators will submit to the review committee a final report in which they describe their original or modified specific aims, their research findings, and most importantly detailed information about abstracts submitted and presented, manuscripts submitted and published, and grants submitted and funded."}
{"text": "Training and Career Development The effective replication and spread of evidence-based practices in cancer communication across and within healthcare systems will be facilitated if healthcare providers and researchers understand not just the practices of better patient-provider communication but also underlying ideas of translational knowledge-toaction processes and cycles.193 In the organizational studies literature, such objectives have been termed double-loop learning as opposed to just single-loop learning; that is, understanding the reasons why change efforts can be effective and not only that certain efforts are effective or not. Of course, teaching principles in addition to how-to knowledge is the basis of pedagogy in the service of enlightened life-long learning. In the Training and Career Development purpose of the CRN Clinical Communication Research Center, we will help targeted audiences with learning of both types: How-to knowledge and principles knowledge. We identify the following targeted audiences for proactive dissemination of research results and practical tools: > Second-year doctoral students from communication and public health programs; > Post-doctoral cancer research scholars; > Healthcare practitioners and researchers in Cancer Research Network institutions; > Healthcare practitioners nationally through three professional societies. In addition, we will also disseminate research results and practical tools through the traditional channels of conference presentations, peer-reviewed publications, and hosting of a special issue of a peer-reviewed journal."}
{"text": "Modified Project Summary/Abstract Section PROJECT SUMMARY FAM57B is an autism risk gene, and part of the 16p11.2 disease locus. 16pdel syndrome is a severe and prevalent (1:2000 people) haploinsufficient disorder, caused by deletion of ~600 kb of chromosome 16. This syndrome is tightly associated with autism, language and intellectual disability, seizures, ADHD, hypotonia and obesity, however genetic contributions to each symptom are unclear. Our proposal addresses the novel hypothesis that alteration of FAM57B function is associated with autism and 16pdel syndrome, by changing cellular lipids of the ceramide pathway. Lipid cohort alteration may change neuronal plasma membrane composition and associated proteins, so altering synaptic activity and contributing to 16pdel symptoms. We defined a 16p11.2 functional interactome, and identified FAM57B as a pivotal ?hub? gene (McCammon et al. 2017). FAM57B has been additionally identified as an autism risk gene (Satterstrom et al. 2019). FAM57B contains a TLC domain found in ceramide synthase enzymes (CerS), but residues required for CerS activity are absent. Loss of function of fam57b in the zebrafish model led to significant changes in brain lipid species. Strikingly, we also observed altered plasma membrane architecture, mis-localization of synaptic proteins, depressed movement and decreased brain activity. The Aim will determine the role of FAM57B in ceramide synthesis and its impact on neuronal and brain function. We hypothesize that FAM57B regulates ceramide levels by interacting with CerS. We further hypothesize that FAM57B maintains the synaptic lipid and protein cohort contributing to neurotransmitter exocytosis. We will determine activity of FAM57B human and zebrafish homologues; delineate molecular changes at the neuronal synapse after loss of FAM57B and determine activity of fam567b in the zebrafish brain. This Aim will solve the function of FAM57B, giving insight into mechanisms underlying autism and 16pdel syndrome phenotypes."}
{"text": "Through the proposed training in his K01, I will pursue the additional mentorship that I need and the additional research time to enhance my passion for intestinal epithelial biology. During this award period I will have the opportunity to acquire and refine fundamental skills of becoming a developed scientist. Career development will be fostered through twice-weekly meetings with Dr. Rustgi, convening an expert interdisciplinary advisory committee twice annually, and taking advantage of opportunities in career and professional development through UPenn, the NIH and the AGA. These skills include (but are not limited to) manuscript and grant preparation, lab meeting presentations, participation in the seminars and courses described herein, and presentations at international research conferences (DDW, Keystone). Training will also entail course-work in biostatistics (two semesters), short courses in RNA biology and basic bioinformatics, ongoing training in bioethics, weekly participation in the seminar series (where I will meet visiting professors), journal clubs (presentations semi-annually), and twice yearly floo lab meetings through the NIDDK P30 Center for Molecular Studies in Digestive and Liver Diseases and Division of Gastroenterology. The overarching hypothesis of this project is the following: LIN28B plays a critical role in regulating growth and proliferation in the intestinal epithelium via cooperation with c-MYC, with key roles for intestine stem cell (ISC) maintenance. Two potential interrelated mechanisms will be explored: 1) LIN28B binds to target mRNAs associated with cellular growth and cooperates with the transcription factor c-MYC, which is a master regulator of cell growth. and 2) LIN28B affects the maintenance of the ISC by promoting stem cell identity and growth. LIN28B is normally expressed in the intestinal epithelial crypt where cell division occurs. LIN28B represses the maturation of miRNAs such as Let-7, which is restricted to villus epithelium, where post- mitotic differentiated cells are located. The spatial restriction of LIN28B and Let-7 expression in the intestinal epithelium is likely crucial for maintaining sufficient but limited compartments of differentiation and proliferation. C-MYC is also restricted to the crypt epithelium and is required for epithelial proliferation and growth. W have evidence to support a Let-7-independent function of LIN28B entailing intimate cooperation with c-MYC. The Specific Aims of this proposal to test the hypothesis are the followin: 1) We will explore the functional relationship between LIN28B and c-MYC, in vitro. 2) We will assess how LIN28B cooperates with c-MYC through in vivo studies of Lin28b in the intestinal epithelium. 3) We will examine the potential of LIN28B to modulate the intestinal stem cell compartment. LIN28B function may be relevant to ISC homeostasis and epithelial proliferation. Determining how LIN28B functions in the intestinal crypt will likely provide an important link among the cellular mechanisms governing epithelial proliferation, differentiation, and/or mucosal regeneration. PUBLIC HEALTH RELEVANCE: This project will explore the regulation of cell growth in the lining of the intestine in stem cells and other dividing cells. Stem cells are required to constanly replenish cells of the intestinal lining throughout the entire life of an individual. This study wll provide key insights into how intestinal cell growth is maintained, controlled, and perturbed in disease states."}
{"text": "The primary means by which nerve cells communicate with each other is through the release of neurotransmitter at chemical synapses. The ability of the brain to process information depends on synaptic connections forming precisely and reliably between many different types of neurons. This proposal is directed towards developing a molecular understanding of the signaling between synaptic partners that regulate synaptogenesis. It is well established that even in simple metazoans like the worm C. elegans changes in synaptic activity induce compensatory changes in synaptic strength and structure. We propose to use a combination of genetics, cell biology, molecular biology and live imaging to identify and characterize the role of molecular components of the signaling pathways that coordinate synaptic development at nerve-nerve synapses. First, we aim to describe the order of cellular events in nascent synapse formation by visualizing the recruitment of fluorescent-tagged components to newly forming synapses. We will define the order in which mitochondria, synaptic vesicles, active zone components and adhesion molecules appear at synaptic sites. We will also define the cellular mechanisms that mediate subsequent growth of the presynaptic specializations. Second, we will define the role of novel molecular components that were identified as mutants that fail to form synapses. Using a variety of molecular, genetic and protein interaction studies we will position the genes within the current molecular models of synapse assembly. Third, we will use genetic approaches to isolate and characterize genes which disrupt signaling between mechanosensory neurons and their synaptic partners in C. elegans using a novel synaptic tag which can be easily detected in live animals under a fluorescent dissecting scope. Together these approaches will help define mechanisms that cells use to identify and communicate with one another during the process of synapse formation and synaptic maintenance. While synaptogenesis is undoubtedly less complex in C. elegans than in vertebrates, it is already clear that similar pathways operate in both systems. Thus, analysis of the molecules participating in the process in C. elegans should help define a set of general and likely conserved principles that are common to synaptogenesis mechanisms in general. PUBLIC HEALTH RELEVANCE: Synaptic connections are the primary neuronal communication structures in the brain. In Alzheimer's disease, it is now well established that changes in synaptic density (i.e. loss of synaptic connections) correlate better with cognitive impairment that the hallmark plaque and tangle lesions that are also associated with the disease. Our work is focused on understanding how synaptic connections are formed. Such basic scientific understanding of brain development and function will aid in developing therapies that intervene early in disease hence slowing or arresting synaptic loss."}
{"text": "The purpose of the Scientific Core is to make a data set rich in information about substance use, risk and protective factors, and other variables readily available to the five individual research projects in the proposed Center, so that important questions about drug use can be addressed by means of state-of-the-art methodological procedures. From 1986 to 1992 the NIAAA-funded Adolescent Alcohol Prevention Trial (AAPT) study collected data on substance use, risk factors, and numerous other variables on four cohorts of Southern California schoolchildren (approximately 12,000). Data were collected annually during the junior high and high school years. The AAPT participants are now entering young adulthood. The result will be a cohort-sequential data set on individuals ranging in age from ten to approximately 19. This data set will provide an exciting opportunity for the five individual research projects to examine risk for substance abuse across adolescence."}
{"text": "Experiments are proposed to investigate the role of abnormal early experience in producing abnormal neural pathways linking the left and right hemispheres of the brain. The interhemispheric pathway matures quite late during development, and its pattern of development is particularly interesting. There is an initial over-production of connections (mistakes?) followed by a gradual narrowing of the zone containing neurons which send axons to the opposite hemisphere. This narrowing process can be altered when there is unusual experience, resulting in persistence of developmental mistakes, which could provide a substrate for abnormal behavior. We will use anatomical techniques involving retrograde transport of horseradish peroxidase and anterograde transport of isotope to address a number of questions related to the role of abnormal experience in producing abnormal interhemispheric pathways. We will use a model system chosen to allow complete control of early experience. In addition, the anatomy and physiology of the interhemispheric pathway are well understood in this system. The goal of the experiments is to increase our understanding of the link between abnormal early experience and abonormal behavior. It has been known for many years that abnormal experience in childhood can result in severe mental health problems in adulthood. Since abnormal experience can result in retention of developmental mistakes in brain connectivity, this link may form a structural basis of many psychiatric disorders. Interhemispheric pathways may be particularly vulnerable to abnormal early experience. Indeed, recent computed tomography studies of schizophrenic patients have shown that they have a high incidence of abnormal hemispheric specialization. The proposed experiments will explore the circumstances and mechanism for retention of developmental mistakes in interhemispheric connectivity resulting from abnormal early experience."}
{"text": "Two approaches were taken for cloning the gene for type IV collagenase from a tumor cell cDNA library. A human melanoma (A2058) gamma gtll library (custom made for Dr. Liotta by Clontech Co.) was first screened with a polyclonal antibody against type IV collagenase. The collagenase was purified from culture supernatant of the A2058 cell line which was used to make the cDNA library. The second approach was to screen the melanoma library with a mammalian collagenase cDNA clone (supplied by C. Brinkerhoff). After the first screening with the antibody, 18 clones were isolated, of which four were plaque purified after 4-5 rounds of screening. All four clones produced clear plaques, which indicates an insert-bearing phage, but failed to demonstrate an insert by restriction analysis; the restriction pattern was identical to that of a wild type gamma gtll phage DNA. Likewise, negative results were obtained when the same melanoma cDNA library was screened with the mammalian collagenase clone. Six clones which were strongly positive through four rounds of screening turned out to produce no-recombinant blue plaques without an insert as judged by restriction analysis of phage DNA. Since there may be problems with the melanoma library, we will start the cloning process all over again using another tumor cell cDNA library. Presently, we are beginning to focus our attention on the role of endothelial cells in metastasis. Therefore, we are in the process of screening a human endothelial cDNA library, purchased from the Clontech Co. Using the mammalian collagenase cDNA clone under low stringent conditions, we expect to pull out mammalian collagenase and other related genes, expressed by endothelial cells."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The ARF-p53 tumor suppressor pathway is one of the cell's major defenses against the stimulation of uncontrolled cell division induced by activated cellular and viral oncogene. ARF and/or p53 are mutated in over 70% of human cancers. The inappropriate activation of growth promoting cellular signaling pathways by oncogenes can result in the induction of ARF. The expression of ARF can activate p53 leading to apoptotic cell death or cell cycle arrest. The mechanisms by which the ARF-p53 pathway is regulated remains to be precisely elucidated. The expression of ARF can activate p53 leading to apoptotic cell death or cell cycle arrest. We have shown that the polyoma virus oncogene, PYMT, activates an ARF-induced p53 mediated block. We find that the polyoma virus small T-antigen, PYST, via its ability to bind to cellular protein phosphatase 2A (PP2A), can negate the ARF-induced block to cell division induced by PYMT. We intend to use the PY induction and inhibition of ARF signaling to p53 to better define this important tumor suppressor pathway. Our hypothesis is that the polyoma virus proteins are revealing an important new aspect of the ARF-p53 tumor suppressor signaling circuit, and we plan to use these viral proteins as tools to study its molecular basis. To better define the role of PYMT in activating ARF and as an oncogene, and to define the role of PYST in blocking ARF signaling to p53 we plan to characterize the proteins complexed to PYMT, PYST and members of the ARF-p53 signaling pathway. In the first instance proteins bound to TAP fusion constructs would be identified by Mass Spectrometry."}
{"text": "The Pilot Core (PC) will work with the Management and Administrative Core (MAC) to support small-scale and innovative pilot projects that are consistent with the overall theme of the Northwest Roybal Center (NRC). Specifically, NRC pilot studies will focus on innovative approaches to improve the health, wellbeing, quality of life and productivity of older adults with cognitive impairment, midlife and older adults at risk for cognitive impairment, and their caregivers through the translation of basic behavioral and social sciences research. Furthermore, the PC will work with the MAC to ensure that each Pilot Study: (1) addresses one or more of the thematic areas of focus identified in the current FOA, (2) moves basic behavioral and biobehavioral research toward the ultimate goal of translation, and (3) leads to additional, larger research efforts to further implement translation activities into a program or product. Finally, the PC will work with the MAC to involve both junior and established researchers from a variety of disciplines in the work of the NRC, and will foster interaction and collaboration among the pilot investigators and outside agencies and corporate partners."}
{"text": "Although we probably know more about the genetics of Drosophila then any other multicellular animal, we do not understand how genes control the development of any organ. The logic behind gene deployment will become more evident once we start classifying genes into those with controlling roles and those carrying \"executive\" functions. Homoeotic mutants are the prime candidates for controlling elements in Drosophila. These mutants transform one body part into a different body part and represent single loci which determine entire developmental pathways taken by groups of cells. Recently it has been discovered that specific groups of cells are set aside at early stages of development, and their descendants form very specific regions referred to as \"compartments\". Evidence suggests that these compartments are under the control of homoeotic genes. Until recently most evidence for compartments was derived by examination of adult cuticle. We propose (1) to expand our mapping of developmental compartments within the imaginal discs by using mitotic recombination (Minute technique); (2) to map by histochemical techniques the distribution and Compartment affiliation of isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in normal and homoeotically transformed imaginal disc tissue; (3) to adapt recently developed procedures for use in establishing enzyme, protein and/or polypeptide profiles for transplanted differentiating normal and homoeotically transformed imaginal tissue; and (4) to study the transdetermination characteristics of homoeotically affected imaginal disc tissue and to record the biochemical changes occurring at each step of the transdetermination process."}
{"text": "The HIV-envelope protein gp120 plays a critical role in triggering the apoptosis of T-lymphocytes, which is a central pathogenic feature of HIV-mediated immune dysfunction. T-cell apoptosis is also a physiological process that regulates antigen receptor repertoire selection during homeostasis and the maturation of T- cells. We hypothesize that gp120-induced cross-talk between T-cell receptor (TCR) and chemokine receptor signaling pathways leads to apoptosis. Our preliminary data on the role of the TCR signaling components CD45 and SLP-76 in HIV-gpl20-induced apoptosis support this hypothesis. In addition, we have recently shown that cross-talk between TCR and chemokine receptor CXCR4 regulates apoptosis via a novel mechanism that is mediated by AKT/Protein Kinase B (PKB), heat shock protein-70 (HSP-70), caspase-1 and RIP2 (caspase recruitment domain-containing serine/threonine kinase). To analyze mechanistically how the TCR components and chemokine receptors CXCR4/CCR5 regulate gp120-induced apoptosis, we will pursue the following specific aims: Aim 1) we will assess the interaction between CD45 and CXCR4/CCR5 signaling molecules by defining the domain of CD45 and characterizing the CD45 signaling responsible for apoptosis. Aim 2) we will perform structural and functional analyses of the TCR-mediated downstream effector SLP-76 that mediates Ca2+dependent apoptotic pathways. Aim 3) we will analyze the role of the AKT/HSP-70 apoptotic pathway that may lead to the induction of forkhead transcription factors, and will characterize the activation of caspase-1 and its regulation by RIP2. We are also exploring innovative strategies to inhibit gp120-induced apoptosis. In this regard, we have shown that a novel protein, Slit, which binds to the Robo receptor and modulates CXCR4 function can inhibit gp120-induced apoptosis. Aim 4) we will identify which domains in the Slit/Robo complex regulate the observed anti-apoptotic effects. These studies are designed to identify apoptotic signaling molecules and effector pathways involved in T-cell loss, and thus to provide novel therapeutic targets to combat immune deficiency in AIDS. [unreadable] [unreadable] [unreadable]"}
{"text": "This study is being done in order to determine safety and potential beneficial effects of a new investigational drug, CP-424,391, in patients with congestive heart failure. CP-424,391 may have value for the treatment of congestive heart failure by causing increased release of a natural hormone in the body, growth hormone. Increasing growth hormone levels could improve the pumping of the heart and thus help the condition. This study will be the first use of CP-424,391 in patients with congestive heart failure. Because this is a research study, CP-424,391 will be available to the patient only during their participation in this study and not after."}
{"text": "New gene expressed in prostate (NGEP) is a prostate-specific gene encoding either a small cytoplasmic protein (NGEP-S) or a larger polytopic membrane protein (NGEP-L). NGEP-L expression is detectable only in prostate cancer, benign prostatic hyperplasia and normal prostate. We have identified an HLA-A2 binding NGEP epitope (designated P703) which was used to generate T cell lines from several patients with localized and metastatic prostate cancer. These T cell lines were able to specifically lyse HLA-A2 and GEP-expressing human tumor cells. NGEPP703 tetramer binding assays demonstrated that metastatic prostate cancer patients had a higher frequency of NGEP-specific T cells when compared with healthy donors. Moreover, an increased frequency of NGEP-specific T cells was detected in the peripheral blood mononuclear cells of prostate cancer patients post-vaccination with a PSA-based vaccine, further indicating the immunogenicity of NGEP. These studies thus identify NGEP as a potential target for T cell-mediated immunotherapy of prostate cancer. Tumor-associated antigens are weakly immunogenic. Human carcinoembryonic antigen (CEA) is overexpressed on a wide range of human carcinomas and represents an attractive target for cancer immunotherapy. A concurrent multicenter, randomized Phase II trial employing a recombinant poxviral vaccine provided evidence of enhanced median overall survival (OS) (p = 0.0061) in patients with metastatic castrate-resistant prostate cancer (mCRPC). The study reported here employed the identical vaccine in mCRPC to investigate the influence of GM-CSF with vaccine, and the influence of immunologic and prognostic factors on median OS. Thirty-two patients were vaccinated once with recombinant vaccinia containing the transgenes for prostate-specific antigen (PSA) and three costimulatory molecules. Patients received boosters with recombinant fowlpox containing the same four transgenes. Twelve of 32 patients showed declines in serum PSA post-vaccination and 2/12 showed decreases in index lesions. Median OS was 26.6 months (predicted median OS by the Halabi nomogram was 17.4 months). Patients with greater PSA-specific T-cell responses showed a trend (p = 0.055) toward enhanced survival. There was no difference in T-cell responses or survival in cohorts of patients receiving GM-CSF versus no GM-CSF. Patients with a Halabi predicted survival of less than 18 months (median predicted 12.3 months) had an actual median OS of 14.6 months, while those with a Halabi predicted survival of greater than or equal to 18 months (median predicted survival 20.9 months) will meet or exceed 37.3 months, with 12/15 patients living longer than predicted (p = 0.035). Treg suppressive function was shown to decrease following vaccine in patients surviving longer than predicted, and increase in patients surviving less than predicted. This hypothesis-generating study provides evidence that patients with more indolent mCRPC (Halabi predicted survival greater than or equal to 18 months) may best benefit from vaccine therapy. Adenoviral transduction with CD40L and poxviral transduction with B7-1, ICAM-1, and LFA-3 (TRICOM) have been used to enhance the antigen-presenting capacity of chronic lymphocytic leukemia (CLL) cells. This study compares the same vector (modified vaccinia virus strain Ankara (MVA)) encoding CD40L or TRICOM for its ability to enhance the immunogenicity of CLL cells. CLL cells from some patients showed differential responses to each vector in terms of induction of autologous T-cell responses. This study supports the rationale for the use of CLL cells modified ex vivo with pre-specified recombinant MVA vectors as a whole tumor-cell vaccine for immunotherapy in CLL patients. In chronic lymphocytic leukemia (CLL), malignant B cells and nonmalignant T cells exhibit dysfunction. We previously demonstrated that infection of CLL cells with modified vaccinia Ankara (MVA) expressing the costimulatory molecules B7-1, ICAM-1, and LFA-3 (designated TRICOM) increased expression of these costimulatory molecules on the surface of CLL cells and thus augmented their antigen-presenting capability. Here, we evaluate the effect of MVA-TRICOM-modified CLL cells on T cells. Following incubation with irradiated MVA-TRICOM-modified CLL cells, allogeneic and autologous CD4(+) and CD8(+) T cells expressed significantly higher levels of B7-1, ICAM-1, and LFA-3. We show that this increase was the result of physical acquisition from the antigen-presenting cells (APCs), and that purified T cells that acquired costimulatory molecules from MVA-TRICOM-modified CLL cells were able to stimulate the proliferation of untreated T cells. These results demonstrate for the first time that T cells from CLL patients can acquire multiple costimulatory molecules from autologous CLL cells and can then act as APCs themselves. Given the immunodeficiencies characteristic of CLL, enhancing the antigen-presenting function of CLL cells and T cells simultaneously could be a distinct advantage in the effort to elicit antitumor immune responses."}
{"text": "These experiments are designed to determine whether adolescents at risk for substance abuse (SA) exhibit impaired cognitive performance on a task that reveals a deficit in performance by adults with a history of SA. Abnormal cognitive performance in adults after long-term exposure to drugs of abuse can reflect either vulnerability to SA (primary effect) or drug effects on the brain (secondary effect). This study tests the hypothesis that a predisposing cognitive deficit in adolescents contributes to risk for substance abuse. To date, 23 adolescents completed the initial phase of the study. Adolescents at risk for SA showed worse performance on a Gambling task (GT), which targets impulsivity and judgment and depends on orbitofrontal cortical function, and on the Wisconsin Card Sorting Task (WCST), which targets executive function and activates the dorsolateral prefrontal cortex in healthy control subjects. Task performance on the WCST was correlated predominantly with global functioning, a measure of psychiatric symptom severity, and performance on the GT correlated with antisocial and aggressive behaviors. Although both tasks differentiated adolescents at risk from controls, the WCST may be less specific to a neurobiological vulnerability for substance abuse because it relates more to psychiatric status. In contrast, the GT task may be more relevant for substance abuse problems as it is influenced by aggressive and antisocial behaviors, which are known to predict later substance abuse. These results are preliminary and await confirmation by studying larger samples, and assessing directly the significance of the findings in follow-up data. - adolescents, cognitive performance, gambling task, wisconsin card sorting task, substance abuse - Human Subjects: Interview, Questionaires, or Surveys Only"}
{"text": "We shall continue to analyze the ontogeny of thymocyte and T cell responsiveness in both neonatal mice and in radiation chimeras. The loss of thymocyte function with age will also be rigorously examined. Particular emphasis will be placed on determining whether or not suppressor T cells are playing a regulatory role in the thymus of both chimeric and normal, aging mice. Preliminary experiments with cyclophosphamide indicate that this will be a useful approach. The responder profiles of T cells and thymocytes from neonatally tolerized (to H-2 angigens) baby mice will be explored in detail. All experiments will utilize the vaccinia virus and influenza virus cytotoxic T lymphocyte systems."}
{"text": "It is well known that increasing an individual's exercise endurance can ameliorate multiple disease processes. We have identified possible quantitative trait loci (QTL) on Chromosomes 2, 8,13, and X that appear to be involved in the genetic regulation of maximal exercise endurance. Therefore, the overall objective of this proposal is to determine the specific genes involved in the control of inherent exercise endurance. This objective will be fulfilled through the following Specific Aims: 1) To complete a genetic linkage analysis and generate high-resolution linkage maps of the high exercise endurance (HEE = Balb/cJ) and low exercise endurance (LEE = DBA/2J) phenotypes; 2) To examine the mechanisms that confer differential maximal exercise endurance phenotypes, we will develop congenic strains of mice that contain the candidate regions from Specific Aim 1; 3) To characterize the maximal exercise endurance, central physiological response, and peripheral physiological response in congenic, HEE, and LEE parental mouse strains; and 4) To characterize and compare the gene expression of the congenic, HEE, and LEE parental mouse strains. The Specific Aims, when fulfilled, will be significant given the health risk of hypokinetic diseases. Understanding the genetic factors that predispose an individual to a certain fitness level could lead to better methods of improving the physical health and well-being of individuals. Unlike other past attempts, this project is unique in that we are proposing to use common, but powerful, genetic research techniques to investigate this area. This will be the first project investigating the genetic basis of exercise endurance to use these techniques that have already proven successful in other fields."}
{"text": "Abstract Simultaneous PET/MR can be considered as an integrated imaging modality only if the information of both modalities is integrated together. In current routine PET/MR applications, the PET and MR scans are performed separately, and the images are reconstructed separately as well. The information is integrated only at the application level. Here we propose unified methodologies of joint PET/MR image reconstruction, a paradigm shifting new way to integrate information of PET and MR to significantly maximize the outcome of PET/MR. The PET and MR scanners indeed measure different physical or physiological signals, but there are still redundant information (e.g. tumor boundary and mutual information) between the images obtained with the two modalities that can be utilized to build connection between PET and MR images in a potential joint reconstruction. In addition, if the compartmental model is taken into account, the physiological parameters estimated from PET and MR can have overlaps, and therefore the parametric image (voxel-wise kinetic parameters) estimated from one modality could be directly used to help the estimation of the parametric image of the other modality. Therefore, there are inter- connections between these two modalities that we can use to develop elegant methods of joint reconstruction. We will first take advantage of the simultaneous acquisition of PET/MR to develop a static image reconstruction with anatomic prior derived from MR images, and to develop methods to jointly reconstruct gated PET images using a motion field computed from MR images. We believe in both cases, the quality of PET images will be significantly improved compared to traditional approaches. For PET/MR, there are many novel ways to jointly model the dynamic PET and MR images. We will thus develop an alternating direction method of multipliers (ADMM) to directly estimate the voxel-wise kinetic parameters of dynamic PET and dynamic MR together from raw data. This will achieve the maximum signal noise ratio of parametric images for both dynamic PET and MR. We will also investigate novel approaches to parametric imaging of non-stationary kinetic modeling in which not only the images are estimated but also the uncertainty on those estimates of the parametric images. The knowledge of uncertainty is important when making decisions about progression/regression of the disease, signal detection, etc. We will use a method developed in our laboratory in which the noise in raw PET data will be transferred to parameter images using origin ensemble algorithm."}
{"text": "The Program Project Application is concerned with basic phenomena of conception and early pregnancy in mammals, including man. The general objectives are: (1) To study in depth the stages of conception and early pregnancy in mammals and man whose failure has been identified as possibly leading to infertility, or whose vulnerability may lead to new methods of controlling fertility. Since it is virtually impossible to study in detail these phases of human reproduction, we propose to study mechanisms which are specific to the reproduction processes of mammals, and pursue their regulation in depth. Where possible, critical experiments will be done on human material. (2) The topics to be investigated include some aspects of spermatogenesis, oogenesis, fertilization, cleavage and implantation. Emphasis is placed on the kinetic aspects of early development in the mammal and the interactions of the preimplantation stages with the local environment where the preimplantation embryonic stages develop within the female genital tract. BIBLIOGRAPHIC REFERENCES: Biggers, J.D. (1975) Development of the mammalian oocyte in vitro. In Vitro 10:362. Ducibella. T., Albertini, D.F., Anderson, E. and Biggers, J.D. (1975) The preimplantation mammalian embryo: characterization of intercellular junctions and their apperance during development. Devel. Biol. 45: 231-250."}
{"text": "A major responsibility of cardiac care unit (CCU) nurses is to monitor patients following coronary angioplasty for signs and symptoms of cardiac ischemia which may signal the complication of sudden coronary artery reocclusion with subsequent acute myocardial infarction. The rationale for interventions to re-establish blood flow following reocclusion is that patients who develop extensive infarction often have numerous repeated hospitalizations for congestive heart failure and chronic, debilitating symptoms such as shortness of breath, inability to perform daily activities, and fatigue. A noninvasive technique more sensitive than the patient's symptoms for detecting recurrent cardiac ischemia is the patient's 12-lead electrocardiogram (ECG). However, because the ECG abnormalities are transient, cardiac ischemia is often missed by the patient's daily 12-lead ECG. The proposed study seeks to determine the sensitivity and accuracy of continuous bedside cardiac ischemia ST segment monitoring using a \"derived\" 12-lead \"ECGD\" compared to the routinely- monitored dual-lead method for detecting recurrent cardiac ischemia following coronary angioplasty. A secondary aim is to determine whether there are gender differences in the sensitivity and accuracy of differences between the 2 lead methods in these patients. 416 subjects will have 12-lead ECGDs recorded with balloon inflation, during coronary angioplasty to record the patient's ischemic pattern during \"controlled\" ischemia. Information will be elicited from the patient at the same time as to their symptoms of cardiac ischemia. Patients will serve as their own controls and be monitored with both lead methods in the CCU following angioplasty. The sensitivity will be analyzed using a 2-factor repeated measures analysis of variance, where the dependent variable is defined as the proportion of true ischemic events detected by each method. To compare the accuracy of the 2 lead methods, 2 nurse experts will independently determine whether both methods contain the same pattern of ST elevation, depression, or isoelectric ST segments compared to corresponding leads of the patient's 12-lead ECGD recorded during coronary angioplasty. Nurse expert ratings will be placed into a 3 X 3 contingency table, where the table rows will contain \"same,\" \"related\" and \"different\" ratings for Method I (routinely-monitored dual leads) and table columns will contain ratings for Method II(12-lead ECGD). The contingency table will be analyzed under a non-parametric \"correlated proportions\" statistical model. The Stuart extension of the McNemar test will be used to test for the equality of the correlated marginal probabilities."}
{"text": "PROJECT SUMMARY/ABSTRACT The goal of this R35 proposal is to uncover novel genetic discoveries and biological mechanisms underlying association with devastating cardiovascular diseases. This proposal builds on strengths in high-throughput genetics and genomics and development and application of innovative computational and statistical methods and genomics technology to maximize the benefits of genetic studies of cardiovascular disease. We will continue our discovery efforts to uncover genetic variants associated with a variety of cardiovascular diseases including atrial fibrillation, aortic aneurysm and dissection, and myocardial infarction and coronary artery disease. Building on our previous work where we identified a number of new genes for coronary artery disease and lipids, we also propose to uncover the mechanisms underlying association at known loci using genetics and epigenomics. We propose to assess the phenotypic impact of the ~19 million variants and 20k indels and SVs identified from whole genome sequenced samples by imputing them into 70,000 new GWAS samples with many cardiovascular phenotypes. We will perform integrated analyses with epigenomics data to highlight clusters of loci with related function. We also propose to perform targeted sequencing of 300 genes in 30,000 CAD cases and controls to search for loss of function variants at CAD loci that implicate CAD genes. We will continue to search for mechanistic insight by performing a PheWAS for all CAD-associated variants identified, disentangling multiple independent signals and correlated traits and clinical endpoints using conditional testing. Completion of these studies will provide new insights into disease mechanisms that have the potential to catalyze breakthroughs in cardiovascular disease prevention, treatment, and diagnosis."}
{"text": "Project Summary/Abstract Despite significant efforts, our understanding of clinically relevant mechanisms underlying human pituitary tumorigenesis remains ashamedly meager. My lab has focused on defining the precise molecular mechanisms by which ubiquitous components of growth factor/Ras/MAPK signaling pathways result in pituitary lactotrope cell-specific transcription responses. We discovered an elegant tri-partite molecular code, involving the protooncoprotein c-Ets-1, the POU-homeodomain protein Pit-1, and a composite DNA element containing an Ets binding site adjacent to a Pit-1 site. Pit-1, Ets-1 and this composite site were shown to be required not only for the oncogenic V12Ras response of the rat (r) prolactin (PRL) promoter, but also to reconstitute lactotropespecific rPRL promoter activity in HeLa nonpituitary cells. During the past funding cycle we made important advances, showing that Ets factors, particularly GABP, are critical for basal and growth factor-induced endogenous rPRL gene expression in GH4 cells and lactotrope ontogeny in transgenic mice;in dissecting the TAD subdomains of Ets-1 and Pit-1;and in identifying co-activators and co-repressors governing basal and Ras-regulated PRL gene expression. In this application, we propose to move our Ras work to a more translational level, and address the role of Ras/MAPK in pituitary prolactinoma tumor formation, and whether PTTG-1 and Menin act as downstream effectors in this pathway. Thus, the main goal of this proposal is to develop pre-clinical GH4 cell line and transgenic mouse models that target V12Ras to pituitary lactotropes, to determine the functionally relevant role of persistently-activated pMAPK and downstream effectors (eg, Menin, PTTG-1 and Mediator components) in regulating lactotrope gene expression, cell proliferation and tumorigenesis. Our unifying hypothesis is that persistent MAPK activation in lactotropes regulates specific Mediator components leading to the stimulation of prl and pttg-1 gene expression, and inhibition of menin gene expression, which leads to increased cell cycle progression, cell proliferation and tumorigenesis. Specific Aims: (1) To use Dox-inducible V12Ras to determine the role of distinct levels of activated MAPK in mediating cell proliferation and differentiation in GH4 somatolactotropes. (2) To use ChIP analysis to define the MAPKdependent chromatin alterations on the PRL, GH, PTTG-1 and Menin promoters and on recruitment of specific Mediator components to these genes. (3) To use shRNA and over-expression to identify the key MAPK downstream effectors required for MAPK-regulated promoter activity and GH4 cell proliferation. (4) To target a V12Ras-IRES-Luc transgene to pituitary lactotropes and determine whether persistent MAPK activation results in tumorigenesis and if drug induced lactotrope hyperplasia enhances tumor formation. Knowledge gained from these studies will provide critical insights into the mechanisms by which pMAPK links to the Mediator complex, and how this link regulates prl, pttg-1 and menin gene expression, thus providing a mechanistic framework for how the MAPK pathway intersects with clinically relevant proteins to contribute to lactotrope tumorigenesis"}
{"text": "The long-term objective of this proposal is to provide new and meaningful structural-functional correlative data as it relates to solute transport along the mammalian renal tubule. The experiments described focus our attention primarily on the collecting duct and place a major emphasis on the role of angiotensin II (All) in the regulation of intercalated cell structure and function and specifically in the control of transepithelial H+ and HCO3- transport. The proposal includes four sections with clearly delineated specific aims and hypotheses to be tested. In Specific Aim I we will examine the effect of All on the ultrastructure of intercalated cells to identify specific intercalated cell subpopulations that respond to All, and determine the effect of All on the subcellular distribution of H+ATPase by immunogold cytochemistry. Hypothesis to be tested: Specific intercalated cell subtypes are responsible for the effect of All on H+-ATPase activity and acid-base transport in the collecting duct. In Specific Aim II the effects of All on H+-ATPase activity and the signalling transduction pathways involved in mediating these effects will be studied in microdissected collecting duct segments. Hypothesis to be tested: The inhibitory effect of All on H+-ATPase activity in the collecting duct is mediated via specific AT1 receptors and activation of distinct signalling systems which will be identified. In Specific Aim III we will determine the expression and cellular location of All receptor isoform mRNA in the collecting duct by quantitative RT-PCR and in situ hybridization and determine the effect of acid-base perturbations on All receptor mRNA expression. Hypothesis to be tested: All receptor mRNA expression varies between different populations of intercalated cells and is influenced by acid-base perturbations. In Specific Aim IV we will determine the effect of All on proton and bicarbonate transport in the isolated perfused rabbit cortical collecting duct and identify the subtype(s) of intercalated cells that responds to All. Hypothesis to be tested: All stimulates HCO3 secretion by type B intercalated cells and inhibits H+ secretion by type A intercalated cells in the cortical collecting duct. The experiments described in this proposal are intended to continue our long-term commitment to delineate the structural and functional characteristics of the renal tubule and specifically to establish the role of intercalated cells in the \"fine-tuning\" of acid-base regulation by the kidney. A better understanding of the mechanisms that control H+ and HCO3, transport in this region of the renal tubule will enable physicians to manage complicated clinical problems of acid-base balance that are often life-threatening with greater expertise and success."}
{"text": "The proposed research explores a synthetic strategy which will provide access to analogs of the tumor promoter thapsigargin (1). New adaptations of two metal-mediated synthetic methodologies will be examined. First, the synthesis of cycloheptene template 10 will extend available protocols for the selective functionalization of the known chiral allylmolybdenum complex 2. Second, palladium-catalyzed cycloisomerization of enyne 10 will be investigated as a means for forming a highly substituted bicyclo[5.3.0]decane system. Functional group manipulation of the resulting product will be pursued to provide tricyclic compound 21. The specific goals for the postdoctoral appointment are the acquisition of this synthetic target, as well as the validation of the methods mentioned above. However, the enantiomerically enriched target 21 represents a potentially general precursor for the preparation of analogs of thapsigargin and other guaiane natural products."}
{"text": "This application requests support for the 11th Symposium on Cochlear Implants in Children. The symposium will be held April 11-14, 2007, in Charlotte, North Carolina. This symposium has been held biannually since 1986. The meetings have focused on numerous pediatric cochlear implant topics that are of importance to surgeons, audiologists, speech-language pathologists, hearing scientists, engineers, and educators. This ongoing series of symposia affords a valuable mechanism to keep the field abreast of advances in research and technology, and provides perspectives from a wide range of relevant fields. The symposium will be sponsored by the University of North Carolina at Chapel Hill and will highlight a number of topic areas that are of current importance to pediatric implantation. Areas of focus will include: 1) cochlear implantation for the very young child; 2) surgical, habilitation and candidacy issues related to auditory neuropathy; and 3) issues that are related to bilateral cochlear implantation and the provision of binaural hearing cues to deaf children. These areas of focus will be explored in the morning sessions via thirteen invited presentations, contributed talks from the scientific community, panel discussions, and audience participation. In addition to these focus areas, a range of other topical pediatric cochlear implantation issues will be examined in afternoon contributed oral and poster sessions. Based upon recent symposia in this series, submission of approximately 250 poster presentations and 75 oral presentations is expected. Pediatric cochlear implantation is greatly benefiting the life possibilities and expectations of children with profound hearing losses; however, we are still far from realizing all of the potential benefits of this approach to the treatment of childhood deafness. Continued progress hinges upon continued interaction and exchange of ideas among surgeons, hearing scientists, engineers, audiologists, speech/language pathologists, and educators. The purpose of this symposium is to continue this necessary and productive interaction. [unreadable] [unreadable] [unreadable]"}
{"text": "The human interleukin-2 receptor is being studied to understand critical components of the T cell immune response in normal and neoplastic cells. Following T-cell activation, IL-2 and IL-2 receptors are induced; the magnitude and duration of the T-cell immune response is controlled by the amount of IL-2 produced, the levels of receptors expressed, and the time course of these events. Three chains of the IL-2 receptor exist, IL-2Ralpha, IL-2Rbeta, and gamma-c, with IL-2Ralpha and IL- 2Rbeta being significantly regulated at the level of transcription. The laboratory has focused primarily on the types of signals induced by IL-2, particularly the activation of STAT proteins, and the mechanism of regulating IL-2Ralpha in response to mitogen and IL-2. The group previously identified two enhancer-like regions regulating the IL-2Ralpha gene, and shown that proteins binding to these regions could physically interact. A critical element binds the Ets family protein Elf-1, which associates with NF-kappaB p50, c-Rel, HMG-I(Y), and TFIIB. Based on a binding site selection analysis, it was reported that the optimal site for ElF-1 binding is to an A(A/t)(C/a)CCGGAAGT(A/S) motif, a motif which interestingly is not found in any Elf-1 regulated genes. Instead, Elf-1-regulated genes, including IL-2Ralpha gene, have lower affinity sites, thereby allowing efficient association of Elf-1 only in the context of accessory proteins, and allowing for finer regulation of these genes. Considerable progress has been made in analyzing the STAT proteins (signal transducers and activators of transcription) induced by IL-2. IL-2 can activate both Stat5a and Stat5b (two closely related proteins with>90% amino acid identity) in fresh peripheral blood lymphocytes (PBL) and additionally activates Stat3 in PBL preactivated with phytohemagglutinin. Both human Stat5a and Stat5b have been cloned and it was reported that the genes encoding these two proteins are located on chromosome 17. It was also reported that IL-2-induced Stat5a and Stat5b binding activity could be reconstituted COS-7 cells. Interestingly, however, IL-2-induced transcriptional activation of a Stat5-responsive reporter construct was not induced, suggesting that either a lineage-specific modification or accessory factor was required for activation. This system should prove valuable in clarifying what additional components are required for successful reconstitution. An IL-2 response element was also delineated in the 5' regulatory region of the IL-2Ralpha gene. This element contains binding sites for Stat5 as well as an Ets family protein and provides an understanding of how the IL-2Ralpha gene can be stimulated by antigen and mitogen (via the two enhancer-like element previously described) or by IL-2 (via a mechanism requiring this newly identified IL-2 response element). Thus, these studies provide a molecular basis for IL-2 upregulation of the IL-2Ralpha gene. We have produced Stat5a and Stat5b protein in baculovirus so that the requirements for binding and activation of these proteins can be studied with purified reagents, and have performed the yeast two hybrid analysis to identify factors that can interact with Stat5. Overall, these findings extend our understanding of IL-2 receptor gene regulation as well as other T-cell activation genes regulated Elf-1 and Stat5."}
{"text": "Group of 12 weanling Osborne-Mendel rats were fed diets containing 0, 0.1, 021, 5 or 56% sucrose. Each rat was caged singly with a donor rat that was infected with Streptococcus mutants 6715-15. The recipient and donor were swabbed daily and the bacteria were cultured on Mitis Salivarious agar. Recipient rats fed 56 and 5% sucrose rapidly became infected with high levels of S. mutants. Recipients receiving low sucrose diets remained free of infection for much longer and when infected harbored lower numbers of organisms. The donor rats fed no sucrose failed to retain infection with S. mutants."}
{"text": "The purpose of this study is to 1) compare glycolytic muscle metabolism in patients with chronic fatigue syndrome (CFS) with normal humans, 2) test the feasibility of using near-infrared spectroscopy (NIRS) measurements to evaluate patients with chronic fatigue syndrome, and 3) to determine if exercise training improves muscle metabolism in CFS patients. To address these aims we tested 27 subjects controls and patients with CFS as identified by Dr. Natelson's research group. The medial gastrocnemius was studied and repeated plantar flexions were performed using a isokinetic device. CFS subjects had oxidative capacity (Vmax determined from PCr recovery) compared to control subjects, consistent with our previous studies. Seven of the 21 CFS patients had abnormal rates of H+ production during the 64 s maximal test. Three had slower H+ rates and four had faster H+ rates. NIRS measurements of the rate of oxygen resaturation showed close agreement with PCr recovery rates in control subjects (32.5 + 11 for NIRS compared to 29.9 + 4.8 s for PCr). However, NIRS recovery after exercise was slower the CFS patients than PCr recovery (48.2 + 21 for NIRS compared to 35.4 + 14 s for PCr). In addition, NIRS recovery of oxygen saturation was slower after cuff ischemia in CFS patients compared to controls (20.8 + 13 for CFS compared to 11.3 + 2.8 s for controls). Four CFS subjects have completed the training and testing protocol. Future studies will continue to test CFS patients and control subjects to complete our aims. We plan to continue testing of CFS patients and normal subjects who are enrolling in the exercise program. We also plan to follow up the NIRS findings of reduced oxygen delivery by measuring muscle blood flow in CFS patients with alternative means (Duplex Doppler flow measurements). In one CFS patient with slow NIRS recovery, Doppler measures showed normal peak blood flows, but reduced sensitivity to submaximal hyperemic stimuli."}
{"text": "The field of dermatology is riddled with many problems that expect solutions from modern biomedicine. Several biologicals/biotherapeutical approaches such as anti-TNFalpha antibodies have already made their impact in the daily treatment of dermatology patients. However, many challenges are still ahead. We still have to fill many gaps in our understanding of biological processes in the skin. For example until recently, an entire class of small regulatory RNA molecules, microRNAs, had remained undetected. Their emergence as important players in virtually all tissues and signaling pathways has led us to explore their significance for skin biology. In our proposal we will focus on one microRNA, miR-31, with an exceptional expression pattern in many skin diseases. This finding prompted us to address the functional significance of miR-31 in epidermal homeostasis, aberrant growth control, wound healing, stress response and hair growth. Its involvement in the regulation of major skin signaling pathways such as TNFa, TGFb and BMP signaling makes it a central node for the control of epithelial-mesenchymal transitions. We have identified a crucial role of miR-31 in hair folliculogenesis, hair growth, nail growth, and the response to wounding and the phorbol ester, TPA. TNFa, EGF and FGF7 can induce miR-31 expression and TGFb stimulates the expression of miR-31 precursors. Based on our existing mouse model of miR-31 overexpression and our preliminary data, we have developed a research plan aimed to establish miR-31 as an important regulator of such processes as EMT, proliferation and motility in keratinocytes. Thereby, we will be able to judge the potential of miR-31 as a therapeutic target for skin diseases with defects in these processes. We plan to use our existing mouse model and establish novel tools in this proposal to apply to our career-long goal to translate this knowledge into therapeutic potential of miR-31 using miR-31 inhibitors in vivo. In Specific Aim 1 of this current research plan, we will test our hypothesis that miR-31 is a key regulator of hair growth and the response of the skin to challenges such as wounding. In Specific Aim 2, we will address the important issue of identifying miR-31 target genes to understand miR-31 mediated processes. And in Specific Aim 3, we will test our hypothesis that miR-31 regulates epithelial-mesenchymal interactions and transitions, EMI and EMT. Furthermore, we will test whether miR-31 buffers certain expression patterns and signaling pathways against unwanted fluctuations and define which pathways exactly are influenced by miR-31 in addition to EMT and hair growth regulating signaling networks."}
{"text": "An identified class of neurons in the cerebral cortex respond characteristically to both acetylcholine and norespinephrine. The increase in cell firing produced by acetylcholine is closely mimicked by cyclic GMP while the depression of cell firing produced by norepinephrine was mimicked by cyclic AMP. Other nucleotides (e.g. 5'AMP and adenosine) also produce profound depression of these same neurons which can, however, be selectively blocked by theophylline. In addition, simultaneous application of both norepinephrine and either 5'AMP or adenosine by microiontophoresis result in a synergistic action of both depressants, suggesting that these two substances (norepinephrine and 5'AMP' or adenosine) have an interaction in the cerebral cortex which may be quite important in modulating the output of these neurons. BIBLIOGRAPHIC REFERENCE: T.W. Stone and D.A. Taylor: Microiontophoretic studies of the effects of cyclic nucleotides on the excitability of neurones in the rat cerebral cortex. J. Physiol. 266: 523-543, 1977."}
{"text": "The objective of this research is twofold: first, to analyze the mechanisms by which an adenovirus positive regulatory element activates the expression of viral genes, and second, to see how these regulatory elements influence the initiation and maintenance of cellular transformation. Methods for introducing mutations into early region I, the transforming region of Adenovirus 5, have been developed. One temperature-sensitive mutant has not been isolated, and we plan to isolate others. These temperature-sensitive mutants will be used in pulse labeling experiments after shifting from the permissive to the nonpermissive temperature. We hope to determine from these experiments whether early region functions are continuously required for expression of other viral genes. The mutants will also be used to transform rodent fibroblasts at the permissive temperature. We will determine if the mutant function is required for maintenance of the transformed state by shifting to the nonpermissive temperature and analyzing cellular properties. Those cellular properties that regress will be considered under continuous control of the temperature-sensitive viral product."}
{"text": "This proposal outlines a multidisciplinary effort by four principal investigators to continue development of a series of cyclic and linear conformationally constrained peptide analogues which have high receptor specificity high agonist or antagonist biological activities, high stability in vivo, and prolonged biological activity at kappa (kappa) opioid receptors. This multidisciplinary approach combines modern computer assisted molecular modeling, organic amino acid and peptide chemistry, conformational analysis and molecular mechanics and dynamics calculations, and biochemical, biophysical, physiological and pharmacological studies. The specific aims of this investigation include: a) continues development in the design, synthesis, and evaluation of novel peptides and peptide mimetics derived from dynorphin and related peptides which possess high kappa opioid receptor potency and specificity; b) comprehensive examination of the potency and specificity of all new ligands for kappa vs. mu and delta opioid receptors using radioligand binding techniques. The most selective and potent ligands will be radiolabeled to high specific activity and used in extensive radioligand binding studies and for receptor localization using autoradiography; c) opioid agonist or antagonist activities will be evaluated in vitro using the GPI, MVD, LVP and HVD assays to establish receptor selectivity and potency; d) in vivo kappa receptor properties will be evaluated with the most selectivity compounds using among other assays, the mouse abdominal stretch test following intrathecal administration, and the standard mouse hot plate test; 5) careful examination of the conformational and dynamic properties of the most potent and selective analogues using 1D and 2D nuclear magnetic resonance spectroscopy, molecular mechanics calculations, and other biophysical techniques; and 6) using all of the above results of the design and synthesis of novel compounds with more potent and selective biological activities, and to test conformational models proposed for kappa receptor selective ligands. The long term goal of this research is to develop an understanding of the physiological roles of the kappa opioid receptor in comparison to other opioid receptors, and to develop ligands for the kappa receptors that can be used for the treatment of disease."}
{"text": "DESCRIPTION (provided by candidate): A Scientist Development Award for New Minority Faculty is being requested to support five years of career development and research activities in developmental psychopathology (substance use and violence) among African American adolescents of maternal substance users. The K01 will provide 80% release time from an academic appointment at UM-St. Louis to collaborate with a three-mentor panel available locally. The panel and consultants will provide mentoring and consultation in developmentally psychopathology, clinical observation, advanced statistical analysis, the responsible conduct of research, and guided research activities. The career development activities will be enhanced through coursework in developmental psychology and qualitative research, training in assessment of adolescent psychopathology, and workshops specific to adolescent substance use and violence. The K01 fuses two research focal points that have dominated the candidate's research career: substance use and violence in African American adolescents and psychopathology in adult African American female substance users. The purpose of the K01 award is to prepare the candidate for an independent research career in African American adolescent mental health. The goal of research activities is to collect pilot data that will provide the basis for an R01 study that longitudinally examines the intervening role of maternal substance use on adolescent normal or abnormal development. The research will occur in two phases to maximize research and career development experiences. Phase I of the research will qualitatively assess the role of fathers and the extended family network in adolescent development and in the home environment from a sample of adolescents of maternal substance users. Phase II of the research will gather pilot data on 150 African American adolescents (15-17 years of age) of active substance using mothers (and 50 demographically matched comparisons). Mentors and consultants will provide expertise and oversight in all career development and research activities."}
{"text": "Cognitive decline is a common, but not inevitable, accompaniment to aging. While much research is currently being directed at Alzheimer' s Disease -- one of the most severe expressions of cognitive decline -- relatively little work is currently aimed at identifying risk factors and mechanisms associated with early (i.e., subclinical) cognitive decline, even though this may be the stage most amenable to prevention. An important issue that needs to be clarified in relation to the etiology of cognitive decline is the role of environmental lead exposure and the interaction of lead with specific candidate genes. In this application we discuss how four candidate genes -- APOE, the HFE (hemochromatosis) gene, transferrin, and Tau protein -- may interact with lead burden to increase the risk for oxidative cell damage leading to neuronal cell loss and cognitive deficits. We review the last 11 years of our research on low-level lead toxicity and describe compelling preliminary data. We then propose a new study that calls for new data collection in our established Bostonarea cohorts [Normative Aging Study (NAS), Nurses Health Study (NHS), and Community Lead Study (CLS)]. Our specific aims are to test hypotheses related to the impact on cognition of lead burden and its potential interaction with our four polymorphisms of interest. We will look at cognition cross-sectionally and longitudinally, using a validated battery of telephone cognitive function assessment tools as well as a battery of in-person cognitive tests that we have been administering in person since 1993. Results of this research promise to shed further light on lead's potential impact on society and may also give rise to tools for identifying susceptible individuals as well as early brain effects."}
{"text": "The Vector Core is a critical resource to members of the Institute for Human Gene Therapy providing access to a wide array of gene transfer technology. This dedicated facility is run by Dr. Guang-ping Gao, who has extensive academic based experience in recombinant viruses. The Core has assembled a comprehensive inventory of plasmids, cell lines, and viruses useful to the development of vectors. More comprehensive service is provided in the design, creation, production, and analysis of recombinant viruses. A number of vector systems are available through the Core, including murine-based retroviruses, adenoviruses, and adeno-associated viruses (AAV). The Vector Core maintains strong links to key laboratories in the Institute for Human Gone Therapy involved in novel vector development. Relevant innovations are quickly brought into the Vector Core, where they are validated and distributed to faculty. In the context of this P01, the Vector Core will play an important role. Methodology for creating chimpanzee adenoviruses will be developed in Project 1 and transferred to the Core who will provide services in terms of vector creation and expansion for all four projects."}
{"text": "This proposal is for a conference grant for the Twelfth Microbial Genomes Conference (formerly the E. coli and Small Genomes Conference) to be held September 26-30, 2004, at the UCLA conference center at Lake Arrowhead, California. The genomics landscape has changed now that numerous genomes are available and that microarrays and bioinformatics methods are advancing rapidly. It is a critical time to foster multi-disciplinary approaches to making sense out of all the genomic sequence information. Considerable integration of different aspects of microbiology will undoubtedly take place, including new perspectives on evolution and the requirements for life. To achieve these goals, particularly of integration and synergy, scientists from diverse disciplines must interact. Thus, this conference will include active genome researchers, such as sequencing experts and informatics specialists working in database design, sequence analysis, or simulation; geneticists developing new methods to take advantage of genomic information, e.g. for functional analysis; and cell biologists and biochemists who have specialized in areas now ripe for genomics such as metabolism, chromosome structure, or gene regulation. One focus of this conference will also be on microbial communities and biodiversity. An additional focus will be on modeling and constructing circuits.and pathways. This meeting has become established as a major annual microbial genomics conference during the last twelve years and has assured status and quality. [unreadable] [unreadable] [unreadable]"}
{"text": "PROJECT SUMMARY The available evidence suggests that intrauterine and/or maternal bacterial infection is a major cause of early and late pregnancy complications. Current antibacterial and anti-inflammatory drugs used to treat bacterial infection during pregnancy are considered harmful to maternal and fetal health. Thus, continued research to look for better treatment options to avoid bacterial infection-induced pregnancy complications is alluring. The decidua and placenta are uniquely positioned at the maternal-embryonic interface to serve as a first line of defense against bacterial toxins, but their defensive mechanisms against bacterial toxins are poorly understood. Lipopolysaccharide (LPS) is an endotoxin of Gram-negative E. coli that is associated with infection-induced pregnancy defects. LPS recognition by cell surface proteins is an important step required for the initiation of its inflammatory signaling or inactivation by cell surface molecules. Our preliminary results establish that: 1) the uterine luminal epithelium, decidua and placenta express the LPS sensing and signaling TLR4/CD14/MD2 complex; 2) LPS injection on day 5 of pregnancy terminates pregnancy by day 8 via selective-activation of the MyD88-dependent TLR4 signaling pathway at the embryo implantation site (EIS); 3) LPS induces expression of proinflammatory cytokine genes such as Tnf-? & Il-1?, chemokine genes such as Cxcl1&2, and neutrophil recruitment to the EIS; 4) cell surfaces of the decidua and placenta express tissue- nonspecific alkaline phosphatase (TNAP) isozyme that is capable of dephosphorylating LPS; 5) dephosphorylated LPS is non-inflammatory and non-toxic to murine pregnancy; and 6) AP isozyme treatment alleviates LPS-induced early pregnancy loss in mice. These preliminary findings have led to the hypothesis that LPS-induced microenvironment disruption at the early EIS is a result of concerted action of resident decidual cells and recruited neutrophils, and LPS detoxification by supplementation and/or induction of TNAP production/activity may abrogate LPS-mediated early pregnancy defects/loss. To test our hypothesis, we have proposed three mechanistic aims. In Aim 1, we will use Cre-lox and antibody- mediated neutralization technologies to establish that an important step in the development of LPS-induced unwanted inflammation at the early EIS is decidual cell-type-dependent recruitment of inflammatory neutrophils. In Aim 2, we will generate novel female mice with uterine deletion of TNAP gene Alpl to determine whether endogenous TNAP deficiency in the uterus augments the response to LPS. In Aim 3, we will examine the potential of TNAP and its activator or inducer in mitigating LPS- or E. coli-induced early pregnancy defects/loss. Upon completion of these aims, we hope to gain: 1) insights into the mechanisms of infection-induced inflammation at the early EIS; and 2) develop a novel LPS-detoxification therapeutic strategy to avoid bacteria- induced early pregnancy complications."}
{"text": "The objectives of this project are to determine the effects of 2450 MHz microwave radiation on biological material at the cellular and macromolecular level and relate the amount of microwave energy absorbed to the effects. Various cellular and macromolecular systems will be tested by exposure to microwaves under carefully controlled temperature and dose conditions. Parameters such as viability, cell membrane permeability and denaturation will be used as indicators of microwave effects."}
{"text": "Clonal evolution is a key feature of cancer progression and relapse. Our recent study, which utilized a newly developed pipeline that estimates the fraction of cancer cells harboring each somatic mutation within a tumor through integration of whole-exome sequencing and local copy number data, linked the presence of subclones harboring putative driver mutations with adverse clinical outcome in chronic lymphocytic leukemia (CLL) and suggested that CLL therapy may accelerate the process of clonal evolution (Landau et al., Cell 2013). We propose that presence of subclonal mutations that are putative drivers are indicative of an active evolutionary process. We now seek to definitively establish the impact of subclonal mutations on CLL biology, the development of disease relapse and clinical outcome. This will be achieved by longitudinal analysis of clonal structure of serial samples collected from patients enrolled on phase II and phase III clinical trials (and hence uniformly treated) that address the treatment landscape of CLL. In particular, we will perform detailed genetic analysis of samples from patients receiving standard-of-care first line fludarabine-based chemotherapy (Aim 1). In parallel, we will examine patient samples exposed to ibrutinib, a highly promising irreversible inhibitor of Bruton's tyrosine kinase which i anticipated to be a cornerstone of future CLL therapy (Aim 2). Analysis of samples exposed to both these types of therapies will include characterization of subclonal structure as well as assessment of the dynamic phenotypic changes (detected by single cell RNA-sequencing) to validate mutation analysis and determine the transcriptional networks of drug resistant cells in order to reveal potential novel and effective treatment combinations. To causally link the impact of putative drivers and therapy on CLL clonal evolution, we will generate an in vivo model to study interclonal dynamics in the setting of therapy (Aim 3). We will use transformative genome-editing techniques to generate cell lines that model leukemic subpopulations bearing representative CLL driver mutations and thereby mechanistically dissect the contribution of individual genetic lesions to the evolutionary landscape. By creating an animal model of clonal evolution, we will have the potential to more effectively evaluate preclinically the impact of nove therapeutics on clonal selection. In total, these studies are designed to establish a framework for understanding the role of the dynamic evolutionary landscape of CLL on the diagnosis, prognosis and treatment of this currently incurable disease."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Aging is associated with increased risk for heart disease, diabetes and physical disability. In women, the incidence of these age-related conditions increases dramatically after menopause. This has led to the hypothesis that ovarian hormone deficiency contributes to these adverse health outcomes. However, the effect of ovarian hormone deficiency, per se, on risk factors for disease and disability has not been clearly defined. Thus, the primary goal of the proposed studies is to characterize the effect of ovarian hormone deficiency on glucose, insulin, fat and protein metabolism. Our overall hypothesis is that ovarian hormone deficiency alters substrate turnover and utilization in a manner that increases the risk for developing chronic disease and disability. Specifically, alterations in glucose, insulin and fat metabolism increase the risk for developing heart disease and diabetes and changes in protein metabolism contribute to reduced lean tissue mass which, in turn, promotes disability. To address our hypothesis, we will measure substrate metabolism using stable isotope tracer methodology in healthy, premenopausal women before and after pharmacological ovarian suppression. Women will be randomized to receive the gonadotropin-releasing hormone agonist leuprolide acetate or placebo. Measurements of substrate metabolism will be performed during both the follicular and luteal phases of the menstrual cycle prior to treatment and 2 months after the initiation of treatment. Experiment 1 will investigate the effect of ovarian hormone deficiency on the glucose and insulin response to hyperglycemia. Experiment 2 will examine the role of ovarian hormone deficiency in the regulation of whole-body lipolysis under postabsorptive and epinephrine-stimulated conditions. Experiment 3 will examine the effect of ovarian hormone deficiency on whole-body protein metabolism under postabsorptive and simulated-postprandial conditions."}
{"text": "Two strains of mice were used in this project. Female C3H and CBA mice were exposed to a total body dose of radiation of 3 Gy with or without Tempol supplementation in the animal's food. Immediately following the radiation exposure, animals were be placed on either control or TP-containing food. The groups include: a) no radiation, control food, b) 3 Gy, control food, c) no radiation, TP food, and d) 3 Gy, TP food. For C3H mice, one additional group was added receiving 3 Gy total body irradiation where the administration of the TP containing food was delayed one month post-irradiation. Preliminary data show TP food supplementation after radiation: a) did not alter food consumption compared to animals on a control food diet, b) compared to animals on control food diet, the TP diet resulted in decreased weights in both mouse strains (40% for C3H and 20% for CBA), and c) TP food supplementation post-radiation significantly enhanced the survival of both mouse stains. Median survival values for 0 Gy, 3 Gy, 0 Gy TP, and 3 Gy TP for C3H mice was 706, 434, 764, and 670 days, respectively. For CBA mice median survival values for 0 Gy, 3 Gy, 0 Gy TP, and 3 Gy TP for C3H mice was 901, 660, 939, and 782 days, respectively. The incidence of hematopoietic neoplasms (predominantly lymphomas) was significantly reduced in both mouse strains by TP treatment and both the onset and incidence of solid neoplasms was significantly reduced in CBA mice treated with TP. These preliminary data would encourage further research and development of TP as a chemopreventive agent. The second hypothesis is also being tested that mice protected from lethal total body irradiation by administration of a radioprotector immediately before radiation exposure will experience an elevated risk of cancer induction. Mice were exposed to a total body radiation dose of 10.8 Gy, a radiation dose that results in 100% lethality. Ten minutes prior to the 10.8 Gy exposure the animals will be injected with a radioprotector. The control for this group, another set of animals was exposed to 5.4 Gy total body irradiation. This radiation dose was derived from the radiation dose modification factor (2) when the radioprotector is administered 10 min before total body irradiation. These animals will also be followed for their entire lifespan for tumor induction as outlined above. Preliminary data show that the median survival for mice receiving 0, 5.4 or 10.8 Gy were 706, 460, and 491 days, respectively. There was no difference between the 5.4 and 10.8 Gy groups (p = 0.42);however, the median survival of both irradiated groups was significantly shorter compared to unirradiated mice (p &lt;0.0001). Cancer incidence (hematopoietic plus solid tumors) was similar between the 5.4 and 10.8 Gy groups and was significantly greater than for unirradiated controls. However, the ratio of hematopoietic to solid tumors differed between the two groups, with the 5.4 Gy group having a higher incidence of hematopoietic neoplasms compared to the 10.8 Gy group (1.8 fold). A greater incidence of solid tumors was observed in the 10.8 Gy group. These preliminary results suggest that mice protected from lethal whole body radiation have a shortened lifespan, due in large part, to cancer induction post-radiation compared to unirradiated controls. Lastly studies have been initiated to determine if metabolites in the urine of mice receiving whole body radiation can predict for radiation-induced cancer induction prior to the observation of tumor mass."}
{"text": "Older adults often struggle to communicate in adverse listening situations, especially when they must understand one talker when there are other voices in the background. Current rehabilitation of older adults with hearing loss focuses primarily on restoring audibility via hearing aids. Unfortunately, this is of limited benefit in dificult listening environments because both speech and background noise are amplified. Restoring audibility is not enough to ensure successful communication in situations where listeners must ignore the distracting speech of other talkers. The long-term goal of this research is to develop evaluation and treatment methods that focus on both the sensory and cognitive age-related changes that mediate comprehension in these situations. The proposed experiments are designed to provide vital information about why older adults experience problems in the presence of more than one talker. Past research has yielded conflicting conclusions about the relative importance of age-related peripheral/sensory factors and age-related cognitive changes in explaining deficits in speech understanding. The studies in this proposal will help to clarify tis issue by examining how listener-related factors (specifically, working memory, processing speed, attention-switching, inhibitory control, and hearing loss) interact with stimulus-related factors to create problems understanding speech in multi-talker situations. Two specific aims will be addressed: 1.) To understand why competing speech signals are so disruptive for older listeners, and how hearing loss and cognitive processing affect their ability to cope with this interference; and 2.) To determine the degree to which hearing loss and cognitive function impact speech recognition in more realistic communication situations. The proposed experiments use both established tasks (e.g., simultaneous speech-on-speech masking, ratings of effort) and innovative techniques (temporally interleaved speech, eye tracking, limitations on response time) to address these questions. The performance of middle-aged as well as older listeners will be measured in order to provide information about which functional abilities begin to decline early vs. later in the aging process. The end result of this project will be an enhanced understanding of the factors that limit older adults from fully participating in conversations in everyday listening conditions. This, in turn, may drive improvements in rehabilitative protocols that incorporate treatment of both bottom-up and top-down contributors to age-related speech understanding problems. PUBLIC HEALTH RELEVANCE: Many older adults struggle to understand speech in adverse listening situations. The purpose of this project is to identify why they experience these problems. The long-term goal of this research is to develop effective and comprehensive rehabilitation programs aimed at improving older adults' functional communication ability in difficult listening environments."}
{"text": "It has been shown that T lymphocytes are primarily responsible for the cell-mediated immunity of the host and the most important cells in the body to eliminate the neoplastic cells. The development of these cells depends upon the thymus gland. We have shown that there is a decrease of T cells in patients whose thymus glands were irradiated during early infancy. This population of patients also has a higher incidence of cancer. It is possible the decreased T lymphocytes may account for the increased incidence of cancer. Our present proposal is to extend our studies to include other populations of thymus-irradiated patients, especially the prenatal fetus, the premature infants and adult patients treated with radiation to their lung or breast. We hope these studies will determine the contributions of the thymus gland upon the T cell development during the different periods of life and the radiation effect to this gland at various thymus gland maturation stages as expressed by the proportion of T cells in the peripheral blood. Also, we will determine the radiation-induced chromosomal abnormalities in the peripheral blood lymphocytes in these patients."}
{"text": "Current studies have indicated that antibodies directed against the stalk region of CD23 cause enhancement of IgE synthesis in both the human in vitro and mouse in vivo systems. CD23 transgenic mice, which overexpress CD23 on all lymphocytes and FDCs, exhibit drastically reduced IgE production in both helminth and alum/ag models. The data suggest a model where the role of CD23 is initially to serve as a component of innate immunity to signal for IgE production by becoming destabilized and cleaved and later by overexpressing at the cell surface and modulating IgE production. This continuation application proposes to investigate the mechanism of these effects. Aim#1 examines the mouse system where the destabilizing mab 19G5 gives enhanced IgE synthesis in vivo. In the current funding, the metalloprotease, ADAM10 has been identified as the primary CD23 sheddase in mouse and humans. The role of ADAM10 in allergic disease will be modeled by making transgenic mouse that overexpress ADAM10 or dominant negative ADAM10. In addition, we will examine the mechanism for the 19G5-induced IgE production by investigating the association of CD23 with another negative signaling molecule, LAX, which has recently been shown to both modulate CD23 expression and regulate IgE levels. Aim#2 will investigate the affect of CD23 overexpression and CD23 destabilization on the mouse asthma model with respect to both modulation and exacerbation of disease. We will utilize both IgE and the new ADAM10 transgenics in order to evaluate the mechanism of the suppression of eosinophilia as well as the capacity of CD23 to modulate the asthma phenotype. Aim#3 will investigate the human in vitro IgE synthesis models with respect to the mechanisms involved in IgE synthesis enhancement, seen with anti-stalk antibodies and synthesis suppression, seen with certain anti-lectin mabs. The importance of ADAM10 in human CD23 cleavage and IgE production will also be explored as will the involvement of LAX. Finally, we will determine if IgE production by B cells obtained from normal and allergic subjects is affected differently by destabilization or stabilization of CD23. In summary, these studies examine the mechanism of action of a natural regulator of IgE production, CD23, with the objective of developing protocols to enhance CD23 expression and thereby regulate IgE, and by analogy, allergic disease in which IgE plays a dominant role.Project Narrative: This project examines mechanisms involved in control of IgE synthesis by a natural regulator. The latter is CD23, a low affinity receptor for IgE. Accumulated evidence indicates that cleavage of CD23 by the metalloprotease ADAM10 increases IgE production in both mouse and humans. This application proposes to study mechanisms involved in this regulation in order to develop new protocols to control allergic disease."}
{"text": "The proposed research plan seeks to determine the morphological changes in cells comprising the Japanese quail oviducts and testes during and after ingestion of various levels of the estrogenic insecticide Kepone. The morphological observations will be accomplished by gross, microscopic and ultramicroscopic (transmission and scanning electron microscopy) means. The first study will determine whether the damages recorded in cytoplasmic organelles of magnum and shell gland regions of the quail oviduct are reversible after ingestion of different concentrations of Kepone was discontinued. This study will involve the examination of cytoplasmic organelles in cells of different oviduct regions during their progressive atrophy after Kepone stimulation ceases. The second study will determine the detrimental effects of various concentrations of the estrogenic pesticide Kepone on the internal organization and ultrastructure of cells in quail testes. This study will determine the severity of low, moderate and heavy Kepone concentrations on spermatogenic process, sperm maturation and structure of interstitial cells. The third study will determine whether the damages in the testicular cells of the quail can be reversible and whether recovery of normal testicular morphology is possible after Kepone ingestion has been discontinued. This study will determine whether recovery of testicular morphology is complete after ingestion of mild Kepone dosage as well as whether damage is severe and permanent after ingestion of heavy Kepone concentration is discontinued. The data from this project will provide some important clinical and ecological information concerning the fate of reproductive capabilities of organisms, including man, exposed to various Kepone concentrations."}
{"text": "FOXO transcription factors extend lifespan and delay age-related disease in animals, and many studies have now linked FOXO3A DNA variants to exceptional longevity in humans. Thus, the time seems right to look for human genes that are likely to regulate FOXO-dependent, or other, longevity pathways. FOXO proteins can be activated in many ways to extend animal lifespan. For example, C. elegans FOXO can promote longevity in response to reduced insulin/IGF-1 signaling, altered serotonin signaling, and elevated AMP kinase activity, elevated heat-shock factor activity, elevated lin-4 microRNA activity, elevated Jun kinase activity and other inputs. Thus, there could be many gene perturbations that can extend healthy lifespan in humans; and some of these perturbations may be safer and more effective than others. Because it is not possible to do genetic screens for long-lived humans, we are doing genetic screens in human cells instead. Our experimental strategy is based on the observation that all FOXO- dependent life-extending pathways tested so far (as well as many other life-extension pathways) increase resistance to oxidative stress. Although the role of oxidative stress resistance in life extension is not clear, the correlation is tight enough that in many model organisms, screens for oxidative stress resistance have yielded long-lived mutants. Therefore, to obtain a set of potential human longevity genes, the Kenyon lab has carried out a genome-wide siRNA screen for oxidative stress resistance in a human primary cell line. The gene hits include known C. elegans FOXO regulators, regulators of other longevity proteins such as TOR and NRF2, and new genes as well. From this set, the Kenyon lab will identify good candidates for new human longevity and healthspan genes. To do this, they will determine which knockdowns trigger other correlates of longevity, such as xenobiotic resistance or autophagy. In addition, they will ask which knockdowns perturb the activities of FOXO3A, TOR or NRF2. Finally, to link these genes to longevity, they will test for their ability to influence lifespan in C. elegans and for their altered expression in centenarian families. This fresh approach will define new potential drug targets for extending the youthful and productive years of human life, and for delaying age-related diseases such as cancer, heart disease and/or neurodegenerative disease. PUBLIC HEALTH RELEVANCE: The wonderful finding that changing specific genes can extend healthy lifespan and counteract age- related diseases in animals makes a search for human longevity genes imperative. This study takes a fresh approach, by looking for genes that, when altered, give cultured cells from human's properties characteristic of cultured cells from healthy, long-lived animal mutants and from very long-lived animal species. This study will define new entry points for extending the youthful and productive years of human life, and for delaying age-related diseases such as cancer, heart disease and neurodegenerative disease."}
{"text": "We are requesting the continuation of a training program in theoretical neuroscience with funds to support 4 predoctoral trainees. Our goal is to train students who combine exceptional skills in mathematics, statistics, modeling and machine learning with a deep understanding of neurobiology. The complexity of neural systems and of the data that we can now obtain demands researchers with these skills if we are to realize the neuroscience community's goals of achieving a mechanistic understanding of nervous system function and making significant progress in the treatment of neural disorders and mental illness. Training will occur at the Center for Theoretical Neuroscience at Columbia University, supported by the 6 faculty at the Center, faculty visitors to the Center, and 26 other faculty from the Departments of Neuroscience, Psychology, Biology, Biochemistry, Biomedical Engineering, and Statistics at Columbia. The Theory Center provides an exceptional environment in which pre-doctoral, post-doctoral and faculty researchers interact and collaborate extensively both within the Center and with researchers in experimental laboratories. Most trainees are members of the Columbia graduate program in Neurobiology and Behavior (with a small number drawn from other graduate programs) and take the courses that satisfy the requirements of that program. The required courses are augmented by a large selection of electives, including courses in theoretical neuroscience, statistics and machine learning. The goal of this training program is twofold: 1) To produce theoreticians who combine outstanding skills in analysis and model-building with a deep understanding and sense of biological neuroscience; and 2) To train experimentalist who are skilled at applying theoretical and computational methods in their research. This will be accomplished through extensive collaborations with outstanding experimental laboratories both at Columbia and elsewhere combined with training in state-of-the art theoretical methods. Whenever possible, students will be co-advised by both a member of the Theory Center faculty and a researcher from our associated experimental faculty, and they will be given desk space both in the Theory Center and in the laboratory of their experimental co-advisor. The opportunity to be involved in the operations of an outstanding laboratory and in the activities and intellectual atmosphere of a world-class theory center provides an exceptional training experience."}
{"text": "The maternal-fetal interface is critically important since is at this level that uteroplacental and fetoplacental blood flows are regulated and dysfunctions are seen in diseases of pregnancy, e.g. preeclampsia. Vasodilation is controlled by PGI2 and Nitric Oxide (NO) of endothelial origin and locally shows obligatory requirements for cell-cell communication via Gap junctions, a common theme in this PO-1. Uterine Artery Endothelial Cells in Pregnancy (P-UAEC) and HUVECs undergo Programmed Adaptation to maintain NO production via a Connexin (Cx) 43 mediated mechanisms. HYPOTHESIS Aim 1: Endothelial Programmed Adaptation is modulated by the Gap junction protein Cx43 that is elevated directly by Shear Stress via cAMP and/or cGMP to maintain vasodilator (PGI2 and NO) production. Purpose Aim 1: To determine in P-UAECs, HUVECs and HUAECs the effects of: 1) acute vs. prolonged Laminar Shear Stress on levels of phosphorylated and total Cx43 & eNOS and vasodilator production (PGI2 and NO) relative to cyclic nucleotide (cAMP and cGMP) adaptation; 2) inhibition of PKA, PKG, Adenylate Cyclase and sol Guanylate Cyclase; and 3) Indomethacin and L-NAME on these shear stress responses. HYPOTHESIS Aim 2: These important shear stress-mediated vasodilator mechanisms are abrogated by patho-physiologic levels of representative cytokines elevated in preeclampsia [VEGF and Tumor Necrosis Factor] via functional Gap junction disruption. We hypothesize that shear stress elevates cAMP /cGMP which is protective for maintaining vasodilator production via Gap junction assembly. Purpose Aim 2: To determine in P-UAECs, HUVECs and HUAECs if these cytokines disrupt the shear stress-associated cyclic nucleotide Gap junction-mediated vasodilator mechanisms we will evaluate: 1) effects of treatment with VEGF and TNF on the acute and prolonged laminar shear stress responses; 2) the therapeutic protective effects 8-Br-cAMP and 8-Br-cGMP on VEGF and TNF disruption of shear stress responses; and 3) the therapeutic protective effects cAMP phosphodiesterase (PDE)2, cAMP PDES, cAMP PDE4, or cGMP PDE5 inhibitors on VEGF and TNF disruption of shear stress responses. These data provide the first mechanistic framework for understanding the interactions between shear stress-mediated cyclic nucleotides for therapeutic protective actions and Gap junctions to regulate endothelial vasodilator (PGI2 & NO) production and it's abrogation via cytokine (VEGF & TNF) mediated disruption of cell-cell communication."}
{"text": "Antimacrophage serum and indomethacin are each capable of blocking the ability of endotoxin to inhibit the hormonal induction of certain liver enzymes of short half-life such as tryptophan pyrrolase and phosphoenol pyruvate carboxykinase. These studies will continue in an effort to identify the role macrophages play in the process. The ability of endotoxin to interfere with the binding of glucocorticoids to liver cells will also be investigated. These experiments will be carried out first in intact mice and subsequently in hepatoma cell cultures. The contribution of endotoxin to the pathogenesis of experimental murine typhoid will be evaluated by determining changes in infected mice that parallel alterations characteristically induced by endotoxin."}
{"text": "Fibrinogen (340 kDa) is a multifunctional plasma protein that after activation spontaneously polymerizes to form a fibrin gel. Fibrin prevents the loss of blood upon vascular injury and serves subsequently as a provisional matrix on which various cell types adhere, migrate and proliferate during wound healing and other physiological and pathological processes. The ability of fibrinogen to polymerize and degrade in a controllable fashion and to adhere to different cells makes it an ideal bioadhesive (fibrin sealant) that has been used increasingly in numerous surgical application for arrest of bleeding, gluing of tissues, delivery of bioactive substances, etc. Better understanding of the mechanisms underlying fibrin-dependent processes and more effective application of fibrin sealant require comprehensive knowledge of the structure and interactions of this complex molecule. Recent studies established a high resolution structure of about two-thirds of the molecule. The first aim of this application is to complete the high-resolution structure of the remaining regions and to further clarify conformational changes upon conversion of fibrinogen to fibrin. Our recent discoveries that fibrinogen alphaC-domains interact with high affinity with plasminogen, tPA and apo(a) suggest their involvement in fibrinolysis and atherogenesis. The second aim is to characterize these interactions and to clarify their role in regulation of these processes. Interactions of the other fibrin(ogen) regions, gamma-modules and the betaN-domains, with receptors on leukocytes and endothelial cells was shown to stimulate inflammation and angiogenesis. The third and fourth aims focus on further establishing the mechanisms of these interactions. These aims will be accomplished by preparation of various fibrin(ogen) domains and combinations thereof by limited proteolysis and recombinant techniques, and studying their structures and interactions by biochemical and biophysical methods. These studies will generate basic knowledge that will have an impact on the understanding of and ability to control the above mentioned fibrin(ogen)-dependent processes. They will also contribute to the development of improved fibrin sealants with desirable properties."}
{"text": "The overall goals of this grant proposal is to determine role(s) of lens proteinases, their cognate inhibitors and proteolytic products of crystallins in the formation of high mo. wt. (HMW) proteins during the development of senile cataract. In this study, human lenses of different ages and cataractous lenses will be utilized. The following four specific aims will be pursued: (1) Regulation of proteolysis in lens: The regulation of proteolysis in the lens at the enzyme and protein substrate levels will be determined, i.e. by searching for zymogens and/or proteinase-inhibitor complexes of two lens proteinases, i.e.a 25 kDa and a membrane proteinase, and examining the effect of oxidation on endogenous proteinase inhibitors and the preferential proteolysis of native vs. oxidatively modified crystallins by endogenous proteinases. (2) Origin of degraded polypeptides from crystallins: The identity of \"parent crystallins\" from which the three in vivo found degraded polypeptides of human lenses, i.e. 5.5 kDa, 9 kDa and 15 kDa, originated will be established by comparing their amino acid sequences. Lens crystallins will be proteolyzed in vitro with the two lens proteinases (25 kDa and a membrane proteinase) and proteolysis products compared with similar polypeptides found in vivo for their Mr's and partial N-terminal amino acid sequences. In addition, the origin of the degraded polypeptides from crystallins by a non-enzymatic reaction (Fenton reaction) will also be examined. (3) Cross-linking of degraded polypeptides to form HMW proteins: The mechanism of cross-linking to form HMW proteins in vitro by degraded polypeptides per se or between these polypeptides and individual crystallins will be determined. (4) Search for and quantitation of cross-linked products of degraded polypeptides in HMW and water insoluble proteins of normal aging and cataractous lenses. Antibodies raised against the specific cross-linked degraded polypeptides will be utilized to identify and quantitate the levels of aggregates of these polypeptides by Western blot analysis and a radioimmunoassay method respectively. The above studies will show: (i) how the lens proteolytic process is regulated in vivo; (ii) whether the degradation of crystallins in vivo is enzymatic or non-enzymatic, and (iii) if the degraded polypeptides cross-link per se and with individual crystallins to form the HMW proteins that exist in increasing levels in aging and cataractous lenses."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project focuses upon Infrastructure development in Computational Biology and Bioinformatics as part of the Core Resources at the Center for Biomedical Research (CBR) at Tuskegee University. Research involving advanced information technologies, the Internet and databases is crucial in examining and analyzing patterns of disease occurrence, genetic traits and molecular level dynamics including AIDS research."}
{"text": "As a national resource for molecular biology information, NCBI's mission is to develop new information technologies to aid in the understanding of fundamental molecular and genetic processes that control health and disease. More specifically, the NCBI has been charged with creating automated systems for storing and analyzing knowledge about molecular biology, biochemistry, and genetics; facilitating the use of such databases and software by the research and medical community; coordinating efforts to gather biotechnology information both nationally and internationally; and performing research into advanced methods of computer-based information processing for analyzing the structure and function of biologically important molecules. To carry out its diverse responsibilities, NCBI: conducts research on fundamental biomedical problems at the molecular level using mathematical and computational methods maintains collaborations with several NIH institutes, academia, industry, and other governmental agencies fosters scientific communication by sponsoring meetings, workshops, and lecture series supports training on basic and applied research in computational biology for postdoctoral fellows through the NIH Intramural Research Program engages members of the international scientific community in informatics research and training through the Scientific Visitors Program develops, distributes, supports, and coordinates access to a variety of databases and software for the scientific and medical communities develops and promotes standards for databases, data deposition and exchange, and biological nomenclature"}
{"text": "Numerous cell surface receptors transduce signals through heterotrimeric GTP binding proteins (G proteins). The alpha subunit of these proteins is a molecular switch, cycling between GDP-bound (inactive) and GTP-bound (active) forms. The purpose of this study is to characterize the intracellular regulation of G-protein-mediated signal transduction. GTPase activity of the alpha subunit is enhanced by a novel family of regulators of G protein signaling (RGS proteins), resulting in inhibition of Gi and Gq-coupled signaling. This project studies specifically the interaction between RGS proteins and G proteins and the resultant control of G protein function. RGS proteins demonstrate little specificity for Gi and Gq subunits in cell-free systems, yet they may discriminate between G-protein-coupled receptors (GPCRs) linked to the same G-alpha. To address the issue of receptor specificity directly, fusion proteins consisting of different GPCRs fused to a single G-alpha subunit were constructed and expressed in cell lines, and receptor-stimulated GTPase activity in the presence of RGS proteins is studied. Although no GTPase accelerating (GAP) activity of any RGS protein for Gs-alpha has been detected, RGS16 interacted unexpectedly with Gs-alpha and inhibited Gs-induced cyclic AMP (cAMP) generation in mammalian cells. Regulation of RGS16 activity was studied by examining whether the protein underwent post-translational modification. RGS16 shares amino terminal cysteine residues with RGS4 and RGS5 that were hypothesized to be sites of palmitoylation. RGS16 underwent palmitoylation, and palmitoylation-defective RGS16 mutants demonstrated impaired ability to inhibit both Gi- and Gq-coupled signaling in mammalian cells. RGS16 also underwent tyrosine phosphorylation in response to both GPCR or receptor tyrosine kinase (Epidermal Growth Factor, EGF) stimulation. Mutation of a conserved tyrosine residue blocked tyrosine phosphorylation of RGS16, and the tyrosine mutant was quantitatively less able to inhibit Gi and Gq- induced mitogen activated protein (MAP) kinase activation. RGS16 co- immunoprecipitated with the EGF receptor complex and inhibited EGF- induced MAP kinase activation. A novel RGS protein with a divergent RGS box was cloned, D-AKAP2, which also contains a protein kinase A anchoring (AKAP) domain. Expression of D-AKAP2 in human embryonic kidney (HEK)293 cells resulted in inhibition of signaling induced by Gs-coupled GPCRs but not those coupled to Gi or Gq. - GTP, G proteins, RGS proteins, signal transduction"}
{"text": "Funding is requested in partial support of the 7th Biennial Conference of the American Society for Matrix Biology (ASMB), which is to be held from Oct. 12-15, 2014, in Cleveland, OH (ASMB2014). ASMB is a national society whose mission is to promote basic, translational, and clinical research on the extracellular matrix (ECM), cell-ECM interactions, and ECM-based therapies and devices, and to support professional development of the matrix biology research community. Its biennial conference provides an in-depth forum integrating new information on the genetics, molecular biology, biochemistry, pathobiology, and translational relevance of all aspects of ECM. Additionally, this conference provides a continuum with cell biology and intracellular signaling via an emphasis on cell-ECM interactions and ECM-driven signaling. This all-encompassing, matrix-immersion aspect of the ASMB conference is lacking in large society conferences and is distinct from the single molecule- or process-focused conferences available to the ECM community, including Gordon, Keystone and FASEB conferences. ECM and matrix receptor gene mutations lead to inherited connective tissue disorders, skeletal dysplasias, muscular dystrophies and blistering skin diseases, and ECM dysregulation is universally a major factor in acquired diseases, both acute and chronic, affecting all organ systems. ECM is especially relevant to skin and the musculoskeletal system, where it is a primary component of bone, cartilage and tendons. Among acquired conditions, the pathology of osteoarthritis, osteoporosis, aneurysms, cancer and metastasis involves ECM destruction, and all fibrotic conditions and aberrant wound healing feature an excess or dysregulated ECM. ASMB2014 has considerable translational relevance, especially to tissue repair and engineering, and provides the basic science foundation upon which to advance these fields. The ASMB Conference brings together scientists, clinician-scientists, post-doctoral trainees, and graduate students from diverse research fields and medical specialties to discuss the most recent advances in ECM biology and their relevance to human disease. The exciting 3-day program comprises a keynote address on protein phosphorylation by Dr. Jack Dixon, five plenary sessions and 18 concurrent sessions on focused topics that provide many opportunities for short talks by trainees. In addition to advancement of the matrix biology field, ASMB2014 demonstrates a commitment to providing opportunities for in-training and women matrix biologists and has specific mentoring components. These goals and activities of ASMB2014 are fully consistent with the mission of NIH and the intent of the R13 mechanism."}
{"text": "Despite recent progress in medical therapy, heart failure is one of the most common causes of death in western countries. Understanding the molecular mechanism mediating growth and death of cardiac muscle is fundamentally important and can potentially lead to better medical treatment for heart failure. This laboratory has been working on an enzyme termed mammalian sterile 20-like kinase 1 (Mst1) which plays an essential role in regulating growth and death of cardiac myocytes. Although this enzyme is activated in the heart in response to high blood pressure and during heart failure, the molecular mechanism by which Mst1 is activated in the heart is not well understood. The aims of this project are to investigate the role of putative regulators of Mst1, termed Ras-association domain family 1, isoform A (RassfIA) and K-Ras, in mediating the activation of Mst1, growth and death of cardiac myocytes, and the resulting impact on heart function. Genetically altered RassfIA and K-Ras mice will be subjected to transverse aortic constriction and used to study the role of RassfIA in the development of cardiac hypertrophy and heart failure. Echocardiography, hemodynamic analysis, post-mortem organ measurements, histology and biochemical analysis will all be used to determine and compare resulting cardiac phenotypes. The possibility that RassfIA can modulate cardiac fibrosis will also be addressed with a focus on NF-KB as a potential mediator. Cultured cardiac myocytes and fibroblasts will serve as tractable systems to allow for the examination of signaling pathways involving RassfIA and Mst1. This work will also seek to evaluate K-Ras as an upstream activator of this signaling pathway in the heart. The knowledge obtained from this investigation will further elucidate the mechanism of Mst1 regulation in vivo and should be useful for the development of better treatment for heart failure."}
{"text": "Project Summary: This application seeks to support the career development of Dr John H. Stewart, IV. His long-term goal is to attain the necessary skills to become a well-trained clinician-scientist with a focus in the treatment of metastatic colorectal cancer and eventually become competitive for independent funding support. This goal will be met by a structured career development plan as well as a well-designed research project. Dr. Stewart's career development strategy will involve continued engagement with his present mentoring committee and participation in a structured didactic course of study to fill gaps in his scientific background. Dr. Stewart will present his research findings at national meetings and publish his work in peer reviewed journals during the proposed career development period. In addition to the aforementioned career development plan, Dr. Stewart and his mentoring committee have devised a research project titled \"Oncolytic Vesicular Stomatitis Virus (VSV) for the Treatment of Colorectal Cancer\". The overall hypothesis of this research project is that local delivery of oncolytic VSV can effectively treat properly selected, metastatic colorectal tumors of the liver and peritoneum. This hypothesis will be tested by three specific aims. In Specific Aim 1, he will seek to identify the molecular determinants of host-cell permissiveness to VSV in colorectal cancer cells. In Specific Aim 2 he will define the apoptotic pathways activated by VSV in colorectal cancer cells. In Specific Aim 3 he will delineate the efficacy of loco-regional delivery of VSV in small animal models of metastatic colorectal cancer. The research proposed in this application will set the stage for further translational research by advancing the novel paradigm of local delivery of oncolytic VSV for the treatment of metastatic colorectal cancer. The Comprehensive Cancer Center of Wake Forest University provides an excellent environment for a mentored training program. This center incorporates expertise from a broad variety of disciplines and has extensive resources. Dr. Stewart has laboratory space that is fully funded by the Department of Surgery. He has the full support of his Division Chief to devote at least 75% of his time to the career development plan outlined in this proposal. Relevance: It is projected that 153,760 individuals will be diagnosed with colorectal cancer in 2007. Despite advances in early diagnosis, surgery and systemic therapy, an estimated 52,180 patients will die of metastatic disease this year. Clearly, new therapies are needed to treat advanced colorectal cancer. This project will attempt to address the need for novel treatments of metastatic colorectal cancer by exploring the efficacy of VSV in the treatment of this disease."}
{"text": "A set of comprehensive neuropsychological test batteries is used to provide a complete assessment of various cognitive and sensory function that can be related to damage or dysfunction in different regions of the e brain. The adult battery comprises tests designed to tap the following aspects of behavior: attention, executive function, language, memory, motor functions, orientation, selected sensory and perceptual functions, vigilance and visual-spatial functions. In addition, adults are given a test of general intelligence and. In some studies, subjects are administered a structured psychiatric interview. Modified batteries have been developed for the assessment of infants, preschool children, and children ages 6-16. The data provided by these batteries are being used to construct a neuropsychological theory of the elements of attention that may be applied to the neurological and psychiatric diagnostic groups under study in the LPP. The LPP has as its major focus disorders involving impaired attention, including schizophrenia, epilepsy, eating disorders, affective disorders, head injuries, and AIDs dementia complex. Comparisons are being carries out between the neuropsychological profiles of various groups of psychiatric patients and those of patients with known cerebral lesions in specified brain regions. Our data are also being used to delineate neurobehaviorally-defined subgroups within diagnostic categories, an undertaking aimed at reducing variability in psychiatric diagnosis, treatment, and outcome. The data provided by this protocol provide a complete behavioral assessment that may be integrated with concurrently gathered electrophysiological, neuroradiological, and biochemical information."}
{"text": "Some aspects of the karyotype are highly conserved in certain mammalian groups, while in other groups the karyotype can undergo extensive repatterning in a short period of time. While it is clear that chromosomal mutation represents a reorganization of the genetic material, the molecular basis and effects of such change are largely a matter of speculation. The suggestion has frequently been made that mobile genetic elements play a role in chromosomal rearrangement. This proposal investigates the role of repetitive sequences in karyotypic repatterning by studying the relationship between hypervariable repetitive sequences and karyotypic plasticity in different mammalian groups. Three mammalain groups will be investigated: cetaceans, which are karyotypically conservative; deer mice, which have undergone karyotypic orthoselection for pericentric inversions and heterochromatic additions; and equids, which have shown an extremely rapid and varied repatterning of the karyotype. Hypervariable DNA will be isolated from one species from each group by phylogenetic screening, and approach for identifying rapidly diverging repetitive sequences. The correlation between the amount and nature of hypervariable DNA and the karyotypic stability of each group will be investigated. Because the equids are so karyotypically unstable, the most derived equid genome will be further investigated. Repetitive elements from this group will be more extensively characterized. Equid DNA will be factionated by density gradient centrifugation, and these fractions will be used to determine whether repetitive families from the Hartmann's mountain zebra are non-randomly distributed in the genome. A clear perception of genome organization may be central to understanding development and the regulation of gene expression."}
{"text": "Hepatitis B Virus (HBV)is a hepadnavirus that is a major cause of acute and chronic hepatitis in humans. Three hundred million people globally are chronically infected with HBV, making HBV one of the most common human pathogens. HBV replication is not cytopathic, and evidence has shown that hepatic pathology is immune-mediated. Thus, understanding the pathogenesis of acute and chronic hepatitis B virus infection mandates understanding the immune responses underlying these processes. Natural hepadnaviral infections occur only in humans and other outbred species whose immune systems are poorly characterized and difficult to study. This proposal seeks to use a new transgenic mouse model of hepatitis B virus infection to identify mechanisms involved in immunopathogenesis of acute and chronic hepatitis B virus infection, including understanding the role of the innate immune response in these disease processes. It is our hope that in identifying mechanisms underlying these disease processes, we will be able to design specific immunotherapies to prevent and treat HBV-related liver disease. Our specific aims are: 1. To determine the mechanism by which non-classical NKT cells mediate acute experimental hepatitis in our transgenic mouse model of primary HBV infection; 2. To test the hypothesis that the early activation of non-classical NKT cells and/or NK Cells in our model of primary HBV infection can significantly influence the chronic phase of hepatitis in our disease model; and 3. To develop an in vitro model of non-classical NKT cell activation in response to Hepatitis B virus, which will establish a fundamental experimental system for identifying the mechanisms of non-classical NKT cell activation."}
{"text": "The overall goal of this proposal is to define new molecular and cellular mechanisms that enable vertebrate photoreceptors to adapt to constantly changing conditions of ambient illumination. We will focus on the massive light-driven translocation of major signaling proteins between the subcellular compartments of the photoreceptor cell, a phenomenon which has attracted overwhelming interest throughout the visual community over the past few years. We will continue to explore the mechanistic aspects and the physiological role of protein translocation and will extend our efforts to address the potential involvement of protein translocation in photoreceptor neuroprotection. The first two Aims are devoted to the translocation of arrestin. Our recent findings indicate that arrestin translocation is triggered at a critical threshold of light intensity by a specific signaling mechanism. The nature of this mechanism will be elucidated in Aim 1. The fact that arrestin is a key player in inactivation of the visual signaling cascade suggests that its light-driven translocation to the light- sensitive compartment of the photoreceptor cell, the outer segment, contributes to light adaptation by making light responses less sensitive and more rapid. This hypothesis will be directly addressed in Aim 2. The other two Aims are focused on the translocation of transducin. In Aim 3 we will follow up on our recent finding that a protein called phosducin serves as a trafficking chaperone for transducin translocation. We will test the hypothesis that the light-dark cycle of transducin translocation is regulated through the concurrent cycle of phosducin phosphorylation and dephosphorylation. Finally, in Aim 4, we will use the mechanistic information learned during the previous grant cycle to generate a mouse model in which transducin translocation is impaired but other transducin functions are preserved. We will utilize this model to explore the putative role of transducin translocation in the neuroprotection of the rod from metabolic and oxidative stress imposed on rods during the many decades of their regular exposure to light. The proposed experiments are relevant to understanding the molecular and cellular mechanisms that regulate normal photoreceptor activity, mechanisms that may be perturbed in several degenerative retinal diseases, both inherited and associated with aging. The studies proposed in this application address the molecular and cellular mechanisms that allow our visual system to efficiently adapt to the drastic variations in light intensity encountered during a normal day. We will explore the role of these mechanisms in the neuroprotection of the photoreceptor cells from the metabolic and oxidative stresses they experience during the lifetime of light exposure. Because these mechanisms are likely to be perturbed in a variety of degenerative diseases of the retina, both inherited and associated with aging, addressing these questions on a molecular level opens doors for developing strategies for disease prevention and future therapeutic interventions."}
{"text": "This study examines whether reductions in air pollution are associated with reductions in the prevailing mortality rates. The areas considered are census tracts that have \"high\" residential stability; this restriction reduces the effect of migration into and out of the area. Analysis of data from Buffalo will be completed by the end of the first year of the project. Next year, data from Detroit, Seattle, and the major cities of Ohio will be analyzed. Results to date from Buffalo support the hypothesis of an association between reduced air pollution levels and a reduction in the prevailing mortality rate; however, these results are not statistically significant. Consideration of the larger data sets will clarify this point. In addition to time trend analyses, the study is examining whether there are any unusual geographical clusterings of mortality rate changes and performing other exploratory analyses. The deaths are analyzed by cause of death to specify the nature of the putative air pollution and mortality association. Primary attention, of course, is given to lung and chronic heart disease."}
{"text": "Perfluorochemicals attracted widespread attention in 1966, when Clark and Gollan showed that mice could survive for prolonged periods when completely immersed in liquid perfluorochemicals equilibriated with O2 at atmospheric pressure. The exceptional O2-carrying capacity of perfluorochemicals has provided the basis for using physiolologic emulsions of perfluorochemicals as O2-transport fluids after hemorrhage or during surgery when blood transfusions are impossible or refused. Treatment with perfluorochemical emulsions (PFCE's) and O2-breathing also reduces myocardial damage after coronary artery occlusion, protects against acute focal cerebral ischemia after ligation of the cerebral artery, and minimizes injury after spinal cord compression, reflecting improving O2 delivery into ischemic tissues. Solid tumors have abnormal, insufficient vasculatures and, as a result, contain areas which are hypoxic because of transient or chronic deficiencies in blood flow. Hypoxic cells are very resistant to the cytotoxic effects of ionizing radiation and limit the efficacy of radiotherapy in achieving local control of some solid tumors. We found that administration of a perfluorochemical emulsion and a high-oxygen atmosphere increases the response of BAlll2 rat rhabdomyosarcomas and EMT6 mouse mammary tumors to radiation. This was shown to reflect a decrease in the proportion of hypoxic tumor cells, rather than a direct toxic or radiosensitizing effect of the PFCE. This project will examine further the clinical potential of this effect, using solid EMT6, KHJJ, and BAlll2 tumors to test the effects of PFCE's and O2-breathing on tumor cell survival curves, tumor growth, tumor cure, and tumor oxygenation. The effect of treatment with PFCE's on the efficacy of fractionated radiotherapy and chronic low dose rate irradiation will also be examined. The effects of exchange transfusions, producing high flurocrits and low hematocrits, will be tested. The toxicities and physiological effects of the PFCE/O2 treatments and the effects of the treatments on tumor growth and metastasis will also be examined. The effects of PFCE's on the radiation responses of several normal tissues (including lung, skin, liver, and the hematopoetic system) will also be studied. These data will be used to determine whether therapeutic gain can be obtained by adjunctive treatment with PFCE's and oxygen, to assess further the clinical potential of PFCE's as adjuncts to radiotherapy, and to identify any problems which may be encountered in the clinical use of these adjuvants."}
{"text": "The specific aims of the study are to recruit 200 obese individuals with type II diabetes and 200 obese individuals who have a positive family history of diabetes. These subjects will be randomly assigned to usual care or to behavioral weight control programs which include either structured diet, structured exercise or the combination. All subjects will be assessed at baseline, 6 months, 1 year and 2 years on measures of weight, glycemic control, coronary heart disease (CHD) risk factors, and eating and exercise behaviors."}
{"text": "The Division of Cancer Treatment and Diagnosis (DCTD) supports the development of novel diagnostic and therapeutic approaches for cancer by expediting the initial and subsequent large-scale testing of new agents, biomarkers, imaging tests, and other diagnostic and therapeutic interventions (radiation, surgery, immunotherapy) in patients. Within DCTD, eight major programs and a patient clinic work together to bring unique molecules, diagnostic tests, and therapeutic interventions from the laboratory bench to the patient bedside. Activities supported under this contract include: isolation and evaluation of natural products; molecular pharmacology; high-throughput screening of compound libraries; pharmacodynamic marker and pharmacokinetic assay development and utilization in clinical trials; drug toxicology research; information systems, technologies, and data science; drug discovery; radiochemistry and radioactive drug production; archival of clinical trial images, in vitro and in vivo translational research; repository support for natural products, patient-derived xenografts, tumors and biologic anti-cancer agents; a biopharmaceutical production facility; medical writers; nursing support services for clinical trials performed in the NIH Clinical Center, correlative studies in collaboration with biopharmaceutical companies; support for precision medicine trials; advanced genomics capabilities supporting tumor evaluation in pre-clinical models and clinical trials; patient biorepositories for NCI-funded clinical trials; nanotechnology characterization and evaluation."}
{"text": "The overall goals of this proposal are to determine the dynamic properties of the intermediate filament (IF) cytoskeletal networks in fibroblasts and epithelial cells as well as to determine the nature of the interaction between IF and the cell surface. IF dynamics will be studied in mitotic cells in which the IF system is depolymerized and in daughter cells in which IF are repolymerized. Specifically, studies of the molecular basis of the extensive remodeling of Type III IF networks containing vimentin or vimentin/desmin will be undertaken. The explosive depolymerization which typifies early-mid prometaphase and the rapid reassembly of IF which takes place during anaphase-telophase can be explained, for the most part, by the hyperphosphorylation of IF structural proteins. Two mitotic kinases have been found in BHK cells which are involved in the phosphorylation reaction. We are undertaking an extensive analysis of one of these kinases termed vimentin protein kinase (VPK). VPK is a complex of three proteins: a 65kD component, a 110kD component, and p34cdc2 which is the catalytic subunit of maturation promoting factor (MPF) and is known to play a major role in triggering mitosis. We plan to carry out experiments aimed at determining the functional significance of the specific site(s) phosphorylated by VPK and other kinases which act on Type HI IF proteins using a variety of cell physiological (e.g., microinjection), morphological (immunofluorescence, confocal, and electron microscopy), biochemical and molecular biological (the use of bacterially expressed proteins and transient transfection of cultured mammalian cells) techniques. We will also attempt to study the interphase dynamic properties of IF. For these studies we will utilize the microinjection of derivatized IF proteins such as biotinylated vimentin and keratin into live cells and to determine their fate in situ using confocal and electron microscopy. Similar approaches using x-rhodamine labelled IF proteins will be used to determine whether or not a steady state or dynamic equilibrium exists in interphase cells through the use of fluorescence recovery after photobleaching (FRAP) experiments. Studies are also described which are aimed at determining the biochemical basis of the binding of IF to cell surface associated desmosomes in epithelial cells. These studies involve the isolation and biochemical characterization of bovine tongue desmosome components and the determination of how keratin polypeptides bind to them. The information derived from our investigations should shed new light on the properties and functions of IF in mammalian cells. A better understanding of IF structure and function is basic to understanding the pathogenesis of a variety of diseases in which changes in IF occur, such as Alzheimer's disease, Parkinson's disease, various cancers, and alcoholic cirrhosis."}
{"text": "Oral clefts (including cleft lip, CL; cleft palate, CP; and cleft lip and palate, CLP) represent a complex and heterogeneous group of crainofacial defects that constitute a major public health burden due to their high prevalence. We propose an international collaborative study to test specific candidate genes and their potential interaction with environmental risk factors using the case-parent trio design. Since this case-parent design avoids the problem of confounding due to population stratification, it is most suitable for international collaborative studies where samples from genetically distinct populations are collected. We feel it is necessary to extend genetic epidemiologic studies of oral clefts into international collaborative efforts to accumulate adequate samples sizes in the shortest time possible. This will require extensive planning to insure similarity in ascertainment of cases, comparability of exposure data collected by each center, and high levels of quality control in the processing of DNA and typing of genetic markers. We are proposing to develop an international study of genetic epidemiology of oral clefts based on case-parent trios (plus sib controls, where available) at 5 centers: Johns Hopkins (USA), Singapore, Taiwan, Hong Kong and Japan. During the 2 year planning phase proposed here, we will hold two meetings among co- investigators to develop standardized research protocols to collect DNA and environmental exposure data from cases and their parent (plus available sib controls). The first meeting will be held in Baltimore, where the Hopkins group will share their experience in the genetic epidemiology of oral clefts. The second meeting will be held in Singapore to review how research protocols should be modified for maximum effectiveness in the Asian centers. After this planning phase, this group of collaborators will prepare and submit a grant to fund a full scale international study of oral clefts."}
{"text": "Prostate cancer (PCa) is the second most common cause of cancer-related death in men in the United States. While multiple molecular events contribute to prostate cancer progression, it has become increasingly evident that epigenetic changes play pivotal roles in regulating cancer development. Polycomb repressive complex (PRC) members maintain the gene expression status of a cell by epigenetically modifying histone proteins. Histone methyltransferase EZH2, an oncogenic PRC2 member, initiates the transcriptional repression by trimethylating histone H3 at lysine 27. We have shown that EZH2 is overexpressed in aggressive prostate and breast cancers, predicts disease outcome, and is required for cancer cell survival. In addition, our studies have shown that EZH2 down-regulates multiple tumor suppressors and a genomic loss of microRNA-101 results in unregulated expression of EZH2 in aggressive tumors. While the role of EZH2 in regulating protein-coding genes is known, its role in regulating microRNA (miR) expression has not been studied. MiRs are critical regulators of cellular functions and are commonly altered in cancers including down regulation of several tumor suppressor miRs. Thus, we hypothesize that transcriptional repressor EZH2 plays a key role in regulating microRNA expression by epigenetic silencing and EZH2-regulated miRs play critical roles in PCa progression. Our preliminary data suggest that EZH2 down regulates multiple miRs, including miR-203 and the miR-200a, miR-200bc family. MiR-203 and mir-200a, bc in turn regulate PRC1 members, BMI1 and RING2. Thus the aims of this proposal are to extend these findings and gain further insights into the regulation of miRs by EZH2 and the role of these miRs in PCa development. In order to accomplish these goals, in Specific Aim 1, we will investigate the role of EZH2 in regulating miR expression in multiple cell types. We will first perform miR profiling using RNA from prostate cell lines in which EZH2 expression is modulated. We will validate the EZH2 regulated miR expression in prostate tumor tissues and correlate them with EZH2 expression. In Specific Aim 2, we will investigate the role of EZH2-regulated miRs in PCa and their role in targeting the PRC1 members. In Specific Aim 3, we will investigate the role of EZH2-regulated miRs in prostate tumorigenesis. We will use select EZH2-regulated miRs that are directly repressed by EZH2 and characterize the consequences of their modulation using both cell line and in vivo models of PCa. Relevance to Public Health: Successful completion of the proposed work will provide evidence for an intricate network of miR and epigenetic regulators in PCa development and identify potential diagnostic and prognostic markers, which in turn may improve PCa therapy through better diagnosis and disease monitoring. Our results may ultimately provide credence for therapeutic \"re-introduction\" of EZH2-repressed tumor suppressor miRs in cancer. PUBLIC HEALTH RELEVANCE: Prostate cancer is one of the most common causes of cancer related death of men in the United States, yet the etiology of this disease is relatively unknown and current therapies have a significant failure rate for advanced disease. Oncogenic histone methyltransferase EZH2 which is overexpressed in aggressive prostate cancer is known to play essential roles in aggressive prostate cancer and our preliminary studies have found that it regulates critical tumor suppressive microRNAs in prostate cancer cells. Our proposed investigations will provide evidence for the role of EZH2 in regulating critical tumor associated microRNAs, suggest biological role of EZH2 regulated microRNAs and will identify potential prostate cancer biomarkers and valuable therapeutic targets for prostate cancer."}
{"text": "This proposal will determine the intracellular signaling pathways that are important for the replication of SMC after arterial injury. The first group of experiments will document the activation of the MAP kinase pathways, namely p42/44erk in the ballooned injured rat carotid arteries. Activation of this pathway will be inhibited in vivo with a topically applied MEK1 inhibitor and the effect on SMC replication quantitated. Furthermore, the effect of activation of the p46sapk on SMC growth by transfecting cells with the upstream activator of p46sapk and measuring their ability to replicate in response to known mitogens. The second specific aim will determine the role of mitogen-activated protein kinase phosphatases (MKP-1) on SMC replication in rat carotid arteries. Initially MKP1/2 expression will be documented at various times after injury. MKP-1 will be inhibited with pervanadate and its expression blocked with an antisense oligonucleotide. The replication of carotid artery SMC to known agonists will then be quantitated. The third aim will determine if the MAP kinases, p42/44erk are activated by FGF2 and PDGF. Rat arteries will be denuded of endothelium in a manner known to induce a minimal SMC replication, and then FGF2 and PDGF will be administered. The activity of p42/44erk and the expression of MKP-1 will be measured in the SMC of these arteries. The final group of experiments will document the time course of cyclin D and E activity and the presence of cyclin inhibitors, p21 and p27 at times after carotid injury. Further studies will evaluate the effect of p27 on SMC replication in vivo by inhibiting its expression with an antisense oligonucleotide and then measuring the ability of intimal SMC to respond to known mitogens. In other studies the inhibitor, p27 will be over expressed in SMC and its role in suppressing SMC replication, when challenged with a mitogenic stimulus, quantitated. These studies will provide important data on the signaling pathways thought to be active in SMC and determine if their activation is critical for SMC replication."}
{"text": "DESCRIPTION (provided by applicant: Diabetes is a disease of multiple organs responding to complex genetic and environmental factors. A complete understanding of insulin resistance and type 2 diabetes mellitus (T2DM) requires an integrative approach that asks how different cell types influence each other through hormonal, neural, and metabolic signals all in the context of extra-organismal stresses including overnutrition and disruptions in normal circadian rhythms. A goal of all studies proposed in this application is to explain normal and pathological metabolism in molecular terms, emphasizing both cell autonomous processes as well as those that depend on organismal integration. The Program Project brings together five outstanding investigators, each with considerable past success as an independent investigator, but each also with a genuine belief in the value of scientific collaboration. Each PI focuses on a specific organ system and how it interacts with other tissues and external stresses, and works in close communication other PIs who study related problems. In Project 1, Lazar addresses how resistin coordinates the multi-organ response to nutritional overload, in which an inflammatory response leads to adverse consequences in insulin target tissues and the cardiovascular system. In Project 2, Stoffers focuses on the response of the beta cell to the stress of peripheral insulin resistance, testing an intriguing model that connects transcriptional regulation to endoplasmic reticulum biosynthesis and cell growth. In Project 3, Ahima uses a mouse feeding entrainment model that mimics circadian disruption in humans to examine how the central nervous system influences hepatic metabolism. In Project 4, Kaestner also studies the response to a perturbed central clock, but in the context of how the hormonal milieu influences the transcriptional control of liver glucose metabolism. Lastly, in Project 5, Birnbaum also considers how hepatic metabolism responds to the stress of obesity, but also asks how it is normalized by the antidiabetic drug metformin. The projects are supported by three Cores that provide histochemical analysis, generation of genetically modified mice, and their metabolic phenotyping."}
{"text": "The periaqueductal gray (FAG) and the adjoining dorsal raphe nucleus (DRN) are mesencephalic cell groups that can act to control pain perception. A consensus has arisen that the PAG functions in initiating and implementing behavioral coping strategies to situations involving stress, fear or pain. The DRN is a major source of forebrain serotonin, which modulates many behaviors and has been implicated in the pathophysiology of depression. Control of pain by these two nuclei is likely a single element of a multimodal response pattern to stressful situations. Substance P (SP) is a neuropeptide well known for playing a role in pain transmission. When released, SP binds the neurokinin 1 (NK1) receptor and precipitates receptor activation and internalization. The NK1 receptor is enriched within the dorsal and ventrolateral FAG as well as the DRN. In this region, focal application of SF is antinociceptive, eliciting the local release of endogenous opioids. In addition, SP neurotransmission is associated with anxiety, cardiovascular adjustments and grooming behavior. Therefore the PAG and DRN represent potential sites where SP may influence several individual components of behavioral coping strategies. The proposed experiments will examine internalization of the NK1 receptor produced by exogenous and endogenous SP using immunohistochemical methods to gain insight into the role of SP neurotransmission in the FAG and DRN. The topography of NK1 internalization by these stimuli will reveal the potential overlap of neural circuits used in coping with these stimuli. In addition, the proposed AIMS will broadly establish the neural circuitry that SP engages to modulate these areas. That is, the hypothesis that enkephalin- or serotonin-containing neurons have the NK1 receptor will be tested using light and electron microscopic analysis. The results of these studies will-yield insight into how distinct modes of stressful stimuli impact SP neurotransmission within neural circuits that coordinate defensive coping strategies."}
{"text": "This proposal is an application for the continuation of Public Health Service Grant HL-11119. Its purpose is to make available a common amino acid analyzer and sequencer operation for eight research groups in the Departments of Biochemistry and Molecular Biology and Biological Sciences, by providing support for an operator and the supplies and maintenance for the available equipment. The present research projects dependent on these facilities include the mechanisms of enzyme catalysis (M. Bender); the isolation of antigenic peptides from lactate dehydrogenase C4 (E. Goldberg); the comparative biochemistry of oxygen-carrying proteins (I.M. Klotz); the structure and function of photoreceptor complexes in phogosynthesis (P. Loach); enzymes and proteins of coagulative processes (L. Lorand); the mitochondrial metabolic role of cytochrome c (E. Margoliash); the antigenicity of globular proteins (E. Margoliash); the biosynthesis of bacterial cell wall components (F.C. Neuhaus); the structure and function of prophobilinogen synthase (D. Shemin); and the mechanism of action and design of antitumor agents (R.B. Silverman). These studies are supported by the Public Health Service and the continued existence of a joint amino acid analysis and sequencer operation is the most economical way of providing for their prosecution while obviating the duplication of efforts and its resultant inefficiency."}
{"text": "The Cell Deposition System (CDS) deposits biological cells on predetermined sites on a microscope slide. With this improvement in flow microfluorometry (FMF) and cell sorting technology, it will be possible to associate an arbitrary cell on the microscope slide with its FMF data. Such correlation is possible by using a computer to store a cell's FMF data and concomitantly determine a cell's location on the slide. An initial study (Tyrer et al., Proc. IEEE 67:10, 1979) demonstrated the feasibility of this device. This proposal seeks to develop an improved CDS prototype which can be used in our laboratory for slide preparation of diagnostic samples. This requires a straight-forward instrumentation development task for a laboratory prototype; studies of the interaction between slide substrate, cell fixative, cell stain and cellular morphology; and studies of the interaction between substrate, cell nutrients and cells when dealing with viable cell systems. This will result in the reliable preparation of slides so that human visualization as well as automated cell localization and analysis can be accomplished with confidence. The prototype will be used in two separate applications to determine a wide range of utility in fixed or living cells. Applications in cancer detection include preparation of microscope slides by placement of cells in a highly ordered fashion for human or machine analysis. The slides will have only cells of interest and be relatively free of debris. The other application involves depositing viable cells in a mechanically stable cell culture medium. Individual cells can then be assessed for their tissue culture character. The CDS will permit true single cell analysis since the computerized determination of cell address and storage of FMF values can be used to drive a Scanning Transmission Microscope (STOM). The STOM, a computerized image analysis microscope system, can use the CDS-derived data and microscope slide preparations to locate and further analyze interesting cells."}
{"text": "We propose to develop an integrated microchip platform for rapid, reliable and comprehensive diagnosis of cancer by examining the pathophysiological mechanism of tumor-immune interaction. This will be done at both molecular and cellular level. By applying this platform to analyze a large number of clinical samples, we expect to gain new insights about cancer immunobiology that point to new approaches for cancer prevention and treatment. During the K99 phase, I have demonstrated an Integrated barcode platform that can measure a large number of protein markers from small quantities of whole blood or from single cells. A panel of signaling proteins that have important implication in tumor inflammation has been integrated Into the microchip platform. In the ROO phase, I will further utilize this technology to Investigate the fundamental biology of tumor-immune interaction via measuring the paracrine signaling pathways between tumor and immune cells. I will also study the heterogeneity of tumor microenvironment by combining population dynamics modeling and the microchip-based molecular analysis. Once this model is trained with experimental or clinical data, it will become a useful tool to predict the outcomes or therapeutic responses of cancer patients. In addition, an on-chip culture of tumor cells and immune cells will be developed to emulate tumor microenvironment and then used for rapid, effective anti-cancer drug screening. To accomplish these goals, I will (1) use the Integrated barcode chip platform to study chronic Inflammation as a common mechanism in various human diseases and to reveal their correlations; (2) apply this tool to monitor the Immunological responses of cancer treatment; and (3) design a Tumor-on-a-Chip to re-engineer the tumorimmune Interactions ex vivo and use this platform for effective anti-cancer drug screening with rich feedback."}
{"text": "The long term objective is to study the chemistry of the prosthetic group of visual pigments. For, although the spectroscopy of retinal, N-retinylidene n-butylamine (NRBA) and related polyenes has been examined in some detail, knowledge of the relationship between the chemistry and spectroscopy is not clearly understood. In order to correctly deduce the chemical role of the prosthetic group within the environment of the pigment protein, the chemistry of the prosthetic group in chemically defined experimental systems must first be probed. In this work, the hydrogen bonding (H-bond) and proton transferring reactions of CH3(CH=CH)5C=NC4H9 (compound I) and NRBA are to be studied both qualitatively and quantitatively. Some of the experimental variables to be studied are: i) donor reagents such as phenol, substituted phenols, acetic acid and chloro substituted acetic acids, ii) selected solvents such as hydrocarbons and chlorohydrocarbons, and iii) changing temperature. It was learned in prior studies that, in hydrocarbon solvents, when I is treated with a weak acid such as phenol, and when the temperature is progressively lowered, three separate spectra sequentially appear. These spectra belong to respectively: compound I, H-bonded I, and proton transferred I. This entire process can be placed in the formal framework of acid-base equilibria studies. The degree of conversion from one species to another will be obtained from changes in absorbance that will be used to find equilibrium constants. The resulting values will be used to determine the temperature dependent thermodynamic constants deltaH, deltaS. Compound I and NRBA undergo proton transfer with certain weak acids, and these reactions will be examined quantitatively in the same manner. The obtained thermodynamic constants for both reaction steps and the three excitation energies will link the three species together in a relative energy state diagram. From these results, it will be possible to measure the relative stability contributed to each ground and excited state by the selected donors and solvents."}
{"text": "The overall objectives of this grant are to determine the effect of cell surface alterations on the immunologic behavior of cells. Certain substances all appear to increase cell immunogenicity in appropriate doses so that many studies have involved the design of model systems for tumor immunotherapy using such altered tumor cells. In recent years, attention has turned less and less to the design of model systems for tumor immunotherapy than to the investigation of the mechanisms of the beneficial effects of tumor immunotherapy using active specific methods of immunization in vitro. Attention has particularly turned toward investigation of how these and other substances interact with cells and alter cellular behavior in vitro."}
{"text": "The purpose of this study is to evaluate the benefits of combined Glucotrol XL and acarbose therapy in patients with type II diabetes mellitus who cannot achieve adequate glycemic control with Glucotrol alone. The comparison of treatments will include evaluation of fasting plasma glucose, post-prandial glucose excursions, incidence and frequency of hypoglycemia, free insulin, lipids, and overall metabolic control. This is a randomized, double-blinded, parallel controlled trial involving 26 patients with type II diabetes mellitus who are hyperglycemic on the maximum dose of a commercially-available sulfonylurea. Patients will undergo screening laboratory evaluations (including CBC with differential, UA, OP16, stimulated C-peptide in response to a mixed meal, lipoprotein profile, total insulin, HBA1c, EKG, fasting plasma glucose, and Beta HCG for females of reproductive age) and a brief interview to determine if they qualify for study participation. Patients who qualify will proceed to visit 2 at which time a history and physical examination will be conducted and the patients will be instructed in a 60% carbohydrate diet. At that time, patients will be switched to Glucotrol XL who are not already receiving it. Following a lead in period of 6 weeks, patients must have a fasting plasma glucose >140mg/dl on the maximum dose of Glucotrol XL (20mg/dl) and a 60% carbohydrate diet in order to enter the study. The next visit (visit 3) will consist of an admission to the General Clinical Research Center (GCRC) at Duke University Medical Center at which time the following laboratory tests will be performed: fasting plasma glucose, HBA1c, C-peptide, total insulin, 1 hr post prandial insulin and glucose levels, 2 hr post prandial insulin and glucose levels, and lipoprotein profile. During this admission, patients will be randomized to one of two treatment arms. One treatment will consists of therapy with Glucotrol XL and placebo for 4 months. The other treatment consists of 4 months of Glucotrol XL combined with acarbose therapy. The initial dose of acarbose will be 25 mg tid, increasing to 50mg tid after 2 weeks and, if necessary, to 100 mg tid after an additional 2 weeks. Our end point will be a fasting plasma glucose under 120 mg/dl or maximum dose acarbose. Approximately 2 months following the admission, patients will return for visit 4 as an outpatient. The purpose of this visit is to assess the level of glucose control and to monitor for drug toxicity. Assessment will include examination of home blood glucose monitoring and the following labs: LFT's and HbA1c. Approximately 2 months after visit 4, patients will be readmitted to repeat the laboratory evaluations of the first admission. The study is ongoing, and results are not available to date. SIGNIFICANCE: Type II diabetes mellitus is a syndrome characterized by resistance to insulin and relative insulin deficiency. While some patients can be managed and euglycemia achieved using oral hypoglycemic agents alone, others require insulin (frequently in substantial doses). Correction of fasting hyperglycemia is a main concern, since it is often impossible to correct subsequent daily hyperglycemia without improving the fasting blood glucose. However, the use of insulin therapy in patients with type II diabetes is associated with several problems, including invariable weight gain and facilitation of atherogenesis. It is therefore desirable to develop other forms of therapy which might minimize insulin use in this patient population."}
{"text": "DESCRIPTION: (Adapted from the Applicant's Abstract.) The objective of the proposed research continues to be a comprehensive, systematic and parametric evaluation of the roles that spectral locus and temporal variation play in selected binaural experiments. By producing interaural temporal and intensive stimuli, we hope to extend greatly knowledge of how the human's ability to lateralize, to discriminate and to detect is affected by the spectral region and spectral compositions of sounds that are processed via binaural interactions. One subset of experiments concerns lateralization and will evaluate, as a function of spectral locus and temporal variations, how extent of laterality is affected by combinations of interaural time-delays, phase-shifts, and intensive differences. These data will be used to extend a new model, the \"weighted-image\" model to incorporate interaural intensive differences across spectral regions. Other lateralization experiments will assess the relative potency of onset/offset and ongoing interaural delays in combination with interaural intensive differences and how each affects the intracranial locus of acoustic images. Another subset concerns the detection and discrimination of interaural delay when a narrow spectral region containing the delay is known vs. when the listener is uncertain with respect to its spectral location. Another group is designed to assess discrimination of interaural delay when the base or initial delay is varied. Narrow bands of noise will serve as targets and these experiments are crucial for determining how well relative discriminations can be performed for high-frequency stimuli. The potential health benefits are a better understanding of how the ear and brain process information and a potential for \"better\" diagnostic procedures that may, eventually, have clinical significance."}
{"text": "Purkinje neurons and granule neurons are two major cell populations in the cerebellar cortex. Purkinje neurons are well recognized by the size and complexity of their dentritic arbors. The numerous spines on the arbors serve as postsynaptic sites for reception of afferent input from the parallel fiber axons of granule neurons. The presynaptic axonal membranes, associated vesicles containing neurotransmitters and the thickened postsynaptic spinous membranes form structural complexes called synapses. The total number of synapses on Purkinje dendritic arbors of normally aging rats was known to be significantly reduced after chronic ethanol treatment, an effect that was reversed after recovery from the ethanol treatment. There were no ethanol-induced decreases in the number of parallel fibers because the number of granule neurons did not change. The overall size of the dendritic arbors was unchanged, but dendritic segments within the arbors were longer than normal following ethanol treatme nt, suggesting that some segments and their branch points had been deleted. The purpose of this study was to determine whether the loss of synapses represented a direct effect of ethanol on the density of spines (spines/nm) on the dendritic segments or an indirect effect of segment deletion. Spine densities on terminal dentrites were determined in Purkinje neurons from 40 old male Fischer 344 rats that were assigned randomly to one of three treatments groups, a chow-fed control group (n=8), an isocaloric and isovolumetric (pair-fed) control group (n=16), or an ethanol-fed group (n=16). Brain tissues were prepared by the Golgi-Cox procedure for light microscopy and coded to avoid investigator bias. 14 Purkinje neurons from each rat (a total of 560 cells) were randomly selected for quantitation of spines. In each neuron, spine counts and terminal segment lengths were determined for 14 paired and 14 umpaired terminal dendritic segments (a total of 15,680 measurements). Group averages tested by ANOVA showed no statistically significant decreases in spine density on spiny branchlets of Purkinje ne urons after chronic ethanol consumption. It was concluded that ethanol did not reduce the density of spines on dendritic segments of Purkinje neurons directly and that the decrease in synaptic density after ethanol treatment represented an indirect effect of segment deletion. (Funded by NIH/NIAAA grant AA05592) Keywords: Cerebellar Cortex, Ethanol, Golgi Cox, Purkinje Neurons, Synaptic Spine"}
{"text": "The long range goal of this project is to use functional magnetic resonance imaging (FMRI) to elucidate the brain mechanisms responsible for normal vision and for brain-related visual pathologies. As a new technology for studying brain function, FMRI is potentially revolutionary because it can produce high resolution pictures and movies of dynamic brain activation during sensory input, task performance, or cognitive activity. Since it is non-invasive and has no known health risks, hundreds of images can be obtained from a single subject, thus permitting detailed and thorough studies of brain function in both human and animal subjects. This project seeks to develop and test this technology further and then apply it to the study of visual perception. Fortunately, the neural basis of vision in monkeys is sufficiently well understood to provide a solid background for interpreting the results of FMRI experiments and for comparing and interrelating visual mechanisms in human and simian species. This will permit the knowledge gained in previous animal experiments to be applied more directly to the study of the human nervous system. To this end, the specific aims of the project are: 1) to determine the parametric relationships between the FMRI signal and visual stimulation, 2) to use FMRI to study the functional specialization of cerebral cortex by creating maps of topographic and functionally selective activation within and among different cortical visual areas in both humans and monkeys, 3) to use FMRI to record and analyze the temporal and spatial evolution of cortical activity during prolonged visual tasks, and 4) to explore the potential application of FMRI in the assessment of visual deficits resulting from cortical pathology. To accomplish these aims, a high magnetic field strength MR scanner (Bruker 3 Tesla) will be used to image cortical activation during computer controlled visual stimulation delivered via a custom designed optical system. Using novel computer algorithms, the 3-dimensional patterns of activation will be displayed on unfolded, two-dimensional maps of the cortical surface. Ultimately, these techniques will be used to study abnormal brain activation in a small number of patients with well characterized visual deficits. The goal will be to explore the potential use of FMRI in the assessment of brain pathology. The results of this project will provide new insight into the relationships between physical brain mechanisms and both normal and impaired perceptual experience. In addition, the testing and refinement of the FMRI technology will contribute significantly to the development of a powerful new tool for medical science."}
{"text": "Moderate alcohol intake is thought to be cardioprotective. The postulated mechanism has always been the elevated high-density lipoprotein cholesterol concentration seen in alcohol consumers compared to nondrinkers. Recent studies have raised the possibility that insulin sensitivity may be mediating the effect between moderate alcohol consumption and lowered cardiovascular disease (CVD) risk. This study therefore has the following aims: 1.To assess whether the daily consumption of a moderate amount of alcohol can improve insulin sensitivity in a volunteer group of nondrinkers predefined as insulin resistant. 2. To determine if moderate alcohol intake by improving insulin sensitivity will have the beneficial metabolic outcomes in HDL, insulin and glucose to lower CVD risk. 3. To investigate if measures of endothelial function (asymmetric dimethylarginine and soluble cellular adhesion molecules) as risk factors for CVD are affected by moderate alcohol consumption and insulin sensitivity. Insulin sensitivity/resistance will be measured by the insulin suppression test. Given the morbidity and mortality of insulin resistance and CVD, it becomes important to clarify the role of moderate alcohol consumption to both."}
{"text": "Group A Streptococcus (GAS, also known as Streptococcus pyogenes) is a common human pathogen, most frequently causing pharyngitis or \"strep throat.\" However, severe invasive infections like necrotizing fasciitis and toxic shock syndrome are also caused by this organism, leading to its nickname in the lay press - \"flesh-eating bacteria.\" This work will help explain the process by which a microbe in the throat can invade and cause life-threatening illness. Previous studies found that GAS alter transcription of multiple virulence factors in response to the human cathelicidin, LL-37, an antimicrobial peptide. A membrane-bound sensor kinase (CsrS) and its cytoplasmic response regulator (CsrR) appear to drive this reaction. This application aims to examine the role of LL-37 in stimulating an invasive phenotype in GAS. We will focus on the interaction of GAS with two host cell types commonly encountered early in infection: keratinocytes and macrophages. We will examine the role that LL-37 and CsrRS play in GAS's ability to avoid internalization and killing. Overall, these studies will examine a fascinating evolutionary interaction between a human pathogen and the innate immune system, offering new insights into the pathogenesis of GAS infections. PUBLIC HEALTH RELEVANCE: The public health burden of GAS infection remains high. Sequelae of infection, including rheumatic heart disease, continue to plague especially developing areas, where prompt antibiotic therapy is unavailable. Additionally, there has been a rise in invasive GAS infections as a new, more virulent strain has spread around the globe. For these reasons, improving our understanding of the earliest stages of human infection will be essential to battling this common yet evolving pathogen."}
{"text": "Schistosomiasis is a tropical parasitic disease infecting over 200 million people. Treatment relies on a single drug, praziquantel (PZQ). In the absence of back-up drugs with PZQ's therapeutic spectrum, the risk of resistance to PZQ and eventual drug failure is a major concern. Traditional phenotypic screens, using adult- stage S. mansoni, are low-throughput and incompatible with modern high-throughput screen (HTS) systems. In keeping with the NIAID's mission, the present proposal aims to turn a newly developed, moderate- throughput phenotypic screen (MTS), into a fully automated, quantitative HTS to accelerate drug discovery for this infectious disease. The proposal involves three PIs with ongoing collaborations and respective biological, screening-technology and bio-computational skills who are focused on just this goal. As a first research track for this proposal, we will utilize in-house automation and a high-content screening (HCS) system to significantly increase throughput over our published MTS approach. The proposal will involve;expanding robotic plating of the parasite, developing protocols for bright-field and fluorescence-based microscopy and adapting commercial image-analysis software to identify (segment), quantitatively describe and track the motion of parasites with a view to prioritizing compounds for further pre-clinical development. Because commercial HCS analysis tools are not likely optimized for recording the complex and dynamic phenotypes displayed by this multicellular parasite, we will also pursue a second and parallel track of research. Specifically, we will develop de novo an automated image-analysis screening technology to define, identify, and quantify the range of phenotypic responses (morphological and behavioral) possible in this parasite. Ultimately, both the experimental and computation tracks will together produce a standardized HTS protocol and a comprehensive, quantitative suite of image-analysis programs to categorize parasite phenotypes. Such rigor will facilitate the screening of large numbers of potential compounds and their prioritization into the secondary and tertiary screening assays available in-house. We also intend to make the algorithmic framework including its methods and implementations, publicly available. PUBLIC HEALTH RELEVANCE: The major goal of the project is to turn a moderate-throughput phenotypic screen (MTS) system for schistosomiasis, into a fully automated, quantitative high-throughput screen (HTS). By so doing, the rate of discovery of drugs to treat this global tropical disease will be increased."}
{"text": "Breast cancer is a heterogeneous disease that can be subclassified into several tumor types. Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. IBC is a locally advanced breast cancer that affects women at an earlier age than other types of breast carcinoma. The disease is characterized by a high tumor microvessel density, fast disease progression, and by extremely poor survival. Although the frequency of IBC among breast cancer cases in the US is only 3-5%, it appears to be much more common in other geographic areas, such as North Africa. The causes of IBC are unknown and both environmental and genetic risk factors are thought to be involved. The early onset of the disease, the strong angiogenicity and propensity of IBC to invade vessels, and the large disparity in the IBC incidence among various populations are indicators of genetic predisposition and inherited susceptibility. Both low- and high-risk cancer susceptibility genes may determine the risk for IBC. Two genes, WISP3 and RhoC GTPase, have recently been identified that are differently expressed in IBC when compared with non-IBC breast cancer. WISP3 is a putative tumor suppressor gene for the disease, and may regulate the insulin-like growth factor pathway. RhoC GTPase is an overexpressed oncogene in IBC, and modulates the induction of angiogenic factors in breast cells. As part of our ongoing breast cancer case-control study, we collected seven specimens from inflammatory breast cancer (IBC) patients, 20 specimens from non-IBC breast cancer patients with poor survival and 20 specimens from non-IBC breast cancer patients with good survival. Serial sections of OCT-fixed tissues have been prepared from the 47 fresh-frozen tumors, and ten normal breast tissues from breast reduction surgery. The first slide of the serial sections has been formalin-fixed and stained with hematoxylin and eosin to identify tumor and surrounding normal tissue. The slides have been reviewed by a pathologist for the presence of tumor cells. The sections are currently being microdissected. We hypothesize that IBC has a distinct expression profile in tumor and normal surrounding tissue that predicts the risk of metastasis and poor survival. Our objective is to identify genes that are preferentially expressed in tumors and surrounding normal tissue of patients with IBC by comparing the IBC gene expression profile with the profile of non-IBC breast tumors. This is a novel approach. IBC is a rare disease, and it is difficult to collect flash-frozen IBC specimens that are suitable for array-based gene expression analysis. We have a particular interest in those genes that promote metastasis, and are expressed in the surrounding normal tissue, but not in cancer cells. To date, no report describes an expression profile in surrounding normal tissue that is associated with tumor metastasis and poor survival in breast cancer. However, a recently reported molecular signature of metastasis in primary breast tumors contained genes that are thought to be expressed in normal tissue, and not by cancer cells. Specific aims 1) Array-based gene expression profiles will be generated for all breast tumors and the surrounding normal tissues, and for ten disease-free breast reduction tissues. We will use laser-captured microdissection to separate tumor from normal tissue. Supervised methods for class comparison and prediction will be used to establish gene expression profiles that distinguish 1) IBC tumors from surrounding normal tissue, 2) IBC tumors from non-IBC tumors, and 3) surrounding normal tissue of IBC tumors from surrounding normal tissue of non-IBC tumors. Non-IBC tumors will be subclassified into patient groups with either good or poor survival, and will be analyzed as separate groups. 2) Candidate marker genes for tumor progression and survival will be validated by Taqman, and further studied by immunohistochemistry, Western blotting, and in appropriate experimental models."}
{"text": "The Clinical Trials Office of the UNMC Eppley Cancer Center participates in the activation and conduct of clinical cancer research at the Cancer Center. This includes assistance with protocol development, coordination of initial and periodic reviews submitted to the Institutional Review Board, data coordination and collection, and coordinates supervision of appropriate clinical trials by the Audit Committee and the Data and Safety Monitoring Committee. Since the last review (7/1/99 through 6/30/03), a total of 3171 patients have been entered onto Cancer Center clinical research protocols, of which 1054 patients have been entered onto therapeutic trials and 2117 patients were entered onto correlative clinical research studies, companion studies, and chemoprevention clinical research protocols. During the past four years (7/1/99 - 6/30/03) 385 patients were entered onto investigator-initiated clinical trials. During the past year (7/1/02 - 6/30/03), 41 Cancer NCI-A 40 2 P30 CA036727-20 COWAN, K Center members have utilized this facility, of which 26 had external funding, and 156 clinical research protocols were open to accrual during this period. In the last year (7/1/02-6/30/03), 558 patients were entered onto Cancer Center clinical research protocols, of which 310 patients (or 56% of the total patients) were entered onto therapeutic trials. Over the last year, the Cancer Center has undertaken several initiatives to enhance clinical research activities by increasing the support of the administration of the Clinical Trials Office, consolidation of the administrative staff of Clinical Trials Office in a central location in the Lied Transplant Center and the establishment of a Data and Safety Monitoring Committee."}
{"text": "Selenium has a role in preventing heart disease and is rapidly becoming recognized as a chemopreventive agent against cancer. The beneficial affects of this element are due, at least in part, to its presence in selenoproteins as the amino acid, selenocysteine (Sec). Our program, therefore, focuses on the means by which Sec is incorporated into protein and the role of specific selenoproteins in human health. As Sec tRNA is regarded as the key molecule in selenoprotein biosynthesis, we have produced transgenic mice encoding as few as 2 and as many as 20 extra copies (transgenes) of the Sec tRNA gene to examine effects of its overexpression. We introduced a silent mutation in the transgene that enables us to distinguish its expression from that of the corresponding wild type gene by primer extension. The transgenes and the wild type genes contribute equally to the total Sec tRNA population, but the level of expression is not linear with gene copy number, strongly suggesting that gene expression is regulated by a feedback mechanism. Overexpression does not appear to affect selenoprotein synthesis or protein synthesis as a whole. This observation, coupled with our earlier findings of normal selenoprotein synthesis in cells containing only ? the Sec tRNA population, demonstrate that Sec tRNA is not a limiting component in selenoprotein translation. We have also sequenced the Sec tRNA gene in zebrafish and Chinese hamsters. Zebrafish contains two functional copies of this gene whereas all other higher and lower animals examined to date contain only one functional gene. Chinese hamsters contain three pseudogenes whereas all other organisms examined to date contain only one or no pseudogenes and since the Chinese hamster gene encodes two pyrimidine transtions compared to all other mammals, it seems likely that these transitions occurred by editing of a transcript and re-insertion into its genome. In our studies on examining the role of specific selenoproteins in human health, we have focused on a recently discovered selenoprotein, designated as the 15 kDa protein. We have shown that its levels are normally elevated in prostate as compared to other tissues, but are reduced substantially in prostate cancer. Since selenium is known to have a chemopreventive role in prostate cancer, it would seem that the 15 kDa protein may have a role in preventing malignancy in this tissue. We, therefore, have made the appropriate constructs encoding the 15 kDa protein to examine the affects of its overexpression in mammalian cells in culture and in transgenic mice. These studies are in progress. We have confirmed the occurrence of two polymorphisms in the 3-untranslated regions of the 15 kDa protein gene and 1 of the polymorphisms may be involved in regulating protein expression. There was statistically significant differences in allele distribution between head and neck cancer tissues and controls. In a related study on the effects of selenium on human health, a previous member of our group (V.N.G., see collaborators below), had shown earlier that the incorporation of selenium into protein is diminished in HIV infected cells. We have subsequently shown that the HIV encoded TAT protein binds to the SECIS element which is a specific stem-loop structure in all selenoprotein mRNAs required for insertion of Sec into protein; but TAT also binds to most double stranded RNAs examined and thus shows no specificity. We are presently using in vivo experiments to determine if specificity occurs in selenoprotein synthesis. We have discovered two new human selenoenzyme thioredoxin reductases and characterized the role of a previously identified thioredoxin reductase in redox regulation of cell signaling. In addition, we identified a thioredoxin reductase in C. elegans and this appears to be the only selenoprotein in this organism. Our study on examining the Sec tRNA population in Drosophila has been completed as well as our study on examining the role of hypermodified bases in the anticodon loop of specific tRNAs on ribosomal frameshifting. - Genes, Gene Targeting, Gene expression, Post-transcription regulation, Selenium, Selenocysteine, Transgenic mice, Translation, - Neither Human Subjects nor Human Tissues"}
{"text": "The E3 ubiquitin ligase Mdm2 acts as a key factor in the degradation of p53 tumor suppressor in human cancer cells and inhibition of Mdm2 activity is a validated approach to activate p53 function in cancer therapy. Nevertheless, inhibitors of Mdm2 such as Nutlin-3 has many limitations, suggesting that additional targets in this pathway need to be further elucidated. The proposed studies aim to dissect the mechanisms of two newly identified ubiquitin ligases critically involved in ARF/p53-mediated tumor suppression and provide the unequivocal evidence for proof of concept to develop the inhibitors of these two potential critical targets in cancer therapy. ARF was originally identified as an alternative transcript of the Ink4a/ARF tumor suppressor locus. It is well accepted that ARF plays a major role in activating p53 function, specifically, in respond to oncogenic stress. Surprisingly, by usig a mouse model in which p53 status can be reversibly switched in vivo between functional and inactive states, it has been found that the protection from tumorigenesis is absolutely dependent on ARF-mediated p53 activation in vivo. Thus, ARF apparently plays a much more important role in suppressing tumorigenesis in general, than originally anticipated. Restoration of the ARF-p53 function remains an important goal in the quest for more effective cancer therapeutics. Indeed, like ARF, Nutlin-3, a small molecule inhibitor of Mdm2, is able to activate p53, and exhibits antitumor efficacy in cancer cells that express wild-type p53. Nevertheless, although Nutlin-3 can effectively block the interaction of Mdm2 and p53, Nutlin-3 is also very effective at increasing the synthesis of Mdm2 mRNA and protecting Mdm2 from degradation. These effects of Nutlin-3 on Mdm2 lead to a rapid restore of cancer cell growth upon temporary removal of the compound, sabotaging the efficacy of nutlin-3 in cancer therapy. Thus additional cancer targets aiming at this pathway are clearly needed for more effective therapeutic purpose. Our preliminary studies reveal that targeting two novel ubiquitin ligases (ARF-BP1 and ULF) in the ARF-p53 pathway represents a particularly attractive approach to activate ARF-mediated tumor suppression in cancer therapy. The central hypothesis to be tested here is that inactivation of ARF-BP1 or ULF can effectively activate ARF-mediated function and suppress tumorigenesis in vivo. It includes the following two specific aims. In Aim 1, we will investigate whether inactivatin of ULF activity is sufficient to suppress tumorigenesis by using mouse models. In Aim 2, to validate whether ARF-BP1 is indeed a potential cancer target, we will use the ARF-BP1 mutant mice to examine whether inhibition of ARF-BP1 suppresses tumorigenesis in vivo in a manner reminiscent of ARF activation."}
{"text": "Synaptic connections are formed during development but are continually modified through experience and learning and memory. Any alteration to the normal connectivity leads to behavioral and cognitive changes that underlies psychological and neurodegenerative disorders. Therefore, understanding how functional networks of synaptic connections are established is critical for gaining insights into normal brain function. A mutant screen for defects in synapse in Drosophila has led to the discovery of a mutant line that shows aberrant synapse formation. Named as baha (bad hair day), homozygous mutant photoreceptors and motor neurons project to their correct target area in early developmental stages but subsequently fail to maintain contacts or form mature and functional synapses. Preliminary work thus suggests that baha may function as a critical factor in synapse formation. To investigate baha's role in synaptogenesis, I propose to perform further characterization of its synaptic phenotype, to identify and clone the baha gene, and to investigate the molecular mechanisms of its action. Cloning and characterization of baha gene may thus lead to identification of components of synapse formation, a process that remains poorly understood."}
{"text": "The objective of our Research is to elucidate the mechanism of electron transfer and proton translocation in the cytochrome b-c1 region of the mitochondrial respiratory chain. During the forthcoming project period our approach will be to test which electron transfer reactions in the b-c1 region take place in the absence and presence of the \"g equals 1.90\" iron-sulfur protein, which we have recently purified in a reconstitutively active form from heart mitochondria."}
{"text": "Obstetric practices, evaluative tools, and treatments have been sometimes passed down by tradition or introduced without rigorous study to evaluate their impact on maternal, fetal, and/or neonatal outcomes. Long-term maternal and infant outcomes are particularly understudied, and surrogate markers of morbidity are commonly substituted in studies of smaller size. The impact of pregnancy complications can be devastating, affecting decades of life. The health and financial impacts of adverse maternal and infant outcomes on the family are potentially staggering. With millions of births in the United States each year, even uncommon complications can have a profound societal impact. Since its inception, the Eunice Kennedy Shriver NICHD Maternal-Fetal Medicine Units (MFMU) research network has conducted clinical trials and observational studies that have brought important understanding regarding prediction and prevention of preterm birth (Preterm Prediction Study, 17-OH progesterone in singletons, twins, and triplets, omega-3 supplements, antibiotics for asymptomatic bacterial vaginosis and T. Vaginalis), treatments for those at imminent risk (antibiotics for preterm labor and premature rupture of the membranes, repeated antenatal corticosteroids, magnesium sulfate neuroprotection), cesarean delivery and VBAC, prevention of preeclampsia, and treatment of gestational diabetes, among others. This network played a key role in evaluating technologies used in obstetric practice (ultrasound cervical length, fetal fibronectin, home uterine monitoring, fetal pulse oximetry). In this application, we demonstrate that the Department of Reproductive Biology at Case Western Reserve University (CASE) investigators have the ability to conduct collaborative research, and have successfully participated in and are qualified to continue in the MFMU network. Our investigators have strong backgrounds in clinical and multicenter studies, those involving neonatal and long term infant follow-up, and in the above listed network studies. CASE investigators provide leadership in network study development and administrative committees necessary to study completion and dissemination of their results. There is a strong administrative and research infrastructure to support the MFMU network, including the CASE-Clinical & Translational Science Collaborative. CASE offers the strengths needed to successfully lead and participate in NICHD-MFMU network research which will directly influence obstetric practice and improve pregnancy outcomes nationally and internationally. PUBLIC HEALTH RELEVANCE: Research performed by the CASE Department of Reproductive Biology, within the Eunice Kennedy Shriver NICHD-MFMU network, will inform and change obstetric practice. This research will identify interventions and tools to predict, prevent or mitigate pregnancy complications such as preterm birth, preeclampsia, fetal growth restriction and perinatal infections, among others. Focusing on significant perinatal and long-term outcomes, we will bring to an end procedures and practices that are ineffective or incur undue risks."}
{"text": "Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by gradual synapse and memory loss. While significant progress has been made in understanding Alzheimer's disease (AD) pathology, treatment options for AD remain limited. Therefore, further characterization of signaling pathways involved in the pathology of AD is important in facilitating the identification of new therapeutic targets. Emerging evidence postulates that epigenetic changes play critical roles in dementia. Consistent with this, inhibitors of histone deacetylases (HDACIs), such as SAHA (suberoylanilide hydroxamic acid; vorinostat), enhance neuroplasticity and memory-associated gene expression in the brain. These findings suggest that HDACIs may be novel agents in treating AD. Recently, a drug discovery team at Georgetown University has developed new Class II-specific HDAC inhibitors (mercaptoacetamide-based 6MAQH (W2), and hydroxamide-based H6CAHA (I2)), which have improved solubility and pharmacokinetic properties compared to HDACI SAHA. We have exciting preliminary data suggesting that 1) W2 decreases Ab levels in stably overexpressed APP N2A cells, 2) W2-injected hAPP 3xTg AD mice (9-10 month old, moderate Ab production) decreases Ab levels compared to control treatment, 3) W2 treatment decreases mRNA levels of A synthesis enzymes and increases mRNA levels of A degradation enzymes (MMP2), and 4) W2-injected hAPP 3xTg AD mice (9-10 month old, moderate memory impairment) rescued memory deficits compared to control treatment as measured by Morris Water Maze. Based on these observations, we hypothesize that Class II HDACIs (W2, I2) regulate AD pathological processes and cognitive functions by modulating the gene transcription of A synthesis and degradation enzymes. To test this hypothesis, we will examine the molecular mechanisms by which W2 and I2 regulate AD pathological processes (Aim 1). We will examine the role of Class II HDACIs W2 and I2 on rescuing cognitive deficits in 3xTg AD mice (Aim 2). Results from our study will provide the molecular mechanisms of action of Class II HDACIs on Ab production, Ab plaque formation, and cognitive function for future development of AD therapeutics."}
{"text": "The recent availability of a genetic test (homozygosity for the C282Y mutation in the HFE gene) for the diagnosis of hereditary hemochromatosis (HH) has focused renewed attention on this disorder and has resulted in the diagnosis being made in younger subjects with smaller iron burdens. However, phlebotomy therapy in HH remains hampered by lack of simple, physiologic laboratory monitoring guides. In addition, phlebotomy therapy has been perceived as wasteful because the blood obtained has not been not used for allogeneic transfusion although many HH subjects meet standards for allogeneic blood donation. Recent regulatory changes now allow increased flexibility in establishing policies for transfusion of blood obtained from HH subjects. In studies performed over the last 12 years, we have developed a simple, physiologically based phlebotomy guide as the basis for a protocol to utilize blood from appropriate hemochromatosis patients for allogeneic transfusion. Prospective studies in the DTM have shown that the red cell mean corpuscular volume (MCV), a physiologic, precisely measured indicator of erythopoietic iron availability, when measured in conjunction with the hemoglobin, provides all the information necessary to manage patients during induction as well as maintenance phlebotomy. Phlebotomy was started weekly and transitioned to less frequent intervals when the MCV decreased to 5 to 10 percent below baseline and the hemoglobin and MCV were decreasing n concert. During maintenance phlebotomy, the hemoglobin was targeted at greater than 13 g/dL and the MCV was kept at 5 to 10 percent below baseline (generally 85 to 90 (3). A stable, asymptomatic, iron-depleted state was obtained in all patients after a median of 38 phlebotomies and removal of 9.0 grams of iron. The MCV was stable for a prolonged period during induction therapy, and then decreased in a smooth, consistent manner, indicating the onset of iron-limited erythropoiesis. Transferrin saturation varied considerably during phlebotomy, but remained below 40 percent during stable MCV-targeted maintenance. Ferritin values were not useful to assess the pace of iron depletion or maintenance phlebotomy. The median stable maintenance interval was 7.5 (range 6 to 16) weeks, corresponding to an average daily iron removal of 35 to 67(g/kg. The data in this study indicate that the red cell MCV is a physiologic, inexpensive guide to phlebotomy therapy in patients with HH. In contrast to standard, more expensive measurements such as ferritin levels, the MCV accurately predicts iron depletion, prevents excessive rebound transferrin saturation and guides maintenance according to each patient?s individual iron absorption. The MCV-guided phlebotomy guideline, along with extended rheumatologic and hepatic evaluations, forms the core of a large, omnibus protocol being developed to optimally manage phlebotomy in hemochromatosis patients while utilizing blood from appropriate patients for allogeneic transfusion."}
{"text": "Project Summary The successful management of chronic pain is inadequate in many patients and has contributed to the abuse of and addiction to opioids, a continuing major public health crisis in the United States and worldwide. The development of non-addictive pain therapeutics can help counter opioid addiction and benefit patients, including those who suffer from neuropathic pain, and in particular diabetic neuropathic pain (DNP). Our project?s goal is to develop a safe, efficacious and non-addictive small-molecule drug that activates Kv7 voltage-gated potassium channels to address overactive neuronal activity in DNP. The first specific aim is discover Kv7 activators that favor Kv7 isoforms altered in DNP and found in dorsal root ganglia, namely the Kv7.2/3 isoforms. Through iterative lead optimization studies on our novel series of Kv7 activators, we are targeting Kv7.2/3 activation with selectivity over both Kv7.4 channels and another off target of other Kv7.2/3 activators, GABAA receptors. This approach is expected to decrease off-target side effects observed with the use of earlier non-biased Kv7 activators including urinary retention (mediated by Kv7.4), and somnolence and dizziness (mediated by enhancement of GABAA receptor function). This phase also includes optimization of absorption, distribution, metabolism, excretion, and toxicity profiles and building correlations between in-vitro activities to in-vivo efficacies in a neuropathic rat DNP model. A second animal model, the L5/L6 spinal nerve constriction model, will also be used to test the ability of candidate compounds to generalize to other forms of neuropathic pain. The second specific aim will be to further characterize two to four advanced compounds by assessing additional pharmacological properties including CYP450 induction/time dependent inhibition and in-vitro safety/selectivity panels. The third aim is to select a candidate for study in non-GLP toxicology and pharmacokinetic studies in rodent and non-rodent species. Specific aims four and five involve completing the studies needed to prepare an Investigational New Drug (IND) application. These studies include Chemical Manufacturing Controls activities such as formulation studies and Good Manufacturing Practices synthesis of drug candidate followed by cardiovascular and safety pharmacology studies, and 28-day Good Lab Practices toxicology studies in two animal species. This screening paradigm is intended to establish a clinic-ready, well-tolerated and widely effective product to treat neuropathic pain."}
{"text": "We have previously demonstrated that beta-arrestin1 and 2 form complexes with various prostaglandin receptors during mouse skin tumor development (Chun et al., Carcinogenesis, 9:1620, 2009). Therefore, the goals of the present study were to elucidate the effects of beta-arrestin1 or 2 deficiency on mouse skin tumor development using the multi-stage mouse skin tumor model. Surprisingly, beta-arrestin 1 decreased the number and size of skin tumors using this model; whereas the deficiency of beta-arrestin2 did not effect the number of tumors but in fact significantly increased tumor size. Furthermore, while beta-arrestin1 deficiency decreased p-ERK1/2 and COX-2 induction, beta-arrestin2 deficiency increased p-ERK1/2 but had no effect on COX-2 expression. Further studies are underway to better elucidate the effects of beta-arrestin1 and 2 deficiency on the signaling pathways we have previously demonstrated to be involved in mouse skin tumor development. In the ovarian cancer studies, our results to date indicate that COX-1, but not COX-2, deficiency reduces mouse ovarian cancer development by about 50%."}
{"text": "Project Summary Significant progress in human cancer therapy in the last decade has been driven by conceptionally new approaches to targeting cancer, including cancer immunotherapy, cancer nanotherapy, or new types of biologics and small molecules. Both my dissertation and postdoc research will be focused on the development of fundamentally new approaches to targeting cancer. My dissertation research is focused on the development of photoswitchable lipids for the optical control of lipid metabolism and function. In addition to targeting specific receptors, ion-channels, or enzymes, light-induced structural changes in a lipid nanoparticle (LNP) could serve as trigger for the release of encapsulated drugs. Triggered release could markedly improve the efficacy of clinically approved LNP-based cancer therapeutics, which include Doxil/Caelyx, DaunoXome, Myocet, Lipo-Dox, or Marqibo. I seek to design and synthesize photoswitchable lipids for `photoactivable lipid nanoparticles', herein termed paLNPs, that allow for effective light- triggered release of encapsulated cancer drugs. Two complementary approaches will be developed for small molecule drugs and RNA-based therapeutics and the pharmacological properties of paLNPs will be systematically investigated in vitro and in cell culture. In my postdoctoral research I seek to use my acquired knowledge in chemistry, lipid-biology, and medicine to develop lipid-drug conjugates for biological targets that function at plasma-membrane signaling hotspots. The initial target will be the mutated oncogene KRAS G12C, which is ideally suited for this new approach. Conjugating selective covalent modifier of this oncogene with a lipid will attach an additional lipid tail to the surface of KRAS that could largely alter its membrane-protein interaction and in the best case completely inhibit its function. This could markedly increase the efficacy of the covalent pharmacophores currently in clinical trials. I seek to synthesize and systematically study these lipid-drug conjugates in vitro and in cell culture."}
{"text": "This proposal will investigate virulence mechanisms of cytoadherence/sequestration using Babesia bovis as a natural host model system. Virulent B. bovis infection is associated with development of severe clinical disease and neurological manifestations similar to those observed in human patients with severe malaria caused by virulent strains of P. falciparum. The proposed unique model enables in vivo study of virulence mechanisms using defined and reproducible virulence phenotypes. In this model, the role of cytoadhesion/sequestration in determining virulence and whether specific gene products mediate these phenotypes can be determined. Published and preliminary data from three virulent and derived attenuated B. bovis pairs strongly supports the role of VESA1 proteins in virulence, and the overall hypothesis that differential VESA1 protein expression alone may be responsible for mediating virulence. Clonal lines of virulent and attenuated strain pairs will be used and their differential phenotypes confirmed. The specific VESA1 surface protein dimer associated with sequestration and expressed by virulent versus attenuated strains both in vitro and in vivo will be identified using monoclonal antibodies. Direct demonstration that a specific VESA1 dimer is associated with sequestration and virulence will be determined by complementation and expression of cytoadhesion and virulence associated ves1 gene(s) in the attenuated T2Bo clonal line, followed by an in vivo study to demonstrate acquisition of the virulence phenotype. If the hypothesis is correct, and if similar mechanisms are used in other pathogens, molecular targets for intervention to prevent severe clinical manifestations and death associated with hemoparasite infection can be further examined. Future directions will be to determine specific domain(s) of VESA1 proteins required for virulence, determine whether these domains are present in other VESA1 proteins and if so whether these proteins are also associated with virulence. Once this is established, the design and testing of virulence directed interventions such as peptide analogues or vaccines to reduce the morbidity and mortality associated with hemoparasite infection in animals and humans will be possible. PUBLIC HEALTH RELEVANCE (provided by applicant): By expanding our understanding of mechanisms used by pathogens to cause disease (virulence mechanisms), advances in targeted therapeutics and vaccines to significantly reduce morbidity and mortality will be made. For blood-borne parasites such as malaria and babesiosis, both of which contribute very significantly to the global health burden in resource poor countries, cytoadherence is a virulence mechanism which leads to severe clinical signs associated with increased mortality. This project will use Babesia bovis infection of cattle as a natural animal model of cytoadherence and virulence, and will address a significant knowledge gap to demonstrate the causal relationship of specific parasite derived gene products to virulence."}
{"text": "16.0 Abstract: Hormone Responsive Cancers Program The overarching goal of the Hormone Responsive Cancers (HRC) Program of UMGCC is to reduce morbidity and mortality from cancers of hormone-responsive tissues. To achieve this mission, the program focuses on three specific themes and related aims: Theme 1: Therapeutic strategies against hormone-responsive tumors?identify new agents to target malignancies that remain responsive to hormone manipulation; Theme 2: Mechanisms of innate and acquired hormone resistance?identify and target the mechanisms that confer de novo or acquired resistance to hormone manipulation; and Theme 3: Invasion and metastasis? identify the mechanisms that promote tumor dissemination and identify therapeutic strategies to target these mechanisms. The HRC Program has 47 members representing 16 academic departments and 5 schools/colleges of the University of Maryland. Members of the program conduct cancer-focused research that receives $7.1 million total annual funding, including $3.4 million from NCI and $2.6 million from other peer-reviewed sources. In addition, HRC Program members receive over $1 million annually from non-peer-reviewed funding sources. Between January 2010 and December 2014, HRC members authored 407 cancer-related publications, of which 30 percent resulted from intraprogrammatic and 23 percent from interprogrammatic collaborations. Twenty-two percent of the publications represent collaborations with external investigators. Research efforts of HRC faculty are supported by extensive use of the BSS, FCSS, GSS, ISS, PBSS, and TLSS."}
{"text": "Abstract While inflammation is a normal, protective response to injury, it is becoming increasingly clear that uncontrolled inflammation is integral to most disease processes including cancer and heart disease, the leading causes of death in the United States. The resolution phase of inflammation is an active process involving downmodulation of proinflammatory mediators and clearance of dead or dying cells. Understanding the molecular mechanisms involved in the resolution of inflammation will provide a framework from which to design directed therapeutics to prevent or diminish aberrant inflammation. Rapid and efficient removal of apoptotic cells is critical to the resolution of inflammation. CD93 is a cell surface molecule required for ingestion of apoptotic cells in vivo and is expressed by myeloid cells, platelets and endothelial cells: those cell types principally involved in regulation of inflammation. It belongs to the Group XIV family of transmembrane C-type lectin-like domain (CTLD) containing glycoproteins with shared functions in adhesion, migration and inflammation. We have recently identified a soluble form of CD93 that is elevated in inflammatory fluids. This proposal tests the hypothesis that CD93 regulates monocyte/macrophage activation extracellularly via its membrane tethered or soluble ectodomain, as well as intracellularly via protein and lipid interaction with the intracellular tail. Specifically, the role of CD93 in regulating inflammation in vivo will be assessed in a sepsis model using both CD93-/- mice and mice double deficient in CD93 and TM-D1, a CD93 homologue that downregulates inflammation in sepsis. Soluble CD93 will be tested for its ability to enhance ingestion of apoptotic cells and gram negative bacteria by primary human and mouse phagocytes. Anti-inflammatory properties of sCD93 will be investigated and compared to TM-D1. Membrane tethered CD93 regulates phagocytosis and adhesion in vitro, critical processes in the regulation of inflammation. Previous studies that have identified specific CD93 cytoplasmic tail interacting molecules known to be involved in cytoskeletal dynamics, such as those required for phagocytosis and adhesion, will be extended. Localization and function of these molecules during the phagocytic process will be assessed. These studies will contribute to our understanding of inflammation, and may lead to CD93 directed therapeutics to regulate this process."}
{"text": "Liver is the primary site of alcohol metabolism and is the primary organ that is injured by alcohol. Though it has long been clear that ethanol is metabolized to acetate in hepatocytes, it is known that the acetate isn't burned as fuel in the liver or directly converted to fat in the liver. The knowledge that the calories from ethanol are burned in the muscle may have inhibited people from considering whether any further metabolism of acetate occurs in the liver and whether this could be linked to alcohol hepatopathology. Recently, liver mitochondrial protein acetylation has been described in response to alcohol ingestion. This means that liver mitochondrial proteins are tagged with the exact same molecule that liver produces when we drink alcohol. We also know from genetics that many mitochondrial proteins with the acetylation tag are inhibited by this adduct and that mice that have this modification accumulate in mitochondrial proteins get fatty liver. In this proposal, a straightforward mechanism from ethanol to acetylated liver mitochondrial proteins is proposed and will be tested. Moreover, nicotinamide riboside, a vitamin that has the property of increasing NAD+ in mitochondria, is proposed as an agent to protect against the development of alcohol-derived mitochondrial protein hyperacetylation and alcoholic fatty liver."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. . \" Glyceollins as novel targeted therapeutics for the treatment of metastatic triple negative breast cancer.\" Dr. Collins-Burow obtained her Ph.D. from Tulane in 1998. She then went to medical school at Tulane and carried out her residency and fellowship in hematology/oncology at Tulane as well. Although she stayed in touch with the laboratory somewhat and carried on some research, she needs time to get her laboratory work going again. Thus, we decided that she would be an alternate rather than a PJI and would receive pilot funding from the Cancer Center to begin to establish her model systems. Her outstanding fit in terms of animal models of carcinogenesis, as well as cell survival would make her an outstanding replacement for a graduating PJI when her work will be able to take off. Dr. Collins-Burow has demonstrated in the MDA-MB-231 cell line that glyceollin inhibits proliferation/survival of these cells in clonogenicity assays. Utilizing an in vivo xenograft mouse model she has shown the ability of glyceollin to suppress tumor growth of the MDA-MB-231 cells. A very exciting finding on follow-up of this study was a decrease in visceral lung metastasis in the animals treated with glyceollin. Based upon these preliminary data she proposes to test the overall hypothesis that glyceollins function as a novel targeted agent in triple negative breast cancer suppressing tumorigenesis and metastasis. She propose the following specific aims: Specific Aim #1, To test the hypothesis that glyceollins block triple negative breast cancer migration/invasion and chemokine signaling in vitro. Specific Aim #2, To test the hypothesis that glyceollin regulates key clusters of gene expression associated with a metastatic phenotype and chemokine function in triple negative breast cancer cells. Specific Aim #3, To test the hypothesis that glyceollins suppress in vivo tumorigenesis and metastasis of triple negative breast cancer cells. We believe that the glyceollins represent the parent structure for a new class of anti-cancer agents that can target tumorigenesis and metastasis of triple negative breast cancer while enabling us to begin to elucidate the distinct molecular targets of this disease which will ultimately be translated to a clinical trial for triple negative breast cancer."}
{"text": "The goal of this proposal is to address the growing problem of prescription opioid abuse by examining the effects of oxycodone in a laboratory setting in the presence of clinical pain and following significant pain relief following maintenance on the buprenorphine / naloxone combination (Bup/NX). Participants diagnosed with moderate pain initially will be admitted to hospital and transitioned from their baseline prescription opioid to a standing daily dose of Bup/NX. During the Bup/NX maintenance periods, participants will have the opportunity to self-administer oxycodone and subjective responses to oxycodone as well as analgesic, physiological and performance effects will be measured. In the second phase of this study, the same patients who participated in the inpatient phase will be followed on an outpatient basis while maintained on Bup/NX. Primary outcome variables during the outpatient phase will include percentage of negative urine toxicology samples for opioids and number of weeks in treatment. A major goal of this proposal is to determine which variables collected in the laboratory most reliably predict subsequent relapse to opioid abuse. In addition, the utility of Bup/NX in treating patients diagnosed with both chronic pain and substance abuse will be assessed. Despite the significant increase in prescription opioid abuse over the past 5-10 years, very few studies have systematically examined the issues surrounding prescription opioid abuse in the population of individuals with chronic pain. To our knowledge, no studies to date have examined opioid self-administration in persons with pain who have a history of opioid abuse. Specifically, the current proposal aims to 1) characterize the behavioral effects of oxycodone in Bup/NX-maintained pain patients who are currently abusing their prescription opioids; and 2) examine the relationship between behavioral effects of oxycodone as measured in the laboratory setting, and subsequent relapse to opioid abuse. These studies will provide important information about prescription opioid abuse liability in pain patients and will yield a laboratory model for predicting likelihood to relapse to prescription opioid abuse. These questions are of immediate clinical relevance to the treatment of chronic pain with opioid therapy."}
{"text": "This core provides in vivo model support for the three research projects. Specifically: 1. Support for Projects 1 and 2: Tumor growth delay studies will be carried out in male nude mice bering subcutaneously implanted human prostate carcinoma (PC-3, DU-145, LnCaP or CWR22) or human bladder carcinoma (RT4 or 5637). The animals will be treated with local hyperthermia to the tumor-bearing limb via water bath heating in the presence or absence of thermal sensitizer administration and/or fractionated radiation therapy (daily for 5 days by external beam). The results of these studies will provide an indication of therapeutic efficacy for the findings of Project 2 and provide a basis for translation of potential thermal sensitizes into Project 1. 2. Support for Projects 1 and 3: Large animals (pigs) will be used as a preclinical in vivo system to test the characteristics of new insonation portals using FSUM spherical applicator and urethral thermometry techniques. These animals will be fully instrumented for acquisition and analysis of all temperature and perfusion data, thus allowing calculation of ultrasound power fields, temperature fields, and thermal dose data. The results of these studies will translate into continued instrument development in Project 3 and into clinical protocol development in Project 1."}
{"text": "Enzyme mechanisms are investigated with small molecules which are designed to resemble altered substrates in the process of being transformed into products. The additional purposes of this work are to provide a basis for the rational design of potent antimetabolites, and to determine the dynamic role of enzyme structure in catalysis. Enzymes currently under investiation include asparaginase, papain, cytidine deaminase, adenosine deaminase, and pyruvate kinase."}
{"text": "This research program will study the regulatory mechanisms which control the different functional levels of the microcirculation in skeletal muscle. This work is based on the general concept that different functional levels in the microcirculation are involved to different degrees in the control of oxygen delivery to, and carbon dioxide removal from peripheral tissues, and that a few stimuli principally govern this control in the normotensive animal. The general concept forms the basis for the second more important hypothesis that at least one functional level for control of oxygen delivery is altered through changes in the type of stimuli which provide control of oxygen delivery in the hypertensive animal. This research program will investigate the relationships among oxygen and carbon dioxide content of inspired air, and small artery and vein phenomena for the microvasculature in skeletal muscle. The relative significance of the effects of innervation, intravascular pressures, and blood gas chemistry on small artery and vein diameters will be determined in normotensive rats and in renovascular and spontaneous hypertensive rats. These studies will utilize techniques which include closed-circuit television microscopy and on-line electronic analysis of video signals in order to obtain measurements of small artery (30-120 microns) and small vein (50-200 microns) diameters."}
{"text": "ABSTRACT / PROJECT SUMMARY Dysregulated bone or dentin resorption is associated with a host of oral diseases including periodontitis and external root resorption, to name a few. Currently, standard diagnosis of these conditions relies on radiographic findings or computed tomography (CT) scans. Nevertheless, these methods usually detect the problem at an advanced stage where significant tissue damage had occurred,oftentimes leading to tooth loss. Moreover, imaging techniques may not indicate if `clastic' cell activity is ongoing or historical. Exosomes are small vesicles of endosomal origin that are released by different cell types after fusion of multi-vesicular bodies with the plasma membrane. Due to their protein, lipid and nucleic acids contents, which closely reflect the nature and state of their parental cells, exosomes are considered an important source of information. Here we present preliminary data demonstrating that 1) there is a difference in composition of exosomes shed from clastic cells resorbing bone (osteoclasts) vs. clastic cells resorbing dentin (odontoclasts), and 2) exosomal proteins involved in clastic cell activity can be identified in gingival crevicular fluid (GCF) collected from resorbing teeth. Thus, we hypothesize that there is a difference in the biochemical composition of exosomes released during the processes of bone and dentin resorption that would allow for distinction between odontoclastic and osteoclastic function. We will test this hypothesis by pursuing the following two specific aims: 1) To Isolate and characterize exosomes derived from osteoclasts and odontoclasts in vitro, and 2) To establish the clinical feasibility of an exosome-based assay in the detection of oral conditions where upregulated bone or dentin resorption is present. In summary, the overall goal of this project is to optimize procedures for exosome analysis while at the same time testing the capability of oral-fluid derived exosomes to distinguish osteoclast-active sites from odontoclast-active sites in vivo. The study will provide a basis for the development of new, safer and less expensive tests to diagnose and monitor the progression of periodontal disease and dental root resorption."}
{"text": "Administration Over the past 16 years, the UNC Lineberger's administrative responsibilities have grown dramatically. Physical space has doubled. Dollars directly administered have increased eight-fold from $15.1 million in 1993 to $127.3 million in 2009 -2010 (including the Center's $50M/year University Cancer Research Fund). Over the next five years the Center will continue to grow in space, funding, and personnel. Growth will follow a Research Strategic Plan that proposes continued recruitment, new research initiatives. and development/expansion of core facilities. In all these efforts, the UNC Lineberger must continue to develop and improve its administrative, technology, and communication infrastructure/operation to facilitate cancer research. Cancer Center Administration (62 FTE. $4 million/year) supports the research activities of all 295 members. Administration provides direct grant, fiscal, and other administrative services to 93 Cancer Center members and 18 core facilities. Administrative support includes: laboratory, office, and conference space;common equipment;grant and fiscal management, human resources and other administrative services, computing infrastructure / networking, communications. and imaging / printing / web services. Center support alos includes In addition, the Center provides support by organizing recruitment, developmental pilot funding, seminars, symposia, and retreats. For Year 36, the Center requests a committed level of $489,049 to support 5.80 FTEs plus costs for supplies, travel, and other operational expenses. The proposed Year 36 budget is approximately 3% higher than the current budget. The requested CCSG Administration budget represents 12.5% of the UNC Lineberger's total Administration budget and 8% of the overall CCSG budget request."}
{"text": "As a cardiologist interested in the basic mechanisms of myocardial ischemia, my primary career objective is to gain an understanding of the role of disturbed endothelial function in atherosclerosis and how it contributes to abnormalities of vasomotor tone and to thrombosis. My dual training in cell biology and clinical research places me in an advantageous position in this regard, since few cardiologists have developed the necessary skills to apply new insights derived from vascular biology to patients with cardiovascular diseases. This application comes at a crucial point in my career as I complete my transition toward fully independent research status. For this burgeoning interest in closing the gap between basic and clinical research to continue to evolve will require well structured and protected time. The institutional environment will be conducive to this strategy for career development provided that my salary support comes from research sources. The studies presented here focus on defining how the presence of atherosclerosis in the coronary arteries of patients effects endothelium-dependent vasodilator and vasoconstrictor responses. A program of research is proposed that utilizes new insights from the experimental laboratory to address three specific aims. The first will test the hypothesis that endothelial vasodilator dysfunction in human coronary arteries leads to constrictor hyperresponsiveness to catecholamines. The second specific aim will test the hypothesis that endothelium-derived constrictor factors (the polypeptide endothelin, as well as prostanoid factors) are secreted from human atherosclerotic arteries in inappropriately large amounts in response to stimuli known to cause enhanced vasoconstriction and thereby contribute to the development of myocardial ischemia. The third specific aim will examine in patients the concepts derived from experimental studies that aggressive lowering of cholesterol can improve endothelium-dependent vascular function apart from important changes in vessel luminal size. These studies should expand our knowledge of the mechanisms and treatment of abnormal constriction of human coronary arteries, and facilitate clinical attempts to control active ischemia."}
{"text": "Convergence disorders and refractive amblyopia are among the most common and treatable vision problems seen in general optometric clinical practice. Oce- and home-based vision therapy (OBVT and HBVT, respectively) are standard treatments for such disorders. Recent advances in computerized video therapies that emulate vision therapy exercises performed in the optometrist's oce provide a lower-cost and more convenient alternative to OBVT. The downside of current commercial HBVT systems is that the recommended eye exercises are highly repetitive, monotonous, and uninteresting, which leads to high levels of patient noncompliance. This presents a problem since consistent performance and proper technique are key to achieving therapeutic benet. An alternative approach, which oers strong potential to increase the success of HBVT, is to incorporate vision therapy into fun and interactive computer games that patients will nd more engaging. To address this need, Barron Associates proposes to develop a novel computerized virtual reality HBVT system called iCare that will overcome the limitations of present systems by addressing three obstacles to successful home treatment: (1) patient compliance;(2) procedural accuracy;and (3) the ability of the optometrist to monitor training. The proposed approach takes advantage of advanced human-interface devices (HIDs) (e.g., Wii Remote Controllers) that have been developed for the video gaming market. Due to the popularity of video gaming, HID technology that would have been prohibitively expensive in the recent past is now widely available at low cost (e.g., <$40). These o-the-shelf HIDs permit the creation of virtual three-dimensional (3-D) environments on computer monitors (for \\near-point\" vision therapy) or plasma displays (for \\distance\" vision therapy). Traditional 3-D graphics technology based on \\3-D glasses\" (i.e., red-blue anaglyphs) is static with respect to the frame of the viewing device;that is, the image is the same regardless of the viewer's position relative to the monitor. The proposed iCare technology exploits motion parallax via low-cost tracking of the patient's head position and orientation, creating a dynamic visual 3-D environment. Visually, the technology allows the viewer to perceive objects as if they are coming o of, or receding into, the screen. This enables the creation of virtual xation objects in a nearly innite 3-D space. Users can even see \\behind\" and manipulate virtual objects. Under this SBIR Fast Track grant application, the proposing Team will develop and test prototype computer gaming environments for HBVT using the technology outlined above to improve treatment eectiveness. Phase I therapy will be aimed at treating refractive amblyopia in children 8-12 years of age;Phase II will focus on treating refractive amblyopia in teens and adults, as well as convergence insuciency in both populations. These disorders are clearly amenable to vision therapy and are common in clinical practice. For both vision problems, clinical studies of the comparative eectiveness of the most popular computerized HBVT programs vs. the iCare system's games will be conducted in children to assess the ecacy of the iCare system. PUBLIC HEALTH RELEVANCE: Convergence insuciency is one of the most common binocular vision disorders, and refractive amblyopia a vision disorder for which a very-high percentage of patients seek treatment;both disorders aect millions of U.S. children and adults. Such vision anomalies can cause a variety of chronic symptoms which, left untreated, often degrade academic and work performance;they may even result in learning disabilities in some cases. Computerized home-based vision therapy (HBVT) provides a relatively low-cost and convenient treatment option;however, the available commercial HBVT programs are highly repetitive and dull, leading to poor compliance. The goal of this research is to make HBVT a more eective alternative to oce-based vision therapy by incorporating the eye exercises into fun, interesting, and interactive computer video games that patients will nd more engaging, which should lead to better compliance and improved therapeutic outcomes."}
{"text": "The long-term objectives of the proposed project are to determine whether and how modulation of gap junctional intercellular communication is involved in the process of carcinogenesis. The specific aim of the project is to test the hypothesis that blocking of communication between an initiated cell and surrounding normal cells is an important determinant in clonal expansion of the initiated cell, i.e., the tumor promotion process. Changes in intercellular communication will be examined during the process of cell transformation by measuring intercellular transfer of fluorescent dye which is microinjected into individual cells. Cell cultures, mainly BALB/c 3T3 and C3H 10T1/2 cell transformation systems, will be employed during the earlier phase of the project. Experiments are designed to answer following specific questions: (i) whether phorbol ester-mediated enhancement of cell transformation is related to the block of intercellular communication by the phorbol esters; (ii) whether so-called \"initiated cells\" in which promotion is suppressed by retinyl acetate communicate with surrounding non-transformed cells, (iii) whether transformed cells communicate with each other and/or with surrounding non-transformed cells, and (iv) whether blocked intercellular communication also occurs during the process of promotion that is not triggered by phorbol esters. Variant cell lines that are sensitive or resistant to TPA-mediated enhancement of cell transformation (Syrian hamster embryo cells) and those that have different susceptibilities to UV- and chemically-induced transformation (BALB/c 3T3 cells) will be utilized to pursue these goals. The possibility of using the block of intercellular communication to screen tumor promoting agents will be examined. We plan to study molecular and biochemical mechanisms in detail. Our approach includes studies on (i) the molecular mechanisms by which phorbol esters inhibit the communication, with special emphasis on possible involvement of protein-kinase C activity, (ii) biochemical analysis of the factors involved in cell-cell recognition that influence gap-junctional communication, and (iii) possible oncogene involvement in blocking intercellular communication. Similar approaches will be applied to an in-vivo study using a rat-liver multistage carcinogenesis model. The microinjection technique coupled to electrophysiological measurements will be used to determine intercellular communication between preneoplastic nodules and/or hepatoma and surrounding normal cells."}
{"text": "[unreadable] [unreadable] The ability of a drug to reach and penetrate its intended target within the body is critical to its success in treating disease. However, drug efflux proteins such as p-glycoprotein (pgp) actively pump hydrophobic drugs away from target tissues and are linked to low oral absorption and multidrug resistance in chemotherapy. Protein pumps are of increasing interest to the pharmaceutical industry, most importantly based on new draft FDA guidelines requiring knowledge of whether a drug candidate is a substrate or inhibitor of pgp. Current pgp assays are cumbersome, expensive and unreliable. Our overall goal is to prepare and validate a novel assay reagent - Fluorosome-trans-pgp - which capitalizes on the Fluorosome Technology that we have developed for measuring passive permeability of small molecules through lipid membranes. Fluorosome-trans-pgp will provide a direct, reliable, simple, rapid and inexpensive assay to determine if a drug is a pgp substrate or inhibitor. Toward this goal, in phase I of this project we have: cloned and expressed the human pgp construct containing a 10His tag in HEK293 cells. isolated and purified the protein \"pgp 10His\" in lipid micelles. demonstrated verapamil-stimulated ATPase activity in the purified pgp 10His lipid micelles. reconstituted the purified pgp 10His into liposome membranes. developed the methodology for the manufacture and assay of Fluorosome-trans-pgp. validated the test systems that will be used to screen potential pgp substrates and inhibitors. Phase II of this project will bring the Fluorosome-trans-pgp assay to the state of an important commercial product. To complete this development, the Specific Aims of phase II are to: 1. complete Fluorosome-trans-pgp production and validation. 2. scale up Fluorosome-trans-pgp production to commercial levels. 3. demonstrate the utility of Fluorosome-trans-pgp with representative pgp substrates and inhibitors. 4. optimize the properties of the Fluorosome-trans-pgp system. 5. develop data analysis software. Our overriding goal in Phase II is to bring Fluorosome-trans-pgp and accompanying software to market. Successful completion of phase II will satisfyi the increasing need for a reliable and economical method to measure a drug's susceptibility to efflux by the pgp pump or to act as a pgp inhibitor. This project also lays the foundation for the design of Fluorosome-based systems to test for the susceptibility of drug candidates to other drug transport proteins. Markets include pharmaceutical and biotechnology companies, contract research organizations, and in-house fee-for service assays. This project completes the development of a test which determines if drugs will be extruded from their target tissue by biological pumps. The test thereby allows the pharmaceutical industry to evaluate, at an early stage, the suitability of drug candidates for continued development. The test is reliable, simple, rapid, inexpensive and amenable to robotics. [unreadable] [unreadable] [unreadable]"}
{"text": "The integrated, transformational strategies of the proposed Orange County Center for Community Health Research (OCCCHR) will potentially have a broad impact on health research and in the reduction of health disparities. The OCCCHR will utilize a state-of-the-art web-based resource portal and videoconference technology to improve information dissemination, technical assistance in, networking, training and mentoring between the Academic Health Centers (AHCs) and community based organizations. The web-based video conference training component builds on UCI's considerable experience with training students, faculty, and community providers. Prior projects have demonstrated that the bi-directional web-based training is an accessible, convenient and cost effective approach to improving participation and suggest that the OCCCHR's videoconference training component will be a powerful tool for increasing community and researcher capacity to engage in research. Furthermore, the annual planning retreat and team building workshops will utilize collaborative models that have proven to be successful planning strategies in Orange County. It will focus the collaborative strengths of the community agencies with the AHCs toward the development of an academic-community partnered health agenda and research projects. The proposed infrastructure should have a major public health impact as a model for information dissemination, resource sharing, web-based training and collaboration. Further, the proposed infrastructure provides the opportunity to improve utilization of health data to develop evidenced-based community research priorities, and increase community research capacity to engage in community-based participatory research. This model can be used to engage communities nationwide to improve the quantity and quality of academic-community partnered research that substantially accelerate the diffusion scientific advancements into the community, thereby reducing health disparities. PUBLIC HEALTH RELEVANCE: The integrated, transformational strategies of the proposed Orange County Center for Community Health Research (OCCCHR) will potentially have a broad impact on health research and in the reduction of health disparities. The OCCCHR will utilize a state-of-the-art web-based resource portal and videoconference technology to improve information dissemination, technical assistance in networking, training and mentoring between the two AHCs and community based organizations. This model can be used to engage communities nationwide to improve the quantity and quality of academic-community partnered research that substantially accelerate the diffusion scientific advancements into the community, thereby reducing health disparities."}
{"text": "Abstract Despite the crucial importance of the inter-follicular epidermis (IFE) for the essential body barrier function, molecular characterization of the stem cells (SCs) within the basal layer has not been achieved. This impairs our ability to study the mechanisms that specifically control IFE SCs for proper homeostasis and wound healing and to understand how these mechanisms may be affected in skin disease and aging. The IFE SCs have been traditionally identified in vivo as DNA label retaining cells (LRCs) while transit amplifying (TA) or progenitor cells were considered non-LRCs. However, LRC and non-LRC markers to unambiguously test this model in vivo had been lacking. Using our H2B-GFP pulse-chase transgenic mouse system, we label IFE LRCs and non-LRCs, define mRNA expression profiles, and find that these cells are molecularly distinct. In our first set of preliminary data, we demonstrate that, contrary to the expectation that SC are frequently dividing cells, both of our IFE cellular subsets of LRCs (marked by Dlx1CreER) and of non-LRCs (marked by Slc1a3CreER) act as two independent SCs in long-term lineage tracing. Collectively, our data support a model in which the IFE is a heterogeneous tissue, being composed of molecularly distinct domains or territories, which are spatially patterned relative to each other and to skin landmarks. These territories are enriched in LRCs and non-LRCs, regenerate at different rates, and contain distinct SCs and differentiated cells that can be defined as molecularly discrete IFE cellular subsets. We provide LRC and non-LRC-enriched markers and genetic labeling tools that define novel IFE cellular subsets, which will enable us to rigorously examine the newly uncovered IFE heterogeneity. The specific objectives are to: (i) examine the organization of IFE territories throughout life, and determine relative SC potential of newly uncovered IFE cellular subsets; (ii) validate and refine markers of IFE heterogeneity from our newly uncovered undifferentiated and differentiated IFE cellular subsets; and (iii) unravel mechanism of IFE heterogeneity by focusing on two transcription factors we identified in our LRC versus non-LRC IFE subsets. Collectively, our data will constitute a key for understanding previously un-recognized cellular and molecular heterogeneity within the IFE and provide a new entry point into SC regulation in this essential, yet poorly understood skin compartment."}
{"text": "This education program will develop, implement, evaluate and disseminate a comprehensive integrative medicine curriculum to allopathic and CAM medical students and health professionals. This longitudinal curriculum will enable students and practitioners to acquire the knowledge, skills and attitudes to practice evidence-based medicine that appropriately incorporates best practices from both conventional and alternative medicine. Core competencies in CAM will be taught using web-based, face-to-face, and interprofessional curricula that takes full advantage of the rich CAM resources in the Bay Area. The curriculum also incorporates a student wellness program to help them maintain their own health and promote wellness as a model for their patients.This program will be designed and executed by faculty and staff at the Osher Center for Integrative Medicine in collaboration with CAM community experts under the guidance of an independent Advisory Committee, composed of allopathic and CAM practitioners, educators, and researchers. The program is unique in its emphasis on self-directed evidence-based learning, case-based instruction, and mix of web-accessible modular learning, 'face-to-face' experiential curricula, and collaborative programs with community CAM institutions.The process and outcomes of this program will be rigorously evaluated and the content continuously updated. Because of its web-based interface, this curriculum is highly adaptable and portable to other academic institutions. Dissemination of the curriculum will also be accomplished by CD-ROM, implementation manuals, lectures, and workshops"}
{"text": "A Fully Automatic System For Verified Computerized Stereoanalysis SUMMARY The requirement for a trained user to interact with tissue and images is a long-standing impediment to higher throughput analysis of biological microstructures using unbiased stereology, the state-of-the-art method for accurate quantification of biological structure. Phase 1 studies addressed this limitation with Verified Computerized Stereoanalysis (VCS), an innovative approach for automatic stereological analysis that improves throughput efficiency by 6-9 fold compared to conventional computerized stereology. Work in Phase 2 integrated VCS into the Stereologer\", an integrated hardware-software-microscopy system for stereological analysis of tissue sections and stored images. Validation studies of first-order stereological parameters. i.e., volume, surface area, length, number, confirmed that the color-based detection methods in the VCS approach achieve accurate results for automatic stereological analysis of high S:N biological microstructures. These studies indicate that fully automatic stereological analysis of tissue sections and stored images can be realized by elimination of two remaining barriers, which will be addressed in this Phase II Continuation Competing Renewal. In Aim 1, applications for feature extraction and microstructure classification, developed in part with funding from the Office of Naval Research, will be integrated into the VCS program. The new application (VCS II) will use these approaches to automatically detect and classify polymorphic microstructures of biological interest using a range of feature calculations, including size, color, border, shape, and texture, with support from active learning and Support Vector Machines. Work in Aim 2 will eliminate physical handling of glass slides during computerized stereology studies by equipping the Stereologer system with automatic slide loading/unloading technology controlled by the Stereologer system. This technology will approximately double the throughput efficiency of the current VCS program and support \"human-in-the-loop\" interaction for sample microstructures on the border between two or more adjacent classes. The studies in Aim 3 will rigorously test the hypothesis that fully automatic VCS can quantify first- and second-order stereological parameters, without a loss of accuracy compared to the current gold-standard - non-automatic computerized stereology, e.g., manual Stereologer. If these studies validate the accuracy of VCS II, then commercialization of the fully automatic program will facilitate the throughout efficiency for testing scientific hypotheses in a wide variety of biomedical research projects;reduce labor costs for computerized stereology studies;hasten the growth of our understanding of biological processes that underlie health, longevity, and disease;and accelerate the development of novel approaches for the therapeutic management of human disease. Solid evidence that the SRC and its strategic partners can effectively commercialize this technology is demonstrated by their worldwide sales and support of the Stereologer system for the past 13 years. Key personnel and participating institutions: 7 Peter R. Mouton, Ph.D. (PI), Stereology Resource Center, Chester, MD. 7 Dmitry Goldgof, Ph.D., University of South Florida Coll. Engineering, Tampa, Fl. 7 Larry Hall, Ph.D., University of South Florida Coll. Engineering, Tampa, Fl. 7 Joel Durgavich, MS, Systems Planning and Analysis, Alexandria, VA. 7 Kurt Kramer, MS, Computer Programmer, University of South Florida, Coll. Engineering, Tampa, Fl. 7 Michael E. Calhoun, Ph.D., Sinq Systems, Columbia, MD PUBLIC HEALTH RELEVANCE: Many fields of scientific research require a trained expert to make tedious and repetitive measurements of microscopic changes in animal and human tissues. This project will produce a computer program that performs these measurements with equal accuracy to a trained expert, but with dramatic savings in time and costs. Allowing scientists to complete more research in less time will accelerate our understanding of the factors that promote health and longevity, and hasten progress toward the development of new treatments for human diseases."}
{"text": "Schizophrenia and other psychoses are among the leading causes of the global burden of disease in low-, middle-, and high-income countries alike. As of 2006, and assuming a point prevalence of 4.6 per 1000 population [1], at least 25 million people in low- and middle-income countries (LMIC) were suffering from schizophrenia, a disorder that, according to estimates for 2004, accounted for 1.1% of all Disability Adjusted Life Years and 2.8% Years Lived with Disability [2]. Other research suggests that schizophrenia and other psychoses are associated with excess mortality, suicide in particular, as well as stigma, discrimination, and violations of human rights [2-6]. In low- and middle-income countries the burden of schizophrenia and other non-affective psychoses is made comparatively greater because of a lack of mental health resources that can provide effective treatments and, thus, decrease the symptom severity and disability associated with these conditions [7,8]. This is certainly the case in Nigeria where a recent study found that non-affective psychoses were relatively common (1.1% prevalence) but frequently went untreated [9], undoubtedly because of a scarcity of mental health resources [10]. This application for an exploratory planning grant (R21) proposes to undertake activities whose ultimate goal is the development of a package of care for individuals with psychosis and their caregivers in one catchment area of Nigeria. To achieve this goal, we propose to establish a collaboration between the London School of Hygiene and Tropical Medicine and Institute of Psychiatry, Kings College London, and the University of Ibadan. The collaboration will build on: 1) the long and impressive history of psychiatric and mental health services research in Nigeria [11-52];2) the evidence presented in the landmark Global Mental Health and Packages of Care series in The Lancet [7,8,53-56] and PLoS Medicine [57-63], respectively;and 3) the strategies for scaling up as outlined by the WHO mhGAP programme [2]. Together, the investigators who will lead the proposed project will consider, in a local Nigerian setting, the needs of persons with psychosis and their caregivers, the available mental health resources, and then formulate an evidence-based intervention package of care that will utilize non-specialist health workers (e.g., primary care physicians, nurses, and community health workers) working in partnership with the available psychiatric specialists, to create a program that will deliver services in community based settings [60,64,65]. PUBLIC HEALTH RELEVANCE: Schizophrenia and other psychoses impose a huge burden of disease in low- and middle-income countries. The aim of this exploratory project is to design an evidence-based, feasible, acceptable, and locally appropriate community-based rehabilitation package of care and that can be implemented in two local government areas of Osun State, Nigeria."}
{"text": "The project's objective is to develop enabling technology for a co-robotic navigation aid, called a Co-Robotic Cane (CRC), for the visually impaired. The CRC is able to collaborate with its user via intuitive Human-Device Interaction (HDl) mechanisms for effective navigation in 3D environments. The CRCs navigational functions include device position estimation, wayfinding, obstacle detection/avoidance, and object recognition. The use of the CRC will improve the visually impaired's independent mobility and thus their quality of life. The proposal's educational plan is to involve graduate, undergraduate and high school students in the project, and use the project's activities to recruit and retain engineering students. The proposal's Intellectual Merit is the development of new computer vision methods that support accurate blind navigation in 3D environments and intuitive HDl interfaces for effective use of device. These methods include: (1) a new robotic pose estimation method that provides accurate device pose by integrating egomotion estimation and visual feature tracking; (2) a pattern recognition method based on the Gaussian Mixture Model that may recognize indoor structures and objects for wayfinding and obstacle manipulation/ avoidance; (3) an innovative mechanism for intuitive conveying of the desired travel direction; and (4) a human intent detection interface for automatic device mode switching. The GPU implementation of the computer vision methods will make real-time execution possible. The proposed blind navigation solution will endow the CRC with advanced navigational functions that are currently unavailable in the existing blind navigation aids. The PI's team has performed proof of concept studies for the computer vision methods and the results are promising. The broader impacts include: (1) the methods' near term applications will impact the large visually impaired community; (2) the methods will improve the autonomy of small robots and portable robotic devices that have a myriad of applications in military surveillance, law enforcement, and search and rescue; and (3) the project will improve the research infrastructure of the Pi's university and train graduate and undergraduate students for their future careers in science and engineering."}
{"text": "A patient with hereditary angioneurotic edema (C1-inhibitor deficiency) also has SLE and is secondarily deficient in C4. Patients with hereditary C4 deficiency are subject to severe SLE. She was given infusion of human C1-inhibitor to prevent the destruction of C4 in her serum by the uninhibited C1. This failed to induce a remission in SLE or to elescript submitted, and treatment with C1-inhibitor was stopped."}
{"text": "Core D Cell Culture Project Abstract The goal of the work described in this proposal is to collect epithelial cells that line the surfaces of the nose and upper airways of human subjects with asthma or cystic fibrosis. The airway epithelial cells can be grown in the laboratory under conditions that allow large-scale expansion of cell number and frozen for storage and distribution to project investigators. Each sample can be thawed and further expanded for study and testing. Most human airway epithelial cells currently available for study are from transplanted disease lungs or lung donations not suitable for transplant. Collection of nasal and bronchial cells by brushing the epithelial surface is simple and safe, and allows us to obtain cells from a much larger pool of subjects. The ability to collect, expand, and distribute airway epithelial cells to researchers will have a positive impact on understanding and developing therapies for common airways diseases and provide an opportunity to test newly developed drugs for cystic fibrosis on rare mutations carried by only a few individuals."}
{"text": "Purchase of a 700MHz NMR Spectrometer for Liquid Applications Project Summary Solution nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful spectroscopic tools available to the synthetic chemist for the elucidation of the structure of a molecule. Advances in computing power, high-field magnet technology, and improved electronics have dramatically increased both sensitivity and chemical shift dispersion such that detection of low natural abundance nuclei is now routine. Presently in the Chemistry department at the University of Michigan (UM, Ann Arbor) there is a rapidly growing gap between the existing capabilities offered by our aging/obsolete NMR spectrometers and the demands of our NIH-funded investigators. This demand stems from the fact that not only have the molecular systems under investigation grown in size and complexity, the questions being addressed by NMR are also more ambitious and include atomic characterization of molecular configuration, conformation, dynamics, interactions and mechanisms. The availability of a high field, multi-channel NMR spectrometer dedicated to liquid applications will have a significant impact on the investigation of complex natural products bearing multiple stereocenters, determination of relative stereochemistry of 5-membered carbocyclic ring systems such as cyclopentanes, tetrahydrofurans, and pyrrolidines possessing up to 4 stereocenters, characterization of unstable organoboron and organosilicon intermediates, and time-resolved kinetic studies of reactive organometallic intermediates formed in low concentrations. High-resolution structural information will be highly valuable in designing small molecule isoxazolidine transcriptional activators, as well as probing how enzymes use free radicals to catalyze chemically difficult transformations. Progress in these research projects and the training of chemistry students in cutting-edge NMR techniques are hampered due to the lack of an accessible modern high field NMR spectrometer. Therefore, to close this gap, we propose the purchase of a multi-channel 700 MHz NMR spectrometer outfitted with the latest in cryogenically cooled, triple resonance probe technology. Additionally, access to such a state-of-the-art instrument will be made available to NIH-funded chemistry projects at nearby institutions such as the University of Toledo, where significant benefit would be realized in the area of complex carbohydrate synthesis. PUBLIC HEALTH RELEVANCE The NIH-funded projects described herein will contribute to a greater understanding of areas that impact human health. Advances enabled by the requested instrumentation include the discovery of potential drug leads, the understanding of biological processes involved in disease progression or prevention, and the development of new strategies in synthesis that enable creative solutions to research projects of this type."}
{"text": "The long range goal of this research is to understand the roles played by the superior colliculus (SC) in visual function. This requires a full accounting of the functional properties of the retinal ganglion cells innervating the SC. In the cat, almost all of these cells belong to the W-cell class. W-cells comprise 50 percent of cat ganglion cells and have obvious equivalents in primates and other mammals. In addition to dominating the retinocollicular pathway, W-cells provide most or all of the retinal input to a host of other visual nuclei that subserve visuomotor reflexes. Despite the ubiquity and importance of W-cells, study of their structure and function has been severely limited by technical factors. This project will overcome these limitations by means of a novel in vitro approach, thereby shedding new light on the organization of the retinocollicular pathway. W-cells are extremely heterogeneous in morphology and function. Rather than representing a true class, W-cells apparently constitute a loose grouping of many distinct ganglion-cell types, each as different from the others as it is from the X-and Y-cell types. Work from this laboratory in past and current project periods indicates that individual W-cell subtypes may exhibit distinctive patterns of collicular projection. The retinocollicular pathway is thus far more complex that has been generally appreciated. Functionally distinct W-cell channels may be processed independently by distinct collicular microcircuits, or may undergo a highly orderly integrative recombination by collicular neurons. Clearly, a prerequisite for understanding the functional meaning of this parallel retinocollicular organization is a detailed understanding of individual W-cell types. The proposed project begins this process through an in depth analysis of a single W-cell type -- the zeta cell -- which is a dominant contributor to the retinocollicular pathway. These W-cells will be fluorescently tagged, either by retrograde transport of tracers from the colliculus or by uptake of a vital dye. The retina will then be maintained in vitro and electrodes advanced toward tagged zeta cells under visual control. The visual response properties of these cells will be studied by extracellular and intracellular recording and their morphology revealed in detail by intracellular dye injection. This approach provides the first practical method for making direct correlations between the morphology, physiology and central projections of single ganglion cells. The method will be used to analyze the stimulus selectivities of these cells, to explore the intraretinal circuitry that produces these response properties, and to characterize the anatomical mosaic through which these cells sample the visual scene. The study will expand our understanding of diversity among the output cells of the retina and the parallel channels of visual information they emit to the SC and other brain centers. It will also establish a powerful new experimental paradigm with extremely broad applicability to the study of retinal ganglion cells."}
{"text": "This project will study the mechanisms responsible for microvascular thrombosis in experimental models of sepsis. Sepsis, a systemic response to an infection, is the main cause of death in adult intensive care units in the United States, and microvascular thrombosis is a severe complication of the disease. The main model we will use involves bacterial endotoxin (lipopolysaccharide, LPS), which mediates many manifestations of patients with a common form of sepsis. In addition, we will use a clinically relevant model of human polymicrobial sepsis of abdominal origin, cecal ligation and perforation (CLP). Our preliminary data demonstrate that both sepsis models enhance microvascular thrombosis in vivo; in this project, we will explore the molecular mechanisms involved. Our central hypothesis is that LPS-induced stimulation of toll-like receptor 4 (TLR4) on endothelial cells mediates microvascular thrombosis in endotoxemia, by a mechanism dependent on the platelet adhesive molecule, glycoprotein Ibct. We propose four aims: in aim 1, we will identify which LPS receptors mediate enhancement of microvascular thrombosis in vivo. In aim 2, we will determine whether bone marrow- or non-bone marrow-derived cells mediate LPS- induced responses in vivo. In aim 3, we will use an ex vivo flow system to examine the effects of endotoxemia and CLP on platelet activation and adhesion to specific adhesion molecules (e.g.-vWf, P- selectin, fibrinogen) under physiologic flow. In aim 4, we will use the in vivo model to define the platelet and endothelial adhesion molecules responsible for LPS- and CLP-enhanced microvascular thrombosis. Completion of these aims will broaden our understanding of the mechanisms of microvascular thrombosis in models of human sepsis. This will allow identification of novel therapeutic targets for microvascular thrombosis in this disease. Our long-term goal is to apply the knowledge gained from these studies to allow optimal management of patients with sepsis and their associated microvascular alterations. Relevance to public health: Sepsis, the body's response to an infection, is a major cause of death in the U.S. Our goal is to understand the causes of a severe complication of this illness, clots in tiny blood vessels. This information would help develop new treatments for patients with this devastating illness. [unreadable] [unreadable] [unreadable]"}
{"text": "This project has the aim of understanding the basic problem of radiation induced liver damage with the ultimate goal of solving this problem in humans when radiation therapy is needed for cancer of the upper abdomen and lower thorax. Mongrel dogs are examined, quarantined and vaccinated, then are submitted to laparotomy for liver biopsy. Blood chemistries and liver scan are obtained prior to irradiation at various therapeutic dose levels. Then the liver scan, chemistries and liver biopsy are repeated to detect liver damage. Various combinations of dose and fractionation are being tested. Anticoagulant therapy will be tried with the aim of reducing the radiation induced liver damage."}
{"text": "While human patients and healthy subjects are being imaged at 3T, 7T, and soon, 10.5T, more data and understanding of RF safety at these ultra-high field strengths are needed. The overall objective of this renewal proposal therefore is to extend the RF heating studies in the human head of our previous grant to the investigation of RF heating in the human body for ultra-high field MRI applications. Safety will be better assured and imaging performance improved by developing the means to accurately predict and measure RF temperature contours in human anatomy. Through development of new theory, technology, methodology, and experimental approach, these means will be achieved by accomplishing the following aims. First, a more fundamental understanding of the electrodynamics and thermodynamic nature of RF induced heating and heat transfer in anatomy will be furthered through mechanistic improvement of the empirical Pennes Bioheat Equation. Second, this new theoretical model will be validated at high field Larmor frequencies by invasive, direct measurement of RF heating in anesthetized porcine models, by fluoroptic thermometry. The porcine model is required as an intermediate step toward understanding and measuring RF heating in humans. Toward this end, the third aim is to develop a noninvasive NMR thermometer that is accurately and precisely calibrated by the invasive fluoroptic measurement. Once confidence is gained in predicting and noninvasively measuring temperatures in pigs, these methods will be applied or correlated to monitoring RF heating in humans and so accomplishing the final aim of this study. With new understanding, data, and thermal measurement methods, high field human MRI will become safer."}
{"text": "Systemic lupus erythematosus (SLE) is a chronic life-threatening autoimmune disorder that currently afflicts up to 2,000,000 individuals within the United States each year. Therapeutic options for treating patients with SLE include agents such as steroids, cytotoxic drugs, and anti-malarials. Although these therapies can reduce disease severity, they often have deleterious side effects that limit their extended use. A better understanding of the factors that trigger disease onset could facilitate the development of drugs that specifically target the autoreactive effector cells without the debilitating side effects and general immunosuppression associated with curent treatment protocols. This proposal is based on recent studies that identify a novel pathway involved in the activation of both autoreactive B cells and (auto)antigen presenting dendritic cells. Our group has shown that chromatin-containing immune complexes (chromatin- IC), bound by either the BCR or an FcgammaR, are delivered to an internal compartment where they activate the cells via a MyD88-dependent Toll-like receptor 9 (TLR9) pathway. Thus, inhibition of the TLR9 pathway may specifically block the development and/or progression of systemic autoimmune disease without interfering with immune responses to foreign proteins. To take advantage of this potential therapeutic opportunity, it will be necessary to further delineate the mechanism and general applicability of this dual receptor paradigm. The goal can best be met by combining the research endeavors of program investigators with diverse research backgrounds - B cell regulation (Marshak-Rothstein), antigen uptake and vesicle trafficking (Corley), chromatin remodeling (Viglianti), dendritic cell activation (Rifkin), and transgenic models of autoimmune disease (Shlomchik) - to address the following questions: (a) What are the unique functional consequences of chromatin-IC engagement of B cells and dendritic cells (project 1 and 2)? (b) What aspects of chromatin structure determine immunogenicity in this system (project 1)? (c) How are chromatin-IC processed and how does TLR9 affect the persistence and site of these events (project 1)?; (d) Do TLR other than TLR9 mediate responses to non-chromatin associated autoantigens (project 2, 3,)? (e) What is the in vivo role of TLRs in systemic autoimmunity (project 3)? and finally (f) Can this pathway be specifically targeted therapeutically with agents that block the TLR9 signaling pathway (project 1,2)? The results of these studies should provide important insights that will facilitate the development of non-invasive therapies for the treatment of systemic autoimmune diseases such as SLE."}
{"text": "The project will investigate the structure and function of gap junctions in the lens. For structural studies, the gap junctions will be studied in normal and cataractous lenses using freeze fracture and thin section electron microscopy, and of isolated preparations using X-ray diffraction and electron micrograph image reconstruction techniques. For the functional studies, metabolic cooperation via gap junctions (the ability of adjacent cells to share small metabolites via low resistance pathways) will be studied using whole tissue and tissue culture autoradiography, correlating functional findings with junction distribution and morphology. These studies will be performed under control, and cataractogenic condition, both metabolic and hereditary. Finally, specific monoclonal antibodies will be generated against the lens gap junction principal polypeptide."}
{"text": "DESCRIPTION (Applicant's Description): The overall objective of the proposed studies is to obtain information regarding the role of the bestrophin gene (VMD2) in retinal degeneration. Best Macular dystrophy (BMD, also called vitelliform macular dystrophy) is an autosomal dominant form of macular degeneration. BMD, caused by mutations in the VMD2 gene, leads to a progressive loss of central vision. Little is known about the protein product of this gene, called bestrophin. In the retina, bestrophin is specifically expressed in the plasma membrane of the retinal pigment epithelium (RPE). In the following application, I plan to conduct research toward understanding the physiological and molecular role of VMD2 in the retina. The first specific aim seeks to determine if mutations in the VMD2 gene cause other forms of retinal degeneration such as age-related macular degeneration (AMD) using PCR-based single-strand conformation polymorphism and direct genomic sequencing techniques. In the second aim, I plan to ascertain the subcellular localization of bestrophin in the plasma membrane of the RPE using imunocytochemistry. In the last aim, I propose in vitro electrophysiological studies as well as in vivo studies using mice. These studies include methods such as Ussing chambers, light, electron and fluorescent microscopy, gene transfer both in vivo and in vitro, electrocculography, and electroretinography. Discovering information regarding the function of bestrophin will hopefully provide knowledge about mechanisms involved in retinal degeneration."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objectives Our objective in this core is to address the practical issues of adapting, optimizing and provisioning biomedical applications in drug discovery, cellular and organ modeling, and image reconstruction based upon current and emerging computing, data and network infrastructures. At the same time, we must ensure the accessibility, reproducibility and versatility of the large ensembles of software that are required to carry out multiscale analysis. Specific Aims Aim 1: Scaling and Adapting Multiscale Biology Codes to High-Performance Clusters and Multicore Processors: To allow application codes to take advantage of a new \"balance of machines where individual nodes become large-scale, shared-memory symmetric multiprocessors (SMPs). Aim2: Providing Transparent and Service-Based Access to Multiscale and Multiphysics Biomedical Codes: To hide as much complexity as possible allows scientists to reason more easily about the logic of the systems or workflows required to carry out their research [unreadable]and less about the low-level system details. Aim 3: Defining, Testing, and Supporting a Complete Cyberinfrastructure: To provide a reproducible cyberenvironment by understanding externally-supplied software, assembling into a distribution that sits on top of a standard Linux operating system, and regularly and rigorously testing. Aim 4: Integrating and Delivering Tools for Translational Research: To integrate and test selected \"suites\" of software application pipelines"}
{"text": "The Centers for Disease Control estimate that approximately 1.7 million people per year sustain a traumatic brain injury (TBI), resulting in an estimated 52,000 deaths. The age groups most likely to sustain a TBI are infants (0-4 years old), adolescents (15-19 years old), and the elderly (65 and older). TBI results in the alteration of many signaling pathways and biological processes. These processes contribute to a variety of deleterious pathophysiological sequelae which significantly affect patient morbidity and mortality, including cerebrovascular dysfunctions such as ischemia and increased susceptibility to secondary ischemia, cerebrovascular edema, and increased blood-brain barrier (BBB) permeability. MicroRNAs (miRNAs) are ubiquitous regulatory RNAs that modulate gene expression at the post-transcriptional level by inhibiting protein translation and/or promoting mRNA degradation. MiRNAs play critical roles in regulating many important biological processes, including vascular integrity. We have shown that miRNA expression levels are altered after a controlled cortical impact (CCI) model of TBI, and these altered miRNAs are predicted to regulate the expression of gene products involved in many pathophysiological processes affected by TBI. MiR-223 expression levels in cerebral microvessels are significantly increased 24-72 hr post-TBI, a time period when peak BBB permeability and cerebral edema is observed. Furthermore, preliminary evidence shows that miR-223 directly targets T-lymphoma invasion and metastasis (Tiam1), a central regulator of actin dynamics that impacts endothelial barrier integrity and adherens and tight junction function. We propose to test the hypothesis that increased miR-223 expression in brain microvascular endothelial cells contributes to BBB dysfunction, and blocking miR-223 activity after TBI will improve BBB integrity. The experiments in this grant proposal employ a combination of molecular biology and biochemical approaches using genetic knockout, in vitro, and in vivo model systems to (i) determine the impact of altered miR-223 expression on tight junction protein expression, localization, and barrier function in primary rat brain microvascular endothelial cells, and (ii) evaluate the impact of miR-223 knockout on BBB compromise after TBI. Examining the functional consequences of altered miRNA expression in complex systems is hampered by a lack of methodologies for acutely manipulating miRNAs in vivo. We propose to utilize the rabies virus glycoprotein (RVG) peptide, which selectively transfect cells that express the nicotinic acetylcholine receptor, including vascular endothelial cells, as a novel targeting strategy to transiently manipulate miR-223 in the brain vasculature in vivo. These studies will contribute to our understanding of BBB regulation after CNS trauma, and help characterize a novel method for in vivo manipulation of miRNA levels. Furthermore, if successful these studies will enable future investigations into the effects of transient changes in miRNA expression on protein regulation in vivo, opening up a previously unexplored area of pathophysiological processes initiated after TBI."}
{"text": "Astrocytes are essential for neuronal survival and function. Yet every neurodegenerative disease and every injury to the brain and spinal cord results in \"activation\" and proliferation of astrocytes, a process termed astrocytosis, which adversely affects neuronal survival and function. Thus, the astrocyte is a two-edged sword, supporting homeostasis in health, but, in pathologic conditions, their activation results in neuronal loss. As an example, the scar that forms in the weeks following stroke is caused by astrocyte proliferation, which further damages neurons, preventing recovery and increasing disability. In chronic neurological diseases, such as Multiple Sclerosis, there is a progressive astrocytosis and a corresponding progressive loss of neurons. GliaMed, Inc., a biotechnology company dedicated to using our proprietary, patent-protected technology to treat a range of neurodegenerative diseases and astrocytoma, has taken the approach that understanding The molecular mechanisms of homeostasis will, by definition, identify important and novel therapies. With this as its scientific cornerstone, and supported by more than a decade of federal and foundation research grants awarded to the Company's scientific founder, the PI on this application, GliaMed has identified both cellular and molecular targets for the effective treatment of a range of conditions that result in loss of CNS homeostasis. In specific, we demonstrated a number of years ago that astrocytes, one of the major celt types in the CNS, are sustained out of the cell cycle by contact with a protein component specific to the neuronal cell-surface. We have recently identified the astrocyte-expressed receptor, termed GMg, and its neuronal ligand, NrS1, that mediate both forward and reverse signaling between these cell types, that results in a number of biologies, including astrocyte cell-cycle arrest. In this application, we provide data elucidating aspects of these interactions, and describe our lead compounds. Further, we provide evidence that these compounds which are based on GM9-NrS1 binding, rescue neurons from programmed cell death and promote axogenesis, both in vitro and in vivo. The overall Specific Aim of this application, based on Preliminary Data provided herein, is to optimize these compounds for in vivo stability and saturation of target sites within the CNS. These data will support the transition of the GliaMed lead compounds from preclinical to clinical development."}
{"text": "Inactivation by hydrolysis of cyclic AMP and cyclic GMP is probably catalyzed by two separate cyclic nucleotide phosphodiesterases. Attempts will be made to separate the two enzymes physically with minimal loss prior to assay or to find means of assaying the two activities in crude extracts by use of selective inhibitors. We will search for hormonal regulation of the quantities or kinetic properties of the enzymes. Brattleboro rats (congenitally lacking vasopressin) will be injected either with vasopressin in oil or the oil alone. The amount and characteristics of the phosphodiesterases in whole rat kidney and various subregions will be determined. Other variables which affect renal concentrating ability will be studied in similar fashion. These include serum levels of Ca ions, body potassium, drugs (especially thiazide diuretics), thyroid status, protein intake."}
{"text": "The purpose of the Mathematical and Statistical Analysis and Modeling Core is to provide an efficient organization for the quantitative support required by all projects in the Center. The range of support needed includes experimental design and data analysis, statistical modeling, mechanistic model formulation, stochastic modeling, and model solution. Specific applications include models of fluid flow and contaminant transport, remediation of contaminated environments, exposure assessment, and statistical and physiologically based pharmacokinetic modeling. The Core comprises a quantitative, interdisciplinary team with backgrounds in deterministic and statistical modeling, biostatistics, applied mathematics, and computer science. Collectively, this group will provide quantitative support through application of known methods and approaches. It will also provide leadership in method development for areas of investigation in the Center with particular need for innovative modeling approaches."}
{"text": "This project involves a continuing effort to develop an understanding of the role of cyclic nucleotides (cyclic AMP and cyclic GMP) in Escherichia coli and the mechanism by which the levels of these nucleotides is regulated within the cells under a variety of conditions. Our findings this year have dealt with the regulation of the activity of adenylate cyclase by the phosphoenolpyruvate linked phosphotransferase system for transport of sugars (PTS). Mutants in one of the proteins of this transport system (Enzyme I) lead to a depression of cellular cyclic AMP levels. Under appropriate conditions, the levels of cyclic AMP in these mutants can be restored to normal by exposure to the phosphorylating agent, phosphoenolpuyrvate. These data have been interpreted in the framework of a model for regulating adenylate cyclase activity by interaction with proteins of the PTS, such that the state of phosphorylation of the PTS proteins determines the activity of the enzyme."}
{"text": "Novel Regulators of Vascular Disease Between 20 and 90 years of age, arterial wall intimal-media thickness increases, promoting arterial stiffness. Increased conduit artery stiffness is a key factor in the pathogenesis of aging-associated diseases such as systolic hypertension, cerebrovascular, cardiovascular, renovascular, and peripheral vascular diseases - all represent a significant burden affecting the Veteran population. This VA-PPA is a unique opportunity to bring together four VA investigators to study the pathobiology of conduit artery stiffness. A series of three inter-related projects and a state-of-the-art animal vascular phenotyping core facility will focus on the common theme of vascular dysfunction associated with matrix protein deposition and medial calcification, two factors known to increase arterial stiffness during aging and chronic kidney disease (CKD). The specific objectives of the three Projects and Core are to: 1. Define the mechanisms by which the aging process reduces endothelial nitric oxide (NO) synthase function and alters vascular production of Transforming Growth Factor-ss (TGF-ss) to facilitate the development of arterial stiffness with aging (Project 1: Molecular Mechanisms of Aging on Vascular Function; PI: Paul Sanders). The working hypothesis of this project is the ratio of active TGF-ss to NO is a critical, modifiable determinant of arterial stiffness of aging. 2. Determine the role of Runx2 in regulating arterial stiffening and elucidate the molecular mechanisms, using a novel smooth muscle-specific Runx2 knockout mouse model (Project 2: Molecular Mechanisms Underlying Arterial Stiffening; PI: Yabing Chen). The working hypothesis of this project is that a high salt intake induces the expression of Runx2 in VSMC, which initiates VSMC calcification and promotes arterial stiffening. 3. Define the role of the heme oxygenase-1 (HO-1)/ferritin system in the prevention of vascular calcification through downregulation of Runx2 and TGF-ss ((Project 3: Role of HO-1/Ferritin in Vascular Calcification During Aging; PI: Anupam Agarwal). Experiments will test the working hypothesis that induction of the HO-1/ferritin system prevents vascular calcification through downregulation of Runx2 and TGF-?. 4. Support a state-of-the-art core facility (Animal Vascular Phenotyping Core; Director: Edgar Jaimes) designed to provide detailed structure-function analyses of arterial stiffness and vascular calcification. The core will consist of a vascular physiology subcore for radiotelemetry, high frequency vascular ultrasound, micro-CT and endothelial function (myography); and a molecular pathology subcore for histology, immunohistochemistry and image analysis. The core will bring to the Birmingham VA Medical Center (BVAMC) new technology to study vascular biology. The long-term goal of this research effort is to lay the essential groundwork necessary to translate the findings to improved cardiovascular outcomes in aging and CKD and further build the research capacity at the BVAMC."}
{"text": "The specific etiology of a majority of cases of keratitis and uveitis, as well as the exact immunologic mechanism of corneal graft rejection is unknown. Yet these entities frequently cause significant visual loss and blindness. The purpose of the proposed project is to elucidate the effector mechanisms involved in the immunopathology of anterior segment ocular inflammation (including graft rejection) and to test experimentally the efficacy and side effects of new therapeutic approaches. These studies will deal with two major subjects: 1) the role of soluble mediators (\"lymphokines\") of cellular immunologic reactions in ocular inflammatory disease, and 2) the use of blocking antibody serum (BAS) in the prevention of immunologic corneal graft rejections. Lymphokines will be produced by guinea pig lymph node cells and purified and characterized. The lymphokines will then be placed in contact with eye tissue both in vivo and in tissue culture. The ability of lymphokines to cause tissue injury in the presence or absence of inflammatory cells will be investigated. An antibody will be made to a purified lymphokine preparation and its ability to block or suppress lymphokine-mediated tissue damage will be evaluated. Guinea pig anti-rabbit lymphocyte serum will be prepared and modified chemically so that it will not bind complement. The characteristics of modified BAS and the mechanisms by which it protects corneal grafts from rejection will be studied by in vivo and in vitro studies."}
{"text": "The overall goal of this project is to gain a detailed understanding of the molecular function of integrins, a large family of cell surface receptors. Integrins are involved in many developmental processes and also function in numerous fully differentiated cells. With respect to known pathological conditions, most studies of integrins have focused on the circulatory system, the immune system, and the metastasis of tumors, however it is likely that integrins will prove to be important in pathologies in other tissues and diseases as well. The PS integrins of Drosophila are very similar to vertebrate integrins, and provide a unique opportunity to examine integrin function in situ in a complex developmental system. We propose to: (1) Further define the interaction of PS2 integrin with the fly matrix protein tiggrin, using our cell spreading assay and genetically engineered fragments of the tiggrin protein. Also, begin steps to generate mutations in the tiggrin gene, in order to undertake a genetic analysis of tiggrin function during development. (2) Define the requirements for PS l integrin during development, using newly generated mutations in the mew gene, which encodes the alphaPS1 integrin subunit. This study will focus on Comparisons between the myospheroid (betaPS), inflated (alphaPS2) and mew phenotypes, and include analysis of genetic mosaics. (3) Continue to generate and characterize hypomorphic mutations of myospheroid, and combine this with cell culture and phenotypic analyses to define domains of integrins required for specific integrin functions. (4) Develop methods for the regulated expression of PS integrin transgenes in the developing wing of Drosophila, with the goal of using wing as a model system for the fine dissection of integrin function during morphogenesis in situ. This phase of the project will include use of GAL4 enhancer trap technology as well as dissection of the regulatory regions of integrin genes, in order to find enhancers that can provide the required patterns of integrin transgene expression. (5) Use the transgenes in (4) in combination with the integrin mutants to dissect the detailed functions of integrins and integrin \"parts\" during wing morphogenesis. This will involve the generation of clones of cells mutant for integrin function, and attempts to rescue the mutant phenotypes by expression of normal and genetically altered integrin transgenes."}
{"text": "The goal of the proposed research is to determine the mechanism of electron transfer and thermodynamic properties of the enzyme, assimilatory NADH:nitrate reductase under various environmental conditions. Specific aims are to determine: 1) oxidation-reduction (redox) potentials of the prosthetic groups FAD, heme, and molybdenum; 2) the effect of pH, ionic strength, substrates, and effectors on the redox potentials; 3) the stoichiometry and intermediate redox states in the transfer of electrons from NADH to nitrate; 4) the pathway and transient state kinetics of electron transfer between NADH and nitate; 5) magnetic interactions between prosthetic groups; 6) physical and functional interactions between nitrate reductase and the next enzyme in the pathway of nitrate assimilation, nitrite reductase. For these studies we will utilize EPR spectroscopy, UV/VIS-dual wavelength spectroscopy, microcoulometry, freeze-quench and stopped-flow techniques, and substituted analogues of nitrate reductase. These studies will provide important information on the mechanism of catalysis by nitrate reductase and factors which influence this activity. Since this enzyme catalyzes the rate-limiting step in the process of nitrate assimilation, the proposed research is of importance in terms of plant productivity and the supply of protein. This enzyme also serves as a model system for more complex electron transfer systems."}
{"text": "This project is concerned with the investigation of the mechanism of action of ACTH on the adrenal cortex. Specifically, it relates to the effect of this trophic hormone on the cytochrome P-450 systems of adrenal cortical mitochondria. Methods have been developed for measuring the binding of cholesterol to side chain cleavage cytochrome P-450 in the adrenal cortex and the studies in this project involve measuring how this binding is affected by agents such as ACTH and cyclic AMP. Also under study is the early and late pathway of aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex. Agents to be studied in that project include potassium, ACTH, sodium and angiotensin II. Methods are being developed for the measurement of the late pathway of aldosterone biosynthesis utilizing high pressure liquid chromatography of extracts following incubation of corticosterone with zona glomerulosa mitochondria. In addition, corticosterone binding to the 18-hydroxylase system of zona glomerulosa will be studied by spectrophotometry and electron spin resonance assays. BIBLIOGRAPHIC REFERENCE: Effect of Hypophysectomy on the Volume and Ultrastructure of Zona Glomerulosa in Rat Adrenal. Peter A. Nickerson and Alexander C. Brownie. Endocrin. Exper. 9, 187, 1975."}
{"text": "The goal of this research is to utilize the chiroptical properties of selected chromophoric groups which occur in molecules of biological significance to probe molecular conformation and structure in solution. The general methodology will be to elucidate the relationships which exist between molecular conformation, chromophore environment, and the optical properties of selected model compounds, by calculating the optical properties and the conformational energy as a function of conformation. The method will be evaluated by comparing the calculated results with experimentally determined optical rotatory dispersion, circular dichroism, and absorption sppctra. The model syytems will be chosen to represent various important classes of biological macromolecules, prrmarily proteins. After the technique is proven for a given type of chromophore by appropriate model compound studies, it will be applied to more complex molecules which contain the chromophore of interest. Special attention will be given to proteins such as ribonuclease and lysozyme."}
{"text": "The regulation of promoter activity by nuclear receptors requires the assembly of large multi-subunit complexes that either directly impact the basal transcriptional machinery or modify core histones to affect chromatin structure and remodeling. The mechanisms responsible for maintaining the highly ordered dynamics of protein binding to hormonally responsive promoters have not been definitively established. We have recently developed a novel in situ fluorescence recovery after photobleaching (FRAP) assay that led to the identification of molecular chaperones and their associated co-chaperones as nuclear mobility factors for the glucocorticoid receptor (GR). This assay will be exploited to provide additional mechanistic insights into the role of chaperones in nuclear dynamics of GR. The major hypothesis to be tested in this proposal is that molecular chaperones play a major role in steroid receptor dynamics at target sites within the nucleus via their regulation of receptor nuclear mobility, chromatin exchange and hormone exchange. Specific Aim 1 seeks to identify the role of individual chaperones and the pathway of chaperone complex assembly that mediates their activity as steroid receptor nuclear mobility factors. Chaperone protein assembly will be manipulated in the context of a novel in situ nuclear mobility assay and effects on steroid receptor/chaperone complex formation assessed by co-immunoprecipitation assays. Specific Aim 2 will determine whether molecular chaperones participate in the dynamic exchange of GR and other factors at receptor target sites within the nucleus. The in situ FRAP assay will be used to determine whether specific chaperone complexes are required for the dynamic exchange of GR and various receptor cofactors from a functional target gene. Chromatin immunoprecipitation assays will also be used with permeabilized cells to examine chaperone effects on GR and cofactor recruitment and cycling on the chromatin of target genes. Finally, Specific Aim 3 will determine whether molecular chaperones are required for the exchange of hormone from chromatin- bound GR. The effects of molecular chaperones on hormone exchange on nuclear GRs will be assessed in permeabilized cells using assays that distinguish hormone release from hormone exchange. Chaperone effects on hormone exchange at a specific target site will be revealed using a fluorescent GR ligand. Public Health Relevance: Hsp90 inhibitors such as geldanamycin (GA) are being evaluated for breast cancer therapy due primarily to the their selective action in cancer cells on hsp90 client proteins such as estrogen receptor, p53 and various kinases. However, the development of therapeutic anti-cancer drugs directed against hspQO or other chaperones has not taken into account the newly discovered roles for these proteins in chromatin dynamics or histone modification, which is the subject of this proposal. [unreadable] [unreadable] [unreadable]"}
{"text": "Research is focused on studying the events involved in HIV-1 fusion, the development of fusion inhibitors directed against gp41 and the development of monoclonal antibodies with broadly neutralizing activity that are directed against gp41. The HIV-1 Envelope (Env) proteins that mediate membrane fusion represent a major target for the development of new AIDS therapies. Three classes of Env-mediated membrane fusion inhibitors have been described that specifically target the pre-hairpin intermediate conformation of gp41. Class 2 inhibitors bind to the C-terminal heptad repeat (C-HR) of gp41. The single example of a class 3 inhibitor targets the trimeric N-terminal heptad repeat (N-HR) of gp41 and has been postulated to sequestrate the N-HR of the pre-hairpin intermediate through the formation of fusion incompetent heterotrimers. We have now shown that NCCG-gp41, a class 2 inhibitor, and N36Mut(e,g), a class 3 inhibitor, synergistically inhibit Env-mediated membrane fusion for several representative HIV-1 strains (X4 and R5) in both a cell fusion assay (with membrane-bound CD4) and an Env-pseudotyped virus neutralization assay. We have also succeeded in obtaining a monoclonal Fab (Fab 3674) selected from a human non-immune phage library by panning against the chimeric construct NCCG-gp41 (that comprises an exposed coiled-coil trimer of gp41 N-helices fused in helical phase onto the minimal thermostable ectodomain of gp41) that effectively neutralizes diverse laboratory-adapted B-strains of HIV-1 and primary isolates of subtypes A, B and C in an Env-pseudotyped virus neutralization assay, albeit with reduced potency (25x) compared to 2F5 and 4E10. Alanine scanning mutagenesis maps a novel epitope to a shallow groove on the N-helices of gp41 that is exposed between two C-helices in the fusogenic six-helix bundle conformation of gp41. We have shown that bivalent Fab 3674 and the C34 peptide (a potent fusion inhibitor derived from the C-helix of gp41) act at similar stages of the fusion reaction and to neutralize HIV-1 synergistically, providing additional evidence that the epitope of Fab 3674 is new and distinct from the binding site of C34.More recently we have shown that the class 3 inhibitor N36Mut(e,g) prolongs the temporal window during which the virus is susceptible to neutralization by the bivalent Fab-3674, and that bivalent Fab-3674 and N36Mut(e,g) neutralize HXB2 and SF-162 strains of HIV-1, as well as diverse primary B and C clade HIV-1 strains, synergistically in a Env-pseudotyped virus neutralization assay. N36Mut(e,g) also rescues neutralizing activity of the monovalent Fab-3674 against resistant HIV-1 strains and renders a series of related non-neutralizing Fabs neutralizing. Moreover, N36Mut(e,g) exhibits the same effects on the broadly neutralizing 2F5 and 4E10 monoclonal antibodies directed against the membrane proximal extended region of gp41. Very recently, we have subjected Fab 3674 to affinity maturation against the NCCG-gp41 antigen by targeted diversification of the CDR-H2 loop to generate a panel of Fabs with diverse neutralization activity. Three affinity-matured Fabs selected for further study, Fabs 8060, 8066 and 8068, showed significant increases in both potency and breadth of neutralization against HIV-1 pseudotyped with envelopes of primary isolates from the standard subtypes B and C HIV-1 reference panels. The parental Fab 3674 is 10-20 fold less potent in monovalent than bivalent format over the entire B and C panels of HIV-1 pseudotypes. Of note is that the improved neutralization activity of the affinity-matured Fabs relative to the parental Fab 3674 was, on average, significantly greater for the Fabs in monovalent than bivalent format. This suggests that the increased avidity of the Fabs for the target antigen in bivalent format can be partially offset by kinetic and/or steric advantages afforded by the smaller monovalent Fabs. Indeed, the best affinity-matured Fab (8066) in monovalent format (50 kDa) was comparable in HIV-1 neutralization potency to the parental Fab 3674 in bivalent format (120 kDa) across the subtypes B and C reference panels. We have now solved crystal structures of the N-HR mimetic 5-Helix with two Fabs that represent the extremes of this series: Fab 8066 is broadly neutralizing across a wide panel of B and C type HIV-1 viruses, whereas Fab 8062 is non-neutralizing. The crystal structures reveal important differences in the conformations of the CDR-H2 loops in the complexes that propagate into other regions of the antigen-antibody interface, and suggest that both neutralization properties and affinity for the target can be attributed, at least in part, to the differences in the interactions of the CDR-H2 loops with the antigen. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5 (developed by Merck), derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop. A key feature of fusion inhibitors that target the N-HR trimer, including antibodies directed against the N-HR trimer, is that deactivation of gp41 in vivo is a slow reversible process that is dependent on chemokine receptor binding to Env, and that the exposed N-HR trimer remains accessible to inhibitors until the final conformational changes in gp41 that lead to the formation of the 6-HB have taken place. This suggests that inhibition of fusion will be most effective when two or more Fabs are bound to the exposed N-HR trimer of the pre-hairpin intermediate of gp41. Since multiple Fabs/antibodies bound to the N-HR trimer are unlikely to dissociate simultaneously, the probability that at least one antibody is bound to the N-HR trimer at all times will be increased. Modeling of the complex of an N-HR trimer with three Fabs suggests that the CDR-H2 loop may be involved in close intermolecular contacts between neighboring antibody molecules, and that such contacts may hinder the formation of complexes between the N-HR trimer and more than one antibody molecule depending on the conformation of the bound CDR-H2 loop which is defined by its interactions with antigen. Thus, in the case of the complex with the most potent neutralizing Fab in our series, Fab 8066, the molecules of Fab can readily bind to the N-HR trimer without any steric clashes between adjacent Fab molecules. In contrast, as a consequence of the different mode of binding of the CDR-H2 to the hydrophobic pocket on the surface of the N-HR trimer, binding of three molecules of Fab 8062 to the N-HR trimer may result in steric clash between adjacent Fab molecules involving the CDR-H2, CDR-L1, CDR-L3 loops and loop 71-78. It seems likely in the light of the current structural data, modeling results and neutralization properties of our Fab series, that neutralization is dependent not only on tight binding of a single Fab to the N-HR trimer but also on the ability to bind multiple Fabs to a single N-HR trimer at preliminary step of fusion process."}
{"text": "PROJECT SUMMARY - Regional/National Resource Core for Improving Diabetes Outcomes in Community Health Centers Community health centers (HC) are vanguard providers of health care for vulnerable populations, serving 24 million Americans in 1200 centers in 9000 sites. Forty percent of HC patients are uninsured, 36% have Medicaid coverage, over 60% are racial or ethnic minorities, and 71% are at or below the federal poverty line. Nationally, HCs serve 1 of every 4 people in poverty, 1 of 10 minorities, and 1 of 9 rural Americans. HCs are a critical provider of care to underserved populations, and a vital part of efforts to reduce national disparities. The Chicago Center for Diabetes Translation Research (CCDTR) will serve as a regional and national resource for diabetes translation research in community health centers. We will continue our longstanding collaboration with the MidWest Clinicians' Network (MWCN) of approximately 130 health centers with 300 practice sites in the 10 Midwestern states as well as with partners including the National Association of Community Health Centers (NACHC), Association of Asian Pacific Islander Community Health Organizations (AAPCHO), Migrant Clinicians Network, ACCESS Community Health Network, state Primary Care Associations, America's Essential Hospitals, and the Medicaid Health Plans of America. The CCDTR will have a Regional/National Resource Core for Improving Diabetes Outcomes in Community Health Centers led by MWCN with the collaboration of its partners. MWCN and CCDTR have deep combined research and implementation strengths. In conjunction with other regional and national partners, the collaboration has widespread reach and connections. Tremendous opportunities exist to perform diabetes translation research in health centers and to disseminate findings regionally and nationally. Most MWCN-CCDTR projects capture the heart of translational research - integrating research with implementation science in a collaborative manner that has practical utility to end-users of the information. The Core will improve diabetes translation research and diabetes care and outcomes in safety-net populations. The specific aims of the Improving Diabetes Outcomes in Community Health Centers Core are to: 1) Serve as a regional and national resource to improve diabetes translation research in community health centers and other safety net settings led by the MidWest Clinicians' Network in collaboration with the National Association of Community Health Centers and other partners. 2) Determine best practices for diabetes care in community health centers, identify research voids, and facilitate implementation and evaluation projects."}
{"text": "Autoantibodies to the Golgi complex are found primarily in patients with Sjgren's syndrome and SLE although they are not restricted to these diseases. This competitive renewal application requests funding for the continuation of studies to characterize autoantigens of the Golgi complex and address the mechanisms that lead to the expression of autoantibodies to these intracellular organelle-associated proteins. The P.I.'s laboratory has been responsible for the identification of a family of Golgi antigens known as golgins. Common features of these autoantigens are that they are located on the cytoplasmic face of the Golgi cisternae and they have multiple alpha-helical coiled-coil rod domains flanked by non-coiled-coil and C- terminal domains. The goal of the proposed studies is to determine if the common structure and function for members of this protein family can explain the origin and production of autoantibodies in disease states. Specific Aim 1 will examine common features of golgins that may explain why they are targets of human anti-Golgi autoantibodies. The P.I. will characterize the events associated with the Golgi complex and golgins and their stability during apoptosis and necrosis. Hypotheses on how these autoantibodies may be produced in experimental models will be examined. Specific Aim 2 will examine if Golgi fragments and vesicular structures will induce immune and autoimmune response in experimental mice. Specific Aim 3 will elucidate the target of Golgi complex in lactate dehydrogenase-elevating virus (LDV) infected mice and address mechanism of how autoimmune response to the Golgi complex can be produced in these mice. Earlier studies have shown that LDV infected mice produce anti-Golgi antibodies. Our current data suggest that the autoimmune response to cytoplasmic organelles such as the Golgi complex is uniquely different from other intracellular autoantigens examined to date. It is anticipated that testing of the P.I.'s hypotheses will provide new insights into autoimmunity and autoimmune diseases associated with subcellular organelles."}
{"text": "The research described in this proposal aims to increase our knowledge concerning the fever response in animals other than humans; specifically relating to the occurrence of the fever response in one species of amphibian and two species of mammals living in their natural environment, the generality of the fever response in reptiles, and the energetic cost of the fever response in a free-living reptile. All of the studies concerning the fever response has never been demonstrated or investigated under natural field conditions where the greater heterogeneity of biotic and abiotic factors may lead to a more complex response. Also, although the fever response has been demonstrated in a number of ectothermic vertebrates under laboratory conditions, no systematic study of the fever response has been undertaken with any specific taxonomic group of ectothermic vertebrates to demonstrate the generality of the response and its relationship to life history characteristic of a species. Data on the fever response of the bullfrog, the California Ground squirrel, and the desert cottontail rabbit will first be gathered in the laboratory and than the laboratory baseline data will be applied to experiments on free-living animals. Body temperatures will be determined with the use of temperature sensitive radio transmitters. In the survey of the Class Reptilia for the fever response, II species representing 7 different Families will be given injections of heat-killed Aeromonas hydrophila, a gram negative bacterium known to be pathogenic to reptiles. The reptilian species to be used represent animals from different habitats (aquatic, mesic, desert), with different food habits (herbivorous, insectivorous, carnivorous), and with different activity patterns (diurnal, nocturnal). In the section that pertains to the energetic cost of the fever response, time budget analysis and oxygen consumption measurements will be combined to yield an estimate of the energetic cost of the fever response in a free-living reptile; the chuckwalla."}
{"text": "ABSTRACT c The vasculature endothelium forms a selectively permeable barrier that facilitates transfer of nutrients, oxygen and waste products between the retina and the blood. Therefore, diseases affecting the structure and function of the vascular endothelium, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD), have a devastating effect on the health of the retina and ultimately lead to severe visual impairment. Traditional treatment approaches focus on ameliorating disease symptoms that lead to vision loss, including retinal and choroidal neovascularization. Whilst effective, treatments such as laser photocoagulation are both invasive and destructive, requiring frequent interventions throughout the patient's lifetime, leading to the ablation of neurosensory retina as new blood vessels are cauterized. Moreover, these treatments fail to address the pathologic abnormalities within vascular endothelial cells (VECs) that underlie abnormal blood vessel function in DR. As such, they serve only to temporarily limit progression of the disease. In contrast to existing treatments, gene therapy represents an attractive therapeutic alternative, potentially allowing for the permanent correction of vascular dysfunction prior to the development of sight- threatening complications. The inability to efficiently deliver genetic material to vascular endothelial cells currently prohibits development of any gene therapy treatment aimed at preventing DR. We have recently taken the first step to overcoming this barrier by elucidating a recombinant adeno-associated virus (rAAV) vector mutant with enhanced affinity for VECs. We propose to further develop these vector technologies and optimize their surgical delivery through the following specific aims: 1) Evaluate endothelial cell transduction and maintenance of gene expression in normal and diabetic vasculature; 2) Characterize early stage biomarkers of DR progression and efficacy of endothelial cell gene therapy, and 3) Assess endothelial cell transduction following rAAV administration by selective intra-ophthalmic artery infusion (SIOAI). Utilizing a well-established rat model of type I diabetes (T1D) we anticipate the development of a strategy to effectively deliver genetic material in both normal and dysfunctional VECs. In doing so, we will utilize various advanced imaging modalities to quantin order to maximize the clinical translation of the proposed DR gene therapy, we will optimize key aspects relating to the targeted intravascular delivery of rAAV using a mini-swine model that accurately recapitulates human cardiovascular and ocular anatomy. The Ocular Gene Therapy Laboratory (OGTL) and Advanced Ocular Imaging Program (AOIP) at the Medical College of Wisconsin, together the University of Florida Department of Ophthalmology, provide the perfect collaborative environment to complete the proposed work. Finally, our proposal addresses an emerging need identified in the NEI Publication ?Vision Research: Needs, Gaps, and Opportunities?: ?develop novel, noninvasive imaging techniques for monitoring electrical or metabolic activity of retinal neurons in vivo, ideally at the spatial resolution of photoreceptors or better for early detection of disease and monitoring of therapeutic intervention.?"}
{"text": "Previously we have initiated a large cDNA microarray effort in collaboration with the Human Genome Project and the Cancer Genome Anatomy Project (CGAP) to develop a comprehensive and novel molecular classification schema for human gliomas based on a gene expression profile using cDNA microarray technology. We have constructed our own cDNA microarray chips which will be enhanced for new and selective genes thought to be important in glioma biology. This project will include hundreds of tumor specimens and offer an unprecedented opportunity for gene discovery, dissecting signal transduction pathways, and learning this exciting new technology. Glioma stem cell is a tumor subpopulation that can self-renew in culture, perpetuate a tumor in orthotopic transplant in vivo, and generate diversified neuron-like and glia-like postmitotic progeny in vivo and in vitro. Recently, conventional and array-based CGH (aCGH) profiling of human gliomas have shown a significant number of copy number alterations (CNAs) including gain/amplification (1p34-36, 1q32, 3q26-28, 5q, 7q31, 8q24, 11q, 12q13, 13q, 15p15, 17q22- 25,19q, 20p, and 20q), and deletion/loss (3q25-26, 4q, 6q26-27, 9p, 10p, 10q, 11p, 12q22, 13q, 14q13, 14q23-31, 15q13-21, 17p11-13, 18q22-23, 19q, and 22q) (Kotliarov et al., 2006; Nigro et al., 2005; Phillips et al., 2006). The large number of chromosomal aberrations, and the large number of genes contained therein, have to date made it impossible to identify which genes are in part responsible for driving the biology of these tumors. We have analyzed a large number glioma samples for genetic characterization of recurring CNAs using Affymetrix 100K single-nucleotide polymorphism (SNP) array chips and Genechip HumanGenome U133 Plus 2.0 Expression array (Kotliarov et al. 2006). Based on our bioinformatics data from these array and gene expression profiling experiments, we have found novel genes frequently altered in gliomas. Furthermore, we have explored the new biotechnology such as next generation sequencing, for this project. We have generated sequence-verified gene Gateway entry clones of these genes and cloned them into pLenti/UbC/V5 expression vectors for transduction of various target cell lines. With our candidate gene constructs, we will identify whether candidate genes change the biology of these cells in such a way that may be consistent with a role in tumorigenesis (i.e. clonogenecity, proliferation, apoptosis, tumorigenic potential in immunosuppressed animals). The NOB laboratory is using the DHS-seq method to profile genome-wide transcriptional changes in glioma patient samples. As described above, the DHS-seq will reveal dynamic changes in the chromatin, which are important in the development and progression of brain tumors and allow us to identify novel molecular targets to treat this disease. We have tested the DHS-seq protocol on two glioma stem cell lines (827P12 and 923P9) and corresponding xenograft tissues. Preliminary analyses of these data suggest that in combination with gene expression and copy number data, we will obtain novel insights into the genomics underlying brain tumor biology. To this end, the NOB laboratory has begun testing this method on patient samples, using tumor tissues and adjacent normal brain directly from surgical specimens. The plan is to continue processing additional patient samples as they become available with the ultimate goal of incorporating the transcriptome analysis into the comprehensive genomic analysis that is being planned as a component of the molecular tumor board, described in the Clinical Project."}
{"text": "DESCRIPTION: Glaucoma is one of the leading causes of blindness in the Veterans population, estimated to affect 285,000 American Veterans as well as 60 million people worldwide, and is the most common neurodegenerative disease. The exact mechanism by which vision loss occurs in glaucoma is not fully understood, which hinders development of a true cure for this disease. It is thought that excessive deformation of the optic nerve tissues in the posterior eye under the insult of elevated intraocular pressure leads to an unfavorable biomechanical environment for cells of the optic nerve head. These cells include the retinal ganglion cells, whose axons transmit visual information from photoreceptors to the brain. Based upon an increasing body of evidence suggesting that the stiffness of the sclera (the white outer coat of the eye) may greatly influence the degree of deformation of the posterior eye, we hypothesize that stiffening the peripapillary sclera will protect against vision loss in glaucoma. Whereas current therapies only delay the onset of symptoms, stiffening the peripapillary sclera could be a novel treatment for glaucoma. In this study, our approach is to screen the efficacy of potential chemical agents and engineered viral vector approaches that modulate peripapillary scleral stiffness in a rat model of glaucoma. In the first specific aim, we will use an optical tracking technique called digital image correlation to quantitatively assess the magnitude of stiffening these agents have on rat scleras. We will also develop a surgical technique to deliver these agents to the back of the eye under ultrasound guidance without affecting surrounding structures. Once we determine the most effective agents, in the second specific aim, we will deliver these agents to rats with surgically-induced glaucoma. Our experimental paradigm is a 3x2 study, where we will compare rats with and without induction of glaucoma to rats with and without delivery of peripapillary scleral stiffening agents (plus additional sham controls). Over 8 weeks, we will monitor anatomic changes to the eye and quantify the degree of vision loss by measuring the rats' visual acuity and contrast sensitivity. At the conclusion of the experiment, we will analyze the expression of genes and proteins associated with glaucoma and ocular damage to determine the overall health of the eyes. The expected outcome of this study is that rats with stiffened peripapillary scleras will experience less vision loss under glaucomatous conditions than glaucomatous rats with no peripapillary scleral stiffening. This will provide the first in vivo data regarding the efficacy of this novel approach to treating glaucoma. If successful, these findings will motivate development of a human clinical trial to deliver biocompatible stiffening agents to prevent vision loss in glaucoma. Overall, the motivation for this study is the need for a better understanding of ocular biomechanics towards the long-term goal of developing a cure for glaucoma in clinical use for Veterans and others with this disease. This meets the Rehabilitation Research and Development goal of preventing and treating vision loss. This project represents a method to screen potential glaucoma treatments in an animal model, as well as to train the postdoctoral candidate to become a successful and productive independent investigator in the field of ocular biomechanics within the VA research environment."}
{"text": "Three-dimensional coordinate information representing neuronal processes is obtained utilizing a video version of the computer microscope. Usually specimens are prepared by the rapid Golgi method although the measuring system has the capability of accurately reconstructing and analyzing most biological structures which are histologically prepared and preserved on microscope slides. The opportunity is also presented to serially reconstruct electron micrographs. Neurons from the hippocampal and related areas of the human immature brain (18 to 33 weeks gestational age) are measured and analyzed according to their branching characteristics. Statistical parameters representing the branching characteristics of neuronal processes (e.g. branching/fission angles, polar angle and radial separation of endpoints, etc.) are obtained and deviant patterns are correlated with specific forms of profound mental retardation (e.g. Down's Syndrome, chromosomal abnormalities, etc.) Attempts are also in progress to quantitatively analyze and classify dendritic spines in regard to their density, distribution and topological characteristics."}
{"text": "Neoplasms are commonly characterized by abnormal gene expression. Our studies will utilize a unique murine pituitary tumor model (MGH 101A) to investigate altered gene activity. This tumor evolved in our laboratory from a thyrotropic tumor. It is unique in that it has lost completely the ability to produce the beta-subunit of TSH and now only synthesizes the alpha-subunit in a nonregulated and promiscuous fashion. Our preliminary observations suggest that these defects occur at a pretranscriptional level. The projected studies on this pituitary neoplasm will be divided into two parts. First, we will investigate the molecular basis for absent TSH-beta subunit production in MGH 101A. The production of the TSH-beta subunit is normally a highly tissue-specific function in thyrotropic tissue. Its absence could therefore be related to an abnormality in the TSH-beta gene structure or the presence of a transacting factor altering the expression of this gene. Our preliminary experiments have shown that the TSH-beta gene is present and is not grossly abnormal. We will therefore use recombinant DNA techniques and molecular biology to discover the mechanisms for the absent TSH-beta gene expression. A solution to this problem could provide insights into similar phenomena for eutopic and ectopic malignancies, as well as an understanding of those factors which regulate gene expression in eukaryotic cells. Second, we will perform studies to determine the molecular basis for the absent regulation of the alpha-subunit production in this tumor. These experiments will explore those factors of gene structure and cellular environment which are important to the regulation of gent expression. Preliminary information suggests that not only is the alpha-subunit gene present, but that it is transcribed very efficiently. Knowledge gained on the regulation of this tumor's alpha-subunit production may contribute substantive information on pathogenetic mechanisms as well as the treatment of human pituitary and non-pituitary neoplasms."}
{"text": "Studies of the physicochemical properties of epiglycanin will be continued. Its masking role at the cell surface will be further investigated in relation to its immunochemical and immunological properties. The relationship of the masking mechanism to the type and location of the carbohydrate chains on epiglycanin will be further studied by the process of mapping active sites by electron microscopy. The biosynthesis of epiglycanin will be studied by precursor labeling and by the use of the epiglycanin antibody. Investigation of possible inhibitors of the synthesis of this glycoprotein will be pursued."}
{"text": "Continuous, non-invasive methods of measuring arterial PO2/CO2 are clinically desirable; but it has become apparent that the present method of monitoring O2/CO2 transcutaneously is suitable as no more than an indication of trends, as data amassed by us and by other investigators makes clear that transcutaneous O2/CO2 values do not consistently correlate with arterial PO2/CO2. The research proposed here will attempt to provide a clearer understanding of what transcutaneous measurements actually reflect by analyzing the basic physiologic variables and mechanisms reflected in transcutaneous values. We propose to employ--and thereby to validate--new methods of measuring various components of cutaneous blood flow and of determining the index of peripheral O2/CO2 perfusion and evaluating the effect of temperature on these components, as well as on dermal permeability. The ability to distinguish and measure these components should provide a means of correcting transcutaneous values so that they more accurately reflect arterial PO2/CO2. By means of photoplethysmography and measurement of the power output of the heater, in conjunction with an occlusive method of plethysmography, we will be able to measure two major components of cutaneous flow--papillary flow and deep cutaneous flow--and relate their effects under both normal physiologic and pathologic conditions of hypotension, hypoperfusion, anemia, polycythemia, and diminished oxygen unloading. When the validity of our method of corrected transcutaneous measurement has been established with animal subjects, the method will be applied in clinical situations with normal and diseased newborn and adult patients so as to establish its usefulness under all clinical conditions. The knowledge obtained from this study will help us to understand the meaning of transcutaneous O2/CO2 as an independent physiologic variable, which may have significant clinical applications of its own."}
{"text": "The vision of the University of Cincinnati Academic Health Center (AHC) Institutional CTSA is to create an environment to facilitate translating discoveries to clinical application. Our new academic home for clinical and translational research, the Center for Clinical and Translational Science and Training (CCTST), is beginning to transform the research environment within the AHC and our affiliated partners in other areas of the University, the community, and industry. The CCTST will coordinate and plan the overall direction of AHC research infrastructure and training opportunities;serve investigators'needs from project concept to completion;optimize skills and foster career development of both new and experienced investigators;and ensure that community input informs research processes, and that AHC discoveries are translated to the community. The CCTST will coordinate and leverage our existing strengths and develop new initiatives. Through Research Central, researchers will have easy access to centralized study design, biostatistical, bioinformatics, regulatory, and community engagement support. The new Pilot &Collaborative Studies core will expand the highly successful pilot funding program at Cincinnati Children's Hospital Medical Center (CCHMC) to the entire AHC. Biomedical informatics will be coordinated across the AHC. A new K12 program and greatly expanded educational offerings, including a new Certificate in Clinical and Translational Research, will be developed, building on the success of our Dean's Scholars in Clinical Research and K30- funded MS in Clinical and Translational Research programs. Through our community engagement program, we will further bi-directional research linkages with the local community, breaking down bureaucratic barriers by creating IRBs that can coordinate community-based research. Expanding services such as nursing/coordinator support and sample processing provided by the existing General Clinical Research Center (GCRC) at CCHMC and the Cincinnati Veterans Affairs Medical Center will promote patient-oriented research for populations outside the GCRC in the community. New translational technologies, including proteomics. drug discovery, imaging, nanomedicine, gene transfer and stem cell biology and translational and molecular disease modeling, will be made more accessible to researchers. Throughout, we will utilize a quality improvement model to evaluate our progress. These efforts will facilitate transforming clinical and translational research at the AHC. leading to improved health outcomes in the community."}
{"text": "The Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Photography Reading Center (CPRC) provides objective, independent photographic verification of two important components of the CLEK Study: 1.) the presence, absence, and degree of severity of corneal scarring in all study eyes and, 2.) the rigid contact lens fit (fluorescein pattern) in all study eyes that wear rigid contact lenses, as well for the first rigid contact lens base curve that provides clearance of the corneal apex. This proposal provides documentation of the CPRC's ability to perform these functions with high sensitivity and specificity. This proposal also provides a description of the day-to-day organization of work, quality control and data management procedures required to insure the scientific integrity of the CLEK Study and to insure the orderly operation of the CPRC. Documentation of the capabilities of the proposed CPRC Staff for the performance of the Study in accord with the details of the CPRC Operations Manual and with the CLEK Study Operations Manual is provided."}
{"text": "This protocol seeks to determine whether there are specific behavioral changes or a clinically detectable specific pattern of behavioral improvement that occurs within the first week of treatment for major depression. It will also evaluate whether such improvement is due to the drug used in treatment (desipramine or fluoxetine), or rather to the process of recovery, regardless of the treatment modality."}
{"text": "Bacterial DNA replication is carefully controlled at the initiation stage, possible by the regulation of the essential activity of DnaA protein. The cellular membrane has long been hypothesized to be involved in chromosomal replication, with accumulating evidence that indicates membranes have a profound influence on DnaA protein. The long term goal of this research is to elucidate the physiological significance of the influence of membranes on chromosomal replication. The research outlined here uses both biochemical approaches with defined components and genetic and physiological studies to directly test the hypothesis that acidic phospholipids of the cellular membrane participate in the regulation of DnaA protein activity. The Specific Aims are to; 1. Identify the membrane binding site on DnaA protein by chemical crosslinking. Disrupt the membrane binding function by site-directed mutagenesis and isolate the mutant DnaA proteins. Confirm with defined components that the other replication activities of DnaA protein have not been altered. 2. Examine in vivo the replication activity of the mutant Dna A proteins are viable, and if so, if they initiate replication at aberrant times during the cell cycle. Localize mutant forms of DnaA protein within the cell by subcellular fractionation and immuno-gold microscopy to examine the importance of DnaA protein-membrane association for regulated initiations. 3. Generate and screen randomly mutagenized dnaA genes for the ability to suppress the growth arrest of acidic phospholipid-deficient cells. Map the suppressor mutations in dnaA and examine the replication activities of the mutant proteins in vitro. Determine the cellular location of the mutant proteins and analyze their effect on the cell cycle control of chromosomal replication in vivo. This work should provide insight into the regulation of the initiation of DNA replication , which is a key control point in the prokaryotic cell cycle and in the determination of eukaryotic cellular quiescence or proliferation. Furthermore, knowledge gained from the proposed studies of DnaA protein may guide future investigations of how phospholipids may act as regulators of enzymatic activities."}
{"text": "The incidence of obesity is increasing at an alarming rate in Western cultures, leading to its classification as an epidemic. In light of this epidemic, there is great impetus to better elucidate the processes that regulate fat production, storage, and release. The ability to regulate fat storage is a fundamental process required for survival of species ranging from mammals to invertebrates. It has therefore been suggested that diverse model organisms may be exploited in the study of fat metabolism. Very recent studies have demonstrated that the nematode Caenorhabditis elegans provides a useful model for identification of genes affecting fat storage. An RNAi screen to inactivate more than 16,000 genes in C. elegans and screen for alterations in fat storage identified 305 genes that reduced, and 112 genes that increased body fat upon inactivation (Ashrafi et al., 2003). More than 50% of these genes have mammalian homologs not previously implicated in regulating fat storage, and represent excellent candidates for having potential roles in human fat metabolism. In this proposal, we will evaluate several candidate \"fat genes\" identified in C. elegans by generating and characterizing gene knockout mouse models. To circumvent the traditional time consuming and laborious process of generating gene knockouts via recombination in embryonic stem (ES) cells, we will utilize a resource containing thousands of pre-fabricated ES cell lines containing gene-trap insertions. We have identified 22 existing ES cell lines carrying inactivating insertions within mouse homologs of C. elegans fat genes identified by Ashrafi et al., which are available from the BayGenomics Gene-Trap Consortium. We have confirmed that several of these genes are prominently expressed in mouse adipose tissue, and hypothesize that these genes, which have already been shown to play a role in fat storage in C. elegans, may affect fat development or metabolism in mammals. We will utilize the existing gene-trap ES cell lines to generate mouse knockout strains for several of the putative \"fat genes\", and characterize them for adipose tissue mass, glucose and energy metabolism. This pilot study will reveal whether \"fat genes\" identified in the invertebrate C. elegans have a similar role in mammals, and may identify novel genes involved in fat storage and metabolism."}
{"text": "Child maltreatment is a major public health problem. In addition to the risk of physical injury or death, early maltreatment is associated with impairments in social competence, emotional regulation and cognition, as well as to an increased risk for the development of anxiety, mood, and addiction disorders later in life. While it has been assumed that these vulnerabilities reflect impacts on neurodevelopment, practical and ethical concerns prevent studying these associations during early human development. Yet, such data would aid attempts to intervene and reduce the mental health burden associated with these early adverse experiences. The present proposal addresses this problem by using a unique, naturalistic, nonhuman primate model of human childhood maltreatment. Interestingly, maltreatment is not just a human problem; it has been reported in nonhuman primates as well, with rates and types of maltreatment similar to those seen in human populations. This rhesus monkey model provides a means to characterize the long-term impact of infant maltreatment on neurodevelopment using neuroimaging technology. However, in order to accomplish this important goal, preliminary data using in vivo neuroimaging are needed to justify longitudinal analyses comparing abused and non-abused animals, and normative data on early development to determine where perturbations in neural structure and circuits occur as a consequence of early adverse experience. The overall hypothesis of this proposal is based on the argument that infant maltreatment, via a sustained sensitization of the stress response, disrupts normal neurodevelopment, including neural systems underlying the control of socioemotional behavior and stress physiology. There are two aims in this project, both of them applying cutting edge in vivo neuroimaging technology and approaches to study the organization of brain structure, connectivity and function in animals with different rearing experiences. Aim 1 will define the long-term consequences of infant maltreatment on neurodevelopment, characterizing brain differences between monkeys with early exposure to infant abuse and non-abused controls as they transition from adolescence to adulthood. Aim 2 will define normative changes in neurodevelopment in rhesus monkeys, from the early postnatal period until 18 months of age. Neurodevelopmental changes will be assessed for relationship to socioemotional development and physical growth. Significance Understanding the neurodevelopmental consequences of infant maltreatment in a closely related phylogenetic relative will provide the framework for defining the neurodevelopmental impact of human childhood maltreatment, a reference for understanding its role in the development of socioemotional problems and pathophysiology in children and adolescents, and aid the design of treatment plans seeking to limit the adverse consequences of childhood maltreatment. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]"}
{"text": "Naturally occurring examples of immune system-mediated HIV control provide encouragement for the ultimate development of effective vaccines and immunotherapies that would limit HIV replication.. Identification of the components, targets, and magnitude of an effective immune response to HIV are important steps toward the development of effective vaccines or immunotherapies. Although patients with normal CD4+ T cell counts and low levels of plasma virus are a heterogeneous group, a small subgroup of patients with truly non-progressive HIV infection and restriction of virus replication likely holds important clues to the basis of an effective immune response to HIV. A small subpopulation (fewer than 0.8% of HIV infected individuals) shows no signs of progression over a 10-year period. We have assembled a stringently defined cohort of such patients, termed long-term nonprogressors (LTNPs) or elite controllers. Many of these patients have been infected for 20 years with no CD4+ T cell decline without taking antiretroviral therapy, and maintain plasma viral RNA below 50 copies per milliliter. Cells from these patients are being used to systematically dissect the mechanisms of immune-mediated restriction of virus replication. HIV-specific T-cell responses of these patients have been studied in extreme detail. Through this project, extraordinary progress has been made in understanding the basis of immunologic control of HIV. Our prior work indicates a dramatic association with the HLA B*5701 allele and that the immune response is highly focused on peptides restricted by this allele. This result established a host genetic and functional link between immunologic control and the CD8+ T-cell response of these patients. More recently we have found that this focus is specific to HIV, and is not found in the response to other pathogens such as Hepatitis C virus or Cytomegalovirus. LTNP patients do not differ in the frequency of HIV-specific T cells or in the ability to recognize the autologous virus, when compared to progressors. The finding of high frequencies of CD8+ T cells specific for the patients virus in both LTNPs and progressors strongly suggests that differences between responses of these patient groups are not quantitative, but rather qualitative in nature. One important qualitative difference in the HIV-specific immune response that distinguishes LTNPs from progressors is the maintenance of HIV-specific CD8+ T cells with a high proliferative capacity. This proliferation parallels perforin expression required for effective killing of HIV-infected CD4+ T cells. In 2009 we established the properties of the HIV-specific CD8+ T-cell response that are tightly associated with the LTNP phenotype. Although the HIV-specific CD8+ T cells of LTNPs have a greater capacity to proliferate and produce molecules responsible for killing HIV infected cells, the mechanism(s) by which these properties translate into effective immunologic control of HIV has remained unknown. Most current assays are not sufficiently powerful to establish if differences in HIV-specific CD8+ T-cell function are determined by frequency, CD8+ T cell proliferation, preferential target or effector cell death, or the mechanism of HIV-infected cell elimination. To better understand the mechanisms of immunologic control, we recently devised a method to measure HIV-infected cell elimination on a per-cell basis. Measured on a per-cell basis, HIV-specific CD8+ T cells of LTNPs efficiently eliminated primary autologous HIV-infected CD4+ T cells. This effective killing was clearly distinguishable from responses of progressors over a very broad range of effectors to HIV-infected targets. Progressors cells did not mediate effective killing even at high effector to target ratios. Defective cytotoxicity of progressor effectors could be restored in vitro. These results establish an effector function and mechanism that clearly segregate with immunologic control of HIV. A deeper understanding of the basis of immunologic control in LTNPs and the loss of immunologic control in progressors is likely to provide information that is critical for development of immunotherapies or prophylactic vaccines for HIV. In future work we seek to understand the molecular basis of the difference in killing capacity between LTNPs and progressors. We are also working to understand the mechanism by which such responses arise in LTNP patients, and determine whether they may be exploited in an HIV vaccine."}
{"text": "Bistability is a striking phenomenon found in various systems in which two very different behaviors can occur for the same external conditions. For example, new-born infants can suddenly stop breathing - for unknown reasons - a condition known as apnea, and, several seconds later, resume normal breathing for reasons which are also unknown. Bistability is also found in simpler systems, including squid giant axons. When the pH of the intracellular medium of the axon is[unreadable] elevated to 7.7, or higher, the axon is either quiescent or spontaneously firing action potentials in a rhythmic manner at a rate of approximately 25 Hz. The spontaneous firing can last for several hours. The ion channel mechanisms underlying this behavior are a persistent, tetrodotoxin-sensitive sodium ion channel, INaP, and a acid-sensing-ion-channel (ASIC) which is also sodium selective. This ASIC differs from the ASIC's found in the mammalian nervous system in that the H+ sensitivity is on the inner surface of the membrane. Alkalinization of the intracellular milleau turns off the ASIC, thereby allowing the INaP channel to destabilize the normal rest state of the axon. We have examined transitions between the two stable states in alkalinized axons, repetitive firing and quiescence, by injecting computer generated noise into the axon. The pattern of on-off switching of the pacemaker depends upon the intensity, spectral properties, and phase angle of the noise stimulus current. Our results reveal a distinct form of bistability in which noise can either silence pacemaker activity, trigger repetitive firing, or induce sporadic burst patterns similar to those recorded in a variety of normal and pathological neurons."}
{"text": "ThermoGen proposes to isolate a library of thermostable glycosidases for use in the synthesis of carbohydrate containing therapeutics. Phase I will identify thermostable glycosidases from ThermoGen's large library of thermophiles and Escherichia coli clone banks of themophilic organisms. Identified glycosidases will be characterized and categorized on the basis of their hydrolytic and synthetic substrate specificity. Phase II will involve the further characterization of these and additional thermostable glycosidases for commercial development. This library of thermostable glycosidases will be available to synthetic chemists for the development and synthesis of carbohydrate containing pharmaceuticals. Phase III will focus on commercialization of identified glycosidases at the industrial scale for use in industrial synthetic pipelines. PROPOSED COMMERCIAL APPLICATION: ThermoGen proposes to isolate and characterize a large set of thermostable glycosidases for use by the synthetic community for the development of new pharmaceutical compounds. These enzymes may also be used to streamline the synthesis of existing commercial processes."}
{"text": "The purpose of the proposed research is to determine how plasma lipoproteins and apolipoproteins bound to lymphocyte membrane receptors - termed proliferation-restraint receptors - inhibit mitogenic recruitment of the cells into and passage through the cell cycle, thereby inhibiting mitogen-induced cell proliferation. The suppressor molecules inhibit biochemical process -e.g., enhanced metabolism of phosphatidylinositol (PI) - which occur within the first hours post-stimulation. Inhibition correlates directly with extent of receptor occupancy. The hypothesis to be tested is that PI metabolism is important in the production of growth factors and/or growth factor receptors which are required for the activated cells to undergo mitosis. The hypothesis will be tested in systems containing defined cellular phenotypes and relying on assays in which events can be assessed in idividual cells by flow cytometry. The three specific aims are: (1) to determine the phenotype of the lymphoid cells required to generate the PI response and to modulate suppression of this response by AP/LP, (2) to determine the influence of AP/LP on IL-2 receptor expression and IL-2 production, paying particular attention to the relationship between the PI response, IL-2 receptor expression and IL-2 production, and (3) To characterize the proliferation-restraint receptor by its specificity for well-characterized ligands and its sensitivity to modification-inactivation. The long term goals of the research are (1) to determine the structure of the lymphocyte proliferation-restraint receptor which recognizes apolipoproteins; (2) to elucidate the mechanism by which it functions to restrict lymphocyte proliferation at the molecular level; (3) to identify the cells, lymphocyte and nonlymphocyte, which express it and are therefore sensitive to its function; and (4) to understand the factors which regulate its expression and its activity. Lipoproteins are natural modulators of the functional capacity of the immune system. Suppression of immune responsiveness by lipoproteins is important in diseases such as cancer in which host immune competence may be a primary defense mechanism. In cell biology/physiology, the proliferation-restraint receptor offers the opportunity to define recruitment of lymphocytes from the nonproliferating state in molecular terms."}
{"text": "Mechanism of dopamine actions in cardiovascular and renal systems will be investigated by delineating the dopamine-dependent cyclic AMP system: the effect of dopamine on intracellular cyclic AMP concentration dopamine binding receptor and adenylate cyclase activity in plasma membrane. The effect of interactions between dopamine and other hormones or catecholamine blocking agents on the cyclic AMP system will also be investigated in cardiovascular and renal systems. Lithium inhibits catecholamine-dependent cyclic AMP, but not all physiological effects of catecholamines. Utilizing this unique effect of lithium, the physiological role of catecholamine-dependent cyclic AMP in cardiovascular and renal system will be further investigated by comparing the inhibitory effect of lithium with that of other adrenergic blocking agents on myocardil contractility, distribution of intra-renal blood flow, and the release of renin. Prostaglandin E2 has vasodilatory effects in inner cortical and outer medullary portions of the kidney. Since vasodilatory effect of the beta adrenergic stimulus in other systems is mediated through cyclic AMP, the possible vasodilatory effect of prostaglandin E2-dependent cyclic AMP will be investigated in various portions of the kidney. The mechanism of interaction between prostaglandin E2 and alpha adrenergic stimulation, angiotensin I or II will also be investigated by studying the effect of these interactions on the cyclic AMP system. These studies are essential for the understanding of mechanism of dopamine action, the release of renin, and prostaglandin E2 in cardiovascular and renal systems."}
{"text": "Molecular signatures collected from intact tissue sections by MALDI imaging mass spectrometry (MALDI IMS) have shown high potential for use as a prognostic or diagnostic pathology tool in the clinical setting. A major obstacle to the widespread deployment of MALDI IMS, however, is the difficulty of identifying the proteins contributing to the signatures. Researchers have tried a number of approaches, including in situ digestion, MALDI TOF/TOF tandem mass spectrometry, and top-down proteomics on specific image regions. In preliminary work, we have obtained promising experimental results using top-down proteomics on intact proteins in the 2 - 20 kDa range. However, the lack of successful algorithms and software to identify the proteins in IMS mass signatures poses a major bottleneck. In particular, available top-down proteomics software relies heavily on high-accuracy mass spectrometry. The requirement for high accuracy precludes the use of some of the most sensitive mass analyzers such as linear ion traps, especially useful for these very small and complex samples. Protein Metrics Inc. is a new software company building on six years of algorithms and software research at Palo Alto Research Center. We plan to extend Byonic, our next- generation proteomics search engine, to intact proteins up to about 20 kDa. For proteins larger than 20 kDa, we will also build software for middle-down proteomics, specifically for assembling large peptides (2 - 20 kDa) produced by limited digestion to recover the identity of the intact proteins observed in IMS. The proposed Phase I feasibility study will allow us to perform controlled studies to determine the best experimental and bioinformatics approaches. Phase II will then build commercial-grade software. The proposed project will advance the state of the art in imaging mass spectrometry. Translation of imaging mass spectrometry to routine clinical pathology use will advance the state-of-the-art in disease diagnosis and treatment, and advance medical imaging and public health. PUBLIC HEALTH RELEVANCE: The project will develop commercial software that will improve our ability to identify the proteins and modifications represented in imaging mass spectrometry molecular signatures. Project success will make imaging mass spectrometry much more useful as a clinical pathology tool."}
{"text": "Modulation of cognitive and emotional responses by the limbic system of the brain is studied in patients undergoing cingulomotomy or amygdalectomy for the relief of intractable pain or epilepsy respectively. Autonomic and electrophysiological changes are studied by 1) stimulation through chronic electrodes situated in various limbic structures and 2) pre- vs. post-operative assessment of the effect of therapeutic brain lesions. Modifications in the appreciation of pain (conditioned avoidance response) and certain psychological functions (memory, perception and learning) are related to mechanisms assigned to the emotional brain of man."}
{"text": "The overall goal is to study the licensing policies and practices of 12 academic institutions regarding their DNA-based patents. The proposed project is conceived as a pilot study that will test the feasibility of conducting a more comprehensive follow-up study of such policies and practices. Specific aims are as follows: (1) To provide a clear, concise definition of the phrase \"DNA-based patents\"; (2) to analyze DNA-based patents into subtypes, using categories that are useful for understanding the policies and practices under which they are commercialized; (3) to gather and publish precise, up-to-date information on the number of U.S. DNA-based patents held by all U.S. and Canadian academic institutions; (4) to invite the technology transfer offices of all institutions holding at least 25 such patents to participate in a pilot study of their patenting and licensing policies regarding DNA-based patents; (5) with the aid of a project advisory board, to select a representative group of 12 positive respondents for more detailed study of their licensing policies; (6) to provide the technology transfer offices of the 12 participating institutions with categorized lists of their DNA-based patents, and to solicit input on categories; (7) to gather detailed information about the licensing of DNA-based patents at these institutions through a questionnaire and follow-up interviews; (8) to analyze and publish the data that have been gathered, paying special attention to policies and practices regarding the licensing of DNA-based patents that were based, at least in part, on research supported by federal funding; (9) during the second year of the project, to select 10 patents or clusters of patents and to develop case studies that illustrate technology transfer based on DNA-based patents. At least one of these case studies will be focused on a research tool. (10) In light of the results from the pilot study, to consider the feasibility and utility of conducting a more comprehensive follow-up study of licensing policies and practices regarding DNA-based patents at U.S. and Canadian academic institutions. [unreadable] [unreadable]"}
{"text": "Thyroid hormone (TH) acts through binding to nuclear protein thyroid hormone receptors (TR), which regulate transcription of specific genes. Clinically significant abnormalities related to TR function are believed present in patients with serious illness, thyroid hormone resistance, and possibly in tumors and obesity. With the availability of cloned cDNAs coding for three forms of human TR (TRalphas and TRbeta), semipurified TR preparations, and cloned TH responsive genes, we are in a position to answer important questions about TH function in human health and disease. While three forms of hTR have been identified, it is possible that other forms are present, and we will screen human cDNA libraries to identify these. We will study the expression of hTR alpha and hTR beta in various human tissues by Northern Blot to determine which forms are expressed in all tissues. The RNAse protection assay will also be used to evaluate the forms of hTR expressed and to quantitate the amount in tissues. To fully evaluate hTR expression, we will develop antibodies to hTRs using synthetic peptides or fusion proteins. With these tools, we can determine hTR mRNA levels, TR protein, and functional TR as (131I)-T3 binding capacity. Function of the hTRs will be determined by co- transfection of hTR and rGH-CAT plasmid constructs into appropriate cell lines. Co-transfection studies will allow us to compare specifically TR alpha and TR beta control of gene transcription, and to evaluate both TRs with different responsive genes. We will clone and sequence portions of the alpha and beta TR genes in order to characterize the genes and to explain the apparent formation of multiple TR isoforms from one primary transcript. mRNA, Southern Blots of genomic DNA, and immunoprecipitable TR protein will be studied in fibroblasts of patients with thyroid hormone resistance. To identify if a specific isoform of TR is related to their illness,-cDNAs will be prepared from their mRNA for specific TRs, amplified, and sequenced. Our studies will identify the forms of functional hTR present in human tissues, their ability to control transcription of specific genes, and the molecular abnormality of TR present in patients with thyroid hormone resistance."}
{"text": "Biomechanical factors, chondrocyte viability, and inflammation are important factors in the pathogenesis of arthritis. Nitric oxide (NO) synthesis is increased in arthritis and is a mediator in the response to mechanical compression, chondrocyte apoptosis and in the inflammatory response to the catabolic cytokine IL-1. The most direct effect of NO is the suppression of energy production, which in many cell types is then associated with decreased oxygen (02) consumption, decreased matrix synthesis and the control of cell death. NO and 02 competitively bind to cytochrome oxidase, which is involved in oxidative mitochondrial respiration (OXPHOS). However, articular chondrocytes are highly glycolytic due to their avascular and hence hypoxic environment and OXPHOS only accounts for up to 25% of total steady state ATP production. It is suggested OXPHOS may contribute to more ATP production under conditions of tissue stress, such as inflammation or mechanical stress, or in arthritis where the oxygen tension of the synovial fluid, and hence cartilage, is further decreased. A steady supply of ATP is essential for articular chondrocyte remodeling of the matrix as an adaptation to biomechanical or inflammatory stresses, but also for the high energy requirements of apoptosis. NO is suggested to have both a protective and destructive role in cartilage. Little is known concerning the effect of NO and oxygen on ATP production in articular cartilage under physiological or ipathological conditions. The putative protective effect of NO might be determined by the intracellular redox state since NO and 02 compete for binding to cytochrome oxidase of the mitochondria. Our primary hypothesis is that oxygen tension determines whether NO inhibits ATP production, and if sufficient ATP is available to respond to inflammatory or mechanical stress. We propose to test whether oxygen tension l affects the ability of NO to inhibit the aerobic OXPHOS pathway and hence the source of ATP required to respond to the inflammatory or mechanical stress. We then propose to investigate how these factors affect matrix turnover and cell death. Since mechanical stress is protective to IL-1 induced matrix breakdown we shall investigate if this phenomenon is influenced by oxygen tension."}
{"text": "The long range goals of this proposal are to better understand how neural activity regulates opioid gene expression, and to understand how these processes contribute to environmental and drug induced stable/adaptive changes in the nervous system. A better understanding of these processes will help to define the adaptive biochemical changes underlying addiction, withdrawal, and drugseeking behaviors. The primary objectives of this research proposal have not changed. The major focus is still to identify and characterize transcription factors which mediate activity-dependent regulation of proenkephalin gene expression via their interaction with a well characterized second messenger inducible DNA enhancer. Studies will focus on defining components the AP-l/ AP-4, and NF-l/ ZFX nucleoprotein complexes which mediate regulation of proenkephalin transcription. Functional and biochemical interactions with the proenkephalin inducible DNA enhancer and signals transmitted through intracellular signaling pathways will be investigated. Recent findings indicate that JunD activates proenkephalin transcription in a fashion which is completely dependent upon the cyclic-AMP dependent protein kinase (PKA), an effect which can be effectively blocked by JunB. We have increased our emphasis on understanding regulation by JunD/JunB in light of the above findings, and the recent discovery that drugs of abuse such as cocaine, amphetamine, and morphine regulate AP-1 (fos/jun complexes) expression in brain. Together, this analysis will define at the molecular level interactions between intracellular signaling pathways, transcription factors, and the DNA elements which regulate endogenous opioid signaling in response to neurotransmitters, drugs, and synaptic inputs."}
{"text": "The immediate objectives of this research proposal are to determine which cellular components are responsible for the behavior of normal and tumor-virus transformed cells in vitro. The longer term goals of this study are to gain new insight into the mechanisms and to elucidate new parameters of the cellular basis of cancer including neoplastic growth and metastasis. Since cytoplasmic fibers are considered to be the major components responsible for many aspects of the behavior of cultured cells, we are emphasizing the study of their structure, function and inter-relationships. Light and electron microscopic studies have revealed the presence of three such fibers in several lines of cultured cells, which have been designated as microtubules, microfilaments and filaments. The localization of all three fibrous components in cells in the various states of physiological activity which occur during the cell cycle, suggests that they play major roles in attachment and spreading, division and locomotion of cultured cells. Furthermore, preliminary observations implicate these fibers in aspects of the social behavior exhibited by cells, including contact-inhibition. The correlation of function with structure is achieved through the coordinated use of light and electron optical techniques. In addition, various drugs such as colchicine, cyclic AMP and Cytochalasin B are used to alter cells in a manner which should yield new information concerning the structures involved in cell behavior. More direct approaches to determining function include the use of heavy meromyosin binding to microfilaments, and fluorescent and ferritin-labelled antibodies made against the major structural and contractile proteins of mammalian cells."}
{"text": "The long-term goal of the research project is to determine the molecular mechanism of the maturation-inducing steroid (MIS) receptor in the regulation of oocyte maturation in vertebrates. Little is known about the physiochemical characteristics of this MIS receptor. Recently, we have isolated and purified the MIS receptor from the plasma membrane of Xenopus laevis oocytes and also produced a polyclonal antiserum against the receptor. The objective of this application is to clone and sequence the MIS receptor and to examine its subcellular localization in Xenopus oocyte. Since the MIS in Xenopus is progesterone, our specific aim 1 of the research is to clone and sequence the cDNA encoding the progesterone receptor. Purified receptor protein will be microsequenced to obtain internal amino acid sequences to design the oligonucleotide probes, cDNA libraries will be prepared from oocytes and screened for positive clones. The cDNA of the receptor will be sequenced and analyzed. Our specific aim 2 is to determine the maturation-inducing activity of the progesterone receptor. The progesterone receptor mRNA will be generated by in vitro transcription of the cDNA and microinjected into immature oocytes. The oocytes will be cultured to allow for the synthesis of the progesterone receptor. Maturation of the oocytes in response to progesterone stimulation will be assayed. Our specific aim 3 is to examine the subcellular localization of the progesterone receptor mRNA and the protein in Xenopus oocytes. Labeled RNA probes will be used for in situ hybridization to determine the distribution of mRNA and the polyclonal antiserum will be used in immunocytochemical localization of the receptor protein. It is expected that the findings will contribute to a better understanding of the mechanism of the MIS receptor in the control of gamete development. The new knowledge will have a great potential for clinic applications such as induction of oocyte maturation in anovulatory patients and development of contraceptive agents for inhibition of oocyte maturation and ovulation in humans and animals."}
{"text": "ABSTRACT: Chronic sequelae of long-term HIV infection and treatment including heart, lung, blood, and sleep (HLBS) co-morbidities are increasingly important causes of morbidity and mortality. Latent HIV and maladaptive inflammation also persist despite successful ART. Therefore, we propose a new training program in HIV-related research, the Pittsburgh HIV Mentored Training for Investigation of Co-morbidities and Cure (HIV MeTrICC), led by Drs. Alison Morris, Dr. John Mellors, and Solomon Ofori-Acquah. They have an outstanding training record for PhDs and MDs, and their research experience spans the spectrum from basic to translational to clinical with expertise in lung, blood, and vascular co-morbidities and HIV cure. HIV MeTrICC will be an interdisciplinary, intensive mentored research training and career development experience focused around two interrelated training tracks ? HLBS Co-morbidities and HIV Cure with the following objectives: 1) To provide intensive mentoring in HIV-associated HLBS Co-morbidities and HIV Cure. Our team of established Mentors will offer unique multidisciplinary mentoring and career guidance to Scholars in the conduct of HIV research projects. Scholars will choose Mentors from the MeTrICC Co-morbidities and HIV Cure tracks. Tracks will be highly interactive. 2) To provide didactic training to foster progression to an independent career in HIV-related research. Formal training will encompass a broad spectrum of research methodologies, technologies, and analytical approaches to basic, clinical, and translational HIV research. Scholars with varying levels of prior research experience will be supported by Individual Development Plans geared to their needs. 3) To provide opportunities for training in Inner-city HIV and International HIV settings. Scholars will be able to choose from two different focus areas if relevant to their career plan. For the Inner-city HIV focus, we partner with the University of Maryland and the NIH to provide experience with the District of Columbia Partnership for AIDS Progress, the nation?s largest urban HIV cohort study. For our International HIV focus, we utilize several sites in Africa with ongoing basic, clinical, and translational research. 4) To provide access to large, multi-center HIV studies: Scholars will have access to large cohort studies including the Multicenter AIDS Cohort Study, the Women?s Interagency HIV Study, the Strategic Timing of Antiretroviral Therapy pulmonary substudy, the Pittsburgh HIV Lung Research Cohort, and the Pharmacokinetic and Clinical Observations in People over 50 sleep Substudy. 5) To support scholars in preparation of an independent research proposal: The program will ensure that Scholars rapidly develop grant-writing skills and submit competitive grant applications to help them secure HIV research funding. We will support five post-doctoral Scholars (MDs and PhDs) for a period of 2 to 3 years based on initial level of experience (i.e. post-docs vs junior faculty). The success of the MeTrICC program will be measured by concrete milestones such first author manuscripts, grant funding, and transition to faculty with the ultimate goal to recruit a new generation of researchers to HIV and HLBS complications."}
{"text": "The N.C.I. Biologic Response Modifer Program has undertaken poly ICLC as a drug to study intensively. In preclinical testing a variety of augmentary actions on several immune functions have been noted, some of which do not appear to be interferon mediated. A Phase I study of biological response modifications in humans is under way in two institutions--N.C.I. and the Naval Hospital in Portsmouth, VA. Upon completion a Phase II study is planned. In addition the N.C.I. has agreed to undertake the monitoring of all cancer protocols with poly ICLC. Six clinical therapy protocols are currently ongoing, involving neuroblastoma, breast cancer, renal cell carcinoma, malignant melanoma, dysimmune peripheral paralytic neuropathies, and multiple sclerosis. There were no beneficial effects in neuroblastoma and breast cancer. There was a partial response in 2 of 5 patients with renal cell carcinoma. It is too early to evaluate, even in a preliminary way the malignant melanoma study. In 14 patients with peripheral neuropathy, there were at least 9 with moderate to marked improvement. In M.S. there are indications of positive effects, but it is too early to have much confidence in the results."}
{"text": "Ribonucleotide reductase (RR) is an important therapeutic target. Therefore, understanding the function and regulation of the RR gene, and development of new RR inhibitors may have a direct impact on cancer treatment. Our long-term goal is to better understand the molecular mechanism of RR gene regulation, and the role of RR in chemotherapeutic drug resistance. We have shown that gemcitabine resistant as well as hydroxyurea resistant clones both overexpress the RR small subunit, hRRM2, mRNA and RR protein. Further studies confirmed that these drug-resistant tumor cell clones are associated with nucleus factor Y (NFY) binding to the RR promoter. Therefore, in Specific Aim I we will investigate whether hRRM2 overexpression in drug resistant cells is directed through transcription factor regulation o: the hRRM2 promoter. Promoter binding of NPY and a new transcription factor, band c, will be examined by gel-shift analysis and specificity will be confirmed by affinity chromatography. The functional consequences of NFY and band c in the regulation of hRRM2 expression will be examined in transfection assays. Since p53R2 is an integral part of the RR gene family, we are also intrigued by the possibility that interactions between different subunits of RR are important. In Specific Aim II, we will examine the interactions of p53R2, hRRM2, and hRRM1 (the RR large subunit), identify interacting domains, and examine the biochemical characteristics of such subunit interactions. A mammalian expression system will be employed to examine in vitro recombination of each subunit. Deletion analysis and mutagenesis will be performed to examine interacting domains and results will be confirmed by affinity chromatography. The plasmid vector containing p53R2 will be transfected into cloned drug-resistant cells for study in vivo. Once we clarify the interaction among RR subunits, we will explore the role of p53 in RR regulation. Our immunoprecipitation results have shown that both p53R2 and hRRM2 can bind to wild-type p53. Our hypothesis is that RR subunit composition may be regulated by p53. In specific aim ifi, we will confirm the protein interaction between p53R2 and wild-type p53 using a yeast two hybrid system. These findings will be confirmed by immunoprecipitation and Western blotting. The effect o wild-type p53 on RR activity and subunit composition will also be examined by transfecting wild-type p53 into cell lines expressing mutant p53. These experiments will help to confirm the interactions between p53R2, hRRM2, and p53. Finally, in Specific Aim IV, we will investigate the molecular pharmacology of a new RR inhibitor, HSC 1 that inhibits human cancer cells through an interaction with hRRM2. Taken together, our proposed studies will provide an integrated evaluation of the molecular control of RR-mediated chemotherapeutic drug resistance."}
{"text": "The CCR-Flow Core is an indispensable resource for NCI and contractor investigators at the Frdereick National Lab for Cancer research. Seventy investigators from 35 different laboratories have used the expertise of the flow core staff and the instrumentation available and maintained by the staff. The projects investigators have been studying include tolerization of the high-avidity T cells in the tumor microenvironment. Another study was of the critical role of tumor necrosis factor in stabilizing the CD4+Foxp3+ regulatory T cell. Regulation of the NF-B signaling pathway of pancreatic cancer stem cells was also studied. A project looking at the pivotal role of IKKalpha in spontaneous lung squamous carcinomas was also done. The influence of HLA-C expression on HIV control was also a project of study. Studies have been done on the role of DC cells in the acute infectious inflammatory process. The flow core staff has trained eighteen investigators in the first three quarters of the year to run their own samples. The instruments are used daily by the trained investigators often into the night and on the weekends. This leaves more time for the flow core staff to sort approximately 450 samples and analyze about 10419 samples in the first three quarters of this year. Without the investigators acquiring the data and analyzing their own samples, the government would have to hire full time experienced individuals to perform the same volume of work. At least 15 papers have been published, 8 papers are in press and 14 papers have been submitted in the past year using data obtained in the flow core. The future goals include keeping up with cutting-edge technology in the flow lab by expanding the pool of investigators trained to run and analyze their own samples to all of CCR in Frederick thereby expanding the use, productivity and quality of the science generated using the core's instrumentation and expertise. It is necessary to up-grade all of the instruments so that laser and fluorochrome capabilities are more interchangeable so that investigators have the flexability to use more than one instrument. This would prevent loss of data because the instrument needed was in use by another investigator or down because of needed repairs. It is critical to have this flexability in analysis of samples as well as for sorting of cells. We would also like to stay a cutting edge facility which would require at least one analyzer to be up-graded to analyze at least 28 parameters at once."}
{"text": "It is now recognized that T cells required 2 signals for induction of immune responses. Interruption of costimulatory signals by blocking CD28 or its two ligands B7-1 and B7-2 can induce transplantation tolerance and prevent or reverse autoimmunity in animal models. Similar data exists for blocking the CD40-ligand:CD40 interaction, a potential additional costimulatory pathway. The overall goal of this project is to understand the role of costimulation in graft rejection and determine the strategies and mechanisms by which blockade of costimulatory signals induces transplantation tolerance. Our studies of induction of transplant tolerance through blocking CD28 or CD40L have shown a complex series of relationships between their ligand, B7- 1, B7-2 and CD40 (all expressed on antigen-presenting cells), and the induction of rejection or tolerance. Specific aim #1 will focus on these questions, using selective blocking reagents and knockout animals in a murine cardiac allograft model to: examine the separate roles of B7-1 and B7-2, test the hypothesis (based on preliminary data) that B7-1 can, in select circumstances, promote tolerance interactions with CTLA4, and examine the cellular and molecular regulation B7-1 through CD40. Our data also show that delivery of additional donor antigen, in the form of splenocytes, at the time of transplantation, is required to synergize with costimulatory blockade to induce tolerance. In the absence of donor antigen, animals are transiently immunosuppressed but undergo chronic rejection. Specific aim #2 will identify the cell and MHC type which is required in the donor- transfusion to induce tolerance. We will then test the hypothesis that immunogenic allopeptides can substitute for intact cells to induce tolerance. Lastly, using the allopeptides, we will test the postulate that chronic rejection is linked to failure to tolerize to indirect allorecognition. Finally, based on data that costimulation through CD28 induces the cell survival gene bcl-x which is required for lymphocyte survival, in specific aim #3 we will use bcl-x transgenic mice to test the hypothesis that immunosuppression through CD28- blockade operates by induction of cell death and that bcl-x transgenic mice will be resistant to tolerance induction through this maneuver. These studies will have important implications for the development of implementation of therapies for transplantation and autoimmunity."}
{"text": "This is a Phase II randomized blind, parallel group, multicenter, therapeutic equivalence trial. The Primary Immune Deficiency (PID) patients will be randomly assigned to one of the two treatment groups: experimental IGIV (IGIV-C, 10%) or control IGIV (IGIV-S/D, 10%). A total of 152 patients will be enrolled with a confirmed diagnosis of Primary Immune Deficiency and who are on stable IVUG replacement therapy for at least three months prior to entry into the trial. Nine infusions, with one of the study drugs, will be every three to four weeks for nine months. Patients will remain on the fixed individual replacement dose and dose interval established prior to entry into the study."}
{"text": "PROJECT SUMMARY Emphysema is an important pathologic finding in Chronic Obstructive Lung Disease (COPD), a leading cause of death in the United States and worldwide. Emphysema is characterized by increased DNA damage and the consequences of failed DNA repair: apoptosis, cellular senescence, and inflammation. Cigarette smoke (CS) is the most important etiology for DNA damage in emphysema. Yet, emphysema does not occur universally among smokers and there is little understanding of protective mechanisms that underlie this heterogeneity. Therefore, identifying cellular responses to CS that promote DNA repair may lead to new therapies for this disease. This proposal seeks to understand the role of Macrophage Migration Inhibitory Factor (MIF) in promoting DNA repair and antagonizing the development of emphysema. MIF is a cytokine secreted in response to CS and a critical regulator of multiple signaling pathways. We previously demonstrated that circulating MIF is decreased in patients with COPD, and genetic deletion of murine MIF results in increased susceptibility to CS mediated airspace enlargement. The endothelial cells of MIF-KO mice are particularly susceptible to CS-mediated DNA damage and consequent apoptosis and cellular senescence. The source of protective MIF is unknown. Our preliminary data suggests that MIF promotes DNA repair in endothelial cells exposed to CS extract and that endothelial cells may be an importance source of protective MIF. Based on these findings, we propose the following hypotheses and specific aims: Hypotheses 1. MIF, via its receptor CD74, promotes HR and NHEJ following CS-mediated DNA damage. 2. MIF, via CD74, protects lung endothelial cells from CS-mediated cellular senescence and apoptosis by promoting BRCA1 gene expression. 3. Deletion of MIF in the endothelium is necessary and sufficient to increase murine susceptibility to DNA damage and emphysema. Aims 1. Define the DNA repair mechanism(s) promoted by MIF in lung endothelial cells. 2. Determine the role of BRCA1 in mediating MIF's protective effect. 3. Define the protective role of endothelial-derived MIF in CS-mediated emphysema. Experimental Approach To address Aim 1, we will use primary mouse and human endothelial cells and expose them to CS extract, followed by a recovery period to observe DNA repair. We will characterize DNA damage and repair using single- cell gel electrophoresis, imaging-flow cytometry (Amnis) and host cell reactivation assays. We will use silencing RNA and chemical inhibitors of MIF-CD74 binding to determine if MIF promotes DNA repair via its receptor CD74. In Aim 2, we will characterize the effects of CS on BRCA1 gene expression and protein in primary lung endothelial cells and paraffin-embedded, de-identified human lung tissue samples. We will use chromatin precipitation assays to determine the mechanism via which MIF promotes BRCA1 gene expression, and we will measure consequences of CS extract in endothelial cells, such as apoptosis and cellular senescence, to determine if MIF protects against these consequences in a BRCA1-dependent manner. We will use silencing RNA and chemical inhibitors of MIF-CD74 binding to determine if MIF promotes BRCA1 gene expression in a CD74-dependent manner. In Aim 3, we will assess the development of emphysema in mice exposed to CS with an endothelial specific deletion of MIF. We will also determine if an endothelial-specific lentiviral vector expressing MIF can rescue the susceptibility of MIF-KO mice to CS. This work will provide us with insight into the pathogenesis of emphysema in addition to the novel regulatory functions of MIF in promoting DNA repair. This grant proposes a research mentorship program at Yale University under the primary sponsorship of Dr. Richard Bucala, an expert in immunology and MIF biology, and Dr. Patty Lee, an expert in oxidant-mediated vascular injury and repair in the lungs as co-mentor. We have also enlisted the expertise of multiple Yale investigators, including two experts in DNA repair research, Dr. Joann Sweasy and Dr. Peter Glazer, Dr. Naftali Kaminski, who is an expert on translational pulmonary research, and Dr. Veronique Neumeister, who has significant expertise in immunohistology. The advisory committee members will provide scientific and career counseling, and the proposed career and research program as outlined will provide an extraordinary scientific environment wherein Dr. Sauler can launch his future independent career as a physician- scientist."}
{"text": "A trial to determine if the relatively new procedure called transjugular intrahepatic portosystemic shunt (TIPS) can treat fluid build up in patients who have had cirrhosis and increased pressure in the veins supplying blood to the liver. Cirrhosis effects 3.6 out of every 100 North American adults and is responsible for the loss of 10.6 million work days and 32,000 deaths annually. Fluid retention is the most common complication of cirrhosis and developes in over 60% of patients. There is a need to develop a better treatment for this problem. Fluid build up is a common complication of cirrhosis of the liver. The worsening of symptoms is often complicated by the development of resistence to diuretics (water pills), or complications limiting the use of diuretics. This fluid retention, which does not respond to treatment, is associated with discomfort, difficulty breathing, malnutrition, liver and kidney failure, and death or increased risk of death following liver transplantation. Paracentesis (repeated removal of large amounts of fluid by piercing the organ) or total removal of the fluid along with intravenous administration of albumin are the most commonly used therapies for this problem. Neither of these therapies addresses the problem causing the water retention and may cause the recurrence of retention. Neither therapy has been shown to improve the survival of patients. TIPS is the insertion of a by-pass into the vein system of the liver in order to help the flow of blood and prevent the build up of fluid in the abdomen and esophagus. TIPS avoids the risk of general anesthesia and major surgery and has been shown to resolve the fluid retention problem in most patients. Also, in some patients, the nutritional state and liver function improve, thereby making transplantation unnecessary. But, because all published studies have been done on a small number of patients, this study will directly compare TIPS with the current standard of care, the puncturing of organs. 72 patients with severe fluid retention will be seen at the General Clinical Research Center. There will be two groups with two different combinations of therapy. The patients will be seen 1,2,and 4 weeks after insertion of the shunt, and monthly for 12 months. Then they'll be seen every three months. Special diets will be assigned and lab tests will be done at every visit."}
{"text": "In the past year, we have continued our investigations on some of the factors that control the proliferation of cells, specifically how the microinjection of monoclonal antibodies may affect cell proliferation. In these experiments, we microinjected monoclonal antibodies into the nuclei of quiescent cells to determine their function. We have been able to show that a monoclonal antibody against a p53 protein, a transformation related protein, inhibits serum-stimulated DNA synthesis. Interestingly enough, the inhibition of serum-stimulated DNA synthesis occurs only if the monoclonal antibody against the p53 protein is microinjected in the first 2 hours after serum stimulation. Once the cells have gone for more than 2 hours after serum stimulation, they are no longer inhibited by the microinjection of monoclonal antibodies against the p53 protein. Control monoclones and other antibodies have been microinjected without any effect whatsoever on serum stimulated DNA synthesis. We have also investigated how the microinjection of monoclonal antibodies against the SV40 T antigen may affect the function of the SV40 T antigen. In these experiments, a number of deletion mutants of the SV40 genome were microinjected into quiescent cells, together with monoclonal antibodies. The deletion mutants were chosen so that different domains of the SV40 T antigen coding gene were represented. The monoclonal antibodies were selected from a battery of monoclonal antibodies which included some antibodies that recognized the amino-terminal third of the T antigen and others that, instead, recognized the carboxy-terminal third. Results clearly indicated that monoclonal antibodies against the SV40 T antigen inhibit the ability of SV40 T antigen to induce cell DNA synthesis in quiescent mammalian cells and also that the microinjection of a monoclonal antibody has no effect unless this antibody reacts with a specific antigen. In other experiments, we have looked at the effect that methylation may have on the expression of microinjected genes. As already reported, we have reconfirmed that the methylation of 5' flanking sequences may abolish the transcription of microinjected genes, whereas methylation of genes along their structural coding sequences has no effect whatsoever."}
{"text": "Uterine leiomyomata (UL) are diagnosed in 25-30% of reproductive-aged women, resulting in considerable gynecologic morbidity and billions in U.S. health care costs annually. African Americans have 2-3 times the UL incidence of Whites and greater disease severity. Sex steroid hormones are involved in the etiology of UL. Given that endocrine disrupting chemicals (EDCs) can alter the functioning of hormones, we propose to evaluate UL risk in relation to 3 classes of EDCs: phthalates, polychlorinated biphenyls (PCBs), and phenols, specifically bisphenol A (BPA). Our choice of chemicals is informed by their high exposure prevalence in humans and by in vitro, animal, and human data documenting their effects on reproductive hormones and processes that could influence UL risk. Existing studies of EDCs and UL are limited by small sample size, uncertain temporality due to retrospective design, and suboptimal measurement of EDCs and UL. This study will use data and biospecimens from the Study of Environment Lifestyle and Fibroids (SELF), a NIEHS prospective cohort study of 1,300 African American women aged 23-34 and free of UL at baseline (2010-2012). Questionnaire data were collected at baseline and subjects are being followed for 5 years. Every 20 months, blood and urine are collected, and ultrasounds are performed by trained sonographers to detect UL. We request support for the laboratory and statistical analysis of EDCs in relation to UL incidence. We propose a cost-effective case-cohort study design that includes 400 incident UL cases and 400 controls. We will measure urinary phthalate metabolites, serum PCBs, and urinary BPA, characterize exposures according to UL risk factors, and determine the associations of these chemicals with UL incidence and tumor characteristics. Non-persistent chemicals (phthalates, BPA) will be measured at baseline, 20 months, and 40 months. Persistent chemicals (PCBs) will be measured at baseline only. SELF provides a unique opportunity to assess the association of common EDCs with UL risk. This project has many strengths that overcome the limitations of prior studies: large sample size, prospective data collection, state-of-the-art EDC measurement, serial ultrasound screening for UL, analysis of repeated EDC measures, innovative statistical approaches for evaluating EDC mixtures, and control for multiple confounders. Pilot data show large exposure variability, providing excellent power to test the proposed hypotheses. This study of African Americans, a high-risk population for EDC exposure and UL, will provide informative data on the effects of widespread pollutants on UL, seek explanations for the racial disparity in UL incidence, and provide critical data to the public, scientific community, and policy makers."}
{"text": "The overall objective of this research is to understand the mechanism for the enzymatic assembly of cholesterol, the control of this process, and the relationship of this process to lipoprotein assembly in the liver. Specific objectives include the isolation and purification of Sterol Carrier Proteins and delineation of the relationship of these substances to lipoprotein assembly. Others objectives include the determination of chemical structures of intermediates in cholesterol biosynthesis, as well as, the mechanism of inhibition for hypocholesterolemic drugs."}
{"text": "In collaboration with Todd Golub at the Broad Institute, we have performed a luminex assay to quantitate tyrosine phosphorylation in tyrosine kinases. Active tyrosine kinases are also generally associated with increased overall tyrosine phosphorylation. Our assay captured more than 150 tyrosine kinases in a multiplex assay and detected the level of tyrosine phosphorylation in all these kinases using a pan-phosphotyrosine antibody (4G10). We used 150 human lung adenocarcinoma tumor tissue and corresponding adjacent normal lung tissue to profile tyrosine phosphorylation of these tyrosine kinases. We identified around 30 specific tyrosine kinases differentially phosphorylated in tumor tissue compared to adjacent normal lung. Examples of such potentially active tyrosine kinases are NTRK1, EPHA2, DDR. We then completed a nanostring assay in collaboration with Dr. Marc Ladanyi at MSKCC to measure transcript expression of majority of tyrosine kinases in a multiplex assay. Interestingly, as we had predicted, expression of several tyrosine kinases do not change at least at the level of trasnscription, however there is significant change in tyrosine phosphorylation, which is generally a surrogate of activation. In collaboration with Dr. Paul Meltzer in the NCI intramural program, we have sequenced around 1300 cancer-related genes, including the tyrosine kinases used in our luminex assay to identify specific mutations in a targeted manner. We are currently analyzing the sequencing data to identify somatic changes in the genes selected. The somatic mutation data is being correlated with the luminex data to identify possible somatic changes in kinases resulting in increased activity."}
{"text": "Osteogenesis imperfecta is a heritable disease of connective tissue that results from a wide variety of mutations of type I collagen. This grant outlines a series of protocols designed to define these abnormalities at the molecular level. It is based on the assumption that it is easier to define the mutations in collagen by studying the mRNA rather than its very complex gene. Fibroblasts derived from patients with 0I will be used as a source of RNA. Structural mutation of the alpha 1 or alpha 2 collegen mRNA will be localized using a series of end-labeled S1 probes that encompass the entire mRNA. The probes will be derived from single-stranded cDNAs cloned in M-13. The exact mutation will be defined by dideoxy sequencing of an extended cDNA initiated from a synthetic oligonucleotide primer complementary to a region 3' to the region suggested of having the abnormality as dictated by the S-1 protection experiments. Another oligo-nucleotide coding for the abnormal sequence will be constructed and used as a mutation specific hybridization probe to precisely localize the mutation to the gene. Mutations which alter the steady state level of collagen mRNA will be tested for an error of RNA processing using a novel application of the M-13 derived hybridization probe. This will involve an S-1 protection protocol to detect an accumulation of a specific unspliced mRNA, measurement of half-life of a normal and mutant cytoplasmic mRNA and solution hybridization of radiolabeled RNA from in vitro transcribed muclei. The methods that are developed in this grant will have general applicability to a wide variety of mutations involving type I collagen. The mutation-specific oligonucleotides provide the means to rapidly detect a subtle mutation in genomic DNA. It has the potential to become a very powerful adjuvant for the clinical geneticist who must counsel a family with a connective tissue disorder."}
{"text": "This project will address the therapy of mesothelin expressing tumors by developing and testing engineered T cells with potent antitumor cytotoxicity. Mesothelin is a tumor-associated antigen that is frequently over expressed on mesothelioma, non-small cell lung cancer, pancreatic and ovarian cancers. The strategy to be used is the \"T-body\" approach, which employs genetically reprogrammed, patient-derived lymphocytes transfected with a novel chimeric receptor that contains combinations of the signal transduction domains of 4-1BB (CD137), CD28, and CD3zeta as well as anti-mesothelin scFv (anti-meso-CD28-41BB-zeta). The central hypothesis to be tested is that previous trials of adoptive therapy for cancer have used insufficient numbers of cytotoxic T lymphocytes (CTL) that have shown inadequate engraftment, persistence and effector function to self antigens. Presently, we are the only laboratory in the world that is actively testing lentiviral modified T cells in the clinic, and in that trial we have demonstrated safety and prolonged lentiviral gene transfer. The following three specific aims will test the hypothesis that engineered human T cells expressing an anti-mesothelin-CD28-41BB-zeta chimeric receptor will have potent antitumor activity in vitro and in vivo by: (1) developing and optimizing the anti-meso scFv vector. The avidity and the cytosolic signaling modules will be optimized to obtain highly efficient lentiviral vectors that retarget T cells to specifically kill tumor cells that express mesothelin at low effector to target ratios in vitro;(2) carrying out in vitro experiments to optimize the effector functions of anti-mesothelin scFv CD28-41BB-zeta T bodies. Experiments will determine optimal conditions for redirected T cell serial killing, cytokine production and proliferation, and compare this to natural MHC restricted CTLs;and (3) performing in vivo experiments in immunodeficient NOD/SCID/beta2null mice xenografted with human tumors that express mesothelin. These experiments will test the hypothesis that vectors with high affinity scFv receptors and 4-1BB and CD28 signaling modules will have the most potent anti-tumor effects. Finally, the engraftment, persistence and antitumor effects of chimeric T cells given by intravenous and intraperitoneal routes will be compared using bioluminescence imaging. In summary, an outstanding team of basic and translational scientists has been assembled that will develop and test a universal T cell receptor to target some of the most common and drug resistant tumors. Lay Description. A common reason for failure of immunotherapy of epithelial tumors is that the immune system does not generate sufficient numbers of T cells to eradicate the tumor cells. It is now possible to use lentiviral vector technology to engineer T cells with potent and specific antitumor effects. This project will evaluate engineered T cells that target mesothelin that is overexpressed on both uncommon tumors such as mesothelioma and a variety of commonly lethal tumors including pancreatic, ovarian and non-small cell lung carcinoma."}
{"text": "Logistical support for BEE/Council Working Group on NHLBI Program Project Grant Mechanism"}
{"text": "The isolated ventricular cell must retain the physiologic characteristics of its native tissue if it is to be useful in studying excitation-contraction coupling. A wide variability in isolated cell size, rest sarcomere length, amount and velocity of sarcomere shortening and action potential has been observed. This proposal seeks to understand this variability by correlating the function of individual cells, as examined by sarcomere motion and rest and action potentials, with the structure of the same cell. Cells will be isolated from the right and left ventricle of the rabbit and rat using enzymatic and mild mechanical dispersion. Rabbit myocardiuim will be studied because it has physiologic properties much more like those of other mammals than those of the rat. The rat will be studied because it has such strikingly different physiologic properties and has been used in many isolated cell investigations. The experiments will make use of the prominent effects induced by altering the rate and pattern of stimulation on contractility and the action potential and the characteristic changes in these effects induced by inotropic agents. Timed changes in extracellular ionic concentrations during rest and contraction will take advantage of the short diffusion distance. The proposal will provide a detailed examination of the physiological and ultrastructural properties of the same cell. This will enable us to assess whether intrinsic in situ differences in cells or the effects of the isolation procedure are the basis of cell-to-cell variability. Sarcomere shortening and action potential characteristics will be related to cell shape, size and ventricular site of origin. Species differences will allow structrue-function correlations to be made. The changes in myocardial performance produced by altering the rate and pattern of stimulation have been used in multicellular preparations to study contractility and to characterize the effects of disease. By producing a detailed quantitative description of the changes in sarcomere shortening velocity induced by altering the pattern of stimulation, this investigation will provide a basis at the cellular level for the force-interval relationship in the intact heart. By assessing the importance of extracellular calcium concentration at different times during activation and rest, this proposal will provide new data for testing models of excitation-contraction coupling. This proposal will provide a basis for future isolated cell investigations of disease-induced derangements in excitation-contraction coupling."}
{"text": "The University of Montana School of Pharmacy and Allied Health Sciences has a long standing history and commitment to the recruitment and training of minority students (principally Native American Indians) for degrees in pharmacy and physical therapy. The recently obtained Native American Center of Excellence (NACOE) grant builds on strength from funding obtained for the HHS-Health Careers Opportunity Program (HCOP), the NIH-Bridges to Baccalaureate Program (BRIDGES), and the NSF-EPSCoR Program to provide both the environment and the infrastructure necessary to offer degree training for under-represented minorities in pharmacy (Pharm.D.), physical therapy (M.S. and D.P.T.), and more recently in graduate education (M.S., Ph.D.). Research infrastructure has dramatically increased in the School's Department of Pharmaceutical Sciences in the past ten years to the point where the School is ranked 1lth of 85 schools/Colleges of Pharmacy in NIH funding per Ph.D. faculty. The Department has created two state-approved research centers and offers two M.S. and two Ph.D. programs. The primary goal of the Endowment Fund Program is to add at least two tenure track minority faculty through training and recruitment efforts, while perpetuating recent success in the recruitment, retention, and training of minority students in Pharmacy, Physical Therapy, and Graduate programs. Focus on minority health disparities research will continue to be expanded. These goals will be accomplished as follows: 1) creating new tenure track faculty lines for minority faculty (preferably Native American Indians), 2) Enhancing opportunities for minority students who earn the Pharm.D. or D.P.T. degree to obtain postdoctoral fellowship training in clinically relevant areas, 3) Enhancement of opportunities for minority students to pursue graduate training leading to the Ph.D. degree. 4) Perpetuation of a strong program to recruit and train undergraduate minority students, 5) Use of endowment income to leverage additional institutional/state and federal support, and 6) Progressive growth of the endowment corpus. Priorities for use of the endowment income are A) Recruitment and training of two minority PhD, students and one minority post Pharm.D. Fellow (Years 1-3) and B) Complete training and/or recruitment of two minority faculty with priority given to Native American Faculty candidates, particularly those involved in health disparities research (Years 3-5). [unreadable] [unreadable]"}
{"text": "Chronic exposure to high concentrations of manganese (Mn) results in adverse neurological health effects commonly referred to as manganism. Manganese neurotoxicity is a significant toxicological problem resulting from the use of manganese as a gasoline additive, and in welding, metal industries, pesticide manufacturing, pharmaceutical preparations, infant food formulations, and battery production. Manganese predominantly accumulates in the basal ganglia structures and causes mitochondrial dysfunction, oxidative stress, and apoptosis. However, cellular and molecular mechanisms underlying manganese neurotoxicity are poorly understood. We have been studying mitochondrial-dependent apoptotic signaling in manganese neurotoxicity and found that protein kinase C-delta (PKCd), a member of the novel PKC isoform family, is persistently activated by caspase-3 to promote apoptosis during manganese exposure. While studying the apoptotic signaling pathway in cell culture models of manganese neurotoxicity, we also unexpectedly identified that PKCd is upregulated both in protein and mRNA levels during manganese treatment. Further analysis of the PKCd promoter revealed that two key transcription factors, NFkB and SP1, regulate PKCd gene expression. Thus, our competitive renewal proposal aims to systematically characterize this gene-environment interaction by studying novel molecular mechanisms underlying manganese-induced upregulation of the proapoptotic PKCd gene. We propose to complete the study by pursuing the following specific aims: i) To characterize upregulation of an oxidative stress-sensitive proapoptotic kinase PKCd in mouse primary neuronal cultures and animal models following manganese exposure, ii) To investigate molecular mechanisms of manganese-induced upregulation of PKCd by examining its transcriptional regulation of a PKCd promoter, and iii) To further define the functional role of NFkB and SP1-dependent PKCd upregulation in manganese-induced neuronal damage during chronic manganese exposure. Cellular, molecular, and neurochemical approaches in relevant cell cultures and animal models of manganese neurotoxicity will be used. We anticipate that the proposed study will provide comprehensive information about how environmental exposure to manganese can alter the expression of the key proapoptotic gene PKCd to augment manganese neurotoxicity, and this knowledge may advance the development of novel translational approaches for the treatment of manganese neurotoxicity. PUBLIC HEALTH RELEVANCE: Environmental exposure to excessive manganese impairs basal ganglia function, resulting in a neurological disorder relatively similar to Parkinsonism commonly known as Manganism. Manganese exposure is of serious concern due to the increased incidences of extrapyramidal neurological symptoms among miners and industrial workers, including welders. Manganese predominantly accumulates in the basal ganglia structures and causes mitochondrial dysfunction, oxidative stress, and apoptosis. However, cellular and molecular mechanisms underlying manganese neurotoxicity are poorly understood. The proposed study will elucidate the neurotoxic mechanisms underlying manganese induced upregulation of a proapoptotic kinase PKCd and its functional relevance to manganese neurotoxicity."}
{"text": "The Administrative Core provides the infrastructure and centralized support that will enable all four research projects to be productive and collaborative. This will be achieved by the following aims: Aim 1: Administration and fiscal management. The administrative core will provide oversight of administrative and budget matters including human resources, fiscal management, conflict resolution, institutional regulatory compliance (i.e. IRBs), and all communication with NIH. Aim 2: Communication management. The administrative core will schedule and facilitate monthly project leader and P01 staff meetings for dissemination of scientific findings, organize the annual scientific advisory panel visit and the executive committee meetings, and foster a collaborative scientific environment (advertising seminars, work-in-progress meetings, attendance at meetings to disseminate research findings within the scientific community, etc.). Aim 3: Scientific oversight. The administrative core will ensure scientific integrity such as data sharing plans and data management and assurances. The administrative core will also provide support for laboratory equipment. Aim 4: Statistical data analysis support. The Core will provide statistical support and oversight that include recommendations for experimental design to achieve desired statistical power, power calculations to determine group sizes, recommendations for data transformations to achieve forms appropriate for statistical analysis and advice regarding appropriate statistical tests to employ during the data analysis phase."}
{"text": "Macrophages are distributed throughout the body where they are poised to detect pathogens and to subsequently alert the immune system to the presence of infection through the production of inflammatory mediators. Inflammatory mediators are critical for pathogen control, but if produced excessively, can result in inflammatory diseases. My long-term goal is to understand the regulation of the production of inflammatory mediators by macrophages during infection. I have identified a novel type of negative regulation of the inflammatory response to pathogens through the TREM-2/DAP12 receptor complex. The specific aims of this proposal seek to define the mechanism of TREM-2 and DAP12 inhibition of inflammatory signaling in macrophages both in vivo and in vitro. The specific aims are: 1. To determine the ligand requirements for TREM-2 and DAP12 inhibition of inflammatory responses in macrophages. I will test the hypothesis that low avidity signals through DAP12 result in inhibition of inflammatory responses, whereas high avidity signals result in activation of inflammatory responses. 2. To determine the mechanism by which TREM-2 and DAP12 inhibit inflammatory signaling in macrophages. These experiments will define how DAP12 signaling results in inhibition of ERK phosphorylation and activation leading to dampening of inflammatory cytokine production in macrophages. 3. To determine why DAP12-deficient mice have enhanced innate immune responses in vivo. I will determine the mechanism for the increased innate response to Listeria monocytogenes infection in DAP12- deficient mice. I also will investigate what myeloid populations are inhibited by DAP12 signaling and whether this correlates with TREM-2 and TREM-2 ligand expression. Results from these studies should provide novel insights into how the early innate immune response to infectious pathogens is controlled. This is a topic of significance in the development of vaccines to prevent infection and immunomodulatory drugs to treat infections with pathogens relevant to biodefense, including Listeria monocytogenes, an Ml AID category B priority pathogen addressed in this proposal. [unreadable] [unreadable] [unreadable]"}
{"text": "Despite a wide array of HIV prevention approaches and related research, there is a decade-long trend of 40,000 new HIV diagnoses per year in the U.S. (CDC, 2004; Copenhaver & Fisher, in press). Injection drug users (IDUs) remain a target population as they represent a significant vector for the transmission of new HIV infections (Avants et al., 2004; Margolin et al., 2003), which occur through preventable drug- and sex- related HIV risk behaviors. A number of evidence-based HIV risk reduction interventions are now widely available as complete intervention packages. However, very few evidence-based interventions have been designed for implementation within common drug treatment CBOs, such as methadone maintenance programs (MMPs), where many high-risk drug users seek treatment. Moreover, based on analogous efforts to disseminate evidence-based behavioral interventions for treating drug dependence (Morgenstern et al., 2001; Institute of Medicine, 1998), the few evidence-based interventions that are applicable to drug treatment CBOs are not designed to be \"community-friendly\" and are therefore unlikely to be implemented as intended or durable within these critical settings. Our team of investigators has developed a significantly shortened, community-friendly, version of the comprehensive evidence-based Holistic Health Recovery Program (HHRP; Avants et al., 2004; Margolin et al., 2003). The shortened version, the Community-friendly Health Recovery Program (CHRP), has demonstrated feasibility and acceptability as well as preliminary evidence of effectiveness in an uncontrolled study within a resource-limited drug treatment CBO (Copenhaver et al., in press; see Appendix). In this revised R01 application, we propose to evaluate CHRP in a randomized controlled trial (RCT). If found to be effective in the proposed trial, CHRP has the potential to be fully integrated, as designed, within many other resource-limited CBOs where large numbers of high risk drug users participate in drug treatment. [unreadable] [unreadable] [unreadable]"}
{"text": "AMPK is a fuel sensing enzyme that is activated by hormones, cytokines, exercise, and stresses that diminish cellular energy state (e.g., glucose deprivation). In addition, metformin and thiozolidinediones, agents used to diminish insulin resistance in type 2 diabetes, have been shown to activate AMPK. Activation of AMPK increases processes that generate ATP (e.g., fatty-acid oxidation) and restrains others such as fatty acid-, glycerolipid- and protein-synthesis that consume ATP, but not acutely necessary for survival. Conversely, when cells are presented with a sustained excess of glucose, AMPK activity diminishes and these synthetic processes are enhanced. Investigations by others have demonstrated that prostate cancer cells require high rates of fatty acid and protein synthesis for their invasive growth and survival. In preliminary studies, we have observed an apparently selective inhibition of the growth of prostate cancer cells by three distinct AMPK activators, AICAR, rosiglitazone, and metformin. We have also found that these effects are associated with inhibition of fatty acid synthase (FAS), acetyl CoA carboxylase (ACC) and mTOR. Based on these findings, we will test the hypothesis that AMPK activation restricts the growth of prostate cancer cells by altering an array of cellular events. We will carry out studies with the following aims: (1) To confirm and extend our preliminary finding that AMPK activity is suppressed in prostate cancer cells and to explore to what extent this accounts for their increased growth/survival and altered cellular metabolism;(2) To determine whether agents that inhibit prostate cancer cell growth in vitro prevents the growth of prostate cancer xenografts and/or cause their regression;(3) To assess whether the uptake and oxidation of exogeneous fatty acids are imparied in prostate cancer cells, and if so to determine the mechanism(s) responsible for these abnormalities, and whether they are reversed by AMPK activation or inhibition of ACC and;(4) To explore the molecular mechanisms by which androgen and AMPK oppositely regulate the mTOR signaling pathway. Since AMPK can be activated by several anti-diabetic drugs and by certain adipocyte-derived hormones (e.g. adiponectin and leptin), success in this effort would suggest a novel, yet practical approach for treating prostate cancers in humans."}
{"text": "This Phase II proposal integrates emerging technologies to expand the behavioral health capabilities and technological sophistication of the Journey to Disaster RecoveryTM website. This web-based system is designed to help restore a sense of control (i.e. coping efficacy) and mastery to survivors by providing a) knowledge to assist in understanding the recovery process, b) skill training to manage trauma related distress, and c) assistance in determining the need for and access to professional assistance. This project provides three essential developments with scientific and technical impact. First, it expands the technical sophistication and capabilities of the website to make it easy to customize and more responsive to different disaster/user needs. Second, it provides a critical scientific evaluation of the site's effectiveness to enhance mental health in the acute phase of disaster recovery helping to extend the very limited current knowledge base in this area. Third, it creates important training opportunities and evaluation with disaster response groups in disaster prone areas of the United States. Our ultimate goal is to provide the most reliable, customizable, and culturally based interactive, web system for empowering disaster survivors. The specific aims for this project are: Aim 1: Generate and evaluate the effectiveness of a content management system to enable highly efficient web customization. This system will integrate with a database of stored information rendered for different users (e.g., pictures, video, and text for different ages, genders, and ethnicities). Aim 2: Extend the technological and psychological reach of the site to incorporate Smartphone technology, allowing the system to deliver real time, interactive support and skill development. This will provide extensive avenues for future scientific exploration about human computer interactions related to disaster mental health. Aim 3: Expand the scientific evaluation of Journey to Disaster RecoveryTM by conducting a large randomized controlled trial during the acute phase of disaster recovery. This will broaden the current knowledge base about Internet interventions for disasters. Aim 4: Develop a web-based training and support system that instructs disaster behavioral health counselors about how to use the site in face-to-face counseling sessions. A quasi-experimental wait-list study will test the utility of the website as a training tool with crisis counselors. Aim 5: Complete and evaluate the cultural sensitivity and relevance of the Spanish language version of the site, MI RECUPERACISN. Given the number of native Spanish speakers in disaster prone areas, a culturally sensitive Spanish version of the site is essential. PUBLIC HEALTH RELEVANCE: The research involved in this Phase II STTR application will provide critical information about the efficacy of a web-based support system for disaster survivors to reduce their psychological distress and promote empowerment. At least 50 percent of disaster survivors report experiencing moderate to severe psychological distress and face barriers to accessing care including: cost of services, lack of knowledge about where services are available, and the stigma of seeking care. Journey to Disaster RecoveryTM provides a unique resource to reach beyond these barriers by offering a highly customizable, culturally relevant, anonymous, theoretically- based system with enhanced scalability to support disaster survivors."}
{"text": "The overall goal of the University of Utah Molecular Medicine Translational Research Center in Thrombosis (U2M2-TRCT) is to test the hypothesis that the abnormal metabolic milieu in type 2 diabetes (T2DM) and the metabolic syndrome (MS) leads to genetic and metabolic reprogramming of platelets. This reprogramming directly contributes to the increase in platelet activation that characterizes these subjects who are at increased risk for thrombosis. The specific focus of project 2 is to elucidate the mechanisms that are responsible for the metabolic reprograming that develops in platelets of humans and mouse models of T2DM and the MS, and to determine if the associated changes in platelet metabolism will directly contribute to platelet dysfunction. Our preliminary studies reveal that diabetes alters metabolome profiles of platelets with changes consistent with increased glycolysis and TCA (tricarboxylic acid) flux and accumulation of lipid intermediates. In addition, transcriptional profiling reveals significant changes in the expression of genes that regulate mitochondrial energetics, coupling efficiency (e.g. UCP2) and mitochondrial dynamics (e.g. Mfn2). Thus we hypothesize that the metabolic milieu of T2DM and the MS increases platelet glucose utilization and mitochondrial metabolism that induces mitochondrial oxidative stress, leading to platelet activation. In aim 1 we will test the hypothesis that T2DM and the MS leads to mitochondrial dysfunction, characterized by mitochondrial uncoupling, ROS overproduction and impaired mitochondrial dynamics. In aim 2 we will determine the mechanisms by which T2DM and the MS alter mitochondrial function and will test the hypothesis that manipulation of these mechanisms (i.e., KO of platelet superoxide dismutase 2, UCP2 and Mfn2) are sufficient to modulate platelet activity. In aim 3 we will determine the contribution of increased platelet glucose utilization to platelet hyperactivation in T2DM and the MS. This hypothesis will be addressed in studies of diabetic murine models with genetic reduction or augmentation in platelet glucose transport. Collectively these studies will shed new insight into mechanisms that alter platelet metabolism and function in obesity and diabetes."}
{"text": "The HIV/AIDS Adolescent Risk Reduction Project (HAARP) focuses on the window of opportunity existing for some adolescents and HIV/AIDS. The proposed project is based on the hypothesis that adolescents who receive motivating information related to their vulnerability to HIV and effective practice in AIDS prevention behaviors will have lower HIV risk scores than those who do not receive the coupled interventions. The proposed project is a collaborative venture between AIDS Impact, Seattle and Western Washington University (WWU) in Bellingham, WA. The HAARP study will have three interventions and a control group: (1) a motivating trigger tape aimed at raising vulnerabilities with guided discussion; (2) a skill-building refusal skills modeling video and role play aimed at increasing self-efficacy; and (3) a combined pairing of the previous two interventions. A risk assessment will be developed and administered pre and post to all groups plus a control group. The 200 students choosing to participate will be from a pool of 650 randomly selected undergraduate students. The project will develop two video tapes and facilitator guides as intervention elements: (1) a :07 trigger tape aimed at eliciting vulnerability and providing motivation; (2) an :08 refusal skills tape providing models."}
{"text": "Brain abscesses represent an important medical problem despite recent advances made in detection and therapy. Because of the emergence of multi-drug resistant strains and the ubiquitous nature of bacteria, these CNS infections are likely to persist. The size of a developing abscess normally extends well beyond the original site of infection leading to damage of surrounding normal brain parenchyma. This finding suggests that the CNS antibacterial response is not down regulated in an efficient manner, resulting in chronic inflammation and large abscess lesions. They propose that a balance exists between sufficient and over-compensatory responses to S. aureus in the CNS, which dictates the outcome of brain abscess development; therefore, therapies aimed at attenuating chronic CNS inflammation subsequent to effective bacterial neutralization may result in smaller abscesses and subsequent improvements in cognitive and neurological functions. The objective of the proposed work is to examine the influences of minocycline and PPAR-gamma agonists on the pathogenesis of brain abscess development. Recently, these compounds have been found to exhibit neuroprotective effects in several models of CNS disease; however, their roles in regulating CNS infectious disease has not yet been examined. To address this objective, the following Specific Aims will be addressed: (I) to evaluate the dose- and time-dependent effects of PPAR-gamma agonists and minocycline on S. aureus-induced brain abscess development; (II) to investigate the effects of PPAR-gamma agonists and minocycline on cell migration and neuronal cell death induced by S. aureus-stimulated microglia; and (III) to examine the mechanism(s) responsible for impaired neutrophil infiltration into brain abscesses of CXCR2 KO mice and the potential effects of PPAR-gamma agonists and minocycline in the CNS compartment. In addition to its anti-inflammatory properties, the bacteriostatic activity of minocycline may augment its effects on brain abscess development. The potential multifactorial effects of these compounds suggest that they may be more efficacious compared to traditional therapies developed to counteract a single pathway in CNS diseases. These experiments should provide meaningful insights into how minocycline and PPAR-gamma agonists influence brain abscess development and will reveal whether their ability to modulate non-infectious CNS conditions extends to infectious diseases."}
{"text": "Enzyme-linked immunosorbent assay (ELISA) was used to quantitate interphoto-receptor retinoid-binding protein (IRBP) in retinoblastoma tumors and in human subretinal fluid (SRF) samples. In retinoblastoma tumors there was a direct correlation between the degree of differentiation of the tumors and IRBP concentration. Higher concentrations of IRBP were found only in the more recent rhegmatogenous detachments, and IRBP was absent from SRF of patients with retrolental fibroplasia. Cyanogen bromide peptides from purified bovine IRBP were purified by high performance liquid chromatography (HPLC) and several peptides were found to produce experimental autoimmune uveitis (EAU) in rats. IRBP was shown to be phosphorylated in a crude bovine interphotoreceptor matrix (IPM) wash, and the phosphorylation was of serine and/or threonine residues. Intravitreal injection of radiolabeled retinol or fatty acids into frog eyes showed both retinol and fatty acids to be bound to IRBP."}
{"text": "A total of over one hundred pharmacologically active alkaloids have been characterized in skin extracts of some 50 species of dendrobatid frogs. Five major classes have been eludicated and their pharmacology defined: 1) The batrachotoxins (6 compounds) are highly toxic, complex steroidal alkaloids which prevent the inactivation of voltage-dependent sodium channels in nerve and muscle; 2) The histrionicotoxins (10 compounds) are 3,7-disubstituted 1-azaspiro (5.5) undecanes, which facilitate the conversion of chemosensitive sodium-potassium channels in nerves and effector cells to the desensitized state; 3) The pumiliotoxin-C class (42 compounds) are 2,5-disubstituted decahydroquinolines, which block nicotinic receptors; 4) The pumiliotoxin-A class (24 compounds) are 6-alkylidene-8-hydroxy-8-methylindolizidines which facilitate and then block calcium-dependent excitation-secretion coupling and excitation-contraction coupling in nerve and muscles; 5) The gephyrotoxins (11 compounds) are 3,5-disubstituted indolizidines and tricyclic analogs. Certain members of this class block voltage-dependent potassium channels and muscarinic receptors. An additional 14 compounds cannot be assigned to a particular class: one of these, a pyridine derivative, represents a new class of potent analgesics: Compounds antagonizing the binding of ouabain to Na ion -K ion -ATPase were identified as bufodienolides in extracts of certain bufonid toads and frogs, while a new class of Na ion -K ion -ATPase inhibitor was present in extracts on one bufonid frog."}
{"text": "Despite glaring health and HIV/AIDS-disparities among American Indian and Alaska Natives (AIAN), Native Hawaiians and other Pacific Island Islanders (NHPI), there is a paucity of culturally grounded research addressing their HIV-related biomedical and behavioral health concerns. A strong network of highly trained and productive Indigenous scholars dedicated to research that is culturally grounded would contribute to ameliorating HIV-related disparities among Indigenous populations. This competitive renewal application, in response to PAR-12-273, NIMH Research Education Mentoring Programs for HIV/AIDS Researchers is designed to develop the Indigenous HIV/AIDS Research Training Program-Lauhoe (IHART2) to develop a cadre of culturally grounded Native scholars capable of serving as PIs on extramurally funded HIV/AIDS- related prevention and disparities studies with Indigenous populations. The program is based on the success of the first 5 years of the Indigenous HIV/AIDS Research Training (IHART) R25 program (under PAR-06-494), the only Native-specific HIV/AIDS research training and mentorship program in the US. IHART2 extends the reach of the original IHART program from a primary focus on indigenous North Americans (e.g., AIAN and Native Latinos), to including NHPI (e.g., Native Hawaiian, Samoans). Additionally, where IHART focused on mid-career scientists, IHART2 now targets postdoctoral and early-career scholars. Building upon IHART success, IHART2 deepens the 360 degree co-mentorship approach with flexibility for program Fellows and Mentors to co-identify HIV training, career development, and cultural needs of the Fellow. IHART2 will be housed at the university-wide interdisciplinary funded Indigenous Wellness Research Institute (IWRI; iwri.org) National Center of Excellence (NIMHD; P60 MD006909-02) at the University of Washington. IWRI is directed and staffed almost entirely by AIAN, with three of the leading federally funded AIAN HIV researchers at the helm. IWRI has the infrastructure and environment necessary to promote culturally grounded Indigenous HIV prevention and disparities science training in a manner that is not currently covered by any existing R25s. To achieve the overall objectives of IHART2, we will select 15 Fellows who will undergo a structured 24-month intensive year-round mentorship program that includes: (a) annual research institutes and writing retreats (writing retreat; Spring Research Roundtable; Summer Research Institute; grant writing workshop,); (b) other training (webinars; 4 quarterly seminars; on-site mentor shadowing; and support for attendance to HIV/AIDS training institutes; (c) technical assistance (peer review for grant submission; statistical, editorial, and technical assistance for grant applications and manuscripts for publication); (d) seed funding ($20,000 to conduct pilot studies, travel to conferences, or to buy out time); and (e) network development (via mentor networks and website). By the end of the 24 month program, Fellows will have implemented a pilot study, published HIV/AIDS articles; presented at HIV-related conferences; and developed a NIH HIV-related grant proposal."}
{"text": "Major depression ranks among the leading biobehavioral risk factors for cardiovascular (CV) morbidity and mortality. Research has revealed several pathways through which depression increases CV risk including health behaviors and psychosocial functioning. That depression remains a significant predictor of CV disease (CVD) after intervening to improve health behaviors and psychosocial functioning in adults with depression suggests that additional, important risk factors have yet to be identified. We have developed a conceptual model that extends previous research on depression and CV risk by considering sleep as a novel biobehavioral mediator through which depression increases CV risk. We hypothesize that sleep disturbance, including decreased sleep duration, continuity and depth and increased sleep disordered breathing contribute to the increased CV risk observed in adults with major depression. Importantly, each of these components of sleep is modifiable and may represent promising therapeutic targets for reducing the cardiovascular consequences of major depression. The proposed 5-year study will evaluate these relationships in a well-characterized cohort of 200 adults with a history of recurrent major depressive disorder (MDD) who underwent psychiatric assessments and sleep studies in our laboratory approximately 10 to 30 years ago (T1). Participants, who were medically healthy without clinical cardiovascular disease at T1, exhibited profound sleep disturbances that persisted during remission. Members of this cohort have expressed great interest in the proposed follow-up study (T2). Proposed T2 measures include follow-up psychiatric assessments and sleep studies coupled with assessment of CV risk and subclinical disease including indices of autonomic imbalance, endothelial dysfunction, preclinical atherosclerosis and the metabolic syndrome. We will also assess health behaviors, psychosocial functioning and potential confounding variables. Together, these data will provide the first test of the hypothesized paths in our conceptual model. Our primary aim is to use T1 depression and sleep data in conjunction with T2-assessed CV outcomes to evaluate whether PSG-assessed sleep disturbance attenuates the prospective relationship between depression and CV risk and subclinical disease. Our secondary aim is to evaluate both wake and sleep pathways in the same model, using data collected at T2, including objective assessment of physical activity. The use of multiple-group structural equation models will provide the opportunity to evaluate whether relationships among depression, sleep disturbance and CV risk/subclinical disease differ by age and gender (exploratory aim). In future studies, experimental approaches will be needed to establish sleep as a causal pathway, identify the biological mechanisms through which specific components of disturbed sleep in depressed individuals increase CV risk, and develop evidence-based sleep interventions to prevent or attenuate the cardiovascular consequences of major depression. PUBLIC HEALTH RELEVANCE: The proposed study will be the first to evaluate sleep as a novel pathway through which depression increases cardiovascular disease risk, morbidity and mortality. Insights into the role of sleep disturbance in the link between major depression and cardiovascular disease is essential to our understanding of the pathophysiology of this prevalent and costly disease. Because sleep represents a modifiable pathway through which depression increases cardiovascular disease risk, it holds promise for the design and implementation of effective, evidence-based interventions to prevent and/or treat the cardiovascular consequences of major depression."}
{"text": "We are now practised in the complex surgery of isolating and perfusing kidney from a rat. Oxygen-sensitive crystals of LiPc were placed in the cortex and outermedulla so as to measure the partial pressure of oxygen (pO2) simultaneousl y in both these regions within the same kidney, allowing us to compare these results with our in vivo model. The advantages of using a perfused system are that many parameters can be measured and tightly controlled: 1) The dependence of tissue pO2 on perfusion pressure and/or flow rate in the control kidney, the pO2 in the cortex region is high compared to that in the medulla. However, at a critical pressure, the pO2 in the medulla sharply increases to a level equal to that in the cortex. If the pressure was again increased, we found that this phenomenon was reversed. 2) The effects of nitric oxide on oxygenation- by addition of nitroprusside (a nitric oxide donor) or a blocker of nitric ox ide synthesis, we were able to control the vasodilation of vessels in the medulla, and monitor the effects on pO2. 3) Endotoxin (either by bolus dose or continuous infusion) probably induces NO synthesis in the medulla, since we observed an increase in tissue pO2 with little change in tissue oxygen consumption."}
{"text": "The goal of this proposal is to develop chemosensors using arrays of engineered yeast strains to mimic olfaction. The experiments I propose will lead to a better understanding of the functional organization and evolution of G protein coupled receptors (GPCRs) and to greater understanding of the molecular mechanisms of olfaction. In many organisms, olfaction appears to be based on a mechanism involving combinatorial activation of broad-specificity GPCRs. I propose to utilize the fundamental mechanisms of olfactory chemosensing to construct yeast arrays capable of serving as chemical sensors. I will take two different approaches to construct the arrays, in each case using yeast strains engineered to functionally express human GPCRs. First, I am currently utilizing directed evolution techniques to generate mutant variants of the human muscarinic and melatonin receptors. I plan to use these mutants to create receptor arrays that are capable of analyzing complex mixtures for low levels of certain analytes. I also intend to continue the efforts of our lab to functionally express olfactory receptors in yeast. I will analyze libraries of chimeras between the melatonin receptor and olfactory receptors with known ligands. Once functional receptors are isolated, I plan to develop a generalized technique for generating functional chimeric olfactory receptors."}
{"text": "Project Summary This project will investigate how different environmental, personal and disease related factors contribute to the experience of behavioral symptoms of patients with Alzheimer's disease (AD). Patients with AD will experience behavioral symptoms of dementia (BSD) at some point during their illness, though most patients will not experience all types of BSD or experience them in a predictable pattern. This has made development of effective treatments and symptom management approaches for AD highly challenging. Our pilot data shows a high degree of intraindividual heterogeneity in daily reports of BSD both in type of symptom and frequency of occurrence. Others report significant intraindividual heterogeneity in the rate of change in BSD over a 3-month period. It is currently unclear what mechanisms contribute to these within- and between-person variations in BSD. While BSD subsyndromes have been proposed it is unclear how the types of BSD within the subsyndromes are temporally dependent or distinct from each other. As applied to AD, the Revised Symptom Management Model, points to environmental (i.e., unmet needs, stressors), personal (i.e., genetic, microbiome, ethnicity), and disease (i.e., HSV1 infection, systemic inflammation) factors as mechanistic pathways for BSD. We hypothesize that these factors across multiple systems contribute to the mechanistic development of BSD, and thus the variability in individual BSD expression. Elucidation of these factors across multiple systems will help create personalized approaches to treatment of BSD spanning from caregiving behavioral training to pharmacological interventions. Dyads of co-residing caregivers and persons with AD or mixed-type AD (with vascular involvement) (N=162 dyads) will be recruited. Equal numbers of Caucasian and Hispanic dyads will be enrolled so that the contributing role of ethnicity can be examined. Using a micro-longitudinal design, caregivers will complete daily diaries for 30 days reporting on their observations of environmental exposures, including type, frequency, duration, and severity of BSD. At enrollment and at the start of each new week of diaries, dyads will visit the research clinic where blood and stool samples will be collected from the person with AD (for 5 samples total). BSD will be reported in the daily diary entries, with BSD type measured as presence or absence of 22 symptoms across 17 domains. We will use a multi-level model analytic framework to examine hypotheses outlines in the research strategy in order to complete the following aims: (1) Determine how environmental (i.e., unmet needs, stressors), personal (i.e., genetic, microbiome, ethnicity) and disease (i.e. HSV1 infection, systemic inflammation) factors impact probability of daily BSD, either directly or through interaction effects, and (2) Identify subsyndromes of BSD, and predictors of group membership. Impact: By competitively testing leading hypotheses for BSD, this project will elucidate criteria for distinguishing patients with BSD that are not resolvable via appropriate attention to their unmet needs. Findings from this project will inform targeted interventions to support persons with Alzheimer's disease and their family caregivers."}
{"text": "The capsular polysaccharides of Streptococcus pneumoniae are essential virulence determinants for this organism. They serve as the basis for serologic classification, with ninety serotypes being distinguished to date, and they are the target for protective antibodies. In both human infections and animal models, virulence is related to the serotype of the capsule expressed. The mechanisms underlying the virulence differences between serotypes are not known but may involve both the polysaccharide structures and other factors in the genetic background in which they are expressed. Molecular genetic studies have identified a common organization among the different S. pneumoniae capsule loci. This organization permits the exchange of capsular serotypes during transformation, leading to new combinations of virulence factors and potentially impacting on the efficacy of polysaccharide-based vaccines. Knowledge of the genetic components of the loci has permitted pathways for polysaccharide synthesis to be demonstrated or proposed. Often, enzymes expected to be essential for capsule production are not encoded by genes in these loci, and the necessary products are obtained from cellular pools contributing to pathways for peptidoglycan and teichoic acid synthesis. Thus, capsule expression and basic cellular functions are intimately linked. Recent studies have demonstrated a requirement for capsule in colonization but have suggested that capsule production is reduced in this environment as compared to other host environments. Expression of other factors important for adherence and sustained colonization may be elevated during colonization and reduced at other times. Coordinate regulation of these virulence factors is thus anticipated and recent studies have identified a surface component potentially involved in adherence whose expression is altered in response to changes in capsule production. In the proposed studies, we will continue the genetic analysis of virulence and capsule expression. The specific aims are to: 1) determine the effect of specific alterations in capsule type and structure on virulence, 2) characterize the requirement for capsule in pneumococcal infections and its expression in vitro and in vivo, and 3) characterize regulatory networks and cellular pathways associated with capsule synthesis."}
{"text": "Optimal post-seizure therapy requires anticonvulsant and neuroprotectant therapeutic strategies, as both prolonged seizures (status epilepticus) and repetitive seizures occurring over time (epilepsy) cause neuronal death in brain and cognitive decline. Our data challenge assumptions on the relevant molecular pathways for such neuronal loss, revealing that Death Receptors of the extrinsic pathway are preferentially activated following seizures, which then trigger endoplasmic reticulum (ER) dysfunction. This extrinsic pathway activation occurs before any (intrinsic) mitochondria-linked cell death pathways become activated. We show protection from extrinsic pathway activation by ER-resident anti-apoptotic Bcl-w, which functions through effects on calcium regulation, attenuation of ER based pro-apoptotic Bcl-2 family protein function and inhibition of the integral ER membrane BAP31 complex. The SPECIFIC AIMS of this project are: Aim 1. Determine the significance and mechanism of death receptor complex activation as a cause of ER dysfunction and neuronal death following seizures. Aim 2. Determine how Bcl-w protects against seizure-induced neuronal death via effects on the extrinsic pathway target: Aim 3. Show the relationship between Extrinsic and Intrinsic pathway activation by seizures. To accomplish these aims we have developed a mouse model of seizure-induced brain injury, not previously available, with continual EEC seizure monitoring, permitting electrographic seizure quantitation and distinction between injurious and non-injurious seizure types. We also use an in vitro seizure model permitting single cell, calcium imaging and culture studies. With mouse modeling we will use knockouts for down regulation of apoptosis modulatory genes under study and provide up-regulation of protective gene products with adenoassociated viruses and TAT fusion proteins. We determine the effect of pharmacological and molecular manipulation of the extrinsic cell death pathway on seizure induced cell damage. Finally, using continuous video-EEC monitoring, we will investigate how prevention of neuronal damage following seizures effects the development of an epileptic phenotype."}
{"text": "Two isoenzymes of amine N-methyltransferase from rabbit liver are available in homogeneous form and have been characterized. The enzymes have overlapping specificity for the transfer of a methyl group from S-adenosyl-L-methionine to any one of a very large number of amine acceptors. Acceptor molecules include those of very different carbon skeleton among which are aliphatic, aromatic and heterocyclic compounds that are primary, secondary and tertiary amines. Methylation of the tertiary amines, pyridine for example, results in the formation of a pyridinium ion. It has now become possible to evaluate the role of methylation in the formation of certain hepatocarcinogenic amines. Hepatocarcinogenicity for compounds such as benzidine, N- methylazobenzene and some other aminobiphenyls is well established. Based on work with purified enzymes, N-methyltransferase and a microsomal flavincontaining monooxygenase, it was concluded that carcinogenicity of such aromatic amines required methylation followed by N-oxidation of the resultant N-methyl derivative; the cytochrome P-450 system did not appear to be involved. Thus ben and 4-aminobiphenyl are both methylated and carcinogenic. 4- Aminoazobenzene is neither methylated nor carcinogenic, although the methylated species, 4-methylaminoazobenzene is carcinogenic. All three carcinogenic monomethyl arylamines yield N-oxidized products with the flavin oxygenase. 2-Aminobiphenyl is neither a substrate for the transferase nor does it act as a tumor inducer. It would appear that N-methylation, followed by oxidation with the flavoprotein system, is a means for producing carcinogenic derivatives of the aminobiphenyls."}
{"text": "Reducing Duration of Untreated Psychosis (DUP) is a primary goal for improving long-term outcomes in young people with a first episode of psychosis (FEP). The standard of FEP care within the US focuses on targeted provider education1 regarding signs and symptoms of early psychosis to motivate patient referrals to FEP services, followed by initiation of services within largely clinic-based settings 2. Experience at the Early Diagnosis and Preventive Treatment (EDAPT) FEP specialty program at U.C. Davis in Sacramento has identified two important bottlenecks to reducing DUP, consistent with reports in the literature from other FEP clinics. These are 1) delays in the identification of psychotic symptoms by referral sources, and 2) delays or disruptions of patient engagement in specialty FEP care. Building upon a comprehensive and established referral network of 20 sites across the Sacramento area (schools/universities, ER/inpatient hospitals, outpatient mental health, primary care), we will address delays in patient identification and engagement using a two- phase, cluster randomized design. We will consecutively test the impact of two interventions to reduce DUP, defined in this RFA as time from first onset of psychotic symptoms to engagement in FEP specialty care. To address identification delays, we will examine the use of standard targeted provider education plus novel technology-enhanced screening compared to standard targeted provider education alone, testing the hypothesis that the education plus technology-enhanced screening will identify more patients, earlier in their illness. To address engagement delays, we will compare the use of a mobile community-based, telepsychiatry- enhanced engagement team to standard clinic-based procedures for intake, engagement and initiation of treatment, to test the hypothesis that the mobile approach facilitates earlier and more stable engagement, thereby reducing DUP. The proposed work will provide new specific evidence-based practices for reducing DUP and improving outcomes through specialty care of individuals with a first episode of psychosis."}
{"text": "The Mouse Genetics Facility was established in 1999 in response to the increasing need of Cancer Center investigators for analysis of gene function in mouse models, and has received Cancer Center support since 2002. Since the last renewal, the Facility has changed leadership, with Dr. Anthony Capobianco now the scientific director of the Facility and Dr. Ping Jiang the facility director. In this funding period, the Institute has invested $167,450 for new and upgraded equipment and renovated laboratory and animal facility space. The Facility provides highly efficient production of both transgenic and knock-out/knock-in (KO/KI) mice, plus it provides a unique resource and expertise in embryonic stem (ES) cell cultivation and engineering, which benefits Cancer Center investigators generating both in vivo and in vitro murine models of cancer. Current services are aligned with the research objectives of the Cancer Center programs and include: (1) production of transgenic mice by pronuclear microinjection of DNA transgene constructs;(2) generation of genetargeted 129 and C57BL mouse ES cell clones;(3) production of chimeric mice derived from microinjection of gene-targeted ES cells into blastocysts;(4) rederivation of mouse lines by embryo transfer;and (5) ES cell cultivation and engineering. Since 2006, the Facility has produced 15 lines of transgenic mice, successfully generated 11 lines of KO/KI mouse ES cell clones, and produced 8 lines of chimeric KO/KI mice. In addition, the Facility successfully rederived a line of an engineered mouse strain and will offer a mouse embryo cryopreservation service in 2008. The Facility is actively collaborating with Cancer Center investigators to utilize its mouse ES cell technology, with the aim of using human ES cells as a vehicle to derive mutant or disease specific cell lineages as a new human disease model system. Goals for the Facility over the next funding period include developing lentiviral mediated transgenesis for some strains of mice that are difficult to produce with DNA microinjection, developing use of tetraploid complementation as an efficient approach to produce chimeras from injections of ES cells of C57BL origin into blastocysts, and establishing human ES cell cultivation and engineering as a routine service."}
{"text": "Rigorous biochemical analyses of pulmonary surfactant show that it is comprised of disaturated phosphatidylcholine, monoenoic phosphatidylcholine, phosphatidylglycerol, a unique apoprotein, and small amounts of neutral lipids, other phospholipids, and carbohydrate. The principal physiological function of pulmonary surfactant is well-known -- it lowers the surface tension of the alveolar interface to less than 15 dynes/cm. It has been determined that the disaturated phosphatidylcholine is capable of this process. Still unknown, however, are the physiological roles of the other lipids, and the apoprotein. This project will explore how the composition of these lipids may affect the physical properties of the entire complex, and how this may relate to respiratory mechanics. Significant emphasis will be given to the interaction of the apoprotein of surfactant with well-defined mixtures of lipids, and the effect of this interaction on the physical properties of the recombinant complex. An attempt will be made to find which of the lipid and/or protein constituents are needed to form tubular myelin, and to correlate morphologic structure with physicochemical properties."}
{"text": "PROJECT SUMMARY Social interaction and communication begin in early infancy, and, although these are fundamental human functions, little is known about the underlying neural mechanisms that regulate them particularly in Autism Spectrum Disorder (ASD). ASD is a neurodevelopmental disorder characterized by significant disabilities in language and social skills, and the specific neural mechanisms that lead to these disabilities remain active topics for investigation. Emerging theoretical directions converge on problems with eye-contact as a salient component of these communication and social disabilities. Technical limitations, however, associated with imaging of two or more individuals during natural communication and mutual eye contact have been a primary obstacle to these investigations. To overcome this technical impasse, we employ a rapidly developing brain imaging technology, functional near-infrared spectroscopy (fNIRS) allowing simultaneous neural imaging of two individuals during valid interactions to observe the neural effects of eye-to-eye contact and actual dialogue. Functional NIRS detects active neural tissue based on the blood-oxygen-level-dependent (BOLD) signal by measuring variations in the absorption spectra associated with oxyhemoglobin and deoxyhemoglobin. Because detectors and emitters are surface mounted on the head, they are relatively insensitive to head movement, and, as such, fNIRS is well suited for investigations of neural events engaged during active interpersonal interactions between two participants. The neural mechanisms that underlie atypical interpersonal interactions and eye contact in adult ASD are the focus of this proposal. Pilot studies confirm the feasibility of all aspects of this research project. Dyads consisting of a confederate and a participants with typical development (TYP) or ASD will be compared during neuroimaging while engaged in natural interaction and communication. We introduce a computational approach based on wavelet analysis to quantify regional cross-brain coherence between the two participants and hypothesize that cross-brain coherence associated with speech and eye contact will be reduced in ASD relative to the TYP cohort. Cross-brain computations also form the basis for a model of dynamic neural processes based on neural ?send and receive? functions during communication. We hypothesize that these dynamic ?cross-brain communication? systems unify and coordinate the roles of language production and reception (Broca's and Wernicke's Areas), respectively, with visual reception involving face specializations (fusiform gyrus). Computational comparison of cross-brain connectivity effects as well as conventional functional connectivity and segregation/contrast effects during live communication both with and without direct eye contact provides a transformational technical, empirical, computational, and theoretical advance toward understanding the dynamic neural mechanisms associated with social and communication disabilities in ASD."}
{"text": "DESCRIPTION (Applicant's abstract verbatim): Noble gas isotopes that can be nuclear spin-polarized are very attractive imaging agents because of their high detection sensitivity for nuclear magnetic resonance. In particular, spin-polarized 3He enables fast, gas-space imaging of the lungs and airways for clinical diagnosis and physiological studies. Unfortunately, the limited availability of 3He severely limits the number of high-resolution lung imaging studies that can be performed. We propose to develop a system to recover and recycle the rare helium isotope so that high-resolution lung imaging can become common and inexpensive. The innovation is a cryogenic gas separation system that extracts and purifies the 3He from a patient's exhalation stream and saves it for re-polarization and reuse in other patients. In Phase I we proved the feasibility of the 3He recovery system by (1) performing a proof-of-concept test that demonstrates the feasibility of efficiently separating, purifying, and sterilizing the 3He from exhalation gases, and (2) producing a conceptual design of a complete system to recover and recycle 3He. In Phase II we will build and demonstrate a complete recovery and recycling system. PROPOSED COMMERCIAL APPLICATION: Not Available"}
{"text": "There are a variety of molecular mechanisms by which organisms can regulate gene expression during growth and development. One such mechanism is observed in B. subtilis during the process of sporulation. In response to nutrient limitations a well-defined developmental program is elicited. This developmental pathway which culminates in the dormant cell type known as an endospore is controlled, in part, by regulatory proteins (sigma factors) that alter the transcriptional specificity of RNA polymerase. The core RNA polymerase can interact with at least four different sigma factors each of which confers distinct transcriptional specificity. The central issue which we intend to address concerns the mechanism by which the sigma factors alter the template specificity of DNA-dependent RNA polymerase. In one model it has been postulated that each sigma factor induces a unique conformation in the core RNA polymerase, thereby allowing it to preferentially interact with a particular canonical promotor sequence. We plan to test this hypothesis by introducing chromophores into core RNA polymerase and spectroscopically monitoring putative structural altervations that may occur in the care enzyme when each individual sigma factor binds. Our approach involves replacing the endogenous, spectroscopically inert ZN(II) in the Beta' subunit of RNA polymerase (oligomeric structrure: Alpha2 Beta Beta'Sigma) with either Ni(II) or Co(II), thereby introducing chromophores at the metal binding sites. Similar metal ion substitutions in other systems such as E. coli RNA polymerase have been shown to be innocuous. To establish this for our system, we will examine the enzymic and physical properties of the metal-substituted derivatives. Next we will use spectroscopie techniques such as circular dichroism and difference spectorscopy to monitor putative conformational changes, upon ligand binding, in the subunit of RNA polymerase which has been shown to extensively interact with the DNA template. Similar techniques have been used to monitor ligand-induced conformational changes in E. coli Co(II)-RNA polymerase. Thus, with this approach, we should be able to correlate the transcriptional specificity of each sigma factor with its effect on the conformation of the core enzyme. These results will have important implications in regard to the mechanisms of gene regulation which may be operative in mammalian systems."}
{"text": "Delivery to nerve terminals of cytoskeletal proteins synthesized in the cell body is crucial for growth, maintenance and regeneration of neurons. Despite its importance, little is known about what motors are responsible for slow transport or in what forms these proteins are transported. We have shown that tubulin moves down the squid giant axon as an unpolymerized protein by both diffusion and slow axonal transport. Additionally, it appears as if the soluble motor that transports tubulin is loosely coupled to its cargo, since separation of the axon from the cell body leads to the rapid loss of slow transport, but not tubulin. To test this hypothesis directly, we are now measuring the binding coefficients of proteins within the transported complex. We have also developed a new 2-photon technique for studying slow transport by selectively disrupting individual cytoskeletal tracts. Moreover, we have shown that myosin, one of the candidate motors for slow transport, is also responsible for the purse-string constriction of the axon following transection. Finally, we have sequenced and characterized a number of the different myosins in the squid giant axon, and are now developing antibodies to determine the functional role of myosins in slow axonal transport."}
{"text": "The Cystic fibrosis transmembrane conductance regulator (CFTR) is the membrane protein product of the gene known to be defective in cystic fibrosis patients. CFTR functions by transporting chloride ions through a transmembrane (TM) domain believed to consist of two sets of six tightly packed and assembled membrane spanning helical segments (TM1-6, TM7- 12, ca, 20 amino acids each). However, channel remain to be deduced in molecular terms. (1) Since packing of TM domains appears to be directed by specific recognition elements contained within the individual TM segments, and the N-terminal portion of CFTR (TM1-6) is known to form a functional channel, we propose to prepare a series of peptides based on the native sequences of the CFTR TM domain segments 1-6 by solid-phase synthesis, and to determine the affinities of the individual TM segments for each other. Individual TM segments will be combined in combinations (e.g., TM5-6, TM6-1, TM1-2, etc.).Affinities would be quantitated by fluorescence energy transfer, circular dichroism spectroscopy, and gel electrophoresis. These experiments will be supported by molecular modeling to identify favorably- packed helical dimers of CFTR helices, and by channel conductance measurements performed on CFTR TM peptides individually and in combinations. Pairs found to have affinity of dimerization would likely be in direct 3-D contact in the native channel. (2) An increasing number o missense mutations within the CFTR TM domain are now being associated with CF disease. We reason that certain 'high-information' residues impair channel function by interfering with helix-helix packing. Peptides prioritized to key TM-domain mutations observed in genotyped CF patients from the in-house database at the Hospital for Sick Children (e.g., G85E, R347H, R347W) ill be prepared and studied as above in combinations with wild type partners. From the combined information thus obtained, we will construct a working model for the molecular geometry of the CFTR channel, and use the epidemiological data to generate hypotheses concerning possible relationships between severity of CF disease and molecular defects in the corresponding mutant CFTR's."}
{"text": "In this proposal we will attempt to further define the mechanistic pathways involved in enhancing tumor immunity using epigenetic agents and improved strategies for enhancing their effectiveness. The long-term goal is to transfer this technology to clinical application. Our specific aims are to determine: 1) the role of enhanced antigen presentation by histone deacetylase inhibitor (HDACi) treated tumor cells in a B16 melanoma vaccine model; 2) the contribution of NK cells to immunity in the epigenetic model; 3) the efficacy of these models in combinations of HDACi with hyperthermia and radiation; 4) the effects of systemic HDACi therapy in combination with other modalities. An additional major aim is to characterize the role of Dicer and microRNA in immune gene regulation. Our studies have identified miRNAs which target several miRNA machinery components including Dicer. We also defined several stresses that downregulate Dicer (ROS, anoxia, dsRNA) and will determine the pathways by which expression of Dicer is regulated. MiRNAs and siRNAs targeting Dicer will be employed to de-repress silenced genes and to determine whether this enhances immune gene expression in combination with HDACi which are currently in clinical trials. The proposed studies are pre-clinical and exploit the B16 melanoma, colon26, as well as the SCC head and neck cancer models. Our studies will employ in vitro immunological assays, standard molecular biology techniques and microarrays for characterization of gene and miRNA expression. Fresh H&N tissue, removed at surgery, will be studied in preparation for a proposed clinical trial. PUBLIC HEALTH RELEVANCE: This application focuses on pre-clinical and mechanistic studies of a new class of agents (histone deacetylase inhibitors) now currently being evaluated in clinical trials. These studies address a novel mechanism by which these agents may improve cancer therapy with low toxicity (compared to standard chemotherapeutics). [unreadable] [unreadable] [unreadable]"}
{"text": "The laboratories and offices of the COBRE PI, Dr. Igor Roninson, COBRE target faculty Dr. Michael Shtutman, the Co-Directors of the COBRE Microscopy/Flow Cytometry Core, Dr Chang-uk Lim and Dr. Gary Schools, are scattered in various inadequate rooms and laboratories between floors 3 and 7 of the Coker Life Sciences Building (CLS) on the Columbia Campus of USC in space that was temporarily provided to Pharmacy from the Department of Biology. This lack of cohesive modern molecular biology laboratory space is disruptive, inefficient, and raises many obstacles for Dr. Roninson's research program and the CTT COBRE. This A&R request is for $300,000 of support toward a $1,150,000 renovation project on the 7th floor of the Pharmacy wing of CLS that will result in 4,857 s.f. of new modern office and research space for the CTT COBRE and Dr. Roninson's entire program. Specific Aim 1. To renovate space on the 7th floor of the Pharmacy Wing of the Coker Life Sciences Building on the USC Columbia campus to house the proposed CTT COBRE. This space will provide modern office and laboratory space for (1) the COBRE PI, (2) COBRE target faculty Michael Shtutman, (3) the COBRE Microscopy/Flow Cytometry Core and its Co-directors Dr. Gary Schools and Dr. Chang-uk Lim, and (4) part of the COBRE Functional Genomics Core. Additionally, this space will house the Administrative Coordinator for the COBRE Center for Targeted Therapeutics, a Conference Room, and a Break Room."}
{"text": "Trifolitoxin (TFX) is a peptide bacteriocin produced by Rhizobium leguminosarum bv. trifoli T24 which is bacteriostatic toward most strains of R. leguminosarum and R. fredii. Sequence analysis of the TFX production and resistance genes strongly suggests that this peptide is produced ribosomally (Breil, et al. 1993, J. Bacteriol., in press). The DNA sequence of the tfxA structural gene and Edman degradation of the purified peptide show that the amino acid sequence of the peptide precursor of TFX is DIGGSRQGCVA. This peptide sequence is post-translationally modified by the tfx genes such that the glutamine residue is cyclized to a UV absorbing chromophore. The purpose of this work was to determine the structure of this chromophore. Two isomers of TFX were isolated by reverse phase HPLC. The mass of the molecules was confirmed by low resolution FAB-MS to be 1037.4 for both molecules. The structure of the chromophore was determined by a combination of modern NMR techniques, such as COSY, TOCSY and HMQC, to be a 2-pyridone derivative which tautomerizes to give TFX isomers. Both isomers of TFX also contain a thiazoline ring which is the result of ring closure between the sulfhydryl group of cysteine and the carbonyl of glycine."}
{"text": "Eye diseases with immunologic properties are common ophthalmic problems. A study of these problems is aided by work on the basic ocular immune mechanisms. Though both cell and antibody mediated mechanisms are important, we have chosen to pursue mostly the role of antibody dependant mechanisms in protecting and in other instances harming the eye, especially the cornea. In the proposed grant period we plan to study by the fluorescent antibody and immunoglobulin quantitation methods 1) the amount, type, distribution and source of immunoglobulins in the cornea; 2) types of plasma cells in the main and accessory lacrimal glands as an indication of the source of immunoglobulin in the tears; and 3) relationship of tear and serum immunoglobulins to serum as a possible source of a part of tear immunoglobulins. Our interest in diseases with immune properties includes study of 1) the possible local production of IgE in vernal conjunctivitis; 2) tear immunoglobulin levels in anterior and posterior uveitis. A continuation of our interest in antibody dependent eye diseases is our planned study of a human model of ocular atopy. The model is produced by dropping small amounts of water soluble pollen antigen into the cul de sacs of persons allergic to this antigen. The amount of reaction, its signs, duration, variation day to day, and variation between subjects is to be observed. The effect of various drug preparations on the model is to be studied. Our interest in clinical diseases with immunologic properties includes the role of tissue antigen in corneal transplantation. We plan to evaluate 1) the role of HL-A typing in corneal transplantation; 2) the role of anti HL-A antibodies before and after grafting; 3) the role of ABO mismatching; and 4) the role of IgM and IgG anti-ABO antibodies. BIBLIOGRAPHIC REFERENCES: Older, J.J. and Allansmith, M.R. Penetrating keratoplasty in a patient with 75 percent third degree burns. Annals of Ophthal., 7:309, 1975; Allansmith, M.R., Drell, D.W., Kajiyama, G. and Fine, M. ABO blood groups and corneal transplantation. Amer. J. Ophthal., 79:493-501, 1975."}
{"text": "Inflamm-aging model is attractive because it derives from the knowledge that aged humans develop low-grade inflammatory state. It is conjectured that the state of low-grade inflammation is a consequence of multiple protective immune responses that develop in order to combat pathogenic infections throughout life. Whereas the notion that chronic inflammatory state could result from multiple immune responses is credible it remains to be demonstrated experimentally. Once this is demonstrated experimentally it will be of interest to demonstrate that experimentally induced immune responses over a period of time correlate with accelerated aging of the whole organism. Long term goals of the experiments proposed in this report aim to establish the role of multiple immune responses in generating a chronic inflamed state, which in turn contributes to accelerated aging in mice. In the short term we will determine if macrophages from young and old mice respond to a common inducer, lipopolysaccharide (LPS), respond differently by measuring waves of gene expression patterns. These studies will set the stage for assaying effects of induced inflamed state in the inflamm-aging model on innate immunity."}
{"text": "Antigen presenting cells (APCs) form the critical link between innate and adaptive immune responses in mammals. The mechanisms of lymphocyte activation in lower vertebrates, however, are largely unknown. Our preliminary studies suggest that the cellular constituents responsible for antigen presentation are remarkably conserved from zebrafish to mammals. In mammals, dendritic cells (DCs) are the most potent cells in priming an adaptive response through direct activation of lymphocytes. We have identified DC-like cells in the lymphoid organs of zebrafish and initiated studies to enable their prospective isolation and functional testing. We will begin our proposed studies in the adult to determine the cellular effectors of antigen presentation. We will develop functional assays to determine which cell types can traffic foreign antigen to secondary lymphoid organs and stimulate proliferation of antigen-specific T lymphocytes. To complement these functional approaches, we will identify markers capable of enriching APCs from hematolymphoid tissues. These basic studies will set the stage for an assessment of the ontogeny of immunity in the zebrafish embryo. We will make use of novel and existing transgenic lines to fluorescently label APCs and the major branches of the hematolymphoid system, and leverage the unique attributes of zebrafish to image the immune response to pathogen in real time in living animals. These studies will help us determine the predominant sites of immune cell interactions. With the ability to specifically identify each of the APC subsets, we will utilize this information to determine the cellular architecture of secondary lymphoid organs under steady-state conditions. We will subsequently ascertain the changes that result in these tissues following immune challenge. Finally, we will ablate APCs using a conditional transgenic toxin to determine the effects upon lymphoid architecture and the overall immune response. Taken together, these studies will provide a new level of precision in the study of the zebrafish immune response, and will lead to a better understanding of the role of APCs in bridging the ancient and acquired arms of the vertebrate immune system. In addition, these efforts will provide unprecedented, real-time study of APC behavior in living animals. Ultimately, this work should lead to new discoveries regarding antigen presenting cells by utilizing the unique imaging and genetic advantages of the zebrafish."}
{"text": "The recent controversial Women's Health Initiative Memory Study (WHIMS) implies that long-term treatment with estrogen and progestin significantly increases the risk of cognitive decline and dementia in menopausal women. These data sharply contrast with studies in women and rodents that have demonstrated a clear ability of estrogen to alleviate age- and hormone-related memory loss. The WHIMS finding that progestin is detrimental to memory is even more surprising because estrogen and progesterone treatment have been shown to be beneficial to memory in laboratory animals. The proposed studies were designed to clarify whether progesterone attenuates the beneficial effects of estrogen and to identify the neural mechanisms underlying the effects of estrogen and progesterone on memory consolidation. Because adverse side effects of hormone replacement such as increased risks of breast cancer and stroke make it impractical to conduct these studies in women, the proposed studies will utilize a female mouse model. The specific aims are as follows: 1) To pinpoint if estrogen and progesterone, alone or in combination, enhance memory consolidation and alter MAP kinase signaling in the hippocampus in young and aging females, 2) To determine if the mnemonic and hippocampal response to estrogen and progesterone is influenced by environmental factors, and 3) To compare the mnemonic and neural effects of estrogen and progesterone treatments that vary in duration (short- vs. long-term) and type (sustained vs. cyclic). To achieve these aims, ovariectomized female C57BL/6 mice (4, 17, and 24 months old) will be used in a series of nine experiments. Spatial memory will be tested in a spatial water maze task and non-spatial memory will be tested in an object recognition task. The goal of this research is to clarify the findings of WHIMS, a study which has greatly impacted the treatment of millions of menopausal women. WHIMS was a limited study, focusing on an older, highly educated population receiving treatment that was not cyclic and included a detrimental type of progestin. Establishing how factors such as age at treatment, degree of cognitive stimulation, type of progestin, and type of treatment (i.e., cyclic vs. sustained) influence the mnemonic and neural response to hormones will be critical to future use of hormone replacement. Our mouse model will enable a more controlled study of these factors than is possible with women. Elucidation of these issues may ultimately lead to the development of safer and more effective treatments to prevent cognitive decline and dementia in women [unreadable] [unreadable]"}
{"text": "This application requests continuing Medical Scientist Training Program (MSTP) support for the Combined MD/PhD Training Program at the University of Minnesota. Originally funded in 1988, the Program remains committed to a single goal: to train physician-scientists equipped to push technology and clinical application to a level of investigative excellence that will have a direct impact on the diagnosis and treatment of human disease. The MSTP provides instruction leading to the MD and PhD degrees in approximately 8 years, although curriculum changes make completion in 7 years a feasible goal. The Program employs a sequential training sequence of medical school (2 years) > graduate school (3-4 years) > medical school (1.5 years). Students conduct a minimum of 3 laboratory rotations during their initial 18 months in the Program, and can choose from over 100 preceptors with research opportunities that span the gamut of disciplines in contemporary biomedical research, as well as biomedical engineering and epidemiology. Future research opportunities will be enhanced by a major expansion of laboratory facilities, including the construction of 4 new research buildings in the East Gateway Biomedical Discovery District at the University of Minnesota. Multiple requirements/opportunities facilitate the essential goal of integrating fundamental scientific training with the development of competent and compassionate physicians. A centerpiece of this integration is a course (Ambulatory Clinics for the Physician Scientist) in which students conduct clinical rotations with the physician-scientist of their choosing. Taken in the final year of graduate training, the course promotes thinking at a scientific/patient care boundary and refreshes students' clinical skills. Program leadership consists of a Director, Associate Director, and a Steering Committee made up of faculty from basic science and clinical departments. The Program recruits from a pool of gifted national applicants who can present a compelling case for a dual interest in science and medicine. The past 5 years have witnessed the complete elimination of any attrition problems, an enhanced curriculum that integrates scientific and clinical training, scientific accomplishments published in premiere journals (eg, Science and Nature), and residency choices that continue to promote academic career development. As of July 2009 there are 56 students in the Program (40% women, 14% under-represented minorities) from colleges and universities throughout the United States. These students are blessed with talent, highly productive (9 have individual F30 awards), and totally committed to a career in biomedical research. Our goal for the next 5 years is to continue our upward trajectory and witness the successful integration of our students into the fabric of biomedical research. RELEVANCE: This program supports the training of physician-scientists (i.e., persons pursuing an MD and a PhD). These individuals will be at the forefront of pioneering new ways to diagnose and treat human disease."}
{"text": "Hypothesis: A subpopulation of prolactinoma patients may have a syndrome of congential malformations consisting of pseudopapilledema and anomalous arterial supply of the pituitary by-passing hypothalamic dopamine inhibition. Exclusion of pituitary tissue from dopamine regulation may predispose a patient to prolactinoma formation. The current proposal proposes to investigate the incidence of pseudopapilledema in prolactinoma patients."}
{"text": "In the past 9 months, we have shown that rats can be operantly conditioned to increase as well as decrease the amplitude of EEG potential components recorded in somatic sensory face area cortex, and evoked by non-aversive shocks to the primary descending trigeminal tract. In 9 of 11 successful training procedures, we found that the latency of face-rub responses to noxious facial heat correlated significantly (p less than .05) with conditioned evoked potential amplitude, yielding correlation coefficients of between plus .5 and plus .9. We have also successfully conditioned potentials recorded in the caudal trigeminal nucleus, but there was no correlate in pain indices here."}
{"text": "This project is concerned with analyzing the differences in the Na-K pump and leak of cells of mammalian hibernators which enable them to retain normal Na and K concentrations at low temperature. Two types of cells are being investigated, erythrocytes and kidney cells, both grown in culture and freshly isolated, from both hibernating and non-hibernating species. With erythrocytes the aim is to determine quantitatively the specific effects of temperature on the kinetics and molecular mechanism of Na-K transport leading to its cessation at low temperature. In kidney cells some of the same analysis may also be possible, but in addition kidney cells should lend themselves to analysis of problems of regulation and turnover of pump proteins and of alterations of properties connected with transepithelial transport. Because of the latter possibility an effort is also being made to characterize the cultured cells in terms of their retention of differentiated epithelial transport activities. The general purpose of the project is twofold, both to understand the process of retention - or loss - of ionic regulation in the cold, which is a factor intimately involved with survival in the cold, and to use cells of hibernators to explore fundamental questions of transport."}
{"text": "Project Summary/Abstract: Evidence-based medicine (EBM) combines the best available practices with clinician experience and patient values. Prior to evidence-based medicine, quality of care was based on adherence to local standard practices given by healthcare providers. This standard was based on what historical practices, and was often anecdotal in nature. Improved means of statistical analysis, especially multivariable analysis, systematic reviews and meta-analysis has helped the advancement EBM. The adoption of EBM tenets has been slow, and acceptance of EBM over eminence-based orthopaedics has suffered. To this end, educating surgeons about the benefits and biases of high level of evidence studies, including randomized control trials (RCTs) and prospective observational studies, will help improve the clinical care of orthopaedic patients. Further, clinical interpretation in practice requires understanding of the Consolidated Standards of Reporting Trials (CONSORT) and Strengthening the Reporting of Observational studies in Epidemiology (STROBE) diagrams. The conflict between EBM and previously accepted standards is evident in several orthopaedic procedures in which EBM has questioned value: viscosupplementation, vertebroplasty, and knee meniscectomy for degenerative meniscal tears. Both RCTs and observational studies have important places in surgical research. A well-done cohort trial may provide greater insight to practical patient care than a RCT that is not applicable to many patients. In addition, observational studies can be particularly useful in addressing questions related to surgical procedures where RCTs cannot be performed or the number of patients necessary to achieve adequate statistical power is too numerous to make the trial practical. Improved education for surgeons reading and reviewing studies is critical to advancing the practice of orthopaedic surgery and the care of surgical patients. Improved understanding of the benefits and biases of studies at the highest levels of evidence, namely RCTs and prospective observational studies, will enable important and much needed advancements in the care of our patients. This symposium will address the strengths and weaknesses of these types of analyses in musculoskeletal care. The recent popularity of registry data for quality purposes will be explored as to how this fits within the hierarchy of evidence and physician/patient decision making. This is especially important given the AAOS emphasis and development of national registries for major orthopaedic procedures."}
{"text": "Dopamine has been implicated as the primary neurotransmitter associated with the psychomotor stimulant and reinforcing effects of cocaine. These findings have resulted in intensive efforts to characterize and elucidate the roles of the various dopamine receptor subtypes in the pharmacology and abuse liability of this drug of abuse. In this pursuit, the dopamine D3 receptor subtype has been recently targeted. However, definitive behavioral investigations have been hampered by the lack of highly selective D3 agonists and antagonists. We have prepared the known D3 antagonist NGB 2904 in multi-gram quantities for acute and chronic behavioral testing. This compound demonstrated in vivo D3 antagonist actions and attenuated cocaine self-administration under a progressive ratio schedule. NGB 2904 also blocked cocaine-induced reinstatement of drug seeking behavior, an animal model of relapse. We then designed and synthesized novel series of compounds, based on NGB 2904. All the compounds included either a 2,3-dichloro- or 2-methoxy-substituted- phenylpiperazine, a four carbon linking chain with varying saturation (butyl, hydroxybutyl, and trans butenyl) and a terminal aryl amide. Evaluation for in vitro binding in HEK 293 cells transfected with human D2, D3, or D4 receptor cDNAs resulted in D3 binding affinities ranging from Ki=0.3-500 nM. The most potent analogs in this series, demonstrated D3/D2 selectivity of >200 and a D3/D4 selectivity of >1000. Functional evaluation in vitro using a mitogenesis assay in D3 or D2 receptor transfected CHO cells demonstrated that these compounds were either potent antagonists or partial agonists at D3 receptors and were selective over D2 receptors, in this function. However, a functional comparison of a series of butenyl and saturated butyl analogues showed that these compounds generally exhibited higher intrinsic activity in the adenylate cyclase assay than in the mitogenesis assay, suggesting the potential of functional selectivity. Furthermore, structure-activity relationships (SAR) were deduced based on function, which was both instructive and provides additional functional data to be compared to in vivo activities for the identification of underlying mechanisms, at the G-protein level. In binding studies, SAR demonstrated that the trans-butenyl linker provided additional D3 selectivity as compared to the saturated linking chain. Moreover, addition of a hydroxy (OH) group in the 2- or 3-position of the butyl linker also gave several highly selective and potent D3 antagonists or partial agonists. Further, replacement of the sterically bulky aryl ring system with various heteroaryl groups served to retain high affinity and selectivity for D3, while decreasing lipophilicity. To this end we have recently discovered very selective D3 antagonists and partial agonists with D3/D2-selectivites reaching 400-fold. In addition, several of these analogues have been further screened for binding in 60 additional receptor and ion channel assays and did not show significant binding affinities at any of these other targets, highlighting that these agents are some of the most potent and selective D3-antagonists and partial agonists reported to date. Further, the (+)- and (-)-enantiomers of one of these 3-OH analogues, PG648, were synthesized and demonstrated enantioselectivity at D3 (>15-fold), but not significantly (<2-fold) at D2 receptors. This was the first demonstration of enantioselectivity of a D3 antagonist and further chimera studies, with these enantiomers, identified a transmembrane region that appears to differ between D3 and D2. Further characterization of these enantiomers and the synthesis of others are currently underway. The latter goal of reducing lipophilicity of the most potent agents was to improve physico-chemical properties that would provide a more favorable pharmacokinetic/bioavailability profile than the currently existing D3 agents. Ten of these analogues are currently being evaluated for pharmacokinetics, blood brain barrier penetration, and for potential metabolic pathways for degradation in vivo, in rats. These compounds are all being tested in a D3-agonist induced yawning model, in rats, to compare their pharmacological and bioavailability profiles in vivo. In addition, several of the most potent and selective compounds of this series have been synthesized in multi-gram quantities and are currently being evaluated in numerous animal models of cocaine and methamphetamine abuse, in both rodents and primates. Chronic studies in these and additional rodent and monkey models of drug abuse and impulsivity are underway, with PG01037 and several other butenyl and hydroxy-butyl linked analogues. In addition, another of these highly selective 3-OH analogues, GCC 3-09 was recently tritiated. 3HGCC 3-09 is currently being developed as a potential radioligand for both rat brain tissue and cell-based binding assays. Our newest series of analogues replaces the 3-OH group in the butyl linking chain with a F-group, and several of these compounds show favorable pharmacological profiles in vivo, with D3/D2-selectivites >200-fold. In addition, we have embarked on an SAR study of novel D2 antagonists, based on the parent ligand L741,626. Structural modification of this molecule has revealed important structural features that impart high affinity for D2 receptors, although improving D2/D3 selectivity has thus far remained elusive. Nevertheless, exceptionally high D2/D3 receptor affinities with some structural motifs have led to the hypothesis that our highly D3-selective ligands maybe bitopic and thus accessing both orthosteric and allosteric binding sites on the D3 receptor. Current synthesis and pharmacological assay development to test this hypothesis is underway."}
{"text": "Fourteen Chinese and sixteen Caucasian normal volunteers were administered a single dose of desipramine (DMI). Plasma and urine samples were analyzed for DMI and an active metabolite, hydroxydesipramine. The total clearance of DMI was lower in Chinese was lower than in Caucasians but the clearance to hydroxydesipramine did not differ between the groups. The renal clearance of hydroxydesipramine was lower in older subjects."}
{"text": "We hypothesize that hypothalamic inflammation associated with high fat feeding arises as a result of Toll-like receptor (TLR) signaling in a specialized cell type lining the inferolateral walls of the third ventricle, the alpha tanycyte. We propose that the activation of NF-?B in these cells releases inflammatory mediators into feeding-related centers in the mediobasal hypothalamus, increasing feeding and/or altering the sensitivity of arcuate/ventromedial nucleus neurons to leptin and/or insulin. To test this hypothesis, RNA-Seq analysis will be used to identify the complete gene expression profile of alpha tanycytes in response to a HFD, with particular emphasis given to the identification of genes and pathways involved in inflammation. Based on this analysis, the effect of a HFD on food intake, energy expenditure, glucose homeostasis, leptin and insulin sensitivity, and other parameters of hypothalamic inflammation will be studied in transgenic animals with conditional KO in alpha tanycytes of selected genes identified in the RNA-Seq analysis. We propose that these studies will demonstrate a critical role of tanycytes in the pathophysiology of hypothalamic inflammation associated with high fat feeding, uncovering new knowledge in understanding the pathogenesis of obesity and a potentially new pharmacologic target for the treatment of obesity."}
{"text": "The central goals of this proposal concern the relationship between protein structure and protein function. The powerful techniques of recombinant DNA technology will be applied by four investigators to study distinct groups of biologically important macromolecules. Most of these molecules are involved in cell-cell communication and in most instances, DNA probes are available to allow a more global approach to a full appreciation of the synthesis, expression and activity of a particular protein. The Program Project mechanism is requested as the technologies required to carry out each individual project require not only the intense intellectual interaction presently taking place, but, in addition, the pooling of resources and equipment. All the studies largely involve protocols in the mouse and rat, although the results of these projects will have clear applications to man."}
{"text": "The Case CCC Imaging Research Core Facility provides researchers with state-of-the art facilities and the services for in vivo imaging. The facility provides cancer researchers its extensive imaging research infrastructure and expertise of -26 imaging faculty in Radiology, Chemistry, Physics, and Biomedical Engineering to enhance and expand ongoing research efforts. The advent of dedicated small animal imaging systems allows the integration of in vivo physiologic measurements with microscopic measurements of structural and cellular activities. The collaborative, multidisciplinary approach provides a wealth of new information in elucidating the complex relationship between structure, genetics, replication, and function in genetically manipulated animal models of disease that have been used widely in cancer research with grounding in cellular/molecular-level understanding. The Imaging Research Core Facility is the centerpiece of the Case Center for Imaging Research, which encompasses all aspects of the much broader imaging research program at Case/UH. This includes molecular imaging, small animal imaging, and clinical imaging thrusts. The Core provides services to Case CCC members from 7 of 8 research programs, with members from the Imaging Program and Cancer Cell Signaling utilizing the Core's services the most. The Core has been actively involved in studies of the glioma microenvironment where novel cryo-imaging techniques demonstrated migration and dispersal pathways in vivid three-dimensional detail. In addition, fluorescence imaging systems in the Core were used to discover that while activation of EphA2 with its ligand ephrin-AI inhibits chemotactic migration of glioma and prostate cancer cells, EphA2 over-expression promotes migration in a ligand-independent manner."}
{"text": "The Baltimore Cancer Research Program (BCRP) has developed cancer guidance displays (treatment protocols) on the computerized Problem Oriented Medical Information System (PROMIS) in Burlington, Vermont at the University of Vermont School of Medicine, with the aid of a communication linkage between BCRP and PROMIS. The computerized displays for the logic and action of treating cancer problems are defined by the creation and sequencing of the computer display pages or frames. These displays provide an automatic connective device between the four phases of medical action of the Problem Oriented Medical Record. The problem-defining sequence determines the specific plan and it is that plan which is in part a protocol or series of protocols for treating a specific cancer problem. It is through the use of these explicit information and directional displays that the health care provider can couple learning with doing while gathering information and treating patients' cancer problems. The entire systems package has been shipped to BCRP and testing and debugging has begun in conjunction with the Interagency Agreement with the National Center for Health Services Research (Y01 CM 80109). This project ends on May 31, 1981."}
{"text": "Mammalian stem and progenitor cells activate the expression of specific regulatory genes to establish and stabilize different cell fates, but the mechanisms and general principles controlling this process are not well understood. Cells utilize fate-specifying regulatory genes to establish and maintain distinct fate identities during development, but it is not clear how they activate and maintain the expression of these genes to establish fate identity. Here, I propose to study this question in the context of two systems: T-cell fate commitment, which is driven by the activation of the T-cell specific transcription factor Bcl11b (Aims 1 and 2); and macrophage development, which we recently found is driven by the cell-cycle length dependent accumulation of the myeloid transcription factor PU.1 (Kueh et al., 2013)1 (Aim 3). Using these two systems, I will test a number of widely debated ideas in the field of developmental gene regulation. First, I will test the idea that developmental signals directly activate the expression of regulatory genes to instruct cell fate (Aim 1). Next, I will tet two proposed classes of mechanisms for stabilizing regulatory gene expression and fate identity: cis-acting mechanisms involving stable and heritable epigenetic modifications at regulatory gene loci (Aim 2); and trans- acting mechanisms involving self-reinforcing positive feedback loops on regulatory gene expression (Aim 3). My main approach will be to use timelapse live-cell imaging to track the expression dynamics of Bcl11b during T-cell development, and PU.1 during macrophage development. As cell differentiation is a dynamic and intrinsically heterogeneous process, single-cell tracking by timelapse imaging will reveal insights that are difficult to obtain with conventional discrete time-point population measurements. To gain mechanistic insights, I will perturb the mechanisms under investigation, and measure their resultant effects using timelapse imaging. These perturbations will involve over-expression or knockdown of genes; for studies of cis-epigenetic mechanisms, I will also develop a CRISPr-based system for perturbing chromatin marks at specific sites in the genome. To better understand this experimental data, and generate predictions for future experiments, I will then use mathematical modeling to analyze the behavior and dynamics of the different regulatory mechanisms studied. Finally, I will complement these approaches with genome-wide measurements of gene expression states in developing cells using high throughput sequencing, which will provide a more global picture of developmental changes, and potentially yield new directions for future work. Through these studies, I hope to uncover fundamental insights into how mammalian cells establish and maintain their distinct fate identities. These insights will potentially help us develop new therapies for leukemia and other cancers, and help us better manipulate stem cells for regenerative medicine."}
{"text": ": This application proposes research related to NIMH Division of Services and Intervention Research. The aim is to design and test the first prototype of an interactive self-help computer program for binge eating disorder. The prototype will be based on an integrative view of the etiology of binge eating disorder, and will combine elements of various empirically supported treatment approaches. Each user will receive a customized learning experience, resulting in customized feedback from the program. We will conduct the following evaluations of the module: (1) Expert Reviews of Content and Usability, (2) Stage One Usability Test (with eating disorder subjects), and (3) Stage Two Usability Test (with eating disorder subjects). Dependent measures and formal criteria for success are specified. PROPOSED COMMERCIAL APPLICATION: Potential commercial markets for eating disorders self-help technology include psychotherapy clients, mental health professionals, and health maintenance organizations."}
{"text": "The psychoactive component of marijuana, ?9-tetrahydrocannabinol, binds to and activates cell surface receptors called cannabinoid receptors. Activation of these receptors produces intense responses in humans, which suggest that endogenous cannabinoid (endocannabinoid) substances may contribute in important ways to brain functions as diverse as appetite, mood and pain. Two such substances are anandamide and 2- arachidonoylglycerol (2-AG), lipid-derived compounds that are released from neural cells and activate cannabinoid receptors with high affinity. Anandamide and 2-AG undergo a rapid deactivation process, which contributes to arrest of their biological actions. In the case of anandamide, this process is thought to consist of two steps, transport into cells and intracellular hydrolysis by the enzyme fatty-acid amide hydrolase (FAAH). By contrast, much less is known about 2-AG deactivation. Previous work in our lab has provided evidence that the enzyme monoacylglycerol lipase (MGL) - a presynaptic serine hydrolase that converts 2-AG into arachidonic acid and glycerol - participates in 2-AG hydrolysis. Based on these results, we hypothesize that MGL is an essential component of the physiological mechanism by which 2-AG is deactivated at brain synapses. Our first objective is to discover potent and selective inhibitors of MGL activity. In our initial studies, we have identified two promising leads, which will be optimized through a series of experimental and computational approaches, including structure-activity relationships, site-directed mutagenesis, mass spectrometry and molecular modeling. Our second objective is to characterize 2-AG transport into neurons and define the role of MGL in this process. We will combine pharmacological and genetic strategies to test the hypothesis that MGL- mediated hydrolysis provides the driving force for neuronal 2-AG internalization. Finally, we will use MGL inhibitors and mutant mice that overexpress MGL in the forebrain to determine whether 2-AG hydrolysis terminates the behavioral effects of endogenous 2-AG. We will focus our attention on two behaviors - feeding and stress-coping responses - in which as-yet-unidentified endocannabinoid signals are thought to play a crucial role. These studies will help uncover the functions served by the endocannabinoid system in the brain and may open innovative avenues for the treatment of neuropsychiatric and substance abuse disorders. PUBLIC HEALTH RELEVANCE: Marijuana works by mimicking important transmitter substances in the brain, called endocannabinoids. The objective of our research is to understand how these transmitters are produced and eliminated, and discover innovative medicines that target these processes."}
{"text": "DESCRIPTION: The Hsp70 chaperone system of E. coli consists of the Hsp70 homologue DnaK, the Hsp40 homologue DnaJ, and the Hsp20 homologue GrpE. The coordinated action of these proteins is important in the folding of some proteins and in regulating the action of several macromolecular assemblies. This proposal focuses on the DnaJ component of this chaperone system. DnaJ is a co-chaperone that forms complexes with DnaK and also with substrate proteins. It contains four evolutionarily conserved, functionally distinct domains or modules. The structure of individual modules of DnaJ will be determined using 13C/15N/1H heteromuclear multidimensional NMR spectrosocpy, and residues that are important for function will be identified by site-directed mutagenesis. Complexes between modules of DnaJ and fragments or domains of its various protein partners will be characterized by NMR spectroscpy. These studies will provide insight into the roles of DnaJ and its homolgues in this important chaperone system."}
{"text": "Glioblastoma multiforme (GBM) is the most common malignant glioma and has an extremely invasive phenotype. The ability of glioblastoma cells to diffusely infiltrate healthy tissue is the major obstacle for current treatments, including surgery and radiotherapy. Over the years, established human glioblastoma cell lines were used as models to study molecular mechanisms of invasion; however, these mechanisms remain elusive since established lines are not invasive in vivo. In contrast to established lines, primary non-established glioblastomas, including GBM6 and GBM12 cells, are invasive in vivo, and thus can be successfully used to study growth, motility, and invasion. Recently, an elevated expression of sphingosine kinase 1 (SphK1), an enzyme that generates sphingosine 1-phosphate (S1P), was correlated with poor prognosis of patients with GBM. S1P is a potent lipid mediator important for cancer growth, survival, migration, and invasion. In the brain, S1P is present at high concentrations, and has been shown to stimulate migration and invasion of established glioblastoma lines in vitro. The activity of SphK1 is rapidly and transiently stimulated by various growth factors and cytokines; however, mechanisms of the transcriptional regulation of the sphk1 gene are not understood and the factors enhancing SphK1 expression in GBM patients have not been identified. Recently, inflammation has been linked to the development and progression of many cancers with inflammatory cytokines involved in these processes. IL-1 is one of the major regulators of inflammation in the brain, which can induce the secretion of other proinflammatory cytokines and also promote proliferation of glioblastoma cells in vitro. More importantly, glioblastoma cells secrete significant amounts of IL-1. In preliminary studies, we show that IL-1 is coexpressed with SphK1 in GBMs, exogenous IL-1 increases expression of SphK1, and both IL-1 and S1P enhance invasion and proliferation of glioblastoma cells. More importantly, neutralizing anti-IL-1 antibodies inhibit proliferation of glioblastoma cells in vitro, while a novel SphK1 inhibitor (SKI) reduces xenografted glioblastoma tumor growth in mice. It is our hypothesis that the IL-1-mediated stimulation of SphK1 expression leads to the production of S1P, which in turn activates proliferation, motility, and invasion of GBMs. We propose to study SphK1 functions and its regulation in primary GBM cells. Moreover, development of potent and specific SphK1 inhibitors that are active in animal models could pave the way for new therapeutics for treatment of GBM. Our specific aims are as follows: Aim 1. To determine the role of SphK1 expression and the effect of its inhibition on proliferation, motility, and invasion of primary non-established GBMs in vitro, and xenografts implanted in the brains of athymic mice. Aim 2. To identify the mechanisms that control endogenous SphK1 expression in response to IL-1. PUBLIC HEALTH RELEVANCE: Glioblastoma multiforme (GBM) has an extremely invasive phenotype, which is the major obstacle for current treatments. It is our hypothesis that the IL-1-mediated stimulation of SphK1 (sphingosine kinase 1) expression plays an important role in GBM growth and invasiveness, and may be a potential therapeutic target. Therefore, we propose to study SphK1 functions and its regulation in primary non-established GBM cells. Moreover, development of potent and specific SphK1 inhibitors that are active in animal models could pave the way for new therapeutics for treatment of GBM. [unreadable] [unreadable] [unreadable] [unreadable]"}
{"text": "Several hypotheses have been created to account for the neurodegeneration and subsequent cognitive deficits observed in Alzheimer's disease. One hypothesis, in particular, has focused on the effects of inflammation as a mediator of neurodegeneration. To address this possibility, we have initiated studies examining the direct and indirect effects that endotoxin and inflammatory cytokines may have on neural tissue and neuronal cell signaling. Initial studies have demonstrated that the intracerebral infusion of endotoxin produces a significant age-related increase in brain tumor necrosis factor- alpha (TNFa) levels, but does not effect the production of a number of other inflammatory cytokines such as interleukin-1 or interleukin-12. We are currently examining young and old rodent brain homogenates and plasma post endotoxin treatment for the presence of various inflammatory cytokines (e.g., interleukin-6, interleukin-4, interleukin-10) and chemokines (e.g., interleukin- 8, MCP-1). Moreover,animals are also being examined for the effects of direct cytokine administration on inducing brain inflammation, permeabilizing of the blood-brain barrier, and effects on leukocyte trafficking, CNS surface markers, neurodegeneration, and cognitive behavior. We believe that cytokine and chemokine infusion (icv and iv) in rodent brains will have significant biological and physiological effects on neural tissue and will ultimately reveal a relationship between neuroinflammation, age, and cognitive behavior. These studies have been initiated using intracerebral TNF-a infusion and we hope to soon follow these efforts with chemokine and interferon infusions. In addition, additional studies will also be performed to determine whether administration of signaling inhibitors and antagonists and/or through in vivo leukocyte depletion can ameliorate the biological and physiological effects of administered cytokines. It is our hope that these efforts will assist in our understanding of the contribution of cytokines and chemokines in the neuroinflammatory and neurodegenerative effects observed in various neurotrauma models and age-related disease states."}
{"text": "This proposed Research Center in Practice-Based Research is a collaboration of six well-established practice-based research networks (PBRNs) that have joined together to create the Meta-network Learning and Research Center (Meta-LARC.) Meta-LARC's mission is to sustain a consortium of practice-based research networks dedicated to increasing the quality, effectiveness and safety of primary care through accelerated research and collaborative learning. Meta-LARC networks include the Oregon Rural Practice-based Research Network (ORPRN, home of the new Center), the lowa Research Network, State Networks of Colorado Ambulatory Practices and Partners, Oregon's Safety Net West Practice-based Research Network, the Quebec Practice Based Research Network, and the Wisconsin Research and Education Network. These PBRNs are comprised of 533 primary care practices and over 6,000 clinicians who provide care for an estimated three million patients in rural, urban, and underserved communities. Combined, the networks have conducted over 200 studies and published nearly 200 scientific publications. The specific aims of Meta-LARC are described below: Aim 1: Foster the capabilities of six PBRNs and 533 primary care practices through a robust collaboration designed to conduct research to improve the quality, effectiveness and safety of primary care. Aim 2: Accelerate the conduct of PBRN research through a well designed, high functioning common infrastructure that enables the efficient conduct of research. Aim 3: Promote continuous learning and sharing across Meta-LARC networks and practices to accelerate the dissemination of knowledge and bi-directional communication."}
{"text": "The 37 million Americans who live in poverty smoke at twice the rate of other Americans. As a result, those living in poverty bear a disproportionate burden from tobacco-related diseases, including lung cancer, other lung diseases, and heart disease. A number of beliefs serve as barriers to their use of evidence-based treatment for tobacco dependence. These beliefs include: 1) smoking is both normative and acceptable under some conditions;2) willpower is sufficient for quitting rendering outside help unnecessary and irrelevant;3) evidenced-based treatments are not more effective than other methods;4) quitting medicines are ineffective, dangerous, addicting, and/or too expensive;and 5) treatments for quitting are not available, hard to access, and/or too expensive. Also, while the poor have as much desire to quit as others, their motivation to make a quit attempt in the near term is less than that of other smokers. New ways are needed to bring evidence-based treatment to this hard to reach population. Providing a brief tobacco dependence intervention to the poor seeking services from community agencies is one such way. Specifically, this study will examine the effectiveness of a brief cognitive-motivational intervention designed to challenge these beliefs and motivate a quit attempt by smokers not otherwise motivated to quit who are presenting for services at the Salvation Army (N=140) relative to an attention control group (N=140), a no-treatment control group (N=140), and smokers who are motivated to quit (N=100). The primary outcome is acceptance of a brief treatment intervention. Other outcomes are subsequent quit attempts and abstinence through three months of follow up. In addition, community agency staff who administer the interventions will complete a survey to assess its feasibility in the community agency setting. Data will be analyzed to evaluate study aims: 1) Did the brief cognitive-motivational intervention result in greater acceptance of treatment, quit attempts using evidenced-based methods, and abstinence than the control groups? 2) Did the intervention boost treatment acceptance and quit attempts above the rate by smokers already motivated to quit? 3) Is the intervention feasible for use in community agencies and acceptable to its cliental? If these aims are achieved, a brief, practical, and effective intervention can be available from community agencies that motivates smokers living in poverty to make quit attempts using evidenced-based methods. Wide distribution of this tailored intervention will help decrease the health disparity experienced by smokers living in poverty. PUBLIC HEALTH RELEVANCE: People living in poverty are far more likely to smoke than others and, as a result, have more tobacco-related disease. The poor are not as motivated to quit and have certain beliefs that prevent them from using the best methods of quitting when they do try. This research will determine if a brief intervention designed to motivate and change these beliefs increases quit attempts when it is provided by a community agency that serves the poor. If successful, this research will help reduce tobacco-related diseases among the poor."}
{"text": "Family caregivers to elders suffering with dementia are at increased risk for stress-related illness. This is especially true for Latino caregivers, whose burden is exacerbated by a lack of Spanish-language information on dementia, and/or culturally-competent services. Even when such services are available, Latino caregivers often face access barriers related to scheduling convenience, geographic distances, and the inability to leave their dementia-affected elder unattended. This project addresses the need for convenient, culturally-competent, Spanish-language services for Latino caregivers. We propose the development of a multi-literacy level Spanish-English website for family caregivers to dementia-affected elders. We have already established a prototype website for Latino caregivers in the Boston are, and will further develop it in Phase 1. We will do this by expanding the higher literacy Spanish component of the website, as well as developing lower literacy versions in Spanish and English. An evaluation study of the website as a learning tool will be conducted. In Phase II, we will expand the website to include caregiver resources nationally, and add a customized e-care component offering on-line and call-in center advice from dementia specialists. HMOs can be expected to contract with us to provide the e-care component as a member service."}
{"text": "PROJECT SUMMARY: Project 3 The major goal of Project 3 is to provide a detailed structural and mechanistic understanding of human norovirus (NoV) evolution and replication to aid in the design and development antiviral strategies. Human NoVs are the leading cause of epidemic acute gastroenteritis. Susceptibility to these viruses is determined by genetically controlled expression of histo-blood group antigens (HBGAs), which are also critical for NoV attachment to host cells. As a result of sequence changes in the P domain of the capsid protein VP1 that harbors the HBGA binding site, these viruses show strain-dependent variability in HBGA specificity presenting a fascinating case study in how genotypic variations allow for exploitation of the polymorphic nature of HBGAs in host populations to counter herd immunity and cause epidemics. Recent studies have shown human antibodies that block HBGA binding correlate with protection from NoV-associated illness (Project 1). Several studies have suggested a correlated interplay between antigenicity and HBGA specificity in driving the evolution of NoVs. The goal of AIM 1 is to provide the structural basis for such interplay by structurally characterizing how HBGA-blocking antibodies interact with NoV strains using currently available human monoclonal antibodies and those newly developed in Project 1, using X-ray crystallography and cryo-EM techniques. While the emphasis in AIM 1 is on virus-host interactions, the emphasis in our following two AIMs is on two key virus encoded proteins that regulate virus replication, both of which are potential targets for small molecule drug discovery. AIM 2 focuses on the viral protease that is critical for polyprotein processing. Based on a recent exciting finding by us and others that NoV protease binds to viral RNA, we have hypothesized that it plays a hitherto uncharacterized role in genome replication. In the first part of AIM 2, our goal is to determine the structural basis of genogroup-dependent substrate interactions and inhibition in order to provide a rational framework for the design and optimization of inhibitors in collaboration with Project 1. In the second part of this aim, our goal is to structurally characterize NoV protease interaction with viral RNA, and in collaboration with Project 2, probe into the role of this interaction in viral replication. AIM 3 will focus on Norwalk virus NTPase, p41, a member of a highly conserved family of proteins encoded by a wide variety of (+)RNA viruses that plays a critical role in virus replication by remodeling cellular membranes into vesicular compartments in infected cells. During the current grant period, we provided the first structural characterization of such a protein from our studies on p41. The goal of this aim is to take the next step of addressing mechanism-related questions using a combination of crystallography, cryo-EM and cell-based techniques and to correlate our observations to the functional aspects of this protein during virus replication in collaboration with Project 2. We expect our proposed studies will provide novel structural and mechanistic insight that will have significant impact on immunological, translational, and replication-related aspects of human NoVs."}
{"text": "Lung injury associated with neonatal Respiratory Distress Syndrome and Bronchopulmonary Dysplasia requires understanding developmental responses of pulmonary proteolytic activity and defense mechanisms intrinsic to the newborn lung. Utilizing neonatal pulmonary effluent obtained by sequential tracheal aspiration, the aims of this proposal are to: 1. Determine sequentially, the levels of protease activity and protease inhibition in pulmonary effluent of human preterm infants and to relate these biochemical indices of lung injury to radiographic and ventilatory assessments to the severity of acute abd chronic lung disease, 2. Using homologous surface active materials derived from term human amniotic fluid, the proposed investigations seek to define whether surfactant supplementation in 20-30 preterm, infants per year with respiratory distress syndrome alters the clinical, radiological, cytologic, proteolytic activity, and protease inhibition activity in infants treated with surface active material supplemention in comparison to infants receiving conventional mechanical ventilatory support with oxygen supplementation using a multiple logistic regression model. In addition, these investigations will define whether or not evidence of immune complex formation results in infants receiving homologous surface active material using conglutinin assays, as well as, measurement of anti-surfactant antibody formation in the sera of treated infants throughout the first year of life in order to determine whether detectable immunologic reactivity occurs from tracheally instilled surface active material in developing preterm infants, 3. To determine whether active Alpha1 protease inhibitor levels, elastase levels, and alastase inhibitory capacities are altered in neonatal rabbits in graded exposure to varying concentrations of FiO2 previously established to result in lung injury, 4. Using neonatal and young New Zealand White rabbits, describe the effects of graded exposure of intratracheally instilled human derived surfactant on lung histology, presence of circulating antigen, and measures of pulmonary immunoreactivity."}
{"text": "The nature and function of metal atoms in enzymes and proteins has been a keystone of biomedical research since Warburg's initial discoveries in the 1930's on \"heavy-metal enzyme catalysis\" and Keilin's identification of hemoproteins. Many biophysical approaches to the study of these heavy metal enzymes have been in process. Optical and electron paramagnetic resonance studies are good examples of those that give definitive information on many properties but have \"blind spots\" that fail to give incisive information on the metal atoms in all valences or states, i.e., certain properties are \"invisible\" to most techniques. K edge absorption of X-rays by heavy metal atoms occurs in all valence states and gives definitive fingerprints of the nature of such atoms and their electronic configuration and chemical environment. Extended edge absorption studies (exafs) give many other properties, some of which can be interpreted as precise distance measurements to neighbors of the heavy metal atoms. While related data may ultmately be inferred from x-ray or nuclear magnetic resonance data, the great potential advantages of the exafs method are the possibilities of structural studies of the metalloproteins that are time resolved at enzymatically functional concentraions. Optical and epr sample monitoring detects any change of valence or liganding under synchrotron irradiation. This proposal seeks to set up instrumentation development and testing of components that increase the efficiency of photon collection from x-irradiated samples, the ultimate goal being to achieve edge absorption EXAFS and anomalous scattering studies in the range appropriate enzyme activity studies, i.e., less than 100 microns M and eventually extending down toward micromolar concentrations. Concomitant with this development are essential innovations necessary for kinetic studies of the state of mental atoms in enzymatic function. Last but not least are improved methods for on-line sample monitoring to ensure the integrity of the biological material throughout the course of the x-irradiation."}
{"text": "There is growing recognition of the importance of 'place' on individual risk of disease, although most studies fail to capture the dynamic nature of the risk environment and its effect on the sex trade has been understudied. The Mexico/US border region is undergoing profound alterations in the environment in which sex work takes place, providing a 'natural experiment' through which we can explore the relative contributions of individual and structural factors on HIV transmission. The primary goals of this research are to define social, spatial, and physical factors affecting female sex workers (FSWs) in 2 border cities and determine their relevance to HIV epidemiology, drug use, and access to services. Tijuana and Ciudad (Cd.) Juarez have witnessed escalating community-level violence and, for Cd. Juarez, changes in the location and visibility of the 'red light' district. While migration and cross-border interactions are major influences, the recent global economic downturn is dramatically altering migration patterns. Based on the above, the specific aims of this project are to: 1) assess changes in social influences on the sex work risk environment over time in both cities and their effect on risk behaviors, HIV/STI incidence, and access to services; 2) determine the locations where FSWs live, work and engage in other activities and their relationship to risk behaviors, perceptions of violence, and access to services; and 3) determine the extent to which the built environment and other sex work venue characteristics relate to individual-level behaviors and HIV/STI incidence. To meet these aims, we will recruit 600 FSWs (300 per city) and collect sociodemographic, location, and behavioral data through interviews. All will be tested for HIV, syphilis, gonorrhea, and Chlamydia and treated as needed. Follow-up interviews and testing will occur at months 6, 12, and 18. To meet aim 2, we will construct a geographic information system (GIS) of both cities and explore factors in relation to where FSWs live, work, buy/use drugs, and access services. Changing spatial relationships, such as the dispersal of the Cd. Juarez Zona Roja and intra-urban or cross-border mobility, will be analyzed to track patterns of infectious disease spread. We will also conduct in-depth interviews and an activity-travel survey with 30 sex workers per city, stratified by geography and venue (e.g. street, bar, etc.) to create geo-narratives based on time-geographic methods and computer-aided qualitative data analysis in order to explore the impact of recent social, economic, political, and other structural changes on participants' lives. To meet aim 3, field measurements of the built environment and other venue characteristics will be combined with individual-level data to explore their effect on health outcomes. The data collected will provide information vital to reframing HIV and drug use interventions to take into account the structural environment. This project is timely as HIV prevalence is rising along the U.S./Mexico border, presenting a window of opportunity to prevent transition to a generalized epidemic."}
{"text": "The purpose of this trial is to determine if there is a difference between different routes of intermittently administered rhIL-2 (IV vs subcutaneous admininistration) with a highly active antiretroviral regimen compared to highly active antiretroviral therapy alone with respect to the proportion of subjects achieving a 50% increase in CD4 counts above pre-randomization baseline values after one year of rhIL-2 and the rate of change in CD4 counts. Another primary hypothesis will focus on safety, tolerance, and effect on quality of life of these regimens. A third aim will focus on changes in immune cell phenotypes and function and on HIV viral load and rate of antiviral drug resistance development when HAART is used in the setting of rhIL-2 compared to HAART alone. The ACTG 872 Substudy will examine specific and different immunologic effects when comparing the two regimens of rhIL-2 plus HAART vs. HAART alone. The ACTG 874 substudy will determine the cellular source of HIV production in lymph node tissue before and after treatment with HAART, and the changes induced in lymph node tissue with the addition of exogenous rhIL-2, in a subset of subjects (n=12). This is a randomized study to ascertain the effect on CD4 response of high dose, intermittently administered rhIL-2 administered by continuous intravenous infusion versus subcutaneous injections in conjunction with highly active antiretroviral therapy versus highly active antiretroviral therapy alone in patients with HIV and CD4 counts of 50-300 cells/mm3. Subjects must be protease inhibitor therapy naive. All subjects will receive treatment with indinavir and 2 nucleoside analogues; one of which is new to the subject (HAART). Subjects will receive 12 weeks of HAART. At week 10 an HIV RNA by bDNA (real time) will be done by Chiron. Subjects with < 5,000 copies/ml will be continued on study. Subjects with > 5,000 copies/ml will require no further follow-up and should be taken off study. Subjects who have < 5,000 copies/ml of HIV RNA will then be randomized to continue HAART alone or HAART with rhIL-2 at either 9 million units by continuous infusion (CIV) for 5 days every 8 weeks for 72 weeks, or 9 cycles, or rhIL-2 at 7.5 million units by subcutaneous injection twice a day for 5 days every 8 weeks for 72 weeks or 9 cycles. Subjects on the CIV rhIL-2 arm who achieve both a $ 25% increase and at least a 100 cell/mm3 increase in CD4 count above the prerandomization baseline value after 3 cycles or after 6 cycles will switch to rhIL-2 at 7.5 MIU by subcutaneous injection twice a day for 5 days every 8 weeks for the remainder of their treatment course."}
{"text": "The long-term goal of the candidate is to be an independent investigator conducting interdisciplinary studies on neural correlates of substance use disorders (SUDs), especially brain-based predictors of treatment outcomes of SUDs by integrating neuroimaging and clinical trial methods. Brain-based predictors have the potential to guide the optimal patient-therapy matching to improve the efficacy of treatment for SUDs. His short-term goal is to gain required skills for achieving his long-term goal through the training supported by this K01 Award. The candidate received training in Medicine and Diagnostic Radiology in China, a Ph.D. in Neuroscience from the Medical College of Ohio, and T32 Postdoctoral training in functional magnetic resonance imaging (fMRI) on SUDs from the University of California Los Angeles (UCLA). Since 2007, the candidate has been a faculty level investigator in the Division of Substance Abuse at the Yale School of Medicine Department of Psychiatry. His research has focused on using fMRI to identify neural correlates of treatment outcomes of cocaine dependence. The candidate now seeks support to expand the scope of his research on neural predictors of treatment outcomes of other SUDs and to obtain additional training focused on mastering diffusion tensor imaging (DTI) analysis and on learning principles of SUD treatment and clinical trial methods. His preliminary findings indicate that better integrity of white matter in the frontal lobes and anterior corpus callosum positively correlates with better treatment outcomes of cognitive behavioral therapy for cocaine dependence. The candidate seeks to replicate and expand these initial findings in studies that use different therapies and with patients of other SUDs. His research strategy will be to conduct a series of analyses of fMRI, DTI, and treatment outcome data to identify neural predictors and correlates of treatment outcomes of patients dependent on cocaine or other substances participating in 5 clinical trials of several different therapies for SUDs. His training program will cover the following topics: 1) enhancing and expanding his expertise in neuroimaging, 2) learning clinical trial methodologies for SUDs, particularly as they relate to brain measures, 3) sharpening interdisciplinary thinking about brain correlates of addiction treatments, 4) enhancing statistical knowledge for longitudinal research, 5) enhancing knowledge on ethics related to human studies and responsible conduct of research, and 6) training in preparation for manuscripts and grant proposals. His mentors include internationally prominent experts in neuroimaging (Marc Potenza, M.D., Ph.D., Godfrey Pearlson, M.D.) and in SUD treatment research (Bruce Rounsaville, M.D., Kathleen Carroll, Ph.D.)."}
{"text": "Filipinos in Hawaii have high rates of end-stage renal failure and are disproportionately represented on the local transplant waiting list. The goal of the proposed project is to increase cadaveric organ, tissue and eye donation by Filipinos in Hawaii through a comprehensive statewide education program. The project's alms are to increase donor card signing and family discussions among Hawaii Filipinos as well as to increase the number of cadaveric organ donors to 35% of Hawaii donors. The project will partner with Filipino health, social, cultural and religious organizations and will use interviews, review of records, focus groups and surveys to: identify why Filipinos have historically low rates of donation; evaluate Hawaii's previous Filipino education program (MOTTEP of Honolulu); and develop minority-targeted messages delivered by ethnically and culturally-similar and sensitive messengers. The messages will emphasize use of personal experiences, trusted community leaders and mechanisms such as informal networks and Filipino media. Filipino health care workers will also be trained to assist the local organ procurement organization in offering the option of donation to Filipino families of potential organ donors. Because Filipinos will soon comprise the largest Asian-American ethnic group in the United States, Bayanihan may serve as the model for other under-represented minority ethnic groups throughout the United States in the future."}
{"text": "Introduction: The events of September 11, 2001, and the deliberate bioterrorism attack with anthrax that followed, led to profound changes in the biologic research and public health agenda of the United States. The recognition that the United States was vulnerable to international terrorism, and the tremendous impact that a relatively limited biologic weapons attack had on the nation, resulted in major changes in the research focus of government, academic, and commercial entities. Ml AID understood that the United States lacked the research capacity and infrastructure to respond to the threat posed by biological weapons and emerging infectious diseases. The Regional Centers for Excellence in Biodefense and Emerging Infectious Diseases Research (RCE) program was a central component of the NIAID response to this threat, and was designed to \"jumpstart\" research in biodefense within each of the 10 public health service regions of the U.S. In 2003, Washington University in Saint Louis, Saint Louis University, the University of Missouri, Case Western Reserve University, and the Midwest Research Institute received funding to establish the Midwest Regional Center for Excellence in Biodefense and Emerging Infectious Diseases Research (MRCE) to support research on biodefense in Region VII (Iowa, Kansas, Missouri and Nebraska) and parts of Ohio. The goals of the MRCE were consonant with the goals of the RCE program: harness the best scientists in the region for discovery and translational research designed to lead to the next generation of therapeutics, diagnostics and vaccines for biological threats;provide training and career support to create a new generation of scientists working in this field;establish core facilities to support researchers throughout the region;and develop an emergency response plan that can rapidly bring to bear our scientific expertise and research capacity in the event of any new biological threat to the region, the nation or the world. Over the past 5 years the MRCE has made significant progress towards each of those goals, and we will detail our accomplishments in subsequent sections. Given the scope of the program, and our aspirations, it is not surprising that much remains to be done. While the fundamental goals of the MRCE remain the same, this competitive renewal is built upon a research landscape that has been transformed by the RCE program. When we prepared the MRCE proposal 5 years ago, there was strength in basic immunology and in microbial pathogenesis research at the partner institutions, but relatively little was focused on the NIAID priority pathogens. Research collaborations between the MRCE partner institutions did not exist on any substantive level, and even intra-institutional collaborations in this research area were limited. There was no pipeline of young scientists and clinicians interested in research careers in biodefense, and there were no established biosafety training programs within the region. The MRCE now coordinates and supports a vibrant, highly collaborative and closely integrated group that has become the face of biodefense and emerging infectious diseases research throughout most of Region VII. MRCE programs have created a pool of talented young investigators interested in biodefense and emerging infectious diseases research. We have trained nearly 100 individuals to work safely at BSL3, and have educated investigators and public health officials throughout the country. The MRCE has responded to three separate regional emergencies, and has become an integral part of the disaster planning process for a significant part of our region. We have more than doubled the number of participating institutions, and have supported researchers from 10 different academic or industrial organizations. Most importantly, MRCE investigators made seminal basic science and translational discoveries leading to new therapeutic targets for West Nile, Dengue fever, pneumonic plague, Ebola, poxviruses, and a wide sector of RNA and DNA viruses (section A.3). We also initiated a pioneering collaborative effort in pathogen discovery and assessment that led to the recognition of 7 new viruses, one of which (WU polyomavirus), may be an important cause of respiratory disease in children. In our original application 5 years ago, we discussed how one could best assess the impact of a complex and wide ranging program like the MRCE on the research effort in Region VII. We proposed then that the most objective measure would be the change in extramural funding for biodefense and EID research in Region VII (excluding MRCE funds) during the course of the MRCE program. Since 2002, (the baseline pre-MRCE year) total extramural funding for biodefense and EID research (excluding MRCE and regional laboratory allocations) in Region VII has increased more than 2.5 fold, with the greatest increases in 2006 and 2007 despite a flat NIH budget. We believe the MRCE program has had a significant impact on Region VII and the nation, and are committed to continuing this important work."}
{"text": "The active constituents in allergenic extracts of Poison Ivy and other plants of the genus toxicodendron have been generically designated as \"urushiol\", a mixture of penta-or heptadecenyl catechols. Objectives of this project are: a) to develop methods for the quantitative analysis of urushiol, including speciation of individual compounds; b) to develop sample preparation techniques to isolate urushiol from plant oil matrices; c) to develop methodologies to determine oxidized and oligomerized urushiol degradation products; d) to correlate urushiol degradation with factors of concentration, solution type and storage conditions. Urushiol samples have been speciated and quantitated by gas chromatography of trimethylsilyl derivatives. Validation studies have been performed for this method. A normal phase HPLC method utilizing a silica column is to be investigated. Development of a capillary gas chromatographic procedure, providing better resolution of individual compounds is in progress. Procedures for the isolation of urushiol at high (greater than 100 ppm) concentrations from plant oils have been validated."}
{"text": "This proposal requests support for a Keystone Symposia meeting entitled Immunity in the Respiratory Tract: Challenges of the Lung Environment, organized by David L. Woodland, Liise-anne Pirofski and Allen G. Harmsen, which will be held in Vancouver, British Columbia, Canada from February 26 - March 3, 2011. Recently, there has been an explosion in information about the regulation of inflammation, immunity, and immunopathology in the lung. Despite these advances, we still have only a rudimentary understanding of how these responses relate to the lung environment or how and why they translate into beneficial or detrimental effects, health or disease. This meeting will bring together allergists, immunologists, microbiologists and vaccinologists to discuss pulmonary inflammation and immunity in the context of lung biology, and to build on this understanding to promote development of improved vaccines and therapeutic interventions for inflammatory conditions and infectious diseases that affect the lungs. Expert talks in plenary sessions will present the latest research in the field. We anticipate that the meeting will considerably advance our understanding of respiratory immunity. Moreover, opportunities for interdisciplinary interactions will be significantly enhanced by the concurrent meeting on Mucosal Biology: A Fine Balance between Tolerance and Immunity, which will share a keynote address and two plenary sessions with this meeting. Project Narrative: Pneumonia is a leading killer of children worldwide under the age of five years, as well as an important cause of morbidity and mortality in adults in the developed and developing world. In light of this, the importance of the Keystone Symposia meeting on Immunity in the Respiratory Tract: Challenges of the Lung Environment cannot be overestimated: leading scientists from diverse backgrounds and settings will engage in critical discussions about the disease and challenges to understanding immunity in the lung and controlling lung inflammation."}
{"text": "Reconstituted high-density lipoprotein (R-HDL) has been shown in vitro and in vivo to have antiendotoxin properties. The hypothesis is that lipoproteins bind, neutralize, and increase clearance of endotoxin. Being a natural biological product, R-HDL could be used prophylactically to prevent septic shock in high-risk patients and in chemotherapy-induced neutropenia, or therapeutically to treat septic shock. To test this hypothesis, in a controlled, randomized trial, we investigated the effects of R-HDL on survival, endotoxemia, cytokine production, and pathophysiologic and metabolic events in our canine model of septic shock. At 0.5, 8, and 16 h after implantation of an E. coli-infected clot, canines received intravenous R-HDL control lipid, or human serum albumin (HSA). Animals treated with R-HDL had lower levels of circulating endotoxin and tumor necrosis factor (TNF) and a smaller decrease in white blood cell counts than did animals treated with lipids and HSA. Survival times of lipid-and HSA-treated animals were similar and were significantly greater than those of R-HDL-treated animals. R-HDL-treated animals had significantly greater abnormalities in liver function compared to lipid- and HSA-treated animals. In normal animals, R-HDL caused transient elevation of liver enzymes. In animals given sterile clot intraperitoneally, R-HDL caused seizures. Thus, this preparation of R-HDL had antiendotoxin effects, but also had hepatic and neurologic toxicities. We now plan to investigate a different preparation of HDL that has a different chemical composition than that used in this canine experiment. This new preparation was manufactured so as to limit the potential of liver and neurological toxicity. First, we will investigate the new R-HDL preparation in small animal models of live-infection (rats), and subsequently, if no toxicity and beneficial antiendotoxin effects are documented, in our canine model of septic shock. If toxicities associated with R-HDL can be reduced, its antiendotoxin effects may potentially be used prophylactically in high-risk patients, or therapeutically in patients with established septic shock."}
{"text": "Research project CA-03641 deals with pathways and rates of hydrogen flow in intracellular oxidation-reduction reactions. Our studies are based on the concept that, in intact cells, pathways of hydrogen are determined not only by thermodynamic properties of the interacting systems but also by factors which influence the kinetics, such as heterogeneity of distribution of dehydrogenases within intracellular compartments, specific enzyme-enzyme interactions, and the different strength of binding of the pyridine nucleotide coenzymes by dehydrogenases. A primary objective of these studies is to attempt to locate steps which control the rates of biological oxidations in intact cells, so that it may be determined whether control of the reductive biosynthesis of cellular constituents, i.e. fatty acids, cholesterol, etc. occurs at the level of the obligatory oxidation-reduction reactions which are an essential part of the biosynthetic process."}
{"text": "Metastatic melanoma is a deadly disease with an expected five-year survival of only 14%. The goal of this project is to better understand the environmental and genetic factors which cause this often-fatal disease in the hope that we may suggest approaches for disease prevention and therapeutic intervention Epidemiology has suggested that ultraviolet (UV) radiation and the red/blond, fair-skinned pigmentation phenotype are two major melanoma risk factors. However, genetically controlled experiments to establish an unambiguous causative link is lacking. This project aims to create a series of mouse models for two main objectives: (1) to establish a clear link between UV, pigmentation and melanoma in order to provide better public health information, and (2) to use these mouse models to understand the molecular mechanisms by which UV and high risk pigmentation variants produce melanoma. In order to produce a novel melanoma mouse model, red/blond, albino, and black mice which also carry an activatable form of BRAF or NRAS (the most commonly mutated oncogenes in melanoma) are being generated. Preliminary experiments using this system have already revealed the striking observation that a significant number of the red/blond mice develop melanomas, while none of the albino mice and very few of the black mice develop the same lesions. With this data suggesting there is a strong genetic tumorogenic effect of the red/blond phenotype, it is likely that examining the role of UV will provide exciting new information. To pursue the first aim, melanoma rates between UV irradiated red/blond, albino, and black mice will be compared to determine the effects of pigmentation and UV alone. Next, melanoma rates between UV irradiated black mice that carry either of the 2 most commonly mutated oncogenes in melanoma (BRAF or NRAS) will be compared to determine the effects of oncogene activation and UV irradiation. Lastly, melanoma rates after UV irradiation of red/blond and albino mice carrying the same mutated oncogenes will be compared to see how the 3 variables work together to alter melanoma risk. The goal of the second aim is to understand the mechanisms by which UV and the red/blond phenotpye induce disease. Initially it will be investigated if the high rate of melanoma in the red/blond context is pigmentation dependent. Next, it will be investigated if the mechanism of red/blond-melanoma is via reactive oxidative damage, as suggested by previous studies showing that red/blond pigment can promote release of reactive oxidative species (ROS). To investigate this question, the types of DNA damage in UV irradiated red/blond, albino, and black mice will be compared. Finally, the ROS burden in melanocytes will be genetically and pharmacologically altered to investigate if this strategy can block red/blond-melanoma induction. PUBLIC HEALTH RELEVANCE: Metastatic melanoma is a deadly disease with an expected five-year survival of only 14%. Alarmingly, in the United States, the incidence of melanoma is rising at a faster rate than any other malignancy. The goal of this project is to better understand the environmental and genetic factors that contribute to development of this often-fatal disease, in the hope that we may use this knowledge to suggest approaches for disease prevention and nodal points for therapeutic intervention"}
{"text": "This Mentored Patient-Oriented Research Career Development Award proposes a program of tailored mentorship, focused research, didactics and coursework to provide the candidate with the training in sleep medicine and perinatal neuroendocrine functioning necessary for independence as an investigator. Candidate. The candidate's background and foundation in clinical research make her well-poised for this award. She completed her Ph.D. in clinical psychology at the University of Alabama, where she received training rooted in the scientist-practitioner model. An emerging interest in sleep and mood led her to pursue a postdoctoral fellowship at the University of Michigan, where she began to develop a niche in behavioral sleep medicine and perinatal mental health. She was recently appointed to the faculty of the Department of Psychiatry at the University of Michigan. Her goal is to receive additional training in sleep medicine and perinatal neuroendocrinology. The candidate has identified two key training objectives necessary to achieve full independence as a researcher: (1) experience in sleep physiology (including assessment and treatment of sleep-disordered breathing in pregnancy) and (2) neuroendocrinology in pregnancy. Environment. The proposed project will take place within the University of Michigan Department of Psychiatry and the Sleep Disorders Center. Both departments have an extensive history of close collaboration with the Department of Obstetrics and Gynecology. The fifth largest research university in the United States, the University of Michigan provides extensive resources and a rich research environment. The candidate will have access to the large, accredited Sleep Laboratory within the Sleep Disorders Center, the well-established perinatal recruitment infrastructure within the Women's Mental Health and Infants Program (in collaboration with the Department of Obstetrics), and resources provided by the CTSA-funded Michigan Institute for Clinical and Health Research. The proposed primary mentor, Ronald Chervin, M.D., M.S., is a well-funded, highly productive expert in sleep-disordered breathing who has more than 20 years of experience in sleep medicine and a long history of mentoring K awardees and junior faculty. Two carefully selected co-mentors, physiologist Louise O'Brien, Ph.D., M.S., and psychiatrist Juan Lopez, M.D., will provide focused training in perinatal sleep and neuroendocrinology, respectively. The proposal is also bolstered by a tailored consultant team. Research. The goal of this application is to understand the contribution of sleep-disordered breathing (SDB) to one of the most common and debilitating adverse pregnancy outcomes, perinatal depression. It is our hypothesis that positive airway pressure (PAP) treatment of SDB in pregnant women with comorbid depression will result in significant reductions in depressive symptoms and improved neuroendocrine functioning. This proposal will leverage our unique expertise with PAP for SDB in pregnancy and adverse pregnancy outcomes, including perinatal depression. The rationale is that a better understanding of the role of SDB and the impact of PAP treatment on perinatal depression will allow for development of targeted and critically-timed interventions which may have long-term benefits for both women and their offspring. We plan to test our hypothesis by randomly assigning 50 women with SDB to receive either positive airway pressure treatment (PAP group) or treatment as usual in obstetrics (non-PAP group). The following specific aims will be tested: AIM 1: Determine the impact of PAP treatment on prenatal depressive symptoms in pregnant women with SDB and depression. AIM 2: Identify the contribution of PAP treatment on the HPA axis in pregnant women with SDB and depression. An exploratory aim is to assess associations between PAP treatment and adverse pregnancy and neonatal outcomes in women with depression and SDB. With respect to expected outcomes, the proposed work will demonstrate that SDB during pregnancy plays a role in perinatal depression, and that treatment of SDB during pregnancy will ameliorate depressive symptoms and HPA axis functioning, and improve pregnancy and neonatal outcomes. Such findings are expected to have an important positive impact, because they will further our understanding of the role of SDB in adverse pregnancy outcomes and reduce the burden of the most common and debilitating adverse outcomes experienced by pregnant women. This will have long term health benefits for both women and their offspring. Summary. The proposed K23 will provide key additional training for a promising early investigator to become a clinical researcher in perinatal sleep medicine. The project will also address important, testable questions about the relationships between a common sleep disorder and the debilitating adverse pregnancy outcome of perinatal depression, a major public health concern."}
{"text": "Major depressive disorder (MDD) is a debilitating disorder of altered mood regulation that is precipitated by chronic stress, modulated by serotonin and of unknown molecular pathophysiology. Four prominent risk factors have been consistently reported to influence rates of vulnerability to develop depression: sex, genetic make-up, prior MDD episodes, and early life stress;however no single animal model has comprehensively combined these vulnerability factors. Using the unpredictable chronic mild stress (UCMS) protocol and behavioral assessments of anxiety/depression-like behaviors in serotonin transporter (SERT) mutant and control mice, I have modeled the interactions of sex, SERT genetic make-up, and stress in eliciting depressive-like behaviors, including the increased vulnerability to clinical depression observed in human female subjects and the risk that is conferred by low SERT levels. Furthermore, gene microarray studies performed by our laboratory on the amygdala of postmortem human MDD subjects and UCMS-treated mice have identified ~40 genes whose changes are specific to human MDD and rodent UCMS, and reversed by antidepressant treatment in rodents, thus representing a critical pool of genes differentially expressed according to altered mood. Therefore, in addition to modeling aspects of human susceptibility to develop depression (sex and SERT genetic make-up), the rodent UCMS paradigm induces molecular changes that are predictive of ?depressive states? across species. Using a comprehensive UCMS-based experimental design in the mouse, I will first confirm the role of sex and SERT as risk factors to UCMS-induced altered mood regulation and then extend my investigation to two additional factors: disease recurrence and early life stress (Aim 1). Using samples generated in Aim 1, I will then begin characterizing neurobiological phenotypes that underlie the UCMS-evoked depressive-like state (Aim 2), including neuroendocrine changes and quantitative assessment of the amygdala gene expression signature. I hypothesize that while risk factors may differentially increase the susceptibility for developing MDD, the ultimate state of altered mood regulation affects a common set of biological and/or molecular disturbances. Results from these studies will begin characterizing changes in these putative common molecular and neuroendocrine disturbances, will reveal the relative contribution of these susceptibility factors to this common phenotype, and will form the basis of future causative studies aimed at investigating the cellular and molecular basis of MDD."}
{"text": "AIDS stigma, or negative meanings surrounding people living with HIV/AIDS, has been fostered by myths and false notions surrounding the epidemic (Herek & Glunt, 1988, p. 886). AIDS stigma has multiple negative psychosocial consequences including anxiety, depression, disruption of family dynamics, physical and emotional violence, loss of social support and the deterioration of productive relations with health-related professionals (Chesney & Smith, 1999; Kalichman, 1998b; Nord, 1997). Given the growing number of HIV cases among the Latino community in general, and Puerto Ricans in specific, it is necessary to address the eradication of AIDS stigma, particularly among professionals who interact with HIV+ individuals such as those in health related fields (Nord, 1997). AIDS stigma has been documented among health services providers such as doctors, nurses, mental health professionals, social workers and other caregivers (Acuff, et al., 1999; Gordon, et al., 1993; Herek & Glunt, 1988; Rizzo, 2002; Silverman, 1993; Stevenson & Storhm Kitchener, 2000; Trezza, 1994; Ventura, 1999). These stigmatizing attitudes towards HIV+ people are unfortunate, especially when research has reported those who feel stigmatized by health professionals face problems accessing optimal health services (Link & Phelan, 2001; Valdiserri, 2002; Weiss & Ramakrishna, 2001). As recently as 2002 stigmatizing attitudes among health professionals in Puerto Rico have been documented by people living with HIV/AIDS. Findings suggest the need to address AIDS stigma through effective interventions that incorporate other stigmas surrounding the epidemic such as negative attitudes towards sexual orientation, drug users, and women. In order to do so, stigma combinations surrounding HIV/AIDS among health professionals must be explored. The main objectives of this research application are to: 1) to explore stigmatizing attitudes towards people living with HIV/AIDS among health professionals in Puerto Rico, 2) to explore the interaction of other stigmatizing attitudes towards gender, sexual orientation, and drug use with AIDS stigma in the same population, and 3) to refine and pilot test an intervention module for stigma reduction geared towards these professionals. These objectives will be achieved through the implementation of a sequential mixed method study using qualitative in-depth interviews and a quantitative self-administered questionnaire addressing AIDS stigma."}
{"text": "Osteoporosis, a disease characterized by the systemic loss of bone mass and strength resulting in fragility fractures, is rapidly poised to become a major public health threat in the United States. Osteoporosis results from imbalances in bone remodeling, a process characterized by osteoblast-mediated bone synthesis and osteoclast-mediated bone resorption. Because osteoporosis is fairly asymptomatic, and is often detected only after the patient has sustained significant bone erosion, therapies aimed at restoring the eroded bone are equally important as those that target bone resorption. However, unlike the highly efficient repertoire of anti- resorptive drugs that form the mainstay of the current anti-osteoporosis therapy, drugs that stimulate bone formation remain largely underdeveloped. Hence there is a critical need for novel therapeutic targets that will stimulate osteoblast-mediated bone accrual together with the inhibition of osteoclastic bone resorption. Our preliminary studies identify Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) as one such target as its inhibition positively impacts anabolic pathways and negatively impacts catabolic pathways of bone remodeling. Mice null for CaMKK2 possess enhanced trabecular bone mass in their long bones, along with significantly higher numbers of osteoblasts and fewer multinuclear osteoclasts. Moreover, its inhibition offers protection from ovariectomy-induced osteoporosis in mice. The proposed studies will enable us to define the precise mechanism by which CaMKK2 regulates osteoblast and osteoclast differentiation and devise potential strategies of its inhibition in the treatment of osteoporosis. Development of CaMKK2 inhibition as a new generation therapeutic target that promotes robust bone mass accrual while inhibiting resorption will represent a major breakthrough in anti-osteoporosis treatment."}
{"text": "A panel of hybrids of HeLa and a normal human fibroblast differing in tumorigenicity in nude mice was screened for RNA levels specific for 21 oncogenes and for human papillomavirus 18 and for papillomavirus were detected. The levels of three of these oncogenes. Transcripts for 15 of these oncogenes were found to correlate with tumorigenicity. These were fos and ets transcripts, which were present at a higher level in tumorigenic hybrids than in non-tumorigenic hybrids and myb transcripts, which were higher in the non-tumorigenic hybrids. Fos protein levels correlated with fos transcript levels. A non-tumorigenic hybrid, when infected with murine sarcoma virus, did not show alteration in the levels of fos, ets or myb transcripts. A panel of human SV80 cells transfected for the proviral mos gene has been generated. These transfectants differ in tumorigenic potential in nude mice. Explants of the tumorigenic members of this panel do not show any alteration in the size or amount of immunoprecipitable SV40 T antigen consistent with an immunological selection against T antigen expression during tumor formation. RNA has been prepared from the members of the panel for an examination of the levels of mos-specific transcripts. An activated N-ras gene cloned previously from human gastric adenocarcinoma material has been characterized using DNA sequencing and transfection assays of in vitro ligated DNA fragments."}
{"text": "The overall objective of this project is to understand the role of E2F transcription factor in regulation of cell proliferation and how a loss of this regulation can lead to oncogenesis. Indications that the cellular transcription factor E2F plays a role in cell proliferation control was obtained from recent analyses which demonstrated a physical interaction between the tumor suppressor protein Rb and E2F. Further, the loss of Rb function is closely associated with a loss of the Rb-E2F interaction, suggesting that E2F is a functionally relevant target for Rb. Additional proteins involved in cell cycle regulation like p 107, cyclins A and E, and the kinase cdk2 are also found to be associated with E2F, indicating that E2F could be playing a major role in regulating cell proliferation. This project aims at studying the role of E2F in tumorigenesis, differentiation and cell cycle regulation. It will be assessed whether there is correlation between the loss of the Rb-E2F interaction and the generation of a wide variety of human tumors. In addition, the possibility that a mutation of E2F can lead to oncogenesis will be examined. To further understand the mechanism of E2F action, additional proteins that interact with E2F will be identified and cloned. Experiments will also be conducted on the role of E2F and associated proteins in the differentiation process of cells. Finally, attempts will be made to identify the cellular promoters that are targeted by E2F an E2F associated proteins."}
{"text": "Tolerance to nicotine develops with chronic smoking and may be associated with the onset of tobacco dependence. It is unclear how quickly this tolerance dissipates after terminating exposure to nicotine. We propose to longitudinally assess responses to measure doses of nicotine by nasal spray in 30 male and female smokers as they smoke ad lib for two days (baseline), quit for six days, and then resume smoking for two days on the GCRC. We hypothesize that responses to nicotine will become larger over the six days of cessation, showing dissipation of tolerance and then be smaller during the final two days of ad lib smoking, showing reinstatement of tolerance with return to smoking."}
{"text": "PROJECT SUMMARY/ABSTRACT ! There is a looming social and economic liability with a growing elderly population. Annually, a staggering 2.8 million elderly are treated in emergency departments for fall injuries. Falls are the leading cause of traumatic brain injury and the primary cause of accidental death in adults over 65 years. Impaired balance, particularly in the lateral direction, is one of the most important risk factors for falls. Unfortunately, common clinical tests of balance are not sensitive to impairments. This lack of sensitivity makes it difficult to monitor changes in balance control that may increase a person's fall risk (e.g. following a change in medication). Therefore, there is a critical need for more sensitive tests to assess balance. We have shown that delays in neuromuscular control result in instability. This work along with the fact that elderly have slower postural adjustments prompted our research group to develop a test to assess speed of balance control. We have shown that speed of balance control can be reliably assessed, is sensitive to subtle impairments in balance not found with common clinical tests, and has ecological validity as demonstrated by predicting performance in common daily activities known to be predictive of falls. The product of this SBIR is a low-cost, easy-to-use system to assess speed of balance control and predict fall risk. In this supplemental award, three members of the research team will attend the National I-CorpsTM program and perform 100 or more customer discovery interviews. By conducting these interviews, we will increase the breadth and depth of knowledge of the various healthcare ecosystems involved in reducing falls in the elderly and identify pain points and barriers for clinical adoption of our balance assessment technology. This information will be important for learning who and what will drive or resist technology adoption. !"}
{"text": "A systematic investigation to evaluate the effect of ionizing radiation upon liver microcirculation is proposed. By using objective methodology radiation induced changes in oxygen transport to tissue, blood cell aggregation, liver function liver scans, histology and ultrastructure will be determined to ascertain acute as well as late effects. Reported results of a pilot experiment show the feasibility of this approach. After proper quantitation of the inflicted insult, prevention or reversal will be attempted by systmatic testng of peripheral circulation flow improvers, such as low molecular weight Dextran, and drugs known to prevent intravascular red cell and platelet aggregation or adhesiveness such as anti-adhesive drugs and aspirin. Successful completion of these experiments will immediately provide a method to allow radiation treatment of liver cancer, metastatic or primary, a disease today lacking a cure except for chemotherapy palliation."}
{"text": "This study is an extension of the original placebo-controlled trial to determine the safety and efficacy of Copolymer I in patients with relapsing-remitting multiple sclerosis. The controlled phase 3 trial showed that treatment with copolymer 1 was effective in reducing the clinical relapses and in slowing accumulated neurological disability compared to placebo treated patients. Most patients completing the phase 3 placebo controlled study elected to enroll in this open-labeled extension study of copolymer 1 for determination of long term safety and apparent persistence of efficacy. The study will be extended for a minimum of two additional years with the evaluation of all enrolled patients with annual quantitative magnetic resonance imaging, with centralized image analysis conducted at the University of Texas-Houston, Health Science Center."}
{"text": "This proposal focuses on investigating the mechanism of synaptic transmission at the inner hair cell (IHC) afferent synapse in the mammalian cochlea. This most peripheral chemical synapse in the auditory pathway serves as an interpreter translating the hair cell receptor potential into a train of excitatory postsynaptic potentials that activate action potentials in the afferent auditory nerve fibers. The specific features of the IHC afferent synapse critically determine how a sound signal is coded in the inner ear and transmitted to the brain via auditory nerve fiber activity. Our aim is to characterize pre- and postsynaptic mechanism that shape the postsynaptic activity in IHC afferent fibers. We will record postsynaptic activity using the whole cell patch clamp method. The recording sites will be the afferent fiber terminal directly at the inner hair cell synapse and the afferent fiber soma in the spiral ganglion in excised preparations of rat and mouse cochleae. To exactly describe the relationship between presynaptic stimulus and postsynaptic activity, we will make double recordings from IHCs and afferent fiber terminals. To affect transmitter release and define the quantal size, we will record excitatory postsynaptic currents and vary experimental conditions; i.e. changing the temperature or ionic environment and using release-affecting toxins. To understand postsynaptic determinants of synaptic transmission, we will provide a basic description of the waveform of the excitatory postsynaptic potential and its relation to the activation of action potentials. Using pharmacological tools, we will try to identify individual components that shape the postsynaptic potential waveform starting with a focus on different classes of potassium channels. We will also study the role of efferent inputs onto afferent fiber activity. Most recordings will be performed in the already established preparation using postnatal cochleae. In parallel we will extend our recordings to mature cochleae, as our ultimate aim is to understand both development and adult function of the IHC afferent synapse. These studies will further our knowledge of basic mechanisms of peripheral hearing and therefore also will lead to a better understanding of abnormal hearing and deafness."}
{"text": "The Office of Education delivers workshops, programs, and individualized opportunities to a population averaging 300 trainees, including postdoctoral, visiting, and research fellows; clinical fellows and medical students; graduate students; and postbaccalaureate fellows, as well as summer trainees. The activities include, typically: public speaking workshops, job interviewing, writing and editorial services, grantsmanship workshops, career presentations and counseling, teaching opportunities through the NICHD Becoming an Effective Scientist course for postbacs and skills workshops and a training program with the University of Maryland, and management programs. An annual retreat for fellows and graduate students is held each spring to address scientific developments and careers, which includes two keynote addresses, fellow presentations, NICHD alumni leading career round tables, and a poster presentation by each attendee. The program is developed and run by a fellow-student steering committee. Among the office's accomplishments from the past year: Dr. Yvette Pittman continues in the Office of Education as Associate Director. Our TmT (Three-minute Talks) competition, now in its third year, is held in conjunction with NIDCR and NHGRI. Dr. Alex Szatmary in the laboratory of Dr. Ralph Nossal received the third-place award. The office developed an online Annual Progress Review for fellows, launched in 2016, to track scientific and career development and progress; APRs will be provided for BSC site visits of our investigators. The database of NICHD alumni from 2008 to the present continues to be updated, leading to an expansion of our Linked In alumni group. In June 2016, the Division of Intramural Research gave its ninth Mentor of the Year awards to Julian Lui, Ph.D., in the investigator category; and Shlomo Krispin, Ph.D., fellow. Twenty FARE awards were made for the 2017 competition. A Fellows Intramural Grants Supplement (FIGS) continues to recognize and stimulate grant applications among fellows, and the Fellows Recruitment Incentive Award (FRIA) continues to support investigators who recruit postdocs from populations traditionally underrepresented in science. The NICHD Scholars program, in its sixth year, had one PhD graduate and one postbac advance to medical school; of three new postbac fellows, two are continuing for a second year, one is pursuing public health training,and two new postbacs are joining this group. The Scholars program focuses on developing talent and supporting trainees' academic and career progression. The NICHD Connection monthly newsletter published its 75th issue in August 2016 and reaches all members of the intramural division and our alumni."}
{"text": "The principal objective of the research is to develop an ultrasonic dosimetric model or models from which the ultrasonic energy being absorbed by or interacting with tissue can be determined. The emphasis will be to define physical quantities which properly reflect an ultrasonic energy deposition in situ based upon knowledge of the free field distribution and ultrasonic propagation properties of the material. The second objective is to develop a dosimetric format by which patient exposure and particular organ and other body site exposure to ultrasonic energy is determinable. The anticipated format would be in terms of a quantitative parameter, or parameters, and would be useful in any subsequent retrospective or propective epidemiology study. The basic procedure will be to account for all of the ultrasonic energy when a specimen is irradiated. This will be accomplished by measuring the detailed ultrasonic field distribution patterns in the free field, in the field with the specimen in place and within the specimen. The measurement procedures include three absolute techniques and one relative technique. The specimens include phantoms of known acoustical properties and the biological tissues, testis, liver and kidney."}
{"text": "Achieving long-term allograft survival in the absence of ongoing immunosuppressive treatment is an important goal in clinical organ transplantation. Homeostatic mechanisms that regulate T-lymphocyte survival during alloimmune responses are probably central to determining the fate of a transplanted organ. There is evidence to show that T-lymphocytes are susceptible to activation-induced apoptotic death following exposure to high antigen dose or persistent antigenic stimulation. The extent to which activation-induced T-cell death contributes to long-term allograft survival and transplantation tolerance is not known. The underlying thesis in this proposal is that activation-induced death of alloreactive T-cells is necessary but not sufficient for induction of long-term allograft survival and transplantation tolerance; suppression of factors that rescue T-cells from death is required to achieve these outcomes. Specific aim 1 is to study the role of activation-induced T-cell death in the induction of long-term allograft survival and transplantation tolerance to vascularized allografts. Heart transplant acceptance will be examined in gene-knockout mice deficient in membrane molecules or cytokines that mediate activation-induced cell death. Biochemical, histochemical, and flow cytometric techniques will be used to study apoptosis of alloreactive T-cells in vivo and in vitro. Specific aim 2 is to study the role of activated antigen presenting cells in rescuing T-lymphocytes from cell death, thus precluding long term-allograft survival and transplantation tolerance. In vivo and in vitro systems will be used to determine which antigen presenting cell cytokines or membrane molecules can rescue activated, alloreactive T-lymphocytes from apoptosis. An in vivo system consisting of scid mice transfused with T-cell receptor-transgenic lymphocytes will be utilized to study intragraft factors that prevent activation-induced T-cell death. The proposed studies will enhance our understanding of T-cell survival following allostimulation and refine our abilities to design and implement new therapies that induce tolerance to transplanted organs."}
{"text": "This proposal focuses on a brain area that is part of the central extended amygdala: the central nucleus of the amygdala (CEA). The CEA is involved in a number of responses to fear, stress and anxiety and contains distinct cell populations. However, it is not known whether the cell populations have distinct functions, and it is currently impossible to ascertain this in vivo with the tools available. This proposal aims to test the feasibility of developing a selective neurotoxic lesion of a specific neuronal population in the CEA using the neuropeptide B and W type 1 receptor (NPBW1) as a target. This receptor is very discretely expressed, specifically in the lateral CEA, but not in the surrounding areas. Furthermore, this receptor is expressed on cells of the lateral CEA that contain corticotropin releasing hormone (CRH) and dynorphin, but is not expressed on cells that contain enkephalin, or on cells of the medial CEA, a major source of amygdaloid output neurons. A selective ligand for this receptor (neuropeptide W-30) will be custom conjugated with saporin (by Advanced Targeting Systems). Aim 1 focuses on the development of an effective and selective neurotoxic lesion of the CEA. The ligand will bind to the NPBW1 receptor and when internalized will release saporin into the cell, causing the death of the cell. The utility of this approach has been shown for other receptor systems, including selective targeting of cells of the intercalated nuclei of the amygdala, using the mu opioid receptor as a target. The specificity of the localization of the NPBW1 receptor suggests that this lesioning method will selectively destroy cells containing CRH and dynorphin, but spare both enkephalin containing cells in the lateral CEA, and amygdaloid output cells within the medial CEA that are thought to be critical for modulation of several fear and anxiety responses (e.g. fear potentiated startle response). In Aim 2, the potential functions of CRH/dynorphin cells of the central extended amygdala will be assessed in adult male rats. These experiments will initially evaluate the effects of this neurotoxic lesion of the CEA on A) basal anxiety-like behaviors in the elevated plus maze and defensive withdrawal paradigm, and B) fear-potentiated acoustic startle reflexes. It is hoped that the development of a tool to selectively target a specific population of the CEA will help to define its functions, with an initial emphasis on the roles that the cells may play in fear and anxiety-like responses. It is hoped that with time, this work will ultimately lead to the development of neurotoxic lesions of several distinct cell types in the central extended amygdala, including the bed nucleus of the stria terminalis, and collectively, this will lead to a better understanding of the processing of negative emotions that are associated with the development and exacerbation of many fear and anxiety-related disorders. This proposal aims to selectively remove a specific neuronal population in the brain that is thought to be involved in stress, fear and/or anxiety responses. It is hoped that selectively targeting a specific population (rather than the more global approach currently available) will lead to a better understanding of how the brain processes these negative emotions. This in turn will be helpful in the design of treatment strategies for anxiety and fear-related disorders."}
{"text": "Resistance to chemotherapy is a major stumbling block in the treatment of cancer. Recently, a role has been defined for the overexpression of the oncogene bcl-2 in the production of resistance to chemotherapeutic-induced apoptosis in some tumors, including advanced beast cancer, acute myeloid leukemia, prostate cancer, and neural crest tumors such as neuroblastoma. It is therefore critical that new chemotherapeutic strategies be developed to overcome chemoresistance in tumors that overexpress bcl-2. Previous reports have documented that overexpression of bcl-2 is accompanied by an increase in the intracellular concentration of reduced glutathione (GSH). One possible way to overcome chemoresistance in bcl-2 - overexpressing cells would therefore be to use chemotherapeutic drugs whose activity is enhanced by reaction with GSH. We have performed extensive studies of one such group of agents, the anodynes. The anodynes are prodrugs that require reductive activation by thiols for their antineoplastic activity. As such, their activity both in vitro and in vivo and in vivo is directly proportional to the cellular concentration of thiols, including GSH. We have recently shown that, unlike other chemotherapeutic drugs, the enediyne neocarzinostatin (NCS) is a more potent inducer of apoptosis in tumor cells that overexpress bcl-2 than in those that do not. We propose now to determine the mechanisms for both the increase in GSH concentration and the increase in sensitivity to enediyne-induced apoptosis in bcl-2-overexpressing cells. We will then use our understanding of these phenomena and their mechanisms to design, implement, and optimize a chemotherapeutic strategy that exploits the unique molecular and biochemical characteristics bcl-2-overexpressing tumor cells to target them for drug-induced apoptosis. This approach has the benefit of maximizing efficacy in bc1-2-overexpressing tumor cells, minimizing toxicity to normal cells, and identifying those tumors for which this is likely to be an effective regimen. Furthermore, it will establish in a preclinical model the potential of this novel strategy for treatment of patients for whom these is otherwise no reasonable chance of survival."}
{"text": "This program project proposes to investigate the nephritogenic and transplantation activities of the streptococcal membrane and soluble fractions derived from it. Soluble fractions have been obtained from the streptococcal membrane which are on chemical analysis found to be low molecular weight glycoproteins which demonstrate several immunological and biological properties. These materials are immunologically cross- reactive with similar materials obtained from human glomerular basement membrane, induce homograft sensitivity in outbred mice to skin homografts and abrogate the cytotoxic activity of anti-HL-A antisera. These materials are being evaluated in in vitro correlates of cellular immunity in patients with rheumatic fever and post-streptococcal glomerulonephritis. Additionally, they are mitogenic for most human lymphocytes evaluated and may be involved in abrogating or altering MLC in humans. It is our intent to chemically characterize the effector molecules of the soluble fractions and to alter them so that they may be further explored in the above experimental parameters."}
{"text": "When presented with food or cocaine, dopamine (DA) in the nucleus accumbens (NAc) initially signals that the item is rewarding. When behaviors, such as drug taking, become compulsive we hypothesize that this reflects a shift in neural systems maintaining the behavior. There is enhanced DA release in the dorsolateral striatum (DLS) and attenuation of NAc DA release. Furthermore, within DLS, activity in the Direct Pathway from the striatum to the substantia nigra, that is important for initiation of appetitive behaviors is enhanced, while activity in the Indirect Pathway, from the striatum to the globus pallidus, that inhibits competing repetitive or stereotyped behaviors is attenuated. Females (women and laboratory rats) exhibit more rapid escalation of drug taking than do males and estradiol (E2) enhances acquisition of drug taking and motivation for drugs of abuse. Experiments from the Becker laboratory have demonstrated that E2 inhibits GABA release, down-regulates DA D2 receptors (D2DR), and enhances cocaine- or amphetamine-stimulated DA release in DLS but not NAc. The overarching hypotheses for this proposal are: 1) An attenuated cocaine-induced DA increase in NAc combined with an enhanced DA increase to cocaine in DLS is related to the propensity to develop a preference for cocaine over palatable food pellets in both males and females; 2) Estradiol's action in DLS of females enhances the cocaine-induced dopamine (DA) increase, inhibits GABA release, and inhibits D2 DA receptors. This combined effect enhances the rate of preference formation and motivation for cocaine over pellets in females; and 3) Decreased inhibition of the indirect pathway in DLS contributes to enhanced motivation for cocaine over pellets in both males and females. In females D2DRs are down-regulated in this pathway by E2, and this contributes to the more rapid preference formation in females and greater motivation for cocaine over pellets. Determining the mechanism(s) mediating formation of preference for cocaine over a highly palatable food reward, and how individual differences and sex differences contribute to this, are important for our understanding of and treatment of addiction in both men and women."}
{"text": "Stroke is a disabling and often fatal disease that affects all ages, but mostly in the elderly. Stroke incidence is expected to rise in the US as its population ages; therefore, its impact on public health and health resources utilization will continue to be significant. The Stroke Progress Review Group and NINDS have identified a need for a network of stroke clinical trials centers in order to prioritize and efficiently design nd conduct translational and exploratory early Phase I/II clinical trials and large Phase III clinical trials to identify and advance stroke treatments. The NINDS Stroke Trials Network is a multidisciplinary stroke research infrastructure that consists of a National Clinical Coordinating Center, a National Data Management Center (NDMC), and 25 Regional Coordinating Centers. The NDMC's role is to establish a collaborative relationship with all parties involved in the Network and provide efficient and standardized central data management that yields high quality data and statistical support in the planning and execution of the stroke trials. To this en, the Data Coordination Unit (DCU) at the Medical University of South Carolina has developed a web-based comprehensive integrated data and project management system, WebDCU, that enables distributed data entry from the participating clinical sites with extensive data quality control and also provides the necessary tools to efficiently manage operational activities for multiple trials, while ensuring compliance with the NINDS Common Data Elements and FDA regulations. Using the WebDCU? system, we will develop, implement and maintain a central database that streamlines and maximizes efficiency in the management of data collection, processing, and monitoring of clinical, biomarker and neuroimaging data. In addition, the WebDCU will incorporate trial management information system that will provide full support for all study operational activities in the Stroke Trials Network. The Neuroimaging Core of the NDMC will create neuroimaging repository for the stroke trials. In collaboration with the study Principal Investigator and the other parties of the Stroke Trials Network, the DCU biostatistics faculty members will: contribute to the innovative and efficient protocol development (including study design and case report forms development); statistically monitor study progress; generate reports for the DSMB and the study teams; conduct interim and final analysis and dissemination of study results via presentations and publications; and create public use datasets for data sharing. Finally, as the Statistics and Data Management Center for the Neurological Emergencies Treatment Trials (NETT) Network, the DCU offers unique advantage of seamless collaboration between the NETT and Stroke Trials Networks."}
{"text": "Psoriasis is a chronic inflammatory skin disease affecting 1-3% of the population worldwide. Several studies have shown a significant association between psoriasis and hyperlipidemia, a well-established risk factor for cardiovascular disease, suggesting these conditions may share common inflammatory pathways. While multiple immune cell types have been implicated in the pathogenesis of psoriasis, including conventional CD4+ and CD8+ T cells, the potential contributions of lipid autoreactive CD1-restricted T cells to psoriasis pathogenesis remain elusive. CD1 molecules bind and present lipid antigens to T cells. These antigens include mammalian self-lipids and foreign lipids derived from specific microorganisms. In humans, the CD1 family consists of group 1 CD1 molecules (CD1a, -b, and c) and the group 2 CD1 molecule CD1d. Mice lack group 1 CD1, but do express CD1d. The unique binding specificity of CD1 suggests a potential role for CD1 molecules in the presentation of modified lipids to autoreactive T cells in hyperlipidemic conditions. However, due to the lack of a suitable animal model, the role of autoreactive group 1 CD1-restricted T cells in hyperlipidemia-associated inflammatory diseases is unknown. To overcome this limitation, we have generated a double transgenic mouse model that expresses human group 1 CD1 molecules and a group 1 CD1-autoreactive T cell receptor. In this study, we crossed this novel transgenic mouse to the ApoE- deficient background to study the role of autoreactive group 1 CD1-restricted T cells in hyperlipidemia. Interestingly, the presence of group 1 CD1-autoreactive T cells under hyperlipidmic conditions resulted in the mice developing severe psoriasis-like skin inflammation. While this finding suggests that autoreactive group 1 CD1-restricted T cells contribute to the pathogenesis of hyperlipidemia-induced skin inflammation, when, where and how these T cells are activated is unclear. Therefore, in Aim 1, we propose to investigate the mechanisms by which group 1 CD1-restricted autoreactive T cells contribute to skin inflammation and the kinetics of activation and localizatio of lipid autoreactive T cells during the course of disease. In Aim 2, we propose to examine how these T cells are activated by deciphering the nature of the lipid antigens presented by group 1 CD1 molecules during disease progression and further examining whether hyperlipidemia affects DC function, thereby resulting in group 1 CD1-autoreactive T cell activation. Collectively, these studies will lead to a better understanding of how group 1 CD1-restricted autoreactive T cells contribute to hyperlipidemia-associated inflammatory diseases and provide the basis for manipulating these T cells to uncover new strategies for therapeutic intervention for psoriasis and other inflammatory disorders."}
{"text": "There is a very high incidence of treatment resistance in bipolar illness. Some two thirds of patients in academic centers remain highly symptomatic despite aggressive pharmacotherapy. Days depressed exceed days manic by a factor of three. This large pool of treatment-refractory patients has been the focus of study of the Branch both to better understand the pathophysiological mechanisms in recurrent unipolar and bipolar disorders and develop new therapeutic modalities. With the current availability of a series of treatment options, finding clinical and biological markers of which patients respond best to a given treatment, is a critical need for the field. For example, a double-blind, randomized trial of six weeks of treatment with an agent that enhances inhibitory GABAergic function (gabapentin, GPN), versus one that decreases excitatory glutamatergic function (lamotrigine, LTG), versus placebo, found significant benefit of LTG (20/39 or 51% response rate) over GPN (11/40 or 28%) and placebo (8/28 or 21%). Correlates of lamotrigine response included male gender, bipolarity, fewer prior clinical trials, and fewer prior hospitalizations for depression (Obrocea et al). Preliminary evidence suggests that a baseline pattern of hypo-perfusion on 0-15 PET scans was associated with clinical response. In relationship to the assessment of possible predictors of clinical response, depressed patients with global hypermetabolism on PET, especially in the left insula, are more likely to be responsive to carbamazepine (N=26), while those with the more classic pattern of frontal and left insula hypometabolism are more likely to be responsive to the dihydropyridine L-type calcium channel blocker nimodipine (Ketter et al). We have found that nimodipine increases somatostatin in cerebrospinal fluid (CSF) and those with lower CSF somatostatin levels at baseline are more likely to respond clinically to nimodipine (Frye et al). The Branch is now analyzing a series of studies of the use of repeated transcranial magnetic stimulation (rTMS) of the brain for the treatment of depression that we have helped pioneer. A double-blind, randomized, crossover trial showed significant antidepressant effects of active 20 Hz rTMS for two weeks compared with the sham (George et al). The next study assessed the differential responsivity to low-frequency (1 Hz) vs. higher frequency (20 Hz) rTMS vs. sham stimulation over left frontal cortex at 80% of motor threshold (MT). This study found differential clinical and metabolic responses within the same patient to these different frequencies. Moreover, those with a pattern of baseline hypometabolism tend to respond to the 20 Hz stimulation, while those with baseline patterns of hypermetabolism are more likely to respond to the 1 Hz stimulation (Kimbrell et al). Because the incidence and magnitude of clinical responsivity was not adequate for many patients, another rTMS study using higher intensities (100% of MT) was conducted. This study replicated the findings of differential responsivity within individual patients, and strikingly, revealed that 20Hz stimulation increased 0-15 blood flow in a long lasting fashion, while 1 Hz rTMS decreased it (Speer et al). The most recent rTMS study used higher intensities of rTMS stimulation (110% MT) for a longer time (3 weeks) in an attempt to increase the response rate. This study randomized patients to 20 Hz vs. 1Hz vs. sham stimulation without a crossover and confirmed the lasting differential effects of high vs. low frequency rTMS on blood flow. It also revealed superior antidepressant effects of both 20 Hz and 1Hz compared to sham. Thus, a number of promising and mechanistically novel treatment approaches have been pioneered in the Branch and the current effort is aimed at defining better optimal parameters for rTMS response and defining clinical and neurobiological markers of individual responsiveness."}
{"text": "Developmental exposure to bisphenol-A (BPA) at doses within the range of human exposure causes a complex array of adverse effects in animals. These outcomes are also known to be present in human populations and the rise in their occurrence coincides with the massive use of BPA and other endocrine disrupting chemicals in consumer goods. The main hormonal activity of BPA is as an estrogen mimic. Exposure to estrogens throughout a woman's life, including the period of fetal development, is considered a main risk factor for breast cancer. Developmental exposure to BPA altered mammary gland morphogenesis in rodents during the period of exposure and led to the development of pre-neoplastic and neoplastic lesions appearing in adulthood. The goal of this proposal is to identify the molecular, cellular and morphogenetic mechanisms underlying BPA-driven altered mammogenesis that predisposes to neoplastic transformation. To achieve this goal, we will use innovative tools such as a fetal mammary gland explant culture model that allows testing for direct effects of hormones and real-time observation of organogenesis. The Specific Aims of this proposal are to test three hypotheses, namely, 1: that the direct effect BPA on mammary gland development is mediated by ER1 and/or 2. 2: that BPA causes altered ductal morphogenesis i) by altering the composition and physical properties of the ECM and ii) by inducing adipogenesis. 3: that the different mammary gland phenotypes resulting from gestational and gestational plus lactational BPA exposure are due to alterations at the hypothalamic level."}
{"text": "Excessive alcohol drinking is an enormous public health burden in need of more radical therapy. Most health problems associated with excessive drinking are due to two types of drinking: (i) binge (BALs > 0.08 grams % in a 2-hour period) and (ii) relapse (sustained heavy drinking for at least 2 days following a single or multiple abstinence periods). Previously, we showed that alcohol- preferring (P) rats express elevated levels of the GABAA subunits for alpha1, alpha2 and toll-like receptor 4 (TLR4) innate immunity receptors, and using specific siRNA vectors infused into the central amygdala (CeA), demonstrated that regulation of binge drinking at this site is mediated by GABAA a2 regulated TLR4 (a2/TLR4 axis). While the alpha1 subunit was also shown to regulate binge drinking, it was associated with the ventral pallidum (VP) and was independent of TLR4 (Liu et al., PNAS, 2011). In the current grant, we propose to better elucidate the mechanism responsible for the TLR4 effect, focusing on the chemokine monocyte chemotactic protein-1 (MCP-1) and the dopamine rate- limiting enzyme tyrosine hydroxylase (TH)--implicated by data obtained after the application was last submitted--and on brain sites that regulate different domains of the alcohol addiction cycle. The working hypothesis is that neuronal TLR4 induces MCP-1 expression via activation of cell type- specific transcription factors and it, in turn, functions as a neurotransmitter to stimulate dopamine release and excitability (inferred by TH) in select reward loci. The specific aims are: Aim I. Define the expression of GABAA a2/a1, TLR4, MCP-1 and/or TH at alcohol reward loci from P vs NP rats. Aim II. Define the role played by the a2/TLR4 axis that encompasses MCP-1 and/TH at alcohol reward loci in impulsive binge drinking. Aim III. Define the contribution of a1, a2, TLR4 and its downstream targets (MCP-1 and/or TH) at alcohol reward loci in compulsive relapse drinking. Aim IV. Define the mechanism of TLR4-mediated regulation of binge drinking by focusing on downstream signals that upregulate MCP-1 and TH expression, through the use of the pHSVsiMCP-1 amplicon. Better understanding of the role of chemokines in neurotransmission and the regulation of distinct (viz. dopaminergic) neurons will help broaden current concepts of neuroimmune communication and their roles in excessive drinking. This highly innovative proposal will create a paradigm shift in understanding the relationship between innate immunity signals and neuronal responses that impact alcohol addiction. Use of non-toxic herpes simplex virus (HSV)- siRNA constructs to inhibit relevant genes at specific brain loci will define therapeutic gene targets and test the potential of gene therapy approaches for binge and relapse drinking."}
{"text": "It is the purpose of this project to characterize the polysaccharides isolated from certain species of parasitic protozoan hemoflagellates of the family Trypanosomatidae, including the human pathogen Trypanosoma cruzi. Topics under present and future investigation include: 1) the chemical nature of the polysaccharide and its possible linkage to other molecules; 2) subcellular localization of these molecules; 3) the antigenic and immunological properties of these molecules; and 4) possible relationship of these molecules to the stage of the parasite's life cycle. The characterization of the isolated carbohydrate indicates nonglycogen-like neutral polysaccharides in addition to negatively charged molecules. The antigenic nature of the polysaccharide has been shown by immunodiffusion and complement fixation techniques."}
{"text": "Recombinational repair is important for cell survival and stability of genetic material. In humans, inefficiency of repair has been associated with cancer proneness, neurological and developmental defects and premature aging. Recent evidence suggests that, in all cells, every round of replication requires some form of replication fork repair. In this proposed study, we will address several important questions in prokaryotic and eukaryotic recombination that have relevance to replication fork repair in every organism. (1) How do the RecA paralog proteins facilitate recombination? Every organism appears to employ a RecA-/Rad51 orthologous strand exchange protein and at least one other RecA/Rad51 paralog protein. What are the paralogs doing? We will use a combined biochemical and genetic approach to address the role of the RecA paralog protein, RadA/Sms in genetic recombination of E. coli. (2) What branched DNA molecules are intermediates of recombination and which enzymes resolve them? Our knowledge of the enzymes that process branched DNA intermediates of recombination is incomplete. We will assay whether the RuvC-related protein of E. coli, YqgF, is involved in recombinational repair and can catalyze cleavage of branched molecules predicted by recombination mechanisms. (3) What factors mediate template-switch repair? This is a recombinational mechanism that leads to sister chromosome exchange without the requirement for a strand-exchange protein such as RecA. Our previous work identified the first two factors involved in template-switch repair of E. coli, the chaperone DnaK and the gamma/tau subunit of DNA polymerase III, DnaX. What other factors enable this reaction? Using genetic analysis, we will test the involvement of proteins of the replisome and seek factors presently unknown. Replication fork repair is important for cell survival and stability of genes. In humans, inefficiency of repair has been associated with cancer proneness, neurological and developmental defects and premature aging. This proposal seeks to understand the mechanisms of replication fork repair. [unreadable] [unreadable] [unreadable]"}
{"text": "We will develop a novel approach to detecting transgene activity in tumors based on proton MRI of enzyme activated reporter molecules. Specifically, we will synthesize, evaluate, optimize, and apply novel agents to detect (3-galactosidase activity generated by the lacZ gene. Gene therapy holds great promise for treatment of cancer. However, major problems involve assessing delivery to target tissue, the uniformity (versus heterogeneity) of bio distribution and determining whether the genes are expressed. We propose a novel approach for evaluating gene expression using MRI. This research draws on foundations in optical detection of reporter genes, specifically P- galactosidase from the lacZ gene. Recently, 3,4-cyclohexenoesculetin-p-galactopyranoside, marketed commercially as S-Gal(tm), has been shown to generate an intense black color as a precipitate upon activation by p-gal in the presence of Fe3+ ions. This prompted us to consider the paramagnetic properties of the precipitate as a potential 1H MRI contrast agent. Preliminary studies have demonstrated feasibility and we now wish to translate and develop the concept to tumor cells for detection in vivo. Specific aim 1 will explore the utility of S-gal, with investigations to optimize and validate the technique. MRI parameters will be varied to maximize contrast with appropriate spatial and temporal resolution. Agent administration will be evaluated to maximize contrast and minimize toxicity. Specific aim 2 will focus on translation to tumors in living mice. MRI will be validated by comparison with optical methods in vivo and traditional methods such as histology and RT-PCR. Specific aim 3 focuses on the synthesis and development of alternate optimized substrates. We believe this approach can both become a valuable tool for detecting p-gal activity in vivo and serve as a proof of principle for a novel platform technology adaptable to other reporter genes and endogenous enzymes such as proteases. [unreadable] [unreadable] [unreadable]"}
{"text": "This is application proposing to determine whether failure to undergo programmed cell death contributes to the pathogenesis of human neuroblastoma. Specifically, the investigator proposes to determine whether oncogene products which modulate programmed cell death (PCD) influence whether oncogene products which modulate programmed cell death (PCD) influence the malignant phenotype of neuroblastoma. The investigator previously determined that Bcl-2, which suppresses PCD, is present in pretreatment neuroblastoma tumore biopsies, and that, when deregulated, inhibits neuroblastoma tumor biopsies, and that, when deregulated, inhibits chemotherapy-induced PCD in neuroblastoma. Based on these prior findings, the investigator seeks to extend these observations in a system in which it is proposed that neuroblastoma is a disease resulting from failed apoptosis. The first aim will address the issue of oncogene cooperativity in the malignant phenotype of neuroblastoma,, specifically whether Bcl-2, in its capacity to inhibit apoptosis, cooperates with N-myc to transform cells. The second aim is proposed to determine whether Bcl-xs expression will lead to neuroblastoma cell death and enhanced response to chemotherapy (by pirating existing cell machinery). The third specific aim proposes to determine the expression of the death gene and survival gene in normal tissue from which neuroblastoma is thought to arise. The investigator feels these experiments will address the molecular link between the benign lesions and malignant neuroblastoma."}
{"text": "The Postdoctoral Training Program in Hepatology is an integral part of the robust liver-related research effort at UCSF. The purpose of the Program is to provide trainees a comprehensive background in liver biology and disease as well as the investigational skills to address new questions and contribute to new knowledge in hepatology. The Program is staffed by 13 faculty based within the Departments of Medicine, Surgery, Biochemistry and Microbiology & Immunology; collectively these individuals offer balanced expertise in basic and clinical hepatology and have a strong track record of working collaboratively with each other. A subgroup of 5 Program faculty comprise a Steering Committee charged with screening applicants, reviewing the training curriculum and monitoring the progress of active trainees. The top priority of the Program is to train physician- scientists who are admitted to UCSF as Gastroenterology fellows. MD applicants to the Program must have a prior record of research accomplishment and dedication to an independent investigative career in hepatology. PhD applicants are admitted largely from mentor laboratories; they must display similar academic promise and an orientation toward translational liver research. All trainees are placed through a core curriculum covering liver-related biology, liver-related research methods and general academic skills. They then undergo specialized research instruction under an individual mentor, with choices ranging from clinical epidemiology, health outcomes and genetics to cell biology, organogenesis, immunology, metabolism and fibrosis/carcinogenesis. Additional focused coursework is highly recommended for all trainees; for those pursuing clinical investigation, enrollment in a Master's Degree program is mandatory. Importantly, the Training Program benefits from numerous institutional resources including outstanding graduate programs in basic and clinical sciences as well as research support units such as the Liver Center and the Clinical and Translational Sciences Institute. Program faculty are strong figures in each of these units, and accordingly can guide trainees to utilize these resources to maximize the value of their postdoctoral experience. The ultimate goal of the Training Program is to provide sufficient group and individual mentorship to enable graduates to assume a faculty-level position and compete successfully for independent research funding in hepatology. PUBLIC HEALTH RELEVANCE: Postdoctoral Training Program in Hepatology is designed to provide MD and PhD scientists the skills to conduct independent research relevant to the liver. The Program fills a need for basic and clinical investigators who will direct new scientific knowledge to the prevention, diagnosis and treatment of liver-related ailments. The Training Program enables faculty to provide focused research instruction to 4 trainees per year. Importantly, it also enables trainees to enroll in didactic courses and obtain an advanced degree. The ultimate goal of the Training Program is for graduates to become independent investigators, meaningful contributors to the field of hepatology, and hopefully mentors themselves. The expectation is that the trainees' research successes will translate into improved outcomes for patients suffering from liver diseases."}
{"text": "There remains a clinical need for myocardial perfusion tracers that more accurately reflect cardiac blood flow over a wide range of flow rates while simultaneously demonstrating a reduced accumulation and/or rapid clearance from the tissues adjacent to the heart. Despite the excellent myocardial extraction and flow kinetics of thallium-201 for evaluating myocardial perfusion and ischemia, the energy spectrum of thallium- 201 makes it particularly prone to soft tissue attenuation artifacts. While there are two FDA-approved Tc-99m perfusion tracers, having more favorable energy spectra for imaging, Myoview(TM) (tetrofosmin) and Cardiolite(TM)(sestamibi), the first pass extraction fraction for both of these tracers is approximately 50% of TI-201, and they exhibit unfavorable abdominal artifacts. At very low flow rates sestamibi and tetrofosmin overestimate flow and at high flow rates (above 2 ml/min/g) underestimate myocardial blood flow. Because extraction of these Tc-99m tracers reaches a plateau at high flow rates, there is a concern that these tracers may not have adequate sensitivity to detect intermediate coronary artery disease during pharmacological stress, when blood flow is increased several times over baseline. Our plan is to exploit our versatile tridentate chelates by synthesizing a series of novel tridentate etherfunctionalized chelates (TECs) and synthesize the corresponding rhenium-TEC complexes, [Re(CO)3(L)3]+, for structural identification. The carrier-free Tc-99m TEC complexes will be prepared using [99mTc(CO)3(H2O)3]+ followed by determination of the in vivo distribution and pharmacokinetic properties of the 99mTc-TECs in rats. Comparisons with TI-201, 99mTc-sestamibi and 99mTc-Tetrofosmin will be performed as a benchmark for initial heart accumulation. Finally, in vitro partition studies of lead compounds with human blood components will be analyzed. This evaluation in normal resting animals will allow for the evaluation of the Tc-99m complexes as potential myocardial perfusion tracers. Successful completion of this project will enable a more rapid and accurate assessment of myocardial perfusion. Since heart disease is the major cause of death in the United States, accounting for nearly 40% of all deaths, as estimated by the American Heart Association, a more accurate agent to assess myocardial perfusion would warrant further development. The current market utilization is approximately 6 million procedures annually in the USA alone."}
{"text": "Hemophilia B is a severe bleeding disorder characterized by a functional deficiency in the blood coagulation protein factor IX (Christmas factor). Therapy for bleeding episodes in these patients requires the intravenous replacement of factor IX given as fresh frozen plasma or with commercially available concentrates of factor IX in which the protein is only partially purified. Although these commercial materials have proven efficacy in this disease, they contain many impurities, are often contaminated with virus, and their use is associated with a significant morbidity. The goal of this project is the development of a highly purified form of factor IX. The factor IX will be obtained by using a recently described invention which employs conformation-specific antibodies against human factor IX to purify this protein. This factor IX will be homogeneous and thus suitable for intravenous therapy. Published studies of factor IX purified by this methodology indicate that it is free of contaminants and infectious agents responsible for complications associated with the currently commercially available concentrates. In this Phase I study sufficient factor IX will be purified to accomplish studies of acute toxicity in rabbits and the ability to correct the bleeding deficiency in hemophiliac dogs."}
{"text": "Project Summary Autism Spectrum Disorder (ASD) disproportionately affects males (?) over females (?), possibly because of a Female Protective Effect (FPE). Characterizing the FPE may help us to understand and treat ASD in both sexes. Our Network has contributed to understanding sex differences in ASD at the levels of gene structure and expression, neural dynamics, brain function and connectivity. We have curated an unprecedented sex-balanced, age-, IQ- and severity-matched cohort of cognitively-able school-age ?, and ? with ASD, age- and IQ-matched typically developing (TD) children and unaffected siblings (US). At T1, we conducted behavioral phenotyping and measured key neural systems at the levels of brain structure, connectivity, function and temporal dynamics. Genotyping, whole-genome sequencing and gene expression analyses are underway. We now seek to pursue an extraordinary opportunity to assess our participants again (T2) as they make the transition through adolescence and into young adulthood. Our field has failed to generate a sufficient knowledge base to help optimize this transition for people living with ASD and their families. We will leverage the expertise of our Network to identify sex differences in ASD longitudinal brain development during this important transition. We will clarify both temporal and spatial characteristics of developing social perception, emotion regulation, reward and implicit language learning circuits, in addition to neural mechanisms for sensory habituation, creating dimensional, multi- level neural signatures of brain development. We will bridge DNA sequence and brain development and relate neural signatures to behavior and genetics to predict ?real-world? functioning in young adulthood. We will combine multiple levels of biology and endophenotypes?SNVs, CNVs, clinical measures, pubertal status, presence of seizures/epilepsy, sex hormones and multimodal, longitudinal measures of brain development?into one framework using an Integrated Weighted Gene Coexpression Network Analysis (iWGCNA). Finally, we will extend our T1 systems-biology approach through a collaboration*with ASD self-advocates/participants to evaluate the experiential validity of our findings. The proposed research marks the start of a new era in which advanced multimodal neuroimaging and genetic analyses will evolve into an integral part of a translational research chain. Novel behavioral treatment and pharmacotherapies for ASD may be further developed in adolescence and young adulthood with the tremendous benefit of directly and more precisely assessing impairment and change in neural circuits. By providing information about distinct, sex/gender-based developmental pathways in ASD, this study will identify if intervention/prevention strategies should include sex-based modifications."}
{"text": "DNA fragment size analysis by flow cytometry has been applied to the analysis of restriction enzyme digest of 3 bacterial gnomes E. coli, E. herbicola, and E. erwinia. Distinct restriction fragment fingerprints were observed and served as the basis for discriminating among the three species. Details are contained in a Research Highlight and a publication that is in press in Cytometry."}
{"text": "The objective consists of the development of methods for the controlled enzymatic synthesis of large oligoribonucleotides and oligodeoxyribonucleotides of defined sequence. The strategy involves the use of enzymes, which have the capacity to form the internucleotide bond, with substrates that contain specific reversible chemical modifications. The particular enzymes to be exploited in the study are polynucleotide phosphorylase, RNA ligase, and terminal deoxyribonucleotidyl transferase."}
{"text": "Work has continued to obtain information relative to the role of regional lymph nodes (RLN's) in patients with cancer so that a firmer biological basis for the management of such patients may be formulated. Interrelationships between a variety of histopathologic discriminants in mammary and colonic carcinomas as well as their correlation with lymphocyte transformation were determined. RLN's from patients with mammary carcinoma exhibited greater sinus histiocytosis and less lymph follicles than those from persons with colonic carcinoma. These latter tumors contained more lymphoid infiltrate and their component cells had higher nuclear grades than breast carcinomas. Differences in the relationship of these histopathologic parameters with lymphocyte transformation of RLNC's in these two tumor types further indicates the singularity of neoplasms of diverse origins. Lower nuclear grade and mild lymphoid infiltrate of mammary carcinomas were associated with nodal lymph follicle formation and the converse with the absence of such nodal structures. No significant difference in these histopathological discriminants were observed in patients with or without nodal metastases. Lymphocyte transformation of RLNC's from patients with mammary carcinoma was increased in those in whom their carcinomas were of low nuclear grade and contained only mild degree of lymphoid infiltrate. These findings suggest that nuclear grade and lymphoid infiltrates may be differently related to tumor host responses than previously contended. No functional or other relationships could be discerned for sinus histiocytosis. In a second study, RLNC's from patients with breast cancer were evaluated at the time of their removal (not after culture) relative to HT3 uptake. Such an investigation was carried out to determine whether such nodes displayed evidence of a response to the tumor, whether variations observed between nodes and between patients found following RLNC culture and PHA stimulation existed at the time of their removal. Another objective was to ascertain whether findings could be related to histopathologic discriminants in the tumor and nodes. In general, no correlation was obtained."}
{"text": "PROJECT SUMMARY/ABSTRACT The overall goal of this clinical research program is to provide infants with Down syndrome (DS) with the training and technology to independently explore their world to the same degree as their typically developing peers. Moving and mobility comprise up to 80% of a young child's waking hours. Such a high dose of sitting, standing and walking are a lofty gold standard for pediatric rehabilitation. Down syndrome (DS), which delays sitting, standing and walking by up to a year, significantly reduces infants' daily exploration with serious cognitive, language, and social consequences. This proposal specifically tests the feasibility and effects of a novel 'mobile therapy environment' that provides powered mobility, progressive exercise and functional skills training in one assistive technology device. Our modified ride on cars specifically combine the fun and exploration of powered mobility (such as a Segway) with the ability to advance balance, strength and coordination through therapeutic exercises while infants practice sitting, standing and walking. Over the last year, we have developed a series of electrical and mechanical modifications that allow a single car to be progressively driven in sitting, then safely driven as triggered by standing (Figure 1 upper) then triggered by over ground walking (Figure 1 lower). This proposal will allow us to determine the feasibility and effect of ride on car training on sitting, standing and walking delays (Aim 1) and on infants' broader cognitive, language and social-emotional development (Aim 2). This proposal, inspired by our mobile robotics work and supported by preliminary data, is scientifically principled, innovative yet feasible within the funding period. The data and devices from this 2 yr project will support more formal work on the clinical and commercial potential of a novel category of assistive technology: the 'mobile therapy environment'. Figure 1 Modified ride on toy cars provide inexpensive effective mobility and socialization as well as strengthening, balance, coordination during driving while standing (Upper) and driving while walking (Lower)."}
{"text": "Studies of the physiological and pathophysiological characteristics of the stable hypertrophied myocardium will be continued. Attention will be focused on continuing studies of the regional distribution of coronary blood flow by hydrogen polarography and microspheres, on characteristics of thallium uptake and imaging by the hypertrophied heart, and on segmental function in the hypertrophied myocardium at rest, under stress, and with ischemia. Studies of preservation and protection of the hypertrophied ischemic myocardium during cardiopulmonary bypass will be continued, and the effects of coronary ischemia on the hypertrophied heart will be studied and characterized in terms of segmental changes and regional blood flow distribution."}
{"text": "Membrane proteins that function normally are vital to health;their defects are associated with many known disease states. Membrane proteins are the targets of many pharmacologically and toxicologically active substances and are responsible, in part, for the uptake, metabolism, and clearance of these substances. Despite the importance of membrane proteins, knowledge of their high-resolution structures and mechanisms of action has lagged far behind the knowledge of these properties of proteins in general. Theoretical modeling may help in deciphering the structure-function relationship of membrane proteins, however theoretical modeling of membrane proteins also lags behind the modeling of globular proteins. Our long-term goal in this proposed project is theoretical modeling of structure and function of membrane proteins to provide understanding of how the molecular and atomistic events during ion permeation and ligand binding lead to mesoscopic events of channel conductance and regulation. One problem in modeling ion channels is that characterizing their function, i.e. ion current - voltage relationships, requires modeling of processes on at least the microsecond time-scale, inaccessible for current atomistic simulations of proteins. The objective of this application is thus to create and apply reliable yet computationally efficient molecular-level models for ion permeation through open channels, which take into account channel protein molecular structure, polarizability and short time-scale flexibility, yet are capable of predicting observable ion currents (a slow process by Molecular Dynamics standards). In order to efficiently span a wide range of time-scales relevant to the ion permeation, the proposed models are of hierarchical nature. Our aims in this project are: to develop, test and apply hierarchical algorithms to model ion currents through open flexible channels of known or predicted 3D structures. In order to implement this hierarchical approach we will use several levels of resolution (\"graining\") of the system under consideration: from all-atom molecular modeling of the channel protein with its surrounding medium to coarse grained continuum approximate models, capable of spanning longer time-scales and mesoscopic sizes. Using this molecular/mesoscopic hierarchy of models we will study several systems of medical and medical engineering interest. We expect that the outcome of this proposed project will be significant for future direction of theoretical and computational approaches to study membrane proteins because once a functional model based on protein structure has been developed it will become possible to develop specific theoretical methodologies for designing drugs and drug delivery systems for membrane proteins via rational computer-aided design."}
{"text": "To determine the maximum total dose of paclitaxel when given with gallium nitrate and assess the objective response rate, duration of remission and overall survival in patients treated with paclitaxel plus gallium nitrate plus G-CSF."}
{"text": "As a federally qualified community health center, the applicant has as its core function to provide primary healthcare services to vulnerable populations. Access to health information is a basic healthcare need. The goal of this project is to contribute to the development of a free on-line multilingual multimedia digital library of health information. The focus of the work in this project is to build upon existing high quality multilingual written resources that have already been developed and put on-line by the NSW (New South Wales) Multicultural Health Communication Service (MHCS) of Australia. The project work will include the development of a community committee to identify the MHCS written materials that deal with topics and languages that are most critical to meet the health information needs of new refugees in the community. A development team will work with the local partners, MHCS and members of the target refugee community to create web-based multimedia versions of this written material. A minimum of four written documents will be developed into at least four languages for a total of at least sixteen individual multimedia software files. These will be created using Macromedia multimedia software. A website will be created that will link with the community health information website (Healthy Communities Without Borders - www.hcwb.org ) to assist end users in accessing the materials. Training for key staff at the performance sites will be provided to ensure that there is a clear awareness of the new materials and an understanding of how to access other quality multilingual on-line health information. Since this project is a key step to understanding how information technology can be better used to meet the health information needs of non-English speaking community members, a strong emphasis will be placed on evaluation activities. The evaluation will include an assessment of target users engagement in the software and whether any knowledge, skills or attitude changes occurred (Levels I and II of training evaluation). Evaluation of the implementation strategies and perceived usefulness at each of the four performance sites will also be carried out. The results of this project will provide guidance in future efforts to provide critical health information to our increasingly diverse population."}
{"text": "Many experimental vaccines have been developed that show promise in animal models of cutaneous or visceral forms of leishmaniasis, but an effective human vaccine still does not exist. In a series of phase III clinical trials in which killed Leishmania vaccines were tested in individuals exposed to infected sand fly bites in the field, no protection was observed. Remarkably, none of the candidate vaccines tested in animal models have been evaluated under experimental conditions using infected sand flies, and we addressed the possibility that vaccine failure in clinical trials may reflect the more stringent conditions of natural sand fly challenge. We compared needle and infected sand fly challenge to evaluate the efficacy of the only defined candidate vaccine currently in clinical trial, comprised of polyproteins (either KSAC or L110f), containing multiple antigenic epitopes from Leishmania delivered in a stable emulsion (SE) (water in oil) with glucopyranosyl lipid A (GLA), a TLR4 agonist suitable for use in people. Polyprotein-vaccinated mice had a 60-fold increase in CD4+ IFNg+ T cell numbers versus control animals at 2 wk postneedle inoculation of L. major, and this correlated with a 100-fold reduction in parasite load. By contrast, following challenge by infected sand fly bite, polyprotein-vaccinated animals had comparable parasite loads, greater numbers of neutrophils at the challenge site, and reduced CD4+IFNg+/IL-17+ ratios versus non-vaccinated controls. Importantly, mice with a healed primary infection were solidly protected against infected sand fly challenge that was associated with the speed with which effector cells appeared at a site of challenge, providing an immediate burst of effector cytokines that may be required to counteract the down-modulatory environment created by the highly localized, neutrophil-dominated, response to sand fly bite. We employed Ly6C, a T-bet regulated, GPI-anchored, surface glycoprotein expressed on T-bethi CD4+ T cells, to phenotype the rapidly recruited cells, and found that pre-existing, short-lived, CD44+CD62L- T-bet+Ly6C+ effector (TEFF) cells, not memory or memory-derived cells, mediate concomitant immunity in healed mice. Upon adoptive transfer and challenge, non-dividing Ly6C+ TEFF cells preferentially homed to the skin, released IFNg and conferred protection. Despite being short-lived, Ly6C+ TEFF cells were maintained at high frequencies in the mice with healed primary infection, presumably as a consequence of the persistent infection in these mice. The lack of effective vaccines against Leishmaniasis may be because protection against these infections requires concomitant immunity mediated by pre-existing TEFF cells, not memory cells, and is therefore not amenable to conventional, memory inducing, vaccination strategies. As indicated above, primary L. major infection typically produces cutaneous lesions that heal but that harbor persistent parasites. While the opposing roles of CD4+ T cell-derived IFNg and IL-10 in promoting parasite killing and persistence have been well established, how these responses develop from nave precursors has not been directly monitored throughout the course of infection. We used peptide:Major Histocompatibility Complex II (pMHCII) tetramers to investigate the endogenous, parasite-specific primary CD4+ T cell response to L. major in mice resistant to infection, and applied this approach to enumerate the expansion, contraction, tissue distribution, and function of parasite-specific CD4+ T cells throughout the course of the infection. Maximal frequencies of IFNg+ CD4+ T cells were observed in the spleen and infected ears within a month after infection and were maintained into the chronic phase. In contrast, peak frequencies of IL-10+CD4+ T cells emerged within 2 weeks of infection, persisted into the chronic phase, and accumulated in the infected ears but not the spleen, via a process that depended on local antigen presentation. T helper type-1 (Th1) cells, not Foxp3+ regulatory T cells, were the chief producers of IL-10 and were not exhausted. Therefore, tracking antigen-specific CD4+ T cells revealed that IL-10 production by Th1 cells is not due to persistent T cell antigen receptor stimulation, but rather driven early and sustained locally by antigen encounter at the site of infection. In addition to studying the mechanisms underlying parasite persistence in healed mice, mouse models are also being used to study non-healing forms of disease. Infection of C57BL/6 mice with most L. major strains results in a healing lesion with minimal pathology at the site of inoculation in the skin. By contrast, using a strain of L. major (Lm Sd) isolated from a patient with chronic cutaneous lesions, C57BL/6 mice also fail to heal their dermal lesions or effectively control tissue parasite burden despite a strong and polarized Th1 response. In studies designed to identify the earliest cells and mediators that precede and promote the severe pathology, we detected elevated levels of IL-1b mRNA and IL-1b+ cells in the inoculation site 3-4 weeks post-infection, followed by a neutrophil infiltrate that persisted until the onset of the pathology. Whereas no phenotype was observed in IL-17 deficient mice, IL-1R deficient mice, as well as IL-1b, ASC, and caspase-1/11 deficient mice, each showed minimal pathology and healed their Lm Sd infection. These studies are unique in the innate immunity field in identifying inflammasome dependent IL-1b as preventing rather than promoting host defense against a microbial pathogen. The mechanisms underlying the failure to control the growth and systemic spread of Leishmania parasites in human visceral leishmaniasis (VL) are not well understood. A key immunological feature of VL is the inability of peripheral blood mononuclear cells (PBMCs) to proliferate or to produce IFNg in response to leishmanial antigens. IL-10 has been implicated in the suppression of antigen-specific T cell responses in human VL based on the elevated levels of IL-10 observed in plasma and lesional tissue, and its role in preventing clearance of L. donovani in murine models of VL. In unexpected findings from our recent studies we were able to show that in sharp contrast to assays employing PBMCs, a cytokine release assay involving L. donovani antigen-stimulated whole-blood cells was able to detect the secretion of IFNg by cells from the majority of patients with active VL. In 35 patients with active VL, 80% secreted high levels of IFNg as compared to 85% of cured VL patients, and 24% of EHCs with presumed subclinical infections. The findings do not support a severe Th1 response defect in kala-azar. Importantly, only the whole blood cells from patients with active VL also secreted IL-10, which better reflects the response that distinguishes individuals with active disease from cured or subclinically infected, immune individuals. More generally, the findings reveal that PBMCs may not accurately reflect the immune competency of peripheral blood cells."}
{"text": "PROJECT SUMMARY The natural history of high-risk, localized prostate cancer, accounting for a substantial proportion of the 29,000 prostate cancer?related deaths per year in the U.S., is highly variable: there are patients who are cured with surgery or radiation, while others develop metastases and succumb to their disease. To date, attempts to stratify patients within these subsets of high-risk, localized prostate cancer have proven inadequate. There is particular urgency within the African American population, where risk of lethal disease is highest. There is a tremendous need for biomarkers that can reliably distinguish the most aggressive forms of disease within high- risk groups. Specifically, we focus on the DNA damage repair (DDR) pathway, where certain variants appear to be associated with prostate cancer aggressiveness. In this proposal, we will address two important questions in the field: 1) Among patients with high-risk, localized prostate cancer, can we identify genetic aberrations in DDR pathway associated with the most aggressive forms of the disease? 2) Do DDR variants contribute to increased risk of aggressive prostate cancer among African American men? In the first aim, we focus on inherited germline variants in the DDR pathway. Using large retrospective and prospective cohorts of patients with high-risk, localized prostate cancer, we will perform sequencing in DNA derived from blood samples and examine associations with development of lethal prostate cancer over long- term follow-up. In our second aim, we will interrogate the somatic genome and matched germline using DNA derived from radical prostatectomy specimens and corresponding blood samples, and we will study associations with lethal disease. In the third aim, we will determine the prevalence of germline DDR in a larger cohort of African American prostate cancer patients and estimate the extent to which DDR mutations contribute to prostate cancer disparities. Completion of the experiments outlined in this proposal will provide an unparalleled look at the association between DDR and lethal forms of prostate cancer. We have the potential to i) discover specific germline and somatic biomarkers for lethal disease that could be used to determine treatment strategies at the time of diagnosis, perhaps using strategies such as PARP inhibition or platinum chemotherapy that target the DDR pathway; ii) discover germline biomarkers that could be developed as screening tools for the most aggressive forms of prostate cancer; and iii) define the extent to which mutations in in DDR pathway genes may explain racial disparities in prostate cancer. The genetic loci and target genes comprising this dataset together with the other projects will stimulate new targets for therapeutic intervention."}
{"text": "Family-based behavioral modification (FBM) is the \"treatement of choice\" for pediatric overweight. A comprehensive package, FBM includes a core set of intervention strategies (parents skills training; reduced sedentary behavior and increased physical activity; and reduced energy-dense and increased nutrient-dense foods). FBM also has strong theoretical foundations, which has allowed it to be rigorously evaluated. Despite these conclusions, the translation of FBM to primary care has received almost no investigation. FBM has been implemented almost exclusively in university-based clinics, by highly-trained clinical researchers, with motivated families having access to the clinic. This model does not reach the vast majority of overweight children in the popuation, especially those living in rural communities. Thus, there is a need for \"translational\" studies that adapt FBM for implementation by existing clinical staff in a cost-effective manner manner, that is well accepted by staff and patients. To this end, we propose a planning, pilot and feasibility intervention study that will be conducted within Geisinger Community Practice network. Geisinger is a comprehensive health care network that serves more than 2.5 million families across 38 counties of central Pennslyvania, many of whom reside in rural and underserved communities. The proposed pilot study, a 2-group randomized clinical trial, will be conducted out of a primary care clinic within the Geisinger network. Participants will be 60 oveweight or at-risk for overweight children, 4 to 8 years old, and a caregiver, who will be randomized to one of two conditions: Family-based Behavioral Modification (FBM; N= 30) or Minimal Nutrition Information (MNI; N=30). FBM will consist of streamlined parent- and child-group meetings, tailored for primary care and implimented by the staff nurse and pediatrician. MNI will consist of nutritional handouts and fliers. Primary outcomes measures will be 4-month changes in child weight status, diet and physical activity, and cardiovascular outcomes (for effect size estimation), and measures of treatment acceptability by families and primary care staff. This pilot study will provide Penn and Geisinger investigators the time, resources, and pilot data necessary to map out a larger- scale intervention study across the Geisinger primary care nework, using the R18 funding mechanism. [unreadable] [unreadable]"}
{"text": "The aim of this research is to obtain basic information on mitochondrial chain enzyme systems enabling conclusions on sequence, action and mechanisms of electron and coupled energy transfer by the methods of sequential fragmentation, systematic reconstitution and related physiochemical techniques. This type of attack may open up new avenues leading to more advanced designs for experiments in the field of terminal electron transport systems. Indeed, some of the results obtained from the project in the last few years would not have been discovered by conventional methods, but were dramatically and unambiguously revealed by reconstitution experiments. This is a long term project which, of necessity, entails the preliminary isolation and characterization of respiratory components from intra-cellular organelles. BIBLIOGRAPHIC REFERENCES: Chiang, Y.L., Kaminsky, L.S., and King, T.E. A Complex of Cardiac Cytochrome c1 and Cytochrome c. J. Biol. Chem. 251, 29-36 (1976). Ohnishi, T., Salerno, J.C., Winter, D. B., Lim, J., Yu, C.A., Yu, L., and King, T.E. Thermodynamic and EPR Characteristics of Two Ferredoxin-Type Iron-Sulfur Centers in the Succinate-Ubiquinone Reductase Segment of the Respiratory Chain. J. Biol. Chem. 251, 2094-2104 (1976)."}
{"text": "Recent studies suggest that reparative cells which contribute to healing in the lung following injury also possess regenerative capacity. Although the biology of post-natal lung regeneration is not well understood, mechanical pre-stress appears to be an absolute requirement for tissue regeneration to occur. Emphysema, a disease characterized by tissue destruction, is a potential target for cell-based therapy. This disease is associated with: 1) loss of resident reparative cells; 2) loss of the extracellular matrix that transmits pre-stress signaling; 3) and loss of pre-stress itself, the very signal required to trigger regenerative responses. Although these factors represent obstacles to the development of regenerative therapeutic strategies for emphysema, preliminary studies in our lab show that modulation of mesenchymal and epithelial cell proliferation using members of the fibroblast growth factor family complexed to carrier molecules in a biocompatible polymer can promote expansion of parenchymal tissues. The polymer scaffold, an air containing foam with mechanical properties similar to healthy lung tissue, effectively transmits stress to reparative/progenitor cells to promote proliferation and remodeling. Studies proposed here will test the hypothesis that 1) therapeutic post-natal lung tissue growth in emphysema can be achieved by augmenting the lung's innate healing response using growth factors to direct endogenous reparative lung cells following a localized mild injury; and that 2) the magnitude of this response can be modulated by altering pre-stress using concomitant bronchoscopic lung volume reduction therapy to increase transpulmonary pressures. We intend to advance this approach, known as pneumografting, into human trials under a physician-sponsored Investigation New Drug Application."}
{"text": "A Radiotherapy-Oncology Center has been established at the Tufts-New England Medical Center for the treatment of human malignancies, and to conduct research on the effects of irradiation on normal and malignant tissues. Education on clinical and biological aspects of cancer is carried out at all levels. Active efforts are sustained to cooperate with clinicians in affiliated hospitals in providing optimal care to cancer patients at the community level. Special efforts are directed toward the combined use of surgery, radiotherapy and chemotherapy in the management of various tumors. Research is ongoing in the application of the computer to all aspects of the practice of Therapeutic Radiology. Radiobiologic research centers on the role of the cell membrane in neoplastic conditions. BIBLIOGRAPHIC REFERENCES: Munzenrider, J.E. Fernando Bloedorn, M.D. -master radiotherapist. Tufts Health Science Review b:65-69, 1976. Pilepich, M.V., Bloedorn, F.G., Munzenrider, J.E., Rene, J.B., and Lowry, S.W. Split-course radiotherapy in advanced head and neck tumors. International Journal of Radiation Oncology, Biology, Physics Suppl. 1:31, 1976."}
{"text": "The precise inheritance of genetic material in eukaryotes requires that initiation at each of the hundreds to thousands of replication origins be subject to exquisite regulation so that the DNA is duplicated exactly once per cell cycle. When replication control mechanisms go awry, genomic instability is predicted to occur, but the precise molecular consequences of deregulated replication for genome integrity are completely unknown. Additionally, the full battery of regulatory strategies that govern replication has not been defined. Cyclin dependent kinases (CDKs) are key molecular regulators that both stimulate initiation and inhibit re- initiation of DNA replication. To understand the molecular basis of genome integrity, it is essential to develop a more sophisticated understanding of these CDK-dependent regulatory events. Additionally, it is critical to analyze how disrupting this regulation affects faithful inheritance of the genome. To this end, we have developed several innovative tools that allow us to study the genesis and consequences of re-replication in the budding yeast Saccharomyces cerevisiae. Our Specific Aims are as follows: (1) We have discovered that CDKs target polymerase alpha primase to block re-replication, which challenges the prevailing paradigm that the only strategy used by CDKs to inhibit re-replication is to prevent reassembly of a pre-replicative complex. Thus in this aim, we will characterize a novel replication control mechanism by investigating how CDKs inhibit polymerase alpha-primase to prevent re-replication. (2) We will continue to uncover new strategies for replication control by completing our ongoing screen to identify new CDK targets involved in either triggering initiation or preventing re-replication; this screen has already successfully identified five such targets, including polymerase alpha primase. (3) Using a robust copy number assay, we have obtained the first evidence that re-replication causes a heritable genetic change, namely a gene duplication event that represents an early step of gene amplification. We will exploit this unprecedented opportunity to examine the mechanisms by which re-replication promotes gene amplification. Because gene amplification is a primary means of activating oncogenes in cancer cells, these studies will shed light on the molecular triggers of tumorigenesis, and potentially identify targets of therapeutic significance. [unreadable] [unreadable] [unreadable]"}
{"text": "Project Summary/Abstract A compact low cost digital microscope that combines brightfield microscopy and fluorescence is proposed. Innovations in key areas have enabled size and cost reduction, and the merging of brightfield and fluorescent microscopy. The microscope will fit into the CD bay of a personal computer and use a standard interface. An open software architecture will allow others to build applications, methods, and custom interfaces. Development will be complete by the end of Phase I. As proposed, the instrument will have broad applications in the advancement of healthcare technologies. Project Narrative Microscopy forms the underpinning for all life science discoveries. A low cost compact digital microscope will be built that enables wide spread use of this technology, including underserved communities."}
{"text": "Amphetamine and related drugs of abuse elicit species-specific motor responses characterized by repetitive or stereotyped patterns. Research on animals, typically rodents, as models of the human response, has implicated the striatum and related basal ganglia circuitry in the motor-activating effects of these drugs. Critical elements of this circuitry include both dopamine- and glutamate-containing fibers that contact neurons in dorsal striatum. During amphetamine-induced motor activation, these neurons establish a pattern of discharge activity mediated, at least in part, by a complex interaction between dopamine and glutamate inputs. Research in this application extends this line of work on behaving animals in two directions. One involves characterization of the neuronal response pattern to amphetamine in substantia nigra pars reticulata, a major target of striatal neurons and an important output nucleus of the basal ganglia. After basic neurobehavioral correlations are established, further studies will examine the extent to which amphetamine-induced changes in reticulata neurons are mediated by the striatum via descending GABA-containing projections. The aim is to determine how amphetamine-induced neuronal response patterns established in striatum are represented in reticulata neurons. A second focus of the proposed research is to examine at the single-neuron level how the major transmitters altered by amphetamine-- dopamine and glutamate--interact with each other and with GABA to influence the activity of striatal neurons in an intact, normally functioning animal. Dopamine, glutamate, and GABA will be applied directly by iontophoresis to electrophysiologically isolated single units in awake, unrestrained rats. Attention will center on the mechanisms by which synaptic dopamine modulates glutamate- and GABA- mediated responses. A major component of this work also involved iontophoresis of amphetamine and other indirect dopamine agonists in striatum to determine how local changes in dopamine transmission modulate striatal activity and to reveal the synaptic action of these drugs unaccompanied by concomitant activation of other neuronal pathways. Collectively, these lines of research will provide important new information on the neurochemical systems and processes by which amphetamine alters neuronal function and motor behavior."}
{"text": "In mice, many genomic regions contain variation that results in differences in adiposity (Reed 2003; 2006; 2007; 2008). The goal of this research program is to find the gene or genes on mouse chromosome 9 that account for the quantitative trait locus Adip5, which is associated with increased weight of the gonadal adipose depot. Although several lines of evidence suggest that a gene or genetic variant here has the ability to regulate adiposity, the exact gene or DNA sequence that causes this effect is not known. The QTL Adip5 has features that make it a practical target for a positional cloning approach: it is not particularly susceptible to maternal effects or epistatic interactions, and it is associated with a distinct phenotype (gonadal depot weight). Furthermore, the experimental plan is designed to identify Adip5 if the locus is imprinted (i.e., if there are parent-of-origin effects). Within the current Adip5 confidence interval, there are several credible candidate genes (Bbs4, Cpy19a1, Crabp1, Cplx3, Il18, Lipc, Nedd4), as well as dozens of genes and noncoding RNA of unknown function. Using a chromosome 9 substitution strain developed in our laboratory for this purpose (CSS-9), we will backcross these mice to the host strain (C57BL/6ByJ; B6) and conduct a genome scan to reduce the confidence interval of Adip5 (CSS-9 X B6 N2 genome scan; Aim 1). Based on the refined confidence interval provided by the genetic mapping information, we will parse this chromosome into small intervals through successive breeding cycles, and create microcongenic strains (<200 kb), one of which will contain the gene responsible for Adip5 (Aim 2). To identify the exact gene responsible for Adip5, we will genetically engineer one or more mouse strains with a segment of 129 DNA substituted into a B6 background by homologous recombination, and evaluate its effect on gonadal depot weight (Specific Aim 3). The long-range goal of this work is to develop an approach to systematically identify genes that contribute to normal variation in fatness among mice."}
{"text": "6. Project Summary/Abstract The myelination of CNS axons during development and the remyelination of demyelinated axons in adults require oligodendrocyte progenitor cells (OPCs) to migrate to their target axons where they mature into myelinating cells. Although a number of critical factors have been identified for these processes, our understanding of the molecular control of CNS myelination and remyelination remains incomplete. We have identified a zinc finger protein (Zfp191) that when mutated in mice results in the absence of CNS myelin despite the presence of normal numbers of mature, process-extending oligodendrocytes. Zfp191 mouse mutants express an array of myelin-related genes at significantly reduced levels, suggesting that this protein participates in the control of the CNS myelination program. Zfp191 belongs to a family of nuclear proteins whose members contain both DNA binding zinc finger domains and SCAN domains, which are responsible for protein-protein interactions. The goal of this proposal is to gain a better understanding of the role that Zfp191 plays in the myelination process. Zfp191 is expressed in all tissues and cell-types examined, including astrocytes and neurons, and the level of Zfp191 mRNA does not change as OPCs differentiate into mature, myelinating oligodendrocytes. Thus, a critical question that we will address in the studies outlined in this proposal is whether Zfp191 has a cell autonomous function in oligodendrocytes or whether other cell types contribute to the myelin abnormalities displayed by the Zfp191 mutants. Moreover, we will determine if the continued expression of this protein is required for the maintenance of the myelin sheath, and we will also assess if this protein has a similar essential function in the remyelination process. We will also explore the molecular mechanism by which ZFP191 controls the myelination program by determining its DNA and protein binding potential. Relevance: The studies described in this proposal will focus on the molecular control of the final stages of oligodendrocyte maturation, which result in the initiation of the myelination program. A better understanding of the factors that enhance oligodendrocyte maturation is essential in our effort to develop strategies to promote axonal remyelination in demyelinating neurological disorders (e.g. multiple scelrosis)."}
{"text": "Our group will continue our studies directed to the understanding and control of latent human herpesvirus infections. We will focus on herpes simplex and human cytomegalovirus. For herpes simplex infection, we will study herpes genitalis in both humans and the guinea pig model of infection. We will continue to study gamma interferon as a correlate of disease containment in both systems as well as investigating the possibility of augmenting immunity conferred by isolated viral glycoproteins with immunomodulators such as interleukin-2. For human cytomegalovirus, we will continue to study the human immune response to two glycoproteins identified in the previous granting period. One glycoprotein is 86 kDa (p86) and the other is a complex of glycoproteins of 130 and 55 Da (p130/55). Our aim is to establish whether humoral and cellular immune responses to these specific glycoproteins correlate with protective immunity. We will also analyse the importance of gamma interferon in human cytomegalovirus infection to determine whether it correlates, as in herpes labialis, with containment of virus. We will examine whether it correlates, as in herpes labialis, with containment of virus. We will examine whether gamma interferon contributes to immunopathology of human cytomegalovirus infection in allograft recipients. Finally we will develop anti- idiotype monoclonal antibodies to the murine monoclonal antibodies with virus neutralizing activity described in our laboratory. We will use these anti-idiotype monoclonals as possible adjuvants for immunization with either the p86 or p130/55 as well as new probes for studying the regulation of the immune response following human cytomegalovirus infection. We will also use them to attempt to identify a cellular receptor for human cytomegalovirus."}
{"text": "PROJECT SUMMARY/ABSTRACT During dynamic exercise skeletal muscle blood flow increases rapidly and dramatically (exercise hyperemia) to meet the metabolic needs of the contracting tissue. Aging is associated with an attenuated hyperemic response during dynamic exercise. The mechanisms responsible for increasing blood flow at the onset of exercise as well as maintaining it over time in young adults involves a complex interaction between mechanical factors, the sympathetic nervous system and local metabolic and endothelial derived substances that influence vascular tone. The mechanisms responsible for the observed reductions in exercise blood flow in older humans are not completely clear. The applicant proposes two main goals: 1) to identify mechanisms contributing to the altered vasodilator responses to single muscle contractions and dynamic exercise in aging humans, and 2) to examine the effect of aging on the kinetics of skeletal muscle blood flow/vasodilation during exercise. During the K99/Mentored phase of the grant, the applicant will examine the mechanical, endothelial, and neural alterations in vascular function that occur with aging and determine how these changes relate to the attenuated rapid vasodilator response following a single muscle contraction. In the first portion of the R00/Independent phase of the grant, the applicant will examine the kinetics (rest to steady state transition) of vasodilation during rhythmic exercise and quantify the effects of aging on these responses. In the second portion of the R00 phase, the applicant evaluate whether the attenuated vasodilator response to single muscle contractions and slower kinetics of vasodilation during rhythmic exercise are similar in the upper and lower limbs of older subjects. Lastly, in the third portion of the R00 phase, the applicant will determine whether the changes in flow following single contractions and/or the kinetics of vasodilation in older humans is a result of physiological aging or related to training status. Collectively, the experiments outlined in this proposal focus on the mechanical, endothelial, and neural alterations that occur in the skeletal muscle vasculature with aging and how these changes impact blood flow in exercising muscle. Identifying the mechanisms by which blood flow to contracting muscles is altered with advancing age will help in understanding whether these changes are due to physiological age per se or a result of inactivity. During the K99/Mentored phase of the award the applicant will 1) continue to gain expertise in basic integrative physiology studies in conscious humans, and 2) continue to learn pharmacological and biochemical approaches to study the control of muscle blood flow from a mechanistic standpoint. Additionally, the candidate will gain new research skills and knowledge related to advanced cutting-edge ultrasound techniques and measures of arterial properties (specifically, Shearwave Dispersion Ultrasound Vibrometry; SDUV) under the mentorship of Dr. James Greenleaf (co-mentor). Training in an established and productive laboratory such as that of Dr. Michael Joyner along with the help of Dr. James Greenleaf at the Mayo Clinic will provide opportunities needed to achieve the goals listed above. Importantly, this training will facilitate the achievement of the applicant's long-term goal to develop an internationally-renowned independent research program in cardiovascular physiology."}
{"text": "Naltrexone, an opioid antagonist, is currently one of the most promising pharmacotherapies for alcohol dependence. In clinical trials, naltrexone has been shown to be more effective than placebo in reducing alcohol consumption and relapse rates. A number of critical questions remain, however, about the optimal strategies for using naltrexone in the treatment of alcohol dependence. The proposed research is designed to achieve four specific aims: (1) to evaluate whether a more intensive psychotherapeutic strategy provided with naltrexone enhances initial treatment outcomes in contrast to a less intensive intervention that may be used with naltrexone in primary care settings; (2) to evaluate whether long term treatment with naltrexone provides additional benefit in preventing relapse, alcohol-related impairments, and alcohol related psychiatric symptoms among those who respond to short term naltrexone treatment; (3) to explore predictors of response to the psychotherapeutic interventions and to naltrexone, including gender, craving, neuropsychological functioning, and severity of alcohol dependence; and (4) to evaluate the safety of long-term naltrexone treatment in an alcohol dependent sample. In order to address the question of treatment intensity, 192 subjects will be randomized to receive naltrexone and either weekly coping skills therapy or advice and clinical management for 10 weeks. The coping skills therapy is a treatment commonly used in specialty alcoholism treatment clinics and is designed to increase the patient's coping strategies in an effort to reduce the probability of relapse. Advice and clinical management is a less intensive treatment that is consistent with primary care treatment. Following completion of this study, two parallel discontinuation studies will be conducted in which treatment responders in each group are then randomized to receive naltrexone or placebo for six months. One study compares the outcomes of subjects maintained on naltrexone versus those on placebo among those initially treated using the advice and clinical management. The second study compares outcomes for naltrexone and placebo maintained subjects who were initially treated with coping skills therapy. Taken together, these studies should provide critical information about psychotherapeutic strategies to provide in conjunction with naltrexone and the optimal length of naltrexone treatment."}
{"text": "The Dutch Famine ('Hunger Winter') of 1944-1945 provides a unique opportunity to identify early life determinants of life course health and well-being. To date, studies have concentrated on health and mortality outcomes in ~45,000 army recruits who were examined at age 18 in the early 1960s. Famine exposure in these men can be inferred from place and date of birth. Funding is now requested to link a wide variety of socio-economic outcomes (employment, wages, and disability benefits) from government sources to our studied population. The study is collaboration between epidemiologists and demographers and economists (Dr. Heckman, University of Chicago). The newly linked data will be analyzed with state-of-the-art epidemiologic and econometric approaches to reliably identify, estimate, and interpret the effects of an early life shock throughout the lifecycle. This work will integrate currently separated research traditions from the medical and social sciences and lead to a better understanding of 'fetal programming' and its implications."}
{"text": "The Virginia Department of Mental Health, Mental Retardation and Substance Abuse Services proposes to enhance the data collection systems of the State's 40 community services boards. The goals of the project include implementation of the MHSIP recommended data sets and standards, improvement in the quality and reliability of data, and increased use of data and integration of data between community programs and inpatient facilities. The project will employ two full-time staff persons working with a Steering Committee of Community Services Board representatives. Activities will include modifying existing CSB data systems to meet MHSIP standards, developing a human resources data base, developing data audit guidelines and disseminating data analyses and reports."}
{"text": "We are studying regulation of sexuality in the male rat and are carrying out projects in the following areas: (1) Mechanism of action of androgens in the seminiferous tubules: Measurement of concentration and ratios of testosterone and 5 alpha-dihydrotestosterone in cytosol and nuclei, both bound and unbound, of seminiferous tubules at specied periods after hypophysectomy, and comparison with androgen concentrations in plasma, in effort to carrry out successful nuclear exchange assay and to assess role of androgen bindng in spermatogenesis. (2) Hormonal rhythms in the male laboratory rat: We are completing our second year of studying the pattern of matuation of plasma testosterone, LH and prolactin in rats from age of 30 through 150 days, in order to detect basic maturational pattern and influence of season and calendar year on this pattern. (3) Mating and the hormone neuroendocrine reflex: We are using the reproducible rise of plams testosterone, LH and prolactin at time of mating as a model for the study of its restoration in castrates by steroid replacement. (4) Peripheral regulation of plasma testosterone: The testicular-denervated, adrenal demedullated male rat is being tested both with stimulation of the preoptic nucleus and mating in order to evaluate CNS regulation of the denervated testis. BIBLIOGRAPHIC REFERENCES: Frankel, A.I., E.J. Mock, W.W. Wright and F. Kamel, 1975. Characterization and physiological validation of a radioimmunoassay for plasma testosterone in the male rat. Steroids 25:73-98. Mock, Edward J., Freja Kamel, William W.Wright and Arthur I. Frankel, 1975. Seasonal rhythm in plasma testosterone and luteinising hormone of the male laboratory rat. Nature 256:61-63."}
{"text": "The goal of the proposed research is to examine how exposure to parental violence operates within the contest of additional risk factors (low socioeconomic status, maternal depression, ;ack of moth-child relationships in the relation between exposure and children's social behavior. The aforementioned risk factors, aggressive expectations, severity and duration of violence witnessed, aggressive behavior problems, and social competencies of 150 children from violent homes will be compared to those of a community group of children where conflict ranges from verbal discord to low level aggression and does not include violence. Parents, shelter counselors, and teachers will complete several measures regarding family violence and their child's functioning, mothers will complete additional measures assessing their degree of depressive symptomatology and their perceptions of moth-child relationship positivity, and children will complete several measures assessing their aggressive expectations in peer relationships, mother-child relationship positivity, and their perceptions of warmth and conflict in their close peer relationship. This research has implications for improving models of the processes through which parental violence impacts on development and for informing prevention and intervention strategies to help child witnesses adjust to their experiences."}
{"text": "Project Summary/Abstract The overall goal of the Cancer Outreach Core (COC) is to reduce cancer health disparities among vulnerable migrant Pacific Islander populations living in Guam (GU) and Hawaii (HI). They are the Chamorro, Marshallese and Chuukese; the indigenous peoples of Guam, the Republic of the Marshall Islands (RMI), and Chuuk State in the Federated States of Micronesian (FSM), respectively. There are over 16,000 migrant Pacific Islanders from the RMI and from the FSM living in GU and over 17,000 living in HI. They have a disparate cancer health status compared to the rest of the populations in HI, GU and the Continental US. The cancer disparities of interest are preventable cancers which have a significant incidence in the Micronesian populations. As an example, the Pacific Islander women from the RMI and FSM have among the highest incidence rates of cervical cancer in the world: 79.7 per 100,000 and 42.4 per 100,000 (2007-2011, age-adjusted to the 2000 US Standard) in the RMI and in Pohnpei State, FSM, respectively, compared to 9.9 per 100,000 women in the United States (US) (1). The COC will build on the 2003-2015 University of Guam (UOG)/University of Hawaii Cancer Center (UHCC) Comprehensive Partnership efforts to prevent and control cancer in Pacific Islander communities. Through established community partnerships, the COC has three main tasks: (1) outreach to primary care physicians (PCP) to reduce preventable cancers in Chamorro, Marshallese and Chuukese peoples, (2) outreach to the Chuukese, Marshallese and other health disparity communities to increase HPV vaccination rates among adolescents, and (3) assist U54 investigators with recruitment of Pacific Islander populations in support of studies focusing on cancer health disparities. Community Health Educators (CHE) who have worked with the target communities will support the COC outreach strategies and the NCI National Outreach Network's coordinated agenda. The COC activities align with the overall goals of the Comprehensive Partnership to Advance Cancer Health Equity (CPACHE), and support hypothesis-driven research as proposed elsewhere in this application (e.g. Pilot Project I, Full Project II)."}
{"text": "Recent events such as pandemic influenza A (H1N1)pdm09 have demonstrated how mutations in a viral genome can greatly impact disease spread and population health risk. Thus, there is now a greater need to merge viral genetics within state health agency surveillance practice. This is particularly relevant for zoonotic viruse that are transmittable between animals and humans such as influenza, rabies, and West Nile Virus. As an added complexity, there are many potential drivers of virus transmission that need to be considered including climate, population and travel, and ultimately, genetic polymorphisms in the virus itself. Zoonotic disease surveillance at the state level is most often performed using data that originates from passive case reporting by laboratories or clinicians rather than secondary data from resources such as GenBank. While these data are sufficient for federal reporting purposes and basic trend analysis, they only measure the number of suspected or confirmed cases and not the genetic characteristics of the virus. When states and federal agencies do use genotyping, it is often limited to certain pathogens (mostly bacteria) and only for samples that are reported through passive surveillance or during outbreak investigations. The omission of secondary viral genetic data limits the types of analysis by state health agencies. For example, current reportable disease data do not enable epidemiologists to determine the origin of a particular viral strain, trace how it has spread, or identify climate, population, and genetic factrs enabling it to propagate. In this study, we will develop and evaluate an integrated bioinformatics framework to supplement current zoonotic disease surveillance approaches at state health agencies. We hypothesize that a framework that properly merges viral genetic data with climate, population, and travel data can accurately predict the timing of initial peaks of seasonal epidemics caused by zoonotic viruses. Health agencies can then use these trends to prioritize control measures and reduce morbidity and mortality. In addition, we will address the barriers to health agency utilization of bioinformatics resources and secondary data by developing an online portal for accessing and querying of complex viral genetic models. We will measure the perceived usefulness of information from our framework as part of our long-term goal of utilization and adoption by health agencies. In Aim 1, we will develop an automated bioinformatics system that models virus diffusion while testing the significance of climate, population, and genetic predictors. As part of this effort, we will provide a publically available Web portal for health agencies and other users to access our results, and run their own models. In Aim 2, we will use our platform to identify significant climate, population, and genetic predictors of diffusion across different zoonotic viruses including influenza and WNV. In Aim 3, we will evaluate the accuracy of a bioinformatics system that uses statistically significant climate, population, and genetic predictors to identify seasona trends of zoonotic virus epidemics and communicate these findings to different health agencies."}
{"text": "The beta thalassemias are characterized by a deficiency of adult (beta) globin chains of adult hemoglobin (Hb), an excess of toxic, unmatched alpha globin chains, and intramedullary hemolysis. The resulting anemia develops only after fetal (gamma) globin synthesis and Hb F is suppressed in infancy. Induction of fetal (gamma) globin to levels which improve globin chain balance by even 10 percent can prolong red blood cell survival and diminish clinical morbidity. 5-Azacytidine has increased hemoglobin (Hb) levels by 1.8-3 gmd/d1 in thalassemia, but also causes general cytopenias and carries carcinogenicity risks. Fatty acids induce (gamma) globin experimentally. Arginine Butyrate, a prototype fatty acid, has been most effective when given intermittently or Pulsed, inducing Hb F to a mean level of 22 percent in 7/9 adults with sickle cell disease and increasing total hemoglobin by 3 gm/dl over baseline levels in 5/6 beta thalassemia patients. Two clinical pilot studies are proposed to test the hypotheses that therapy with Pulsed Butyrate, or rhu-EP0 + Pulsed Butyrate, will induce gamma globin chain synthesis sufficiently to improve non alpha: alpha globin chain balance and red blood cell survival, and increase total Hb in a significant proportion of patients with beta thalassemia intermedia. Baseline hematologic levels will be assayed four times over a two-month period. Butyrate will then be administered during an Induction Phase, to determine a patient's optimal dose, followed by a \"Maintenance Phase\" of therapy for 3 months. Pulsed Butyrate will also be tested with rhu-EPO. The proportions of patients on each study in whom the following endpoints are achieved, compared to baseline levels, will be analyzed: 1) an increase in total Hb of at least 2.0 grams/dl, 2) an increase in hematocrit of at least 5 percent, 3) a decrease in hemolysis, measured by LDH and bilirubin, 4) improvement in globin chain synthesis by 10 percent. Whether specific genotypes and in vitro response to Butyrate correlate with clinical responses will also be analyzed. These studies should determine the proportion and some genotypes of beta thalassemia patients which can benefit from Pulsed Butyrate plus/minus rhu-EP0 therapy."}
{"text": "This proposal has been targeted at two areas of catalytic antibody research: The obtainment of new antibody catalysts and the development of new strategies for obtaining improved antibody catalysts, including antibodies with altered specificities and chemical reactivities. The development of new antibody catalysts will center around a hapten design strategy which we introduced and termed \"bait and switch\" catalysis. We will apply this strategy for our investigation of antibodies which can catalyze cationic cyclization processes. We will explore two types of cationic cyclization reactions. The first involves antibody catalyzed olefin cyclization processes and thus the formation of carbon-carbon bonds. To augment these studies, we intend to also explore the possibility of utilizing antibody catalyzed cationic cyclization reactions to form carbon-heteroatom bonds. A long-term goal of these two projects is to channel this type of antibody catalysis for the formation of unique steroid and unusual heterocyclic molecules. An equally important reaction in the formation of carbon-carbon bonds is the Aldol condensation. Using our \"bait and switch\" approach, we plan to investigate bimolecular antibody catalysis for an intermolecular Aldol reaction. We will engage a multisubstrate \"bait and switch\" tact for the induction of amino acid residues in an antibody's combining site to act both as a Lewis acid catalyst and, to provide additional binding forces to overcome entropic loss which is seen when two substrates are brought together. An advantage of such catalytic antibodies would be the avoidance of environmentally damaging Lewis acids which are commonly employed in Aldol reactions. Hybridoma methodology has been used exclusively in the obtainment of catalytic antibodies. However, this process of isolating catalytic antibodies suffers from some limitations. Of special concern is the ability to generate a large enough array of monoclonal antibodies to screen for the desired activity. We intend to apply antibody phage display semisynthetic combinatorial libraries in conjunction with panning methodologies which are designed to select for antibody chemical catalysis. To complement these studies, we will also utilize antibody phage display methodologies to examine the potential of altering a catalytic antibody's specificity and chemical reactivity. This arsenal of antibody phage display studies which we have selected to investigate should provide us with a means to access more sophisticated modes of antibody catalysis."}
{"text": "The inflammatory bowel diseases (IBD), ulcerative colitis and Crohn's disease, are histologically characterized by an accumulation of lymphocytes and plasma cells in the lamina propria and foci of polymorphonuclear lymphocytes. An immune mechanism has long been suspected in the pathogenesis of IBD but, the etiology remains unknown. It is also not clear whether alterations in lymphocyte populations or their function mediate the mucosal injury or are epiphenomena of inflammation. Current research efforts investigating the functional properties of intestinal mucosal lymphocyte subpopulations, especially the intraepithelial lymphocytes (IEL), have been hampered by the inability to specifically identify IEL in cell suspension after isolation from intestinal specimens. The major goal of this project is to define possible phenotypic and functional differences in the mucosal lymphocyte subpopulations in IBD and normal intestine. This will be accomplished by using recently developed monoclonal antibodies that are primarily restricted to mucosal lymphocytes and IEL in particular. Monoclonal antibodies that recognize the T cell receptor gamma/delta will also be used to determine the intestinal distribution of these cells in IBD and their functional characteristics. A difference in the distribution or function of lymphocyte subpopulations in the lamina propria or epithelial compartments, may play a role in the pathogenesis of IBD."}
{"text": "The Duke Department of Pediatrics'training program is based on developing pediatric Junior Faculty into physician-scientists who are skilled in cutting-edge methods of laboratory research and who will pursue independent academic careers investigating important issues related to childhood diseases. Our program is based on our pool of outstanding candidates, a strong curriculum of didactic courses, experienced mentors who perform state-of-the-art research, and an excellent research environment. Our commitment to develop future academic pediatricians is evidenced by the academic success of our Junior Faculty. Our Principal Investigator is Dr. Joseph St. Geme, III, Chairman of the Department who is assisted by Dr. Page Anderson, Program Director, Dr. Delbert Wigfall, Minority Recruitment Advisor, and an Internal and External Advisory Committee. Four young scholars will be supported each year. They will be drawn primarily from our fellows and Junior Faculty. Recruitment of underrepresented minorities is a focus of the program. Our faculty, which includes many mentors from other Departments, has strong track records in research, funding, and mentoring. Our research training centers are in the areas of Developmental Biology, Cell Biology and Cell Signaling, Microbiology and Immunology, and Genetics, Genomics, and Proteomics. Didactic courses, including a multi-lecture course on writing, will complement the laboratory research experiences, enabling the trainees to write and submit grant applications to support their career. The trainees will be required to take a five lecture course in Responsible Conduct of Research. The young Scholars will have access to all the research resources of the NCI-funded Comprehensive Cancer Center, the shared facilities at Duke University, the Institute for Genome Sciences and Policy, and the Center for Human Genetics."}
{"text": "Our short-term aim is the development of algorithms for locating, enhancing, and recognizing selected classes of objects in radiographic images. Our long-term aim is the development of a man-machine technology capable of storing, analyzing and retrieving radiographic pictures in greater numbers, with greater accuracy and reliability, and at lower costs than ever before. We also wish to establish a research environment where medical and engineering researchers can work effectively together on the automation of image analysis. To achieve these objectives we will build upon the results of our research over the past several years on computer-aided image processing, pattern recognition, and trainable classifiers. Our plan of research consists of three concurrent branches all three of which are major forces for the success of the project: 1) basic theories and techniques, 2) medical applications, with special emphasis on radiology, and 3) the picture-processing computer facility. It is expected that the techniques and insights developed in this project will be useful not only in radiology -- which receives the major emphasis here -- but also in a wide class of clinical and research activities involving the analysis of images."}
{"text": "It is the hypothesis of this grant application that the pathogenesis of rheumatoid arthritis (RA) depends not upon the absolute numbers of T cells in the blood or the joints, but rather upon the relative percentages of auto-antigen specific T cells that produce and respond to pro-inflammatory and anti-inflammatory signals, including cytokines and chemokines. These T cell populations may be the target for therapeutic intervention. In preliminary experiments that prompted this grant application, we have shown that: i) peptides of bacterial origin (E. coli dnaJ heat shock protein) are strong immunogens in RA patients. dnaJ shares with HLA DRB1*0401 the susceptibility sequence to RA (shared epitope); ii) T cells in the blood RA patients that react with the shared epitope can be identified and isolated, using a novel technical approach; iii) T cell receptors usage and patterns of cytokine production by these shared epitope specific T cells can be analyzed. Based upon these initial results, we now need to test our hypothesis in a larger cohort of RA patients with different disease courses, and responses to treatment. Thus, the aims of this project are: 1. To characterize frequencies, function and phenotypes of T cells in RA patients reactive with the shared epitope. Parameters to be studied will include cytokine production, T cell receptor usage and membrane markers of activation and memory. Chemokine receptors will also be studied. 2. To determine the effects of disease activity and severity, and of slow acting anti rheumatic drugs, on function and phenotype of the shared epitope specific and bystander T cells. 3. To develop methods to modulate the Th1-type phenotype of shared epitope specific T cells, using in vitro immune manipulation with altered peptides. The long term objectives of this project are: i) to contribute to a better understanding of T cell-mediated events in the pathogenesis of rheumatoid arthritis; (ii) to develop paradigms for the prediction of disease outcome, and for evaluation rapidly new models of therapy."}
{"text": "Peripheral neuromodulation has been pursued to control conditions such as chronic pain and dysfunctions of pelvic organs. The expansion of such therapies is challenged by incomplete understanding of peripheral neural circuits, which comprise complex networks of peripheral ganglia and nerves that innervate multiple organs and carry axons of afferent as well as efferent peripheral neurons. Methods for manipulation of neural activity through genetically engineered receptors and channels such as channelrhodopsins and DREADDs (Designer Receptor Exclusively Activated by Designed Drug) create an opportunity for the development of tools for targeted neuromodulation through viral vector-mediated cell-specific gene transfer. The use of cell-specific promoters and combinatorial vector systems for targeted expression of optogenetic or pharmacogenetic transgenes presents a potential solution to the neuroanatomical challenges of peripheral neuromodulation. The long-term objective of this research program is to develop strategies for peripheral neuromodulation through cell-specific delivery of neuromodulatory transgenes to peripheral ganglia using adeno-associated viral (AAV) vectors. Based on our work on the biodistribution of AAV vectors, the objective of this application is to provide proof-of-concept fo site-specific targeting of transgene expression to afferent and efferent systems. We will address the central hypothesis that cell-specific targeting of a neuromodulatory transgene to peripheral neurons by AAV vectors enables organ-specific functional interventions. To address the Specific Aims of the project, we will develop AAV vectors for cell-specific targeting of DREADD, validate neuroanatomically their restricted biodistribution, and demonstrate the neuromodulatory function of the transgenes using behavioral, in vitro and in vivo neurophysiological, and in vivo imaging analyses. Aim 1: Test the feasibility of a strategy for organ-specific neuromodulation of afferent systems through AAV-mediated gene transfer. We will test the hypothesis that combinatorial AAV vector targeting of colon-innervating sensory neurons, using a Cre- dependent vector that carries the gene for the inhibitory DREADD (hM4Di) and an AAV-Cre vector, will enable organ-specific control of afferent activity. Aim 2: Test the feasibility of a strategy for organ-specific neuromodulation of efferent autonomic systems through AAV-mediated gene transfer. We will test the hypothesis that cell-specific AAV vector targeting to parasympathetic bladder post-ganglionic neurons, using a vector that carries the gene for the excitatory DREADD (hM3Dq) under the control of the cholineacetyl transferase (ChaT) promoter, will promote bladder emptying in a rodent model of spinal cord injury. The proposed strategies and vector tools will enable functional dissection of peripheral neural circuits, applicable to multiple organs and across species and animal models. These approaches can be extended to optogenetic neuromodulation, to integration with closed-loop platforms, and potentially to development of next generation neuromodulation therapies as the clinical translation of AAV-mediated gene transfer advances."}
{"text": "The Administrative Core will facilitate and coordinate communications between the members of the Consortium themselves and between them and the 4 members of the Scientific Advisory Board (SAB), all of whom will be Consultants to the Consortium. The major thrust of the proposals made in this application is to investigate and hopefully resolve the problems associated with coagulation dysregulation following organ xenotransplantation in pig-to-primate models. Frequent communication, most likely in the form of conference calls, is likely to be required between members of the Consortium and Consultants, all of whom are experts in the field of xenotransplantation and/or coagulation. The Core will help with travel arrangements and hotel accommodation for Consultants visiting Pittsburgh for the annual workshops at which the results of the Consortium's efforts will be discussed and plans made for the future 12-month period. The Core will also arrange travel and hotels for the members of the Consortium who will be participating in the annual meeting with other scientists funded through this RFA. The immunologic and coagulation assays to be used in the studies in Projects 1 and 2 will, as far as possible, be standardized between the two centers, and this will require exchange of samples, tissues, and reagents. This will be organized and coordinated through the Administrative Core. Data from the various experiments and assays will be collected and collated in the Administrative Core. The Core will also be responsible for assisting with the preparation of manuscripts reporting the scientific work of the consortium to be submitted for publication. The Core will do what it can to ensure timely reporting in peer-reviewed journals of the results obtained by the Consortium. It is the Consortium's plan to invite other scientists who are working in this field to the annual workshops that will take place in Pittsburgh, even if those scientists are neither Consultants to the Consortium nor funded by the RFA (although these scientists will have to fund their own travel expenses). The members of the Consortium believe that it is only through full collaboration and exchange of ideas and information that the problems of xenotransplantation will be resolved in a timely manner. The Administrative Core will provide assistance to visiting scientists with regard to reserving hotel accommodation, etc."}
{"text": "DESCRIPTION: (Applicant's abstract) The recently-determined sequence of the genome of bakers' yeast (S. cerevisiae) revealed that almost two-thirds of the genes of this well-studied organism had escaped detection. Thus, we know almost nothing about the function of most of the genes that comprise this simple eukaryotic cell. To catalyze approaching the ultimate goal of a comprehensive understanding of the function of all the proteins that constitute a eukaryotic cell, we propose to construct quickly the complete set of mutants deleted for each of the approximately 6000 genes of this important model organism. It seems certain that wide distribution of this collection of mutants will greatly accelerate the pace of discovery of gene function. This goal will be achieved by a consortium of 5 laboratories, each disrupting 300 genes in each of two year, yielding 3000 mutants. The other 3000 genes of the organism will be disrupted by our European and Canadian colleagues. The mutants will be screened for a few standard phenotypes to provide necessary information for the users of the mutant collection, then distributed widely to all interested scientists for more detailed and complete analysis. We propose to replace the coding sequence of each gene with the KanMx gene encoding resistance to the drug gentamycin. In addition, a novel sequence tag will be introduced into each of the mutants to uniquely identify them. Non-essential genes will be disrupted in haploids of both mating types; essential and nearly essential genes will be disrupted in diploids. Gene disruption will be accomplished using a facile and popular PCR-based technique that will enable construction of the complete set of mutants in two years. In the final year of the project, we will enhance the value of the mutant collection by constructing conditionally-expressed versions of essential and nearly essential genes, constructing approximately 750 double mutants deleted for both members of a duplicated gene pair, and disrupting small genes as they are revealed."}
{"text": "Abstract A key problem in chronic infections is progressive dysfunction of the infected organ. Importantly, patients with similar risk factors often exhibit marked variation in the rate of disease progression, as do individual patients at different times. The chronic Pseudomonas aeruginosa (Pa) lung infections in people with cystic fibrosis (CF) are a prime example. Pronounced variability in the rate of lung function decline is seen across patients even when host CFTR genotype and airway microbiology are similar. While both bacterial and host factors likely contribute to disease variation, several new findings lead us to focus on genetic variation that evolves in infecting Pa. First, CF Pa strains have been found to genetically diversify in vivo, producing clonally-related variants that differ markedly in virulence, and these variants can co-exist simultaneously inside lungs. Second, our data show that the distribution of genetic variants changes over time and the emergence of highly virulent genetic variants can be temporally associated with changes in disease. Finally, many groups have investigated host genetic and environmental factors that affect disease, but the effects of bacterial genetic variants on disease progression are relatively unexplored Here we exploit unique clinical and technical resources, and our knowledge of Pa pathogenesis to test the hypothesis that some gene variants that evolve in the Pa populations infecting CF patients increase lung injury. We will test this hypothesis in three steps. First, will we measure Pa gene variants in sputum collected at specific time points based on the lung disease phenotypes. Second, we will perform genetic association analysis to delineate which variants/variant groups are most strongly associated with the phenotypes. Third, we will investigate the functional effects of gene variants on Pa virulence, host injury, and inflammation. This work could identify potential cause and effect relationships and provide proof of principle for a disease progression mechanism that may operate in many chronic infections."}
{"text": "The goal of this research is to understand how the nervous system encodes information about odors. The sense of smell presents unique problems to the nervous system in terms of stimulus detection, neural encoding and recognition of complex stimuli; understanding how the brain solves these problems will likely lead to new general insights into how the brain processes information. The initial code for odors consists of patterns of activity across olfactory receptor neurons, which project to glomeruli in the olfactory bulb, the first stage of synaptic processing of olfactory information. Here, patterns of activity across thousands of receptor neurons are transformed into spatially organized maps of glomerular activation - these glomerular patterns are unique for a given odor and odor concentration. The research in this continuing project uses optical imaging methods to visualize these patterns and to investigate how they represent olfactory stimuli. Previous work built on the observation that spatial maps of receptor input to glomeruli are temporally dynamic, and asked how these dynamics participate in odor coding and shape the initial stages of olfactory processing. A key finding was that much of the temporal dynamics of odor maps are organized around the respiratory cycle, which is heavily modulated in the awake, behaving animal and is integral to the act of smelling. The experiments proposed here will investigate, for the first time, how odor sampling behavior (i.e. - `sniffing') shapes early olfactory coding at the level of the olfactory bulb and the transformation of receptor inputs into patterns of postsynaptic activity. The experiments will image receptor input to olfactory bulb glomeruli while an animal is awake and actively performing odor-guided tasks, and relate these patterns to the animal's sniffing behavior. Different patterns of sniffing will also be played back in the anesthetized animal in order to separately evaluate effects of sampling behavior and effects of behavioral state-dependent modulation of receptor inputs. The experiments will also ask how sniffing shapes the transformation of odor representations by the olfactory bulb, using electrophysiological recordings from individual olfactory bulb projection neurons during activation by odorants sampled with different sniffing patterns. In addition to testing, for the first time, several longstanding hypotheses about the role of sampling behavior in shaping odor codes, this work will be important in understanding how olfactory information is encoded and processed in the awake, behaving animal. This work has the potential to lead to new treatments for olfactory deficits or therapeutic approaches to improving odor or flavor perception in individuals with impaired nasal function. PUBLIC HEALTH RELEVANCE: The sense of smell presents unique problems to the nervous system in terms of stimulus detection, neural encoding and recognition of complex stimuli; understanding how the brain solves these problems will likely lead to new general insights into how the brain processes information. This project in particular focuses on the importance of respiratory behavior in determining how the brain represents and processes olfactory information. Understanding this relationship could lead to new treatments for olfactory deficits or therapeutic approaches to improving odor or flavor perception in individuals with impaired nasal function."}
{"text": "PRAM-1 is a phase-II, randomized, open-label, multicenter, multi-arm protocol. HIV-infected, clinically stable children, 24 months to 17 years of age, treated with the same antiretroviral therapy for >16 weeks, will be randomized to one of the three following treatment arms: 1) Zidovudine (ZDV) plus Lamivudine (3TC); or 2) Stavudine (d4T) plus Ritonavir; or 3) ZDV plus 3TC plus Ritonavir. Subjects will be stratified by CD4% <15% or >15%. The first eight subjects randomized to the d4T+Ritonavir and ZDV+3TC+Ritonavir arms will participate in a real-time Phase-I pharmacokinetics study (16 subjects in total). The three arms will be evaluated primarily with respect to change in plasma HIV-1 RNA copy number from baseline to 48 weeks and with respect to safety and tolerance."}
{"text": "Dementia is the most common neurological disease in the elderiy. Experimental, epidemiological, and neuropathological evidence suggest associations between vascular factors and cognitive decline and dementia regardless of subtype, with vascular pathology present in half of all dementia cases. However, the mechanisms which underiy these associations are a topic of debate. While large vessel cerebrovascular disease and cortical stroke have been linked to cognitive decline and dementia, the specific role of cerebral small vessel disease is unclear. Few population based studies have assessed cerebral microvascular structure and function in relation to cognitive decline and dementia, due in part to the difficulty of applying specialized and costly imaging techniques in large population based samples. We propose to apply two non[unreadable][unreadable] invasive, relatively low cost techniques for assessing microvascular status (with retinal phootography) and function (transcranisal Doppler ultrasound (TCD) with C02 challenge) to test the hypothesis that cerebral hypoperfusion and microvascular disease precede the onset of dementia and contribute to cognitive decline. Because retinal and cerebral arterioles have similar anatomy, physiology and embryology, retinal characteristics assessed by standardized grading of fundus photographs provides a surrogate marker for cerebral microvascular status. Similarly, TCD with C02 challenge is a safe non-invasive technique for assessing cerebral vascular function. Recently, these techniques have been studied in relation to dementia, although the majority of studies have been limited by cross-sectional design, restriction to clinical AD cases, or limited neuropsychological data. Little is known of their associations to eariy cognitive decline and preclinical dementia. Applying these techniques in the EAS will allow us to assess whether retinal microvascular status and cerebrovascular function predict cognitive decline and dementia, with a focus on eariy cognitive changes. To elucidate mechanisms that may underly these associations, we will evaluate whether they mediate associations of vascular risk factors with cognition and will assess the relation of retinal and TCD measures to neuroimaging measures of structual brain changes. RELEVANCE (See instructions): Much is known regarding the prevention and treatment of vascular disease. Better understanding the role of vascular status and function in dementia will direct the application of these strategies toward the prevention of this prevalent condition."}
{"text": "The purposes of this study are to collect data on severe headache in order to measure the prevalence and to describe the demographic characteristics of the major types of headache. To this end a survey of the general population has been designed. A survey questionnaire, which includes sections on demography, descriptive headache features, medical information, and history, has been developed. The data will also be used to identify and assess the etiological and environmental factors associated with the major idiopathic headache types. The study was designed in two parts: a feasibility study and an area survey. The feasibility study has been completed. Telephone interviews have been conducted with the patients from four headache clinics. The questionnaire data have been processed together with information abstracted from the physician records about the headaches. The planning and design of the area survey has been completed. The area survey will not be funded and this study is thereby completed."}
{"text": ": The Children's Computerized Physical Activity Reporter: Reliability and Validity, PI: Patricia Flannery Pearce, MPH, PhD, RN, APRN, FNP Assistant Professor University of Utah, College of Nursing Salt Lake City, Utah Purpose: The purpose of this descriptive, comparative study will be to: (a) evaluate the test- retest reliability and (b) concurrent validity of an innovative, computer-based self-report questionnaire, the Children's Computerized Physical Activity Reporter (C-CPAR), compared with a commonly used paper-based questionnaire and objective criterion of accelerometry. Background: Physical inactivity is linked to obesity, metabolic syndrome, and early cardiovascular risk. There are global directives to better understand children's physical activity, but physical activity measurement is difficult, especially with self-report. Currently available self-report questionnaires provide adequate reliability, but less than robust validity. The inconsistent validity of currently available physical activity questionnaires may be due the lack of attention to matching the questionnaire content and format to children's understandings of the underlying constructs, or to the form and structure that children need for precise recall. The C-CPAR was developed with children, for children, in a study aimed at identifying children's understanding of physical activity, their needs for reporting, and their perspectives on currently available questionnaires. Usability and feasibility of the C-CPAR were excellent in the original study. In a pilot feasibility study (N=31), with the National Youth Sports Program (NYSP), comparison of MET minutes of NYSP camp activity reported for two 1-day reports demonstrated strong reliability (r=0.64, p<.0001). Average MET level for scheduled NYSP activities and C-CPAR reported activities for the NYSP time period, correlations were significant, ranging between r=0.66 (p<.01 Day 1) and r=0.37 (p<.01 Day 2). Comparisons of total NYSP activity reports to crude pedometer counts were not significant (r=0.33).77 Time to completion for the C-CPAR was significantly faster on Day 2 of the study. A Spanish language alternative is being developed. However, C-CPAR reliability and validity must be assessed further, to determine the relevance of the C-CPAR for clinical or research venues. Participants: Participants will be 180 junior-high school students from a single, ethnically-diverse public junior high school using, selected with stratified random sampling (grade and gender). Methods: Children will be randomly assigned to two groups in a crossover design to control for questionnaire sequencing effect. Participants will wear an Actical(r) accelerometer for three days (Friday to Monday), complete one self-report (C-CPAR or PD-PAR) on each of the two following consecutive days, followed by crossover to the alternate report daily for two subsequent days. Analyses will include Pearson correlations for test-retest reliability and validity, Bland Altman plots to evaluate associations between measures, and differences in correlations for test of accuracy. Differences in age, gender, and ethnicity will be evaluated. Implications: The C-CPAR may provide the format and content that matches children's understanding and needs for accurately reporting their activities. If reliability and validity are found adequate, the C-CPAR potentially can be used to assess children's activities in clinical and research venues. Improved measurement will translate to improved understanding and provide a stronger foundation for intervention. The long-term goal of this program of research is to develop intervention studies, specifically contributing to knowledge of physical activity and its behavioral components, while maximizing the use of technology. Future research will focus on the utility of the C-CPAR for therapeutic application in research and practice. The World Health Organization, Healthy People 2010, and the Surgeon General encourage the accumulation of at least 30-60 minutes of moderate activity daily for all age groups. Promotion of physical activity and counseling about its importance should be a priority for all health care professionals. Yet there is an increasing trend of inactivity well documented. To understand physical activity in children, measurement is essential. Many of the measures of activity for children are modifications of instruments originally developed for adults, but the study of physical activity of children is complicated by developmental differences, vocabulary, and unique activities in a rapidly evolving technologic environment. Currently available self-report questionnaires are inadequate for children's reporting. The C-CPAR was designed with children, for children, to support children's reporting. The current proposal is designed to examine the reliability and validity of the C-CPAR, and expand knowledge regarding children's physical activity and its self-report measurement. A valid self-report tool will aid researchers and clinicians in understanding physical activity. More in-depth understanding will serve as a strong foundation for interventions. The long- term goal of this program of research is to develop intervention studies, contributing to knowledge of physical activity and its behavioral components, while maximizing the use of technology. Future research will focus on the utility of the C-CPAR for therapeutic application in research and practice. [unreadable] [unreadable] [unreadable]"}
{"text": "The ribonucleases of human pancreas have been less completely characterized than similar enzymes of other organisms. This project is concerned with the isolation and biochemical characterization of human pancreatic ribonucleases, and with the definition of the relationship between the human pancreatic enzyme and ribonucleases found in other human tissues, in serum, and in urine. In addition to enzyme characterization, a radioimmunoassay procedure will be developed to quantitate the pancreatic protein in various tissues, and to estimate cross-reactivity of structurally related RNases in other organs and organisms. Radioimmunoassay procedures will also be used to quantitate nuclease of pancreatic origin in serum of individuals suffering from a variety of diseases, particularly pancreatic carcinoma; this could permit development of a technique for early diagnosis of this malignancy."}
{"text": "Latina women living in the U.S. are twice as likely to be diagnosed with cervical cancer and 40% more likely to die from the disease than non-Latina whites. Lower utilization of Pap smear contributes to greater cervical cancer mortality among Latinas, with recently-immigrated, uninsured women from Mexico at greatest risk of inadequate Pap smear utilization. To reduce this disparity, we propose to: 1. Increase routine Pap smear screening rates among uninsured Latina women living in the southeast using an empowerment-based, communication skills-building intervention. 2. Explore changes in knowledge, barriers, self-efficacy, and shared-decision making after the intervention and evaluate the impact of these factors on Pap smear screening rates. This pilot study uses a quasi-experimental community trial design to address these aims. The proposed community-based intervention uses a lay health advisor model to educate women about cervical cancer etiology and preventive strategies and to empower them with communication skills that will help them negotiate the medical encounter. Our research has several advantages beyond what has been documented to date: (1) The research is targeted to women in need of a Pap smear and who are at highest risk of not having one ; (2) The intervention combines education AND empowerment, a considerable advantage over previous research that has focused exclusively on increasing knowledge. (3) Empowerment, through skills- building, is grounded in theory and is culturally-appropriate; (4) The quasi-experimental community-trial design allows us to move beyond feasibility and actually test the effect of the intervention at the community- level; (5) The intervention will be easily replicated and disseminated given the size and scope of the project; (6) To date, no such interventions have been rigorously tested among newly arrived uninsured Latinas in the southeast. Given that the southeast has the fastest growing Mexican immigrant population in the U.S. and health systems are just beginning to respond to their health needs, this research study is strongly justified. Our research aims address NCI's 2006 strategic investment cancer research to \"overcome the unequal burden of cancer experienced by various population groups by ....developing effective interventions to reduce those disparities, and facilitating intervention delivery.\" [unreadable] [unreadable] [unreadable]"}
{"text": "The repair of extensive bone injuries remains an unmet clinical challenge. By developing two new and complementary models of large-scale bone regeneration in zebrafish and mouse, we aim to understand the role of the cartilage callus in generating large segments of full thickness bone. While the periosteum generates osteoblasts during homeostasis, the specific subpopulation that builds the repair callus remains poorly defined. Using transgenic lineage tracing, we provide compelling preliminary evidence that a rare bi-potent Sox9+/Runx2+ periosteal population generates new cartilage and bone during repair. This newfound ability to label, manipulate, and isolate a specific stem cell population allows us to test whether the remarkable regenerative capacity of the rib is due to the unique properties of its periosteal stem cells. In Aim 1, we team up with an orthopaedic surgeon to test that Sox9+ cells from the rib periosteum can be expanded in culture and used to heal a critical-sized femoral defect. The formation of a cartilage callus is a common feature in bone repair, yet how the periosteum generates cartilage only during repair remains a mystery. In preliminary data, we find that the cartilage callus is severely compromised when either the Ihha ligand is deleted in zebrafish or the Hh receptor Smo is deleted from Sox9+ cells in mice. In Aim 2, we test that this reflects a repair-specific role for Ihh, which is markedly different from its developmental role in osteoblast differentiation and chondrocyte proliferation. Further, our preliminary data suggest that these Ihh-induced repair chondrocytes differ in important ways from those in the growth plate since repair chondrocytes co-express osteoblast genes even at pre-hypertrophic stages. This increase in osteogenic character subsequently correlates with a conversion of chondrocytes into osteocytes. In Aim 3, we test whether repair and developmental chondrocytes represent distinct cell types by comparing global gene expression at different stages of maturation. We also use lineage tracing to quantitate the extent to which cartilage-derived osteocytes preferentially contribute to full thickness bone during repair. Lastly, we use powerful new chromatin accessibility assays to test that as Sox9+ periosteal cells become cartilage, their greater osteogenic character results from the maintenance of poised osteoblast enhancers. Our findings will reveal how a rare population of periosteal stem cells can be induced to make cartilage during injury, and how this specialized repair cartilage can be used to regenerate full thickness bone. A better understanding of these important stem cells will form the basis of future pre- clinical trials aimed at healing large-scale skeletal lesions in other parts of the body."}
{"text": "Chronic ethanol consumption results in microsome proliferation and an increase in the level of a cytochrome P-450 (P-450alc). The relative of this pathway and the alcohol dehydrogenase (ADH) pathway in ethanol metabolism in the liver is subject to debate. The ADH-negative and ADH-positive deermouse stocks have suggested that a substantial amount of ethanol metabolism may occur by non-ADH mediated pathways. The P-450alc has been purified from the deermouse and used to raise antibodies which are monospecific as judged by Western blotting. Antibodies to P-450alc inhibit ethanol metabolism in vivo. These antibodies will be used to identify cloned cDNA for P-450alc in a Lambdagtll expression lkibrary. The P-450alc cDNA will be sequenced. The mechanism of induction of P-450alc will be studied by measuring P450-alc synthesis rates in control and ethanol-fed animals using radiolabeling and specific immunoprecipitation techniques. Levels of P-450alc mRNA in control and ethanol-fed ADH-positive and ADH-negative deermice will be measured. An analysis of P-450alc mRNA levels in baboons and humans will be started. Investigation of possible genetic variation in P-450alc induction in many representative inbred mice will begin as will a survey of P-450alc gene structure in inbred mice. With P-450alc cloned DNA, the possibility exists to study variation in this gene and surrounding sequences in human alcoholic and non-alcoholic populations to identify possible genetic variation which may be of significance in controlling the expression or structure of this physiologically important protein. An analysis of DNA sequences upstream, where interesting regulatory sequences reside, from the structural gene will require the isolation of a genomic clone. This should be made readily feasible by the availability of the cDNA for P-450alc."}
{"text": "The expression of a region of the bacteriophage T4 genome is being studied as a model for examining developmental gene regulation. Throughout infection, T4 uses the host transcriptional apparatus to transcribe its DNA, but with time different regions of the T4 genome are transcribed. To determine signals and factors that regulate this transcription, the expression of the T4 genes uvsX (recombination protein), 40 (stimulates head formation), and 41 (primase-helicase component) have been examined by nuclease Sl protection experiments and Northern analyses. Changes in the transcription pattern of the uvsX-40-41 region are programmed with phage development. Early in infection a heterogeneous population of transcripts is observed, having major 5' starts about 900 and 200 bases upstream of uvsX. The bulk of these RNAs continue through 40 and into 41, but a portion (about 1/4) stop just downstream of uvsX (within gene 40). Later in infection a 5' start closer to uvsX (about 50 bases upstream) is observed, in addition to the ends seen earlier, and a significant fraction of the uvsX RNA ends at the stop. Thus, as infection proceeds, the level of 40, 41 mRNA decreases relative to that of uvsX. Analysis of uvsX-40-41 transcripts expressed by a plasmid with this region reveals different 5' ends from those after T4 infection, but significant utilization of the RNA stop downstream of uvsX. Thus, T4 infection is needed for the 5' starts, while the host can produce the RNA stop. To help determine what host factor(s) are needed for this stop, transcripts were examined after infection of the E. coli transcription termination (rho) mutant, rho026. Previous work has suggested that in rho026, transcription termination may be enhanced, resulting in less expression of regions downstream of a rho-dependent termination site (Stitt et al. (1980) J. Virol. 35, 775). My transcription mapping indicates that infection of rho026 results in several-fold less 40,41 mRNA than infection of the rho+ parent, because of a 2.5-fold greater use of the RNA stop and less transcription overall. Thus, rho factor either acts to terminate transcription at the stop or influences other factors needed for the regulation of this site."}
{"text": "Summary Hypercholesterinemia-mediated cardiovascular diseases can be treated by statins. However, there are many instances of unresponsiveness and intolerance. Thus, there is urgent need for new approaches to lower plasma LDL, preferably that act synergistically with statins. Blocking liver VLDL, the precursor of LDL, secretion has long been recognized as an effective alternative to lower LDL in the circulation. However, this can have unwanted consequences, such as lipid accumulation in the liver. Therefore, targeting VLDL secretion without causing lipid accumulation is challenging but has great promise. Preliminary studies done for this proposal have revealed an important role of liver phospholipid transfer protein (PLTP) in regulating VLDL secretion, and thereby plasma VLDL/LDL levels. However, the mechanism involved is still not quite clear. The establishment of liver-specific PLTP expression (not overexpression)/apoB100-only mice gives us a unique opportunity to evaluate whether liver-generated PLTP plays a critical role in human-like VLDL production. Our working hypotheses are 1) PLTP plays a major role in intracellular VLDL lipidation, transport, and post-translational degradation, thus promotin VLDL production; 2). Cellular PLTP activity can be tightly regulated by some factors, furin (PCSK3) is one of them. We have two specific aims. Aim 1: To investigate the effect of liver-specific PLTP activity on human-like VLDL production. We will attempt to answer the following questions: 1) Is PLTP activity required for the VLDL lipidation in hepatocytes? 2) Is PLTP activity required for VLDL transport vesicle (VTV) formation? And 3) Does PLTP activity prevent VLDL degradation in hepatocytes. Aim 2: To investigate the impact of furin-mediated PLTP regulation on VLDL production. We will characterize the interaction between PLTP and furin, in particular its pro-segment (profurin) in vivo, and determine the effect of such interaction on live VLDL production. We will also investigate why profurin/PLTP-mediated VLDL less production does not cause lipid accumulation in the liver. We anticipate that this project will broaden our understanding of both PLTP and furin biology and provide evidence for a novel strategy for lowering plasma VLDL/LDL levels that will be effective in humans."}
{"text": "Retrospective dietary information of high validity and reproducibility is critical for use in case control studies of the relationship of diet to cancer, cardiovascular and other chronic disease and disability. A cohort of Tecumseh residents completed interviews on frequency of food use and 24-hour dietary recall in 1967-69 as part of a general epidemiologic survey. Approximately 1700 members of the cohort aged 35-64 will be re-interviewed in 1981-82 about their current diet, recollections of past diet and perceptions of dietary change, and the results will be compared to past reports. Within-person comparisons will eliminate the component of variance between persons present in the comparison of matched controls. Characteristics of respondents which influence reproducibilty of historical dietary reports will be investigated. Sensitivity and specificity of the retrospective categorization according to food usage, nutrient intake and dietary practices of interest in the etiology of chronic disease and disability will be determined. Multivariate statistical methods will be used to evaluate quantitative changes, changes in dietary patterns, and reproducibility of retrospective dietary reports within the individual. Recommendations will be made for the design and use of a new form of retrospective dietary instrument which will be based on findings validated by measurements in the same individuals over 13 years."}
{"text": "The present proposal outlines a number of specific projects designed to elucidate significant unanswered questions in the shikimic acid biosynthetic pathway. Synthetic approaches to shikimate-3-phosphate, 5-enolpyruvylshikimate-3-phosphate, chorismic acid, isochorismic acid and bacilysin are described. Mechanistic possibilities from some of the enzymatic transformations of the above metabolites are presented."}
{"text": "Innovative and culturally appropriate multilevel health communications interventions are desperately needed to address the chronic disease epidemic in high-risk populations, such as low-income urban African Americans (AA). However, the vast majority of communications strategies have focused on educating individual consumers about healthy food choices, while in poor urban settings the lower availability of affordable healthy food choices greatly limits the impact of these messages. We will work with 3 wholesalers and 24 small retail food stores to develop and test novel strategies in Baltimore, Maryland, including: 1) multilevel health communications alone directed at wholesalers, retailers and low-income AA consumers intended to enhance willingness to stock and/or purchase healthy foods; 2) pricing strategies (performance based allowances) directed at wholesalers and retailers to increase their stocking of healthy foods at reduced prices; and 3) combined health communications and pricing strategies. Intervention strategies will be tailored to meet the needs of the target populations based on formative research and stakeholder input. Our proposed work is based on significant field experience in this setting, including the development of evaluation tools to assess change in stocking and pricing of key foods (at the store level), and psychosocial factors, dietary intake, and food purchasing behaviors (at the consumer level). Our study has the following aims: 1) Conduct formative research with representatives of multiple levels of the Baltimore food environment (i.e., local wholesalers and retail food store owners) in order to select key foods for promotion, determine appropriate communication strategies (e.g., messages, channels, materials) for each level, and select the most appropriate pricing approach (i.e., performance based allowance structure and stipulations). 2) Pilot the multi-level program with three wholesalers and 24 food stores (6 control, 6 health communications only, 6 pricing only, 6 combined), and assess program implementation through detailed process evaluation. 3) Assess impact of the pilot program on a) the stocking, pricing, marketing, and sales volume of promoted foods at wholesale and retail levels, and b) food purchasing behaviors and associated psychosocial variables (i.e., self-efficacy, intentions, perceived cost) at the consumer level (final sample n=12 consumers/store, 288 total). The proposed research directly addresses the objectives of the PA by seeking to develop effective, multilevel communication strategies to improve diet and reduce risk for diet-related chronic diseases. We anticipate this design will demonstrate the value of a multipronged and multilevel health communications approach to obesity and chronic disease prevention, and will lead to a large-scale trial and informed policies designed to improve food availability and affordability in low-income urban settings."}
{"text": "Despite the detailed understanding of signals 1, 2, and 3 and the critical function of T cells and dendritic cells (DC), clinical transplantation tolerance is almost never achieved. In transplantation, models of immune function, and interventions designed to promote immune regulation, have been based on simplified receptor-ligand and cell-cell interaction models. There has been a lack of studies that place immunological recognition within anatomic contexts and evaluate the critical role of anatomic microdomains in the regulation of the immune response. Our preliminary data now demonstrate that during tolerization there is alloantigen specific clustering of T cells and plasmacytoid DC (pDC) in the T cell areas of the lymph node (LN) near the abluminal surface of the high endothelial venules (HEV). Within these clusters T cells undergo either priming or development into de novo CD4+CD25+ regulatory T cells (Treg). Importantly, B cells presenting specific alloantigen are present in these cell clusters and contribute to Treg development. We hypothesize these multicellular clustered interactions in the LN are key to the induction and maintenance of tolerance. Specifically, we hypothesize that the precise interaction of T-APC (pDC, B cells) determines the outcome of T cell migration, positioning, proliferation, and maturation, and ultimately whether rejection or tolerance are induced. This hypothesis integrates many of the known receptor-ligand and cell-cell interactions, and places these interactions in the context of secondary lymphoid organ structure. To investigate the role of these cellular and structural elements in LN clustered interactions, we propose the following specific aims: Specific Aim 1. What are the T-APC-LN interactions that are important for tolerance? Using a transplant model that allows the tracking of alloantigen specific T cells, specific alloantigen presenting APC, and the positioning of the cells with respect to HEV, we will characterize specific receptor-ligand interactions between T-HEV and pDC-HEV that regulate tolerance, and define the interactions between T-pDC and other DC that determine the generation of Treg and inhibition of effector T cells within the LN. Specific Aim 2. How is the priming of effector T cells altered in the LN during tolerance? This aim will study the specific interaction between effector T cells and Treg in the LN clusters during tolerization. We will determine how Treg-T effector interactions regulate effector T cell migration, proliferation, and differentiation. We will characterize specific receptor-ligand interactions between Treg and T effector cells that determine whether T cell priming and rejection versus suppression and tolerance predominate in the immune response. Specific Aim 3. Determine the role of B cells in tolerance in the LN We will investigate the roles of B cells in the LN during the T-APC clustered interaction that results in tolerization. Analyses will focus on B cell APC function, chemokine production, and immunoglobulin production. PUBLIC HEALTH RELEVANCE: The research will investigate and define the cellular and molecular interactions that are important for generating regulatory suppressor T cells and antigen specific tolerance. The ability to define these interactions and achieve tolerance is important for achieving good graft survival and patient survival for transplant recipients."}
{"text": "This project has continued to focus upon the potential mechanism(s) underlying the selective brain tumor (CNS) cell line cytotoxicity of certain quaternized ellipticine derivatives. Sensitivity of these glial derived human brain tumor cells to N2-methyl-9-methoxyellipticinium acetate (MMEA) correlates with peak cellular concentration of the drug (r=0.976, p<0.0001). Peak cellular concentration of MMEA by sensitive CNS cell lines is achieved 10-15 hours following initiation of drug exposure and represents intracellular drug concentrations several hundred-fold in excess of that found in the extracellular medium. Cellular accumulation is mediated by a high-affinity, sodium-independent transport system which is competitively inhibited by the plant alkaloid reserpine, secondary and tertiary tricyclic antidepressant drugs and, with much lower affinity by catecholamine neuro-transmitters and serotonin."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A series of three studies were conducted in African green monkeys (AGM) to test a proprietary compound, TMC 353121, formulated by Tibotec Pharmaceuticals against RSV in nonhuman primates. In previous studies, the compound, an RSV fusion inhibitor was shown to have efficacy against RSV in vivo in smaller animals. These studies were performed in collaboration with Bioqual, Inc. with animals and veterinary personnel at their facility. My laboratory was to provide not only virus challenge stock but performed all virological testing of samples taken from the animals during each of the three studies. We performed serological testing of 48 AGMs to prescreen and identify 36 RSV seronegative animals to be used for the studies. The first study, NC282, was an infection study and utilized 6 AGM. We prepared 37, 1ml vials of RSV strain at each of two concentrations of 1x10e3pfu/ml and 1x10e4pfu/ml and cryopreserved them for the entire study. The animals were divided into two groups with each group receiving either 10e3pfu/ml (Gp.1) by intranasal and intratrachael administration and the other receiving 10e4pfu/ml (Gp.2) by similar administration. Animals were monitored for clinical signs of RSV infection with nose and throat samples taken daily and bronchoalveolar lavage samples taken every two days and tested for virus loads by plaque assay. Peak viral loads were reached in both groups by day 6 in the throat with 3.7e2 pfu/ml and 1.4 e3pfu/ml and in the BAL with 2e2 pfu/ml and 3.7e2 pfu/ml and for Gps1 and 2 respectively. These results confirmed the model and the decision to utilize the 10e4pfu/ml inoculation dose for the two subsequent efficacy studies was made. Two efficacy studies with the continuous intravenous infusion of the compound were performed. Study 2 had 15 animals divided into three groups with Gps. 1 and 2, therapeutic and prophylactic arms with TMC 353121 was administered at a plasma level of 50ng/ml and Gp 3 the vehicle control arms;and Study 3 had 12 animals divided into three groups with Gps 1 and 2 therapeutic arms only with TMC 353121 administered at plasma levels of either 5ng/ml or 500ng/ml and Gp 3 a vehical control group. Animals were preconditioned to wear the jacket and tethering system, underwent surgery for implanting the catheter, and infusions begun. The animal studies have recently been completed, however, we are still conducting plaque and antibody assays and compiling data."}
{"text": "The transfer of phospholipids between the ER and mitochondria is critical for mitochondrial biogenesis. Mitochondria cannot synthesize many of the lipids they require for membrane biogenesis and yet there is little or no vesicular trafficking to mitochondria. It is thought that lipids are transferred from the ER to mitochondria by a poorly understood nonvesicular mechanism. This transport has been proposed to occur at regions where the ER and mitochondria are closely apposed. We have found that lipid synthesis at contacts between the ER and mitochondria (and other contact sites) promotes lipid exchange and are working to discover the mechanism. In a second project, we have identified a protein that facilitates contacts between the Golgi and the ER and facilitates ceramide transport. Ceramides are key intermediates in sphingolipid biosynthesis and potent signaling molecules. However, excess ceramide is toxic, causing growth arrest and apoptosis. We identified a novel mechanism by which cells prevent the toxic accumulation of ceramides; they promote nonvesicular ceramide transfer from the ER to the Golgi complex, where ceramides are converted to complex sphingolipids. We found that the yeast protein Nvj2p is an ER-Golgi tether that generates close contacts between these compartments and promotes the nonvesicular transfer of ceramides to the Golgi complex. The protein normally resides at contacts between the ER and other organelles but during ER stress it relocalizes to and increases ER-Golgi contacts. ER-Golgi contacts fail to form during ER stress in cells lacking Nvj2p. Our findings demonstrate that cells regulate ER-Golgi contacts in response to stress and reveal that nonvesicular ceramide transfer out of the ER prevents the build up of toxic amounts of ceramides. A third project focuses on sterol transport to the vacuole. We have found that sterol-enriched domains form on the vacuole membrane in response to various cellular stresses. The domains regulate the stress response. We are working to understand how sterol transport is mediated and have found that conserved class of lipid transport proteins is required. The mammalian homologues of these proteins are seem to perform the same function. A fourth project focuses on the biogenesis of lipid droplets in the ER. We have found that lipid droplet emergence from the ER is regulated by a conserved family of proteins called FIT proteins. We think these proteins modulate the lipid composition of the ER and we have evidence that lipid droplet emergence from the ER can be driven the lipid composition of the ER."}
{"text": "The antitumor agents, 6-thioguanine and 6-mercaptopurine, have been reported to produce cellular toxicity as a result of incorporation of either agent into DNA as 6-thioguanine nucleotide. However, there is no data which demonstrates directly that a DNA polymer containing 6-thioguanine has altered functional properties. The purpose of this proposed project will be to ascertain the functional potential of a DNA containing 6-thioguanine in internucleotide linkage, thereby establishing methodology for future studies on the mechanism of action of other purine and pyrimidine analogs. The three methods of approach will be as follows. A. Allow vaccinia virus to incorporate 6-thioguanine into its DNA, and after harvesting viral particles, determine the ability of the genomes to express their information, namely: 1) ability to induce virus-specific mRNA synthesis and 2) ability to induce the synthesis of virus-specific proteins, i.e., thymidine kinase, DNA polymerase and deoxyribonuclease. The study will be controlled by monitoring the extent of 6-thioguanine incorporation into the viral DNA and relating this level to that found to be toxic to mammalian cells grown in tissue culture. B. The significance of incorporation of 6-thioguanine into cellular DNA on induced-protein synthesis will be evaluated as follows. Cultured cells will be treated with 6-thioguanine and washed free of non-incorporated drug. The cells will then be stimulated with an interferon-inducer or with interferon, and the production of induced interferon and induced antiviral activity by or in the cells will be quantitively determined. C. The significance of 6-thioguanine metabolites on DNA and protein synthesis in the cell cytoplasm will be determined by pretreating cells with 6-thioguanine. The ability of the pretreated cells to permit vaccina virus replication will then be assessed. This DNA virus replicates in the cytoplasm independently of the host cell genome."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We will use the recently completed rhesus macaque genome map, to identify approximately 83,333 single nucleotide polymorphisms (SNPs) randomly distributed throughout the rhesus macaque (Macaca mulatta) genome. At least ten copies of each DNA fragments in reduced representation libraries (RRLs) constructed from pooled (n=25)DNA samples from 7 different regional populations will be sequenced, then filtered, parsed and compared to the rhesus macaque draft sequence and to each other using bioinformatics tools we previously developed to identify the locations, flanking sequences and minor allele frequencies (MAFs) of 83,333 SNPs. Genotyping assays on lliumina iSelect chips will be constructed for the 7,600 SNPs whose frequencies differ most between Indian and Chinese rhesus macaques and genotyped in geographically representative samples of 192 Indian rhesus macaques, and192 Chinese rhesus macaques and in 678 Indian/Chines rhesus macaques with varying proportions of Indian and Chinese ancestry. Genotypes for the 7,600 SNPs will be used to 1) confirm Mendelian segregation of SNPs in multigeneration families, 2) assess the ability of the SNPs to accurately estimate proportions of Indian and Chinese ancestry in Indian/Chinese hybrid, pedigreed animals and 3) identify ancestry informative markers for admixture mapping of QTLs and to conduct admixture mapping of a QTL for \"temperament.\". Our SNP map will be made publically available through several outlets including dbSNP. The 384 most potentially informative of the 83,333 SNPs for longtail macaques will be genotyped in 96 samples from six different longtail macaque populations to identify minimum panels of 96 SNPs each for identifying country of origin and for parentage analysis and estiinating parameters useful for genetic management. Assays will be constructed of these two panels and made available to NIH investigators, providing cost recovery. The genotypes of SNPs described above will be used for genomic and population structure analyses of regional populations of rhesus and longtail macaques. PUBLIC HEALTH RELEVANCE (provided by applicant): Rhesus and longtail macaques are the most and second most used models for the biomedical study of human diseases. Genetic resources developed for rhesus macaques are not available for longtail macaques. Mapping disease phenotypes and QTLs is possible with the recent sequencing of the rhesus genome but requires the identification of many known polymorphic loci (e.g., SNPs) throughout the macaque genome. The most practical method for conducting whole genome association studies is admixture mapping."}
{"text": "1. OBJECTIVES/METHODS: Bleed vaccines annually; test serum for rabies- neutralizing antibody by the Rapid Fluorescent Focus Inhibition Test (RFFIT); boost those with <1.0 International Unit rabies-neutralizing antibody/ml serum with HDCS Rabies Vaccine (Institut Merieux). 2. RESULTS: Distribution of human serum quantified for rabies-neutralizing antibody in IU/ml: Employing Agency Number of People Number of RFFIT (serum source) Sampled Tests CBER-FDA 12 24 NIH 90 180 TOTAL HUMAN SERUM TESTS 102 204"}
{"text": "Cell surface receptors coupled to the heterotrimeric GTP-binding proteins are universally responsible for the transmembrane transmission of extracellular messengers such as hormones, neurotransmitters and a variety of sensory stimuli. Because of this direct involvement in the regulation of the most crucial cellular functions, G-protein coupled receptors (GPCRs) are among the most important targets of therapeutic intervention. It's estimated that about 50% of drugs in use act on GPCRs. Thus, understanding of the receptor and the G- protein functions at the molecular level is among the highest priorities of public health research. Several competing models aim at describing the universal mechanism of G- protein activation by GPCRs, but none has presented compelling and conclusive experimental evidence so far. HYPOTHESIS: G-protein 23-subunit complex is a key molecular switch at the center of the gear-shift model of G-protein activation. We will test this hypothesis using the prototypical GPCR rhodopsin (R) and the G-protein transducin (Gt) responsible for phototransduction in retinal rod cells as a model system. Three interconnected Specific Aims will test various aspects of the hypothesis, such as questions of the molecular organization of the receptor- G-protein complex, the high-resolution picture of the receptor-G-protein interface, the mechanism of signal transfer from the receptor, and the roles of individual G- protein subunits, especially the G23 subunit complex, in this dynamic process. This project aims at understanding the universal principles underlying cell- to-cell communications and cellular responses to a variety of sensory stimuli. Several competing theories describing these basic molecular mechanisms will be tested to gain insights into the inner workings of cell surface receptor proteins and specific protein-protein interactions. Because almost half of all therapeutics on the market today target these signaling pathways, knowledge obtained as a result of these studies will be essential in new drug design and fighting a wide range of diseases such as heart problems, asthma and vision disorders."}
{"text": "Abstract Ischemic stroke is a leading cause of death and long-term disability. Current drug treatment is limited to tPA, which has a low success rate and potentially severe side effects. Acidosis is a common feature of neurological disorders such as brain ischemia, and it has been shown to play a critical role in stroke. The mechanisms, however, remained elusive. The discovery that protons activate a distinct family of cation channels, the acid-sensing ion channels (ASICs), has shed new light on acid-signaling and acidosis-mediated brain injury. The studies in our laboratories in the past 10 years have provided convincing evidence suggesting that activation of ASIC1a contributes markedly to acidosis-mediated ischemic brain injury. Following our initial report, others have demonstrated an important role for ASIC1a activation in spinal cord injury, traumatic brain injury, and axon degeneration. Thus, ASIC1a represents a novel therapeutic target. Despite its well-established role in neurological disorders, the detailed mechanisms underlying ASIC1a-mediated neuronal injury in stroke remain unclear. We now have strong evidence suggesting that, besides the well-documented Ca2+ toxicity, a combination of increased ASIC1a surface expression, Zn2+ toxicity, and an ion conducting independent cell death pathway participate in ASIC-mediated neuronal injury in ischemia. The objective of this application is to investigate the detailed molecular mechanisms and pathways underlying ASIC-mediated neuronal injury. Given the limitations of currently available pharmacological inhibitors that target these channels, e.g. the non-specificity of amiloride and large molecule nature of PcTX1, the proposed studies may disclose novel and alternative therapeutic strategies for ischemic brain injury."}
{"text": "This application for an R34 award proposes to refine and conduct a pilot RCT of a peer-led cognitive-behavioral intervention to determine the feasibility, acceptability, and outcomes for preventing depression and other negative outcomes among out-of-school inner-city adolescents and young adults in a community-based employment training program. The specific aims of this study are: (1) to refine the intervention protocol for the peer-led cognitive behavioral intervention, develop an intervention fidelity checklist, develop a training manual for peer leaders who deliver the intervention, and train peer leaders to deliver the intervention;(2) to conduct a pilot RCT of 142 adolescents and young adults, aged 16-22, that compares outcomes for youth involved with our peer-led cognitive-behavioral intervention with outcomes from youth participating in an attention control condition;and (3) to obtain the information and experiences required for designing and implementing a larger randomized trial. To achieve Specific Aim 1, will refine our peer-led intervention, develop an intervention fidelity checklist, develop a training manual for peer interventionists, and train peer interventionists. To achieve Specific Aim 2, we will conduct a pilot RCT of our peer-led cognitive behavioral intervention. 142 adolescents and young adults with elevated depressive symptoms but without a current depressive episode will be randomized to our 9-session intervention or a 9-week life skills attention control. Assessments post- intervention, 6 months, and 12 months post-intervention will measure depressive symptoms, depressive episodes, anxiety and PTSD symptoms, cognitive appraisals, engaged coping strategies, quality of life, and education and employment milestones. To achieve Specific Aim 3, we will use findings from our pilot RCT (e.g., implementation fidelity, recruitment, attendance) to develop a R01 application to be submitted during the final year of our R34 project. The public health significance of this project is substantial. Given consistent evidence that depression is a debilitating and chronic condition, interventions must attempt to prevent initial depressive episodes from occurring. Adolescence and young adulthood is an ideal time for preventive interventions to occur given the high depression incidence rate in this age group. The innovation of this project is also notable, as it brings mental health services to employment training programs that are being promulgated with new federal funding initiatives. These employment training programs serve large numbers of adolescents and young adults who are not reached by existing mental health interventions. This project is consistent with the goal of the R34 mechanism to encourage research on the development and/or pilot testing of new or adapted interventions and with two high priority areas of NIMH'S Division of Services and Intervention Research to develop innovative interventions and personalize them for optimal use in diverse populations and to employ strategic partnerships and community participation to enhance research capacity and infrastructure. PUBLIC HEALTH RELEVANCE: Depression is a significant public health problem, with adolescents and young adults among the most affected. This project tests a peer-led intervention that aims to prevent the onset and worsening of depression in adolescents and young adults (16-22) who are out-of-school and not in the workforce and enrolled in an employment training program-a group that has not been the focus of previous mental health research. Our intervention may have great benefits to society, as preventing depression among this population has the potential to increase the employability, and ultimately, economic self-sufficiency of participants."}
{"text": "Size regulation requires both extrinsic and intrinsic mechanisms that define growth limits. Extrinsic mechanisms sense nutrient availability and control growth systemically. In contrast, intrinsic mechanisms regulate growth in individual organs and tissues in an autonomous manner. It has been postulated that one way intrinsic size regulation of organs is achieved is through organ compartmentalization. The aim of this proposal is to investigate how developmental compartments contribute to organ growth and size. Expression of dMyc in the Posterior (P) compartment of the imaginal disc results in a proportionately normal size wing. We have found that cell death is increased within the P compartment, and also non-autonomously, in the Anterior (A) compartment in this case which might account for the proper size regulation. I propose studies to examine the role of cell death in regulation of the size of each compartment, and of the entire developing wing. Our results point to a possible asymmetry in growth regulation imposed by compartments. I propose experiments to address whether asymmetry exists and the possible mechanisms involved in this potential asymmetry. [unreadable] [unreadable] [unreadable]"}
{"text": "The goals of this project are to develop, evaluate, and disseminate an Internet (web) application, Moderate Drinking (MD). MD will use a behavioral self-control training (BSCT) protocol that helps drinkers learn skills to either moderate their drinking or to abstain. The target population is members of the self-help organization, Moderation Management (MM) and to visitors to their web site. MM (www.moderation.org) describes itself as an organization that can be helpful for problem drinkers wanting to either moderate their drinking or to abstain. Its members tend to be less severe problem drinkers with few signs of dependence. MM offers written materials describing a program of behavioral change, an online mutual-help community, and face-to-face meetings. Their behavioral change program is a version of a current generation BSCT protocol that will be used in the web application. BSCT is an intervention with strong empirical support. The interactive program will be unique on the Internet. The program will help users choose a goal (moderation or abstinence) then learn cognitive and behavioral skills necessary to achieve and maintain their goal. Modules include goal setting, self-monitoring, \"doing a 30,\"increasing motivation and self-efficacy, identifying and managing triggers, developing alternatives, coping with lapses/relapses, and a segue to abstinence if the user is having difficulty achieving and maintaining moderation. Because moderate drinking is an option, the program may to appeal to less severely dependent drinkers who might otherwise avoid a program that is purely abstinence based. This group of less severely dependent drinkers is an under served but growing population. Phase II goals are to complete the development of the program, pilot test it, and conduct a randomized controlled trial with a 12 month follow-up to assess its effectiveness. The experimental group will receive training via the MD web site and will participate in MM's online mutual help community. The control group, will only participate in the online mutual help community."}
{"text": "Beneficial mutations can arise within individuals neutral to the mutation's utility; variants demonstrating the change can then be selectively sorted from the progenitor population by the environment. This observation alone does not exclude the possibility that selective forces can also influence either the generation of genetic alterations, beneficial or otherwise, or the retention of alterations. Inheritable changes can be of at least two types: those that alter the genome by changing its DNA sequence (genetic), and those that establish a self- perpetuating physiological state (epigenetic) in individuals within an otherwise isogeneic population. Both genetic and epigenetic changes that favor a cell's survival may occur in organisms after exposure to an environment. I have demonstrated the existence of mechanisms that allow eukaryotes to adapt to growth-halting environments after exposure of the organism to the environment. The mechanisms result in the change of genomic DNA sequence at specific (selected) loci nonrandomly. These changes are the result of recombination and require only some of the components of the homologous recombination machinery. Environmentally induced changes in microbes could compromise the efficacy of vaccine- based therapies."}
{"text": "Three transparent gels participate successively in the visual process: The cornea, the lens and the vitreous. The molecular composition of these gels are known but to a large extent the way these molecules form aggregates and superstructures in the tissues are not fully elucidated. Optico-mechanical studies on the gels themselves are advantageous because they do not destroy the tissues, they don't introduce any artifacts and furthermore they can be utilized to study the gels under dynamic conditions (either unidirectional or cyclic vibrational motions). The same optico-mechanical techniques such as light scattering, birefringence, dielectric studies, and dichroism can be used to study the gels and solutions of single macro-molecular components of the gels of the eye. In addition, hydration and fluorescence studies will be employed on single components as well as electron diffraction, if microcrystals of macromolecules can be obtained. The same techniques will apply to study 2, 3 and higher number component mixture of macromolecules. Thus, the description of gel state will be done both frm synthetic (components and sums of components) and from the analytical (the whole tissue) point of view."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Maternal smoking is the major preventable cause of intrauterine growth retardation and prematurity. Recent evidence shows that developing lung is also highly sensitive to maternal smoking and that smoking during pregnancy leads to decreased lung function, increased respiratory diseases and increased incidence of sudden infant death syndrome (SIDS) in the offspring. Given that every year more than 400,000 infants in the US alone are born to women who smoked during pregnancy, it is of major importance to find ways to prevent those changes. Our data shows that nicotine is one of the factors responsible for the changes in pulmonary function present in children born to smoking mothers. In this project, rhesus monkeys are used to characterize the effects of chronic exposure to low levels of nicotine throughout pregnancy on lung development and function. The purposes of this project are to 1) characterize the molecular basis for nicotine's actions by determining how nicotine effects the functioning of nicotinic receptors in fetal monkey lung;2) characterize the effect of fetal nicotine exposure on lung development by functional, morphometric, immunohistochemical and molecular analysis;and 3) develop ways to block the effects of nicotine on lung development that can lead to clinical interventions that can be combined with vigorous smoking cessation programs in pregnant smokers in order to help the offspring of smoking mothers."}
{"text": "The Vaccine Pilot Plant (VPP) is a multiuse facility designed to manufacture vaccines for human clinical trials according to current Good Manufacturing Practices (cGMPs) required by the US Food and Drug Administration (FDA). The facility is composed of a warehouse to receive the raw materials, central process support facility to manufacture buffers and media components, manufacturing suites to produce active vaccine ingredients, a filling suite to fill the product into vials for delivery to the clinic, Quality Control laboratories to test the vaccine product, and supporting office and utility space for staff and equipment. These areas all work in concert to take raw materials and turn them into vaccine product that can be evaluated in humans."}
{"text": "Dengue virus and other flaviviruses including West Nile virus, tick-borne encephalitis virus, and Yellow Fever virus, are widespread, mosquito- or tick-borne human pathogens. About 40% of the world's population lives in areas with substantial risk of dengue transmission, and as many as 100 million people may be infected annually, experiencing dengue fever and potentially lethal severe dengue. Development of a vaccine effective against all four dengue serotypes has been difficult because infection with one ofthe four serotypes does not lead to protective immunity against any of the other three and may lead to enhanced risk of severe, life-threatening illness, especially in children. Antivirals against dengue are of interest due to their potential to reduce the severity of dengue-associated disease as well as to reduce transmission. This proposal will contribute to efforts to develop dengue antivirals by developing mechanistically well-characterized small molecule inhibitors of dengue entry and using these to validate the dengue E protein as an antiviral target in vitro and in vivo. We have previously identified three structurally distinct lead compound series and inhibitors that exhibit a spectrum of activities against dengue virus entry. In the proposed work, we will use a combination of x-ray crystallography, medicinal chemistry, direct protein-small molecule affinity measurements, single-virion fusion and live-cell imaging assays, and resistance studies to optimize the pharmacological activity of our compounds and to elucidate the biochemical and structural mechanisms of action that correspond to potent, pan-serotype inhibition of dengue. Key to these efforts are quantitative assays, analogous to enzymatic activity measurements, for studying the biochemical functions of the viral envelope protein and that allow us to link the biochemical mechanism of small-molecule inhibitors with the step(s) in the cell-entry pathway at which they block infection. Optimized dengue entry inhibitors will be tested in a murine model of dengue infection and pathogenesis. Collectively, our efforts are aimed at producing compounds that can be advanced as preclinical candidates for dengue drug discovery efforts. More broadly, this work establishes the template for a rational approach to the development of small molecule inhibitors of other enveloped viruses as potential antivirals"}
{"text": "We propose to conduct the first pharmacotherapy relapse prevention study in body dysmorphic disorder (BDD). BDD, an often-delusional preoccupation with an imagined or slight defect in appearance, is a distressing, impairing, and relatively common body image disorder. It is associated with high rates of functional impairment and markedly poor quality of life. It appears that SRIs are often--and selectively--efficacious for BDD and that many BDD patients receive SRIs. It also appears that most patients discontinue an efficacious SSRI at some point, as the alternative is life-long treatment. However, no relapse prevention studies have been done. Such a study is important from a clinical and public health perspective, because BDD appears to often be chronic and require long-term treatment. It is therefore critically important to investigate the risk of relapse with SSRI discontinuation, and whether continuation SSRI treatment decreases relapse risk. 128 subjects will be enrolled and first treated openly for 14 weeks with escitalopram; 58 escitalopram responders will then be randomized to double-blind continuation treatment with escitalopram or placebo for 6 additional months. Our primary aim is to compare time to relapse and relapse rates in responders to acute escitalopram who are then randomized to placebo versus continuation treatment with escitalopram. Secondary/exploratory aims will explore 1) Whether subjects who receive continuation escitalopram perform better on secondary outcome measures (e.g., quality of life) than those on placebo; 2) Whether subjects taking continuation escitalopram have further improvement in BDD symptoms during the continuation phase; 3) Predictors of relapse after escitalopram discontinuation; and 4) Acute treatment response. Because this study will offer a unique opportunity to investigate the genetic basis of BDD, we will explore BDD's genetic basis and the relationship of selected candidate genes to treatment outcome and side effects. In summary, this study will be the first relapse prevention study in BDD and the first study of continuation pharmacotherapy in BDD. It will provide critically important information on relapse with continuation versus discontinuation of an SRI, whether continuation treatment protects against relapse, and whether patients further improve with continuation treatment. It will explore previously unstudied questions about BDD's pharmacogenetics, which may ultimately guide and enhance medication treatment. This study will yield unique and clinically important data, and will fill gaps in knowledge about this relatively common, severe, and understudied illness."}
{"text": "Myeloid lineage osteoclasts are the sole effective bone-resorbing cells. Osteoclasts are required for the resorptive phase of physiological bone remodeling that maintains musculoskeletal integrity and regulates bone mass. Under physiological conditions the generation and function of osteoclasts is tightly regulated and coupled to the function of bone-forming osteoblast lineage cells. Many pathological conditions associated with excessive bone resorption and bone loss are characterized by loss of normal regulation/coupling, and excessive osteoclastogenesis. The long term goals of this project are to elucidate new mechanisms that suppress osteoclastogenesis, with the associated goal of using this information to develop new therapeutic approaches to suppress pathological bone resorption. Inflammation is an important driver of pathological bone loss. Inflammation decreases bone mass by suppressing osteoblast-mediated bone formation, and concomitantly strongly promoting bone resorption by increasing the differentiation and bone-resorbing function of osteoclasts. Thus, inflammation induces local bone erosion/osteolysis at inflammatory sites in diseases such as rheumatoid arthritis, periodontitis, infections, and orthopedic implant loosening. Recent work, including contributions from our laboratory, has made clear that potent inflammatory factors, such as the inflammatory cytokines IFN-? and GM-CSF and ligands for Toll-like receptors (TLRs) that sense microbial products and tissue damage, also induce feedback inhibition mechanisms that restrain osteoclastogenesis and thus limit the amount of bone resorption that occurs in inflammatory settings. Little is known about feedback inhibitory mechanisms induced by inflammatory factors to limit osteoclastogenesis. Based on our overarching hypothesis that augmenting feedback mechanisms represents an attractive alternative therapeutic approach to suppress pathologic bone resorption, we have investigated mechanisms by which inflammatory signaling restrains osteoclastogenesis. We have discovered two novel and complementary mechanisms by which inflammatory factors, including ligands for Toll like receptors and ITAM-associated receptors, suppress differentiation of osteoclast precursors. These are: 1. Modulation of a proteolytic pathway that generates a biologically active intracellular fragment of the Fms receptor for M-CSF. 2. Induction of the BCL6 transcriptional repressor to inhibit expression of the 'master regulator' of osteoclastogenesis NFATc1. These novel inhibitory mechanisms target two major nonredundant proteins required for osteoclast differentiation, Fms and NFATc1, and effectively inhibit osteoclastogenesis. We will characterize these inhibitory mechanisms to obtain knowledge that can be used to develop new approaches to suppress osteoclastogenesis and pathologic bone resorption by augmenting these mechanisms therapeutically."}
{"text": "Mosquitoes are insect vectors responsible for the transmission of many infectious diseases to hundreds of millions of people worldwide. Females of most mosquito species require blood from vertebrate animals for their egg development. Multiple bloodfeedings enable mosquitoes to transmit disease pathogens, including malaria parasites and dengue virus, from one person to another. Our long term goal of this project is to elucidate the molecular mechanisms that regulate mosquito egg production and identify target molecules that can be utilized for mosquito control. Mosquito egg development is governed by alternating peaks of two major insect hormones - juvenile hormone (JH) and 20-hydroxyecdysone (20E). Deprivation of JH in newly emerged adult female mosquitoes will halt egg maturation. On the other hand, topical application of JH to mosquitoes shortly after blood feeding interferes with the normal responses to 20E and impairs egg production. We have recently demonstrated that the mosquito Methoprene-tolerant (AaMet) protein is a key player in the juvenile hormone signaling pathway in the newly emerged adult female mosquitoes. AaMet protein binds to JH and forms a complex with AaFISC protein, a coactivator of the 20E receptor. AaMet and AaFISC are found to be associated with the promoters of JH target genes and activate their expression. In addition, our preliminary studies imply that AaMet mediates the inhibitory effects of JH on 20E-induced gene expression. Taken together, the results suggest that AaMet and AaFISC are components of a juvenile hormone receptor. The objective of this project is to elucidate the molecular details of how AaMet functions in juvenile hormone signaling that regulates egg maturation in mosquitoes. In Aim 1, we will perform structure-function studies of the juvenile hormone binding domain in AaMet and define the structural determinants required for high affinity binding to JH. In Aim 2, we will investigate how AaMet and AaFISC proteins are recruited to juvenile hormone response elements in the JH target genes. In Aim 3, we will test the hypothesis that AaMet is involved in the crosstalk between juvenile hormone and 20E signals in blood-fed female mosquitoes. The study will significantly advance our understanding of the molecular action of juvenile hormone in mosquitoes, and provide a structural basis for designing new pesticides that specifically target the mosquito JH signaling pathway."}
{"text": "Premitec, Inc., in collaboration with the Doheny Eye Institute at the University of Southern California has developed novel, 3-dimensional wide field microelectrode arrays (3D-WFA) for retinal prosthesis. These devices can double the visual field for retinal prostheses versus the devices currently being tested in clinical trials. The electrode arrays have a 10-mm diameter area of contact with the retina, thus enabling a wide field of vision. This work is funded by the National Institute of Neurological Disorder and Stroke (NINDS) at NIH. A conclusion of our results so far is that the large area polyimide array, although it has a three dimensional curvature to match an \"average\" eye, nonetheless has the tendency sometimes to separate from the retina, particularly at the edges, due to difficulty of the implant procedure, the nature of the polyimide array edge and variation in eye curvature. In addition use of a single retinal tack to affix the prosthesis to the retina not only damages the retina at the insertion site but also leads to greater non-uniformity in distance to the retina across the array. To address these shortcoming we propose to develop a novel hybrid silicone/polyimide wide-field array and eliminate the tack altogether. Recently we have demonstrated that the silicone surface can be modified to bind an adhesive protein which also binds strongly to the inner limiting membrane of the retina, and more generally to brain and other tissues. Replacing the tack with an adhesive will virtually eliminate the distance between the electrode and the retina, and thus reduces the stimulus threshold. By incorporating adhesive-silicone technology into the design of the 3D-WFA, we can address two important issues: increase implant safety by reducing the likelihood of retina damage and improve implant efficacy by reducing the distance between the array and the retina. PUBLIC HEALTH RELEVANCE: Advancements in microfabrication and materials have made possible the development of flexible neural interfaces. These medical devices are targeted at treating incurable neurological disorders and solve difficult technical problems that are limiting the capability of neural implant devices. Such diseases are widespread in the population as a whole and their impact on individual health is profound. The progress made by us so far with support from NINDS through Phase II SBIR demonstrates that our technology has the potential to lead to a successful commercialization of complex and challenging neural interfaces like the retinal stimulator and make a real impact on public health by increasing the capability of implantable neural prostheses."}
{"text": "The Mid-America Cancer Center Program is continuing to develop its strengths in research, education and patient care in order to bring the increasing cancer expertise at four participating institutions to bear on the cancer problems in the portion of the Midwest centered in Kansas City. The Center brings together scientists at The University of Kansas Medical Center, The University of Kansas-Lawrence, Kansas State University and Midwest Research Institute in program of mutual interest in the cancer field. The research strengths shared include drug development from basic design to clinical trials, virology, biology, immunology, carcinogenesis, epidemiology and radiobology. Based at the major tertiary care facility in the region, the opportunity exists to combine these research strengths of the Cancer Center with a rapidly improving clinical capability to bring outstanding care to the cancer patients of the region. The transmission of this knowledge to the medial profession as a whole remains a major goal of the educational and outreach program of the MACCP. At the same time, the MACCP is increasing its total information efforts to insure that all facets of cancer are understood by not only the professionals but by the public through cooperative programs with the Amerian Cancer Society divisions in the area. The Cancer Data Service has become a computerized regional registry providing the broad statistical base necessary for effective evaluation of the efforts of all participating elements of the program and for the conduct of epidemiological studies."}
{"text": "The 2007 UNAIDS report estimated that for each person treated with potent antiretroviral therapy, 4-6 new individuals became infected. Without a foreseeable cure for HIV, prevention measures must control the HIV epidemic and structural/barrier/behavioral methods simply have limited efficacy or applicability. There is a critical need, for coitally-independent dosing strategies which women can initiate and are imperceptible to their partners. It is increasingly clear that antiretroviral pharmacokinetics predict efficacy: using samples from the CAPRISA 004 study, our laboratory demonstrated that subjects who maintained the highest tenofovir concentrations in cervicovaginal fluid (and thus in genital tract tissues) were the least likely to become infected with HIV-1 and HSV2. Although topical formulations such as gels and vaginal rings are being investigated, oral antiretroviral drugs also hold significant promise for prevention. Currently, antiretroviral doses used in prevention studies are those that are FDA-approved for treatment of HIV infection. However, there are no data to confirm that exposures with these standard treatment doses will protect mucosal cells from HIV acquisition, or to inform alternative dosing strategies. We propose a highly significant plan to develop a model for oral antiretroviral prevention strategies. In healthy women volunteers, we will determine the ability of 3 doses of 4 antiretroviral drugs to concentrate in 3 at-risk mucosal surfaces. In explant tissue cultures, the concentration of these drugs, alone and in combination, required to protect tissues exposed to multiple infectious molecular clones will be identified. A new approach to normalizing tissue responses to cell numbers and composition will also be implemented. Once the in vivo tissue pharmacokinetic results and ex vivo target concentration results are known, a mathematical model will be developed to predict the antiretroviral doses/regimen maximally protective at all mucosal surfaces. Finally, a second proof-of-concept study is proposed to dose women with the final antiretroviral regimen and challenge tissue biopsies with HIV to determine risk of infection."}
{"text": "One of the most compelling features of Williams syndrome (WS) is the distinctive social profile that holds promise for understanding the underlying neurogenetic systems that provide meaning to human social interaction. Our studies to date suggest that while individuals with WS typically demonstrate an increased appetitive social drive, the social profile is characterized by dissociations (e.g., overly-friendly with a difficulty in making friends; socially fearless but anxious; positive affect with maladaptive behaviors). The aims of Project V focus on the characterization of the social phenotype of WS, enabling links to the genetic and neurobiological pathways of these dissociations. To this end, the Specific Aims are: Aim 1: Insatiable Appetitive Drive for Approaching Strangers will examine the underpinnings and variability of the increased attraction and approachability towards unfamiliar people observed in individuals with WS. Aim 2: The Unique Salience of Faces will elucidate the nature and underlying mechanisms of the atypically high interest in faces in WS, and its relation to the resultant hypersocial phenotype. Aim 3: Unusual Emotional Sensitivity will investigate both (a) the perception and processing of affect of others by those with WS, and (b) the overall affective style of individuals with WS. Using a multi-method design reflecting multiple levels of explanation (electrophysiology, autonomic function, and eye fixation). Project V studies will produce highly nuanced, quantifiable and independent key dimensions of the unique social behavior characteristic of WS. From a theoretical standpoint, a major thrust of the proposed work is to disentangle the processing of key components of social interaction and their respective underpinnings in the context of the enigmatic, yet paradoxical, WS social phenotype, with the ultimate goal of characterizing the complete system of social behavior and understanding the ways it can break down. Such a multileveled approach has not been previously adopted within this domain of inquiry. Results from these studies will add unique knowledge to our understanding of social behavior, by further defining the pathways implicated in gene-brain-behavior linkages, and are designed to contribute to better-informed treatments."}
{"text": "The ubiquitin-dependent N-end rule pathway relates the in vivo half-life of a protein to the identity of its N-terminal residue. We have previously reported UBR1 and UBR2 as its functionally overlapping E3s and elucidated their in vivo roles using mouse knockout approach. Unexpectedly, although both UBR1 and UBR2 strongly bound to N-degrons, UBR1'^'UBR2^'cells still retained the N-end rule E3 activities, indicating the presence of yet unidentified N-recognins (N-degron-Recognizing E3s). The goal of this study is to identify and characterize N-recognins and their substrates and to elucidate the physiological meaning of their E3-substrate interaction. To identify mammalianN-recognins, we developed a novel affinity-based proteomic approach using synthetic peptides bearing N-degron, yielding a novel 570 kDa-protein named UBR4 and a 300 kDa-E3 ligase EDO (termed UBR5). UBR1, -2, -4, and -5 shared a zinc finger-like domain named the UBR box. Mammalian genome appearsto encode seven UBR proteins, named UBR1 through UBR7. Further, by using a functional proteomic approach, we have obtained -35 candidate N-end rule substrates from -14,000 different proteins expressed in the rabbit reticulocyte lysates. Preliminary characterization of candidate substrates unveiled several novel in vivo N-end rule substrates (RGS4, RGS5, RGS16, and CDC6), the first to be identified in mammals. To further extend our current understanding of the N-end rule pathway, we propose the following Aims. Aim 1. To characterize UBR box proteins as candidate N-recoqnins. We will examine: 1) proteolysis of model N-end rule substrates in UBR mutant cells, 2) the interaction and specificity of UBR box proteins with N-degrons, and 3) in vitro ubiquitylation of model substrates by UBR box proteins. Aim 2. To determine whether UBR box motif is the recognition domain for N-degron. We will determine whether the UBR boxes of N-recognins are required and sufficient for direct binding to N-degrons and identify essential residues for recognition of N-degron. Aim 3. To identify physiological N-end rule substrates. We will dissect proteolysis of candidate substrates in reticulocyte lysates and UBR mutant cells, determine the N-recognin-substrate interaction, and test whether N-recognins support substrate ubiquitylation in vitro. Aim 4. To dissect physiological processes underlying identified N-end rule substrates. We will examine the physiological significance underlying the N-end rule dependent proteolysis of RGS4, -5, and -16, emerging in vivo N-end rule substrates."}
{"text": "High Performance Detectors for SPECT P.I.: Dr. Michael R. Squillante Abstract Single photon emission computed tomography (SPECT) is a powerful, noninvasive medical imaging modality that mathematically reconstructs the three dimensional distribution of a radionuclide throughout the body of a human patient or a research animal. Typically, the collected data are displayed and evaluated as a set of two-dimensional images through the organ or diseased area under investigation. SPECT allows quantitative study of the function in the investigated region and therefore is an extremely useful tool for understanding organ and tissue physiology including that in the heart. SPECT is very commonly used in identifying as well as localizing coronary artery disease and as many as 90% of all myocardial perfusion studies are now performed using SPECT. Thus, SPECT is playing a critical role in cardiac imaging, providing both diagnosis as well as prognosis. However, there is urgent need for improvement in the instrumentation that is currently used for this imaging modality and expand its capabilities in order to exploit its full potential. At present, the performance of SPECT systems often is limited by the detectors used in these systems. Modern SPECT systems consist of scintillation crystals coupled to photomultiplier tubes as detectors. Important requirements for scintillators used in SPECT applications include high light output and high energy resolution, reasonably fast response and high gamma ray stopping efficiency. Ideally, the scintillator should also be inexpensive, rugged and easy to manufacture. Currently, NaI(Tl) is the detector of choice in SPECT systems and it is relatively inexpensive and its light output is fairly large. However, the poor energy resolution of NaI(Tl) often limits SPECT performance. The energy resolution of NaI:Tl is limited by its relatively poor proportionality. If scintillators with higher energy resolution at typical SPECT energies (~140 keV) were available, the essential process of scatter rejection would improve. The goal of the proposed effort is to investigate new high resolution detectors for SPECT studies. Enhanced scatter rejection can be expected from the new detectors, which are also expected to be relatively easy to produce. The Phase I project will be aimed at demonstrating the feasibility of the proposed concept, while the Phase II project will be aimed at optimization of the new detectors, implementation of the prototype modules and detailed performance evaluation. PUBLIC HEALTH RELEVANCE: High Performance Detectors for SPECT P.I.: Dr. Michael R. Squillante Project Narrative - The proposed research will investigate a promising detector technology which should have a major impact in health care, in particular, in the development of detectors for in-vivo imaging. Other areas to which this research could be of benefit are: physics research, materials studies, homeland defense, and non-destructive testing."}
{"text": "Research goals for the coming year: We plan to isolate the endotoxin-binding structures from human platelets and lymphocytes in physico-chemically homogeneous form. We will investigate if both major subpopulations of lymphocytes (B and T cells) have endotoxin-binding properties and if in the affirmative, do they have it to a similar extent. We will determine the minimum number of active binding sites per receptor molecule and define the structures on the receptors which carry the active binding sites for endotoxin. We will determine the mode of action the receptors, especially the extent of reversibility of receptor-endotoxin combination. It is one of our major aims to clearly define the chemical structure of the host receptor sites to which endotoxin attaches. We will use the receptor(s) and active receptor fragments in animals to determine if they prevent fever as well as lethal shock. The undersigned agrees to accept responsibility for the scientific and technical conduct of the project and for provision of required progress reports if a grant is awarded as the result of this application."}
{"text": "Studies of the specificity and efficiency of reactions catalyzed by enzymes and the elucidation of a biological control mechanism are the particular subjects of this proposal. The enzymes chosen are of interest to health-related problems. The serine proteins include enzymes involved in blood clotting, nerve transmission, and digestive processes. Some of these enzymes are used clinically in the treatment of inflammations. A possible role in cancer therapy has recently been suggested for lysozyme, an important enzyme in biological defense mechanisms. BIBLIOGRAPHIC REFERENCES: Rubsamen, H., Hess, G.P., Eldefrawi, A.T., and Eldefrawi, M.E. Biochem. Biophys. Res. Comm. 68, 56-63 (1976) Interaction between calcium and ligand-binding sites of the purified acetylcholine receptor studied by use of a fluorescent lanthanide. Rubsamen, H., Montgomery, M., Hess, G.P., Eldefrawi, A.T., and Eldefrawi, M.E. Biochem. Biophys. Res. Comm. 70, 1020-1027 (1976) Identification of a calcium-binding subunit of the acetylcholine receptor."}
{"text": "The yeast mitotic spindle is less complex than its counterparts in larger eukaryotes and has been intensively studied using genetics, biochemistry, cell biology and ultrastructure approaches, providing an opportunity to develop an understanding of its function and regulation at a level that is not currently achievable in any other organism. The proposed studies are critical for attaining this goal. Correct establishment, function and checkpoint monitoring of kinetochore-microtubule attachments are central to mitotic fidelity. Despite much progress identifying key proteins involved in this physical attachment, and assessing their activities in vivo and in vitro, little is understood about mechanisms regulating attachments, about how microtubule-binding outer kinetochore proteins associate with the central kinetochore, or about emergent properties that result when separate subcomplexes like Dam1 and Ndc80 are together in kinetochores. Building upon discovery and analysis of the Dam1 complex, and structure studies of the Ndc80 complex, new studies will identify their binding partners, determine how Dam1 and Ndc80 complex structures relate to function, how post-translational modifications and binding partners affect function, and how ensembles of kinetochore complexes interact dynamically with microtubules. Previous discoveries that Aurora kinase regulates kinetochore attachment via Dam1 complex phosphorylation, identification of an Aurora kinase consensus site, identification of the fourth yeast Aurora kinase complex subunit, and novel implication of casein kinase 2 in inner kinetochore regulation, provide a robust foundation for studies to reveal how protein kinases regulate critical mitotic functions. Proposed studies will determine how the Aurora kinase complex is targeted to specific cellular locations, will identify its targets at each location, and will determine how phosphorylation affects activity of its targets. Molecular genetics and biochemical analysis of the fully reconstituted, four-protein Aurora kinase complex will determine how this complex is regulated. Having recently identified casein kinase 2 as a mitotic regulator of the inner kinetochore protein Ndc10 and the widely conserved Mif2 (CENP-C) linker protein, and obtained in vivo evidence for dual-regulation of these proteins by CK2 and Aurora kinase, powerful in vivo and in vitro tests of how these two kinases regulate kinetochore function will be conducted. Thanks to unique advantages of yeast, strong inroads into a full molecular dissection of mitotic spindle disassembly have already been made, and will be built upon to fully elucidate pathways and mechanisms. As cells exit mitosis, the enormously complex mitotic spindle must be disassembled rapidly, which involves taking apart stabilized microtubule bundles, disassembling protein subcomplexes, reversing post-translational modifications and destroying proteins. Because spindle disassembly mechanisms are largely unexplored, this is an extremely fertile and important area for investigation. Comprehensive genetic screens and interaction analyses will identify genes and pathways, and focused phenotypic and biochemical studies will reveal detailed mechanisms. PUBLIC HEALTH RELEVANCE: These studies will increase understanding of fundamental aspects of mitotic spindle function and regulation, and since many mitotic proteins and mechanisms are evolutionarily conserved, will provide a framework for elucidating mitotic mechanisms in humans. Chromosome instability is a key contributing factor in cancer and birth defects. Therefore, understanding principles of spindle function may suggest novel strategies for prevention, detection and treatment of human diseases."}
{"text": "Alpha1-Antitrypsin (alpha1-AT) is the major proteinase inhibitor in the human plasma, inhibiting pancreatic elastase, trypsin, chymotrypsin and related proteinases. An important relationship between plasma levels of alpha1-AT and two pathological conditions have been observed in humans: unusually low levels of alpha1-AT are found in the plasma of patients with an adult form of pulmonary emphysema and in a childhood form of cirrhosis of the liver. Moreover, an accumulation of alpha1-AT or alpha1-AT-like material have been noted in the livers of these patients, suggesting the glycoprotein is being synthesized but not secreted into plasma. One likely explanation of the problem of transport is the occurrence of abnormality in the attachment and completion of the carbohydrate moiety to the polypeptide chain of alpha1-AT. We have purified, characterized and determined partially the amino acid sequence of alpha1-AT (PiMM) from plasma of normal individuals. We have also studied its mechanism of proteinase inhibitory activity, as well as its survival in animal circulation after removal of sialic acid residues. We propose to perform similar studies on alpha1-At from normal liver (PiMM) and cirrhotic livers (PiZZ). These studies will include determinations of molecular weight, electrophoretic mobility, isoelectric point, amino acid and carbohydrate compositions, peptide mapping, proteinase inhibitory activities, and survival in plasma. The nature of the bond linking carbohydrate unit(s) to the peptide chain will be studied. Ultimately, the amino acid and carbohydrate sequence will be performed. Characterization of the carbohydrate moiety will be of utmost importance since, as in other glycoproteins, it may be vitally important in the transport or survival of the inhibitor in plasma. From these studies, we hope to gain an insight into the relationship between the function and structure of alpha1-At and thus better understand the pathologies of pulmonary emphysema and cirrhosis of the liver on a molecular level."}
{"text": "The extracellular adenosine receptors have a modulatory role in the nervous, circulatory, endocrine and immunological systems. The prospect of harnessing these effects specifically for therapeutic purposes is attractive. We have synthesized the first selective A3 adenosine receptor agonists and antagonists. We recently characterized the first allosteric modulators of this receptor. Imidazoquinoline and pyridinylisoquinoline derivatives were found to enhance the actions of agonists of the A3 receptor, and thus may prove to be suitable leads for the development of therapeutic agents based on this concept. A3 agonists are under development for treating cancer, rheumatoid arthritis, and other diseases. Allosteric modulators of G-protein coupled receptors are a new approach to receptor activation without the limitations of use of directly-acting agonists as therapeutic agents. We have identified two new classes of allosteric modulators of the A3 receptor and are currently exploring the structure activity relationship (SAR).[unreadable] [unreadable] The potential of A3 agonist therapy is of great interest. We are collaborating with Dr. Bruce Liang and Dr. Asher Shainberg on various aspects of the use of adenosine receptor agonists in protection of the heart. We have designed a mixed agonist of A1 and A3 subtypes, both of which are protective in the heart. This mixed agonist MRS3630 is protective in a model of ischemia in the isolated perfused mouse heart. The adenosine A3 agonist IB-MECA is currently in clinical trials for use in cancer treatment by our CRADA partner Can-Fite Biopharma. [unreadable] [unreadable] One of the issues in the development of adenosine receptor ligands is the species dependence. Some compounds that are very potent at a given human adenosine receptor are weak in rat tissue. We have developed adenosine agonists and antagonists that work generally across species. This is important for testing the same compounds in various animal models before suggesting the feasablity of their use in humans. The key to A3 receptor ligands that are potent and selective across species is the use of the nucleoside structure as the starting point in the design process. Nucleosides tend to bind to that receptor subtype with greater consistency across species than nonpurine heterocycles.[unreadable] [unreadable] Adenosine A3 antagonists may be useful for the treatment of glaucoma. Early efforts to identify antagonists of the A3 receptor in our library involved screening of chemically diverse libraries. One of the limitations of this approach is that the antagonists often bind well only at the human, but not murine A3 receptors. We are currently developing other novel A3 antagonists based on nucleotide structures, that have proven to be generally applicable across species. We are currently studying systematically the SAR of adenosine derivatives that affect efficacy as A3 adenosine receptor agonists. Surprisingly, a commonly used A1-selective agonist, cyclopentyladenosine, was found to act as a pure antagonist at the A3 subtype. Other nucleosides may be chemically modified, especially on the ribose moiety, to have reduced efficacy at the A3 receptor. Some of these analogues derived from highly potent A3 agonists, such as 5-dimethylamides, were found to be A3 antagonists. A novel nucleoside-based antagonist of the A3 receptor MRS1292 was found to lower intraocular pressure a mouse model of glaucoma.[unreadable] [unreadable] We are using mutagenesis to study the determinants of recognition of adenosine within the binding site of the A2A and A3 receptors, and proposing conformational factors involved in receptor activation. Since the four subtypes of adenosine receptors have been cloned it has been possible to conduct molecular modeling of the receptor protein, based on sequence analyses and homology modeling using the high resolution rhodopsin structure as template. We intend to use such a modeling approach for the design of more selective adenosine receptor agonists and antagonists. [unreadable] [unreadable] Recently this project has also focused on the effects of adenosine agonists and antagonists in the central nervous system and in the heart and on the possibility of therapeutics for treating neurodegenerative and cardiovascular diseases. An A3 agonist, administered chronically, proved to be highly cerebroprotective in an ischemic model in gerbils. A3 agonists cause morphological and biochemical changes in astroglial cells. Adenosine is released in large amounts during myocardial ischemia and is capable of activating both A1 or A3 receptors that occur on cardiac myocytes to exert a potent cardioprotective effect. We have shown that synthetic adenosine agonists,selective for either the A1 or A3 subtype, protect ischemic cardiac myocytes in culture and in the isolated perfused heart and thus might be beneficial to the survival of the ischemic heart. An acutely administered A3 agonist, Cl-IB-MECA, was cardioprotective in cell culture, through the selective activation of A3 receptors without side effects, such as bradycardia, associated with the A1 subtype. The protection was blocked in the presence of a selective A3 receptor antagonist. Inn summary, highly selective adenosine analogues may have therapeutic potential in treatment of cerebral ischemiastroke and possibly other neurodegenerative disorders as well. It is proposed that modulation of A2B and A3 receptors may be useful in treating asthma and inflammatory diseases. The pharmacolgical properties of novel xanthines developed in our lab that act as selective A2B receptor antagonists are being explored as potential antidiabetic and antiasthamtic agents. The first highly selective A2B receptor antagonist, synthesized in our section, has now been radiolabeled and is used in assaying newly synthesized analogues for affinity at the A2B receptor. We are also screening chemical libraries for novel leads for A2B receptor antagonists."}
{"text": "DESCRIPTION (taken from the application: The concept of orthopaedic research encompasses a very broad area spanning the field of musculoskeletal research from the clinical aspect to both biological and biomechanical basic sciences. The Bioengineering and Orthopaedic Science Gordon Research Conference has been the premier conference for investigators to combine the three disciplines of orthopaedic sciences. The format of these conference, held in a small private school in New Hampshire every other summer, provides a unique environment for the exchange of new and unpublished information. The cross fertilization that can only come from a conference were clinicians, engineers and biologists meet, is more vital to this field than ever before. For the first time, biological principles are being used in the treatment of orthopaedic injuries and diseases and these treatments must confirm to and meet the biomechanical needs of the skeleton. For example, investigators and clinicians are now beginning to utilize molecules such as parathyroid hormone; parathyroid hormone related peptide, insulin-like growth factors, fibroblast growth factor and the hedgehogs. In addition, physical parameters such as ultrasounds and electrical stimulation are currently being used to stimulate bone growth. Bioengineering has moved from the mechanical design of joint implants to the problems associated with long-term use of implant materials and a general understanding of the biological effects of these materials in the living system. For the first time, a session will be held on skeletal tumors. Two noteworthy changes will be made in the format from previous meetings. In the comments on the 1996 Gordon Conference Program, many attendees felt that the junior members of the audience did not feel comfortable speaking out and that the discussion were dominated by only a few senior scientists. In an effort to increase the comfort level of the junior scientists and to increase their participation in general, the following changes were implemented: (1) five to ten scholarships will be designated for junior faculty and postdoctoral fellows and their availability will be announced in conference literature; (2) One evening will be dedicated to talks by junior scientists. Employing these options to encourage the participation of junior scientists has worked very well for other Gordon Conferences such as the Proteoglycan Gordon Conference which uses the scholarship mechanism and Molecular Genetics which uses the junior scientists evening option."}
{"text": "The inflammatory agents, histamine, bradykinin, and serotoin have been shown to increase capillary membrane transport in various tissues. Other studies have suggested a dependence of capillary membrane transport upon vascular calcium concentration. Evidence has been given that both effects are caused by widening of the capillary membrane inter-endothelial clefts. A logical hypothesis is that inflammatory agents alter capillary transport by interfering with calcium in the membrane, thus altering cell-cell contracts. We will test this hypothesis by: 1) quantifying the dependence of capillary membrane transport parameters upon inflammatory agents, 2) quantifying the dependence of transport parameters upon vascular calium concentratin, 3) testing for antagonism of inflammtory agent on transport parameters by calcium, 4) testing for alteration of calcium binding to the capillary endothelium by inflammatory agents, and 5) testing the effect of calcium channel blockers on alteration of transport parameters by inflammtory agents. The experimental preparation is the isolated, perfused (Langendorff) rabbit heart. Measurement of capillary membrane transport parameters (permeability coefficient, filtration coefficient, reflection coefficient) to several nonelectrolytes (urea, sucrose, inulin) will be made by the method of osmotic transients. Calcium binding by capillary endothelium will be observed under electron microscopy using ionic lanthanum as a marker for calcium binding sites. Long-term objectives of the project are to shed light on how normal intercellular junctions are maintained and to point out possible mechanisms for reversing the deleterious effects of inflammatory agents, e.g., edema formation. With particular reference to the heart, edema formation is known to be detrimental to cardiac function and, further, reversal of edema is associated with improvement of that function. If release of inflammatory agents during cardiac damage or shock states contributes to edema and associated reduction in cardiac performance, knowledge of the mechanism of action of inflammatory agents at the capillary level may prove beneficial to its treatment."}
{"text": "The wild-type Rep proteins of adeno-associated virus type 2 (AAV) have been demonstrated in tissue culture to inhibit the replication of HIV-1, the etiological agent of AIDS. Unfortunately, their potential as a therapeutic agent for AIDS is limited because they also appear to block cell division. Several groups have tried and failed to produce cell lines producing high levels of Rep proteins, although we have achieved high levels in transient expression assays in cells transfected with a plasmid expressing the rep gene from the long terminal repeat promoter of HIV-1 (HIV-LTR). Our objective is to create a mutant Rep protein which can block HIV-1 replication without blocking cell division. We have made a series of mutant rep genes expressed from the HIV-LTR. Our collaborators at the New Jersey Center for Advanced Biotechnology and Medicine have already tested several of these mutants for their ability to block HIV-1 replication. Plasmids containing the mutant rep genes are co-transfected with an infectious proviral clone of HIV-1 into human cells and the cells and culture supernatants are assayed for reverse transcriptase activity, HIV-1 RNA and for HIV-1 gag protein. We have identified several portions of the Rep proteins which are important for this inhibition. Several of these mutants inhibit HIV-1 better than the wild-type proteins. If we can identify a mutant Rep protein that blocks HIV-1 production without blocking cell division then we would have the basis for a novel treatment for AIDS. If Rep proteins truly block cell division then they may also be developed into a treatment for cancer. We will continue to produce and test mutant Rep proteins for HIV- inhibition. We will be concurrently attempting to produce cell lines making these mutant proteins. This will allow us to determine which parts of the Rep proteins are involved in the putative cell division block."}
{"text": "The current thrust of our studies of neonatal drug metabolism lie in the area of transplacental carcinogenesis. The model used is the administration late in pregnancy of 3-methylcholanthrene to pregnant mice. It is known that the offspring of such animals have a high incidence of tumors during late adulthood. We plan to evaluate mixed- function oxidase activity in various organs during different developmental stages of such animals. Also studied will be the response of these animals to inducers such as phenobarbital and 3- methylcholanthrene. Another phase of our studies will be to examine the effect of exposure of rats to intense blue light on microsomal drug metabolism in liver and skin. These studies are designed to evaluate possible responses to high intensity light such as those encountered during the clinical practice of phototherapy of jaundiced infants. Bibliographic references: Lampert, R.P., Robinson, D.S., and Soyka, L.F.: A critical look at oral decongestants. Pediatrics 55:550-552, 1975; Soyka, L.F., Robinson, D.S., Lachant, N., and Monaco, J.: The misuse of antibiotics for treatment of upper respiratory tract infections in children. Pediatrics 55:552-556, 1975."}
{"text": "Autonomic dysfunction including orthostatic hypertension (OH) is a major health problem, causing significant morbidity and mortality. Its pathophysiology remains poorly understood and hence its management lacks a solid scientific base. The PPG focuses on the pathophysiology and treatment of autonomic failure. Project 1 (Low) incorporates a novel strategy of cholinesterase inhibition in the treatment of OH, an approach that promises to improve OH without supine hypertension. A second blinded treatment trial will evaluate if sodium chloride will expand plasma volume and if urinary sodium excretion is a suitable surrogate measure of plasma volume status. A series of studies, including the use of microneurography to measure sympathetic impuls3es, will evaluate the pathophysiology of postural tachycardia syndrome (POTS). A novel approach of amplitude modulation of the EEG in POTS shows a selective reduction of a frequency band of 0.02-0.05 Hz; this component is of particular interest since it may have a brainstem origin. The venous capacitance bed will be evaluated (Projects) to determine if there is excessive transcapillary efflux and changes in compliance in POTS and the effects of aging. The relative importance of the mesenteric, systematic and cerebrovascular circulations in OH will be evaluated. Project (Benarroch) will expand its studies on the neurochemical organization of autonomic control regions of the medulla in multiple system atrophy (MSA) and the parkinsonian syndromes. These include quantitative evaluates of new cellular groups (nucleus ambiguus, nuclease retroambiguus) and new receptors (including angiotensin II) that are likely to provide insights into the pathophysiology of autonomic failure in MSA. Project (Joyner) will undertake a detailed evaluation of the effects of denervation (mild in POTS and severe in neurogenic OH) and aging on the venous capacity and compliance. Project (Brimijoin) will focus on the response of the pre-ganglionic neuron to denervation and will study the mechanism of spinal intermediolateral column cell loss, using he model of immune-mediated pre-ganglionic autonomic neuropathy. The roles of apoptosis, excitotoxicity, growth factors, and aging will be evaluated and related to MSA."}
{"text": "DESCRIPTION (from abstract) Many neuroendocrine peptides are synthesized as precursor proteins; post-translational processing of these precursors is a key step in the production of biologically active peptides. The enzymes involved in this processing are important regulators of these intercellular messengers. A variety of endopeptidases and exopeptidases have been proposed to be involved in neuropeptide processing. In this grant application the applicants describe studies to examine the contribution of various processing enzymes in the generation of opioid peptides using transgenic animals and cell lines. The first specific aim is to examine dynorphin processing in animal models that lack specific peptide processing enzymes. They will assess the extent of involvement of carboxypeptidase E (CPE) in dynorphin processing by comparing the profiles of various dynorphin peptides in fat/fat mice (lacking active CPE) with those in control mice. In addition, they will assess the extent of involvement of prohormone convertase 2 (PC2) in dynorphin processing by comparing the profiles in PC2 K/O mice (that lack PC2) with those in control mice. The second specific aim is to examine the contribution of various peptide processing enzymes to dynorphin biosynthesis using a cell culture system that inefficiently processes dynorphin. The investigators will examine whether a dynorphin processing defect in NIT-3 cells (that lack CPE) can be overcome by endocrine carboxypeptidases or by prohormone convertases. These studies will provide information about the interactions of various processing enzymes that could modulate their function. The studies proposed in this grant application will address the physiological role of the processing enzymes in a neuropeptide biosynthesis. The information gained through these studies will provide insights into the multiple regulatory steps (including cross-talk between enzymes) that govern neuropeptide processing which cold modulate the levels of neuropeptides in normal an dysfunctional cellular events such as those seen in several neurological disorders."}
{"text": "The proposed research aims to elucidate the psychosocial and behavioral characteristics of female adolescents who qualify for DSM-III-R diagnosis of Psychoactive Substance Use Disorder (PSUD) as a relatively simple disorder and comorbid Psychoactive Substance Use Disorders and Conduct Disorder (PSUD/CD) as a more complex drug abuse disorder. The latter group of PSUD/CD individuals is at particularly high risk for multiple personal and social problems (e.g., teenage pregnancy, AIDS, prostitution, sexually transmitted diseases, drug teratogenicity). The specific aims of this research are to: 1) identify the biobehavioral characteristics, psychosocial correlates, family liabilities and differential dyadic interaction patterns between parent and adolescent female offspring and; 2) determine the relationship among age of pubertal onset, sexual activity and drug use. In order to accomplish these objectives, five groups of female subjects will be studied: 1) Adolescents with PSUD and no comorbid diagnosis; 2) Adolescents with PSUD/CD; 3) Adolescents with CD only; 4) Control normal adolescents with out PSUD and CD or any other psychiatric disorder, and, 5) Older sisters of the PSUD/CD and PSUD female who do not present PSUD. The results of this study will elucidate the factors associated specifically with a PSUD outcome in female adolescents, identify factors which could potentially protect against a PSUD outcome, and reveal the processes mediating the progression or development from drug use to a diagnosis of PSUD."}
{"text": "The major goal of this Resource is to construct high-resolution expression and functional profiles for orphan receptors in order to elucidate the basis of their tissue and cell-specific functions. Although there are many reports describing the expression patterns of many individual orphans at a given developmental stage or in a specific tissue, systematic analyses of the expression of individual orphans at all stages of development, in all cell types, have yet to be undertaken. It will therefore be important for the proposed Atlas, through this Resource, to rigorously examine the expression pattern of an initial subset of ONRs at the cellular level. The expression profiles of the selected ONRs in all cell types of different organs will complement the expression profiling of ONRs in all organs proposed in Strand A. In addition, we will use transgenic models to determine the spatiotemporal functional profiles for ONRs. These models will help to clarify the role of ligands, regulators and co-regulators in modulating ONR function, and to better understand the developmental, physiological and metabolic roles of these receptors. The transgenic models and the expression of functional profiles will be distributed by this Resource and Bioinformatics Resource to all investigators in the field to facilitate future investigations of various aspects of ONR biology, including the identification of ligands, upstream regulators, co-factors and signaling pathways that modulate the function of the ONRs. This Resource has two objectives 1. Determination of the cell-specific expression profiles of orphan nuclear receptors. To generate transgenic mouse lines which will faithfully recapitulate the in vivo expression of orphan receptors, we plan to use an orphan receptor gene promoter-EGFP system in the context of bacterial artificial chromosomes (BACs) to map the native expression of these orphans. To construct individual functional maps of orphan nuclear receptors in vivo, we will generate BAC-based transgenic models that utilize a functional chimeric receptor and an integrated responsive reporter, EGFP. These BAC transgenic approaches will be used to directly determine the expression and functional profiles of orphan receptors. The goals of the Resource, although long term, are consonant with the original mandate of accelerate the achievement of the long term goals of the Atlas, and acting as a Resource for the entire ONR research community."}
{"text": "Cells of hibernating animals are compared with those of mammals which do not hibernate to examine basic mechanisms of ion transport and interactions of transport mechanisms which ensure cellular ionic balance. The principal adaptation of hibernators studied so far has been the ability of their cells to function at low temperature. Past effort has documented the involvement of the Na/K pump and of general leak in this adaptation in several tissues (kidney, erythrocytes, brain, liver). Future efforts will focus on erythrocytes. Our aim is to define the points of failure in the cold in terms of the pump's kinetics and its overall scheme in cold-sensitive cells of non- hibernators and to exploit unusual features of the pump in hibernator cells for a better understanding of pumps in general. The second objective is to define the differences in the several components of passive permeability to Na between cold-sensitive and cold-tolerant red cells that permit ion balance with reduced active transport at low temperature and to determine how cytoplasmic factors may be involved in bringing pumps and leaks into balance under widely changing conditions. Another adaptation of many hibernators is seasonal inanition and self-starvation. Some species, such as bears and prairie dogs, exhibit this ability even without the concomittant lowering of metabolism by cooling. Studies in human and mammalian red cells have suggested that reduction in energy-dependent membrane transport may be a factor in the energy conservation of the cell during starvation, but this has not been examined in these species which specialize in starvation-adaptation and previous studies have not considered the contribution of reduced permeability as a possibility. We propose to do this. The perceived areas of health-relatedness are, for the low temperature studies, improved viability of cells during storage and, for the seasonal/inantion studies, cellular adaptation to and effects of under-nutrition."}
{"text": "Immune responses are dependent on the coordinated movement of leukocytes from sites of antigen deposition, to secondary lymphoid organs and to sites of infection or inflammation. Chemokines play a critical role in this process by directing leukocyte trafficking throughout the body. Although it is well known that leukocytes express chemokine receptors and can migrate directionally in response to chemokine gradients, the molecular mechanism(s) that control chemokine receptor responses are still largely unknown. CD38, an ADP-ribosyl cyclase, that catalyzes the production of the calcium-mobilizing metabolite cyclic ADP-ribose (cADPR), appears to be a critical regulator of chemokine receptor signaling and leukocyte trafficking. We observed that neutrophil migration is impaired in CD38-deficientmice resulting in attenuated and reduced inflammatory responses. We also found that the cADPPR produced byCD38 modulates calcium mobilization in neutrophils that have been activated with inflammatory chemoattractants such as peptides derived from bacteria and viruses. Furthermore, we showed that cADPR-specific antagonists block the migratory response of neutrophils to these peptides. The data suggest that small molecule inhibitors of CD38could potentially be used to block neutrophil-dependent inflammatory responses. Recently, we observed that the migratory response of dendritic cells is also impaired in CD38 deficient mice. Specifically, we found that CD38-deficient dendritic cells cannot migrate in response to ELC or SLC, chemokines that direct dendritic cells to migrate from sites of damage or injury to lymphoid tissues. This impaired chemotactic response observed inCD38-deficient dendritic cells results in inefficient T cell priming and significantly reduced T cell-dependent immune responses. Based on our previous data, we now hypothesize that CD38, through its production of cADPR, regulates cell-dependent immune responses by modulating the migration of dendritic cells. To test this hypothesis we have proposed the following Specific Aims: (1) we will determine whether CD38 regulates migration of all mature dendritic cell subsets to ELC or SLC, (2) we will determine whether CD38 regulates the migration of dendritic cells to inflammatory chemoattractants and (3) we will determine whether cADPR production by CD38 controls dendritic cell trafficking in vivo. These experiments will validate whether CD38 antagonists have the potential to be used as immunosuppressive agents that attenuate immune responses by modulating leukocyte trafficking."}
{"text": "Most HIV seropositive persons show humoral and cell-mediated immune responses to HIv antigens and in particular to the env gene products of HIV, the external envelope glycoprotein gp120 (EEG). the overall goal of this proposal is to define the role of the carbohydrates of the external envelope glycoprotein in affecting the immune response to the virus. The simian immunodeficiency virus/rhesus macaque model system will be used to focus on a variety of humoral and cell-mediated immune responses to the wild-type EEG and selected mutant and variant EEGs of the cloned SIVmac239 strain of the rhesus macaque simian immunodeficiency virus. Variant and mutant EEGs will be produced using oligonucleotide-mediated site-directed mutagenesis of the EEg DNA sequence and B' mutations in the pathway of protein glycosylation (Objective 1). Purified EEGs will characterized based on their structural and functional properties (Objective 2), and after immunization of small animals with selected mutant and variant glycoproteins the specific immune responses will be assessed (Objective 3). The strength, nature and extent of the responses will be compared with those induced by the wild-type EEg and will be correlated with the carbohydrate structure of the immunogen. The ability of mutant and variant EEG to provide protection from SIV infection will be determined (Objective 4), by challenging immunized monkeys with infectious clones of SIVmac 239 and 251 and with extracts that produce AIDs-like symptoms to determine whether the immune responses are broadly protective. To enhance development of cell mediated immunity, genes specifying the appropriate EEGs will be used to construct replicating simian adenovirus.SIVenv expression vectors, as has been done previously for an HIV env-adenovirus vector. These recombinants will be evaluated as potential vaccine candidates."}
{"text": "This supplementary request for the above named core function under the immediate supervision of Dr. Walter Korytnyk is being submitted for consideration and, hopefully, funding. This collaborative effort involves a number (8) of ongoing projects from groups of investigators as well as many (12) anticipated projects. All of these interests have in common the requirement that the nuclear magnetic resonance spectrometer is essential for the conduct of these individual research proposals. The instrument (NMR) has been purchased with other funds, but in order to function as a core facility we require the personnel and support to effectively operate it. The Core Grant Committee, composed of: Dr. Lucius F. Sinks, Chairman; Dr. Gabor Markus; Dr. Enrico Mihich; Dr. Arnold Mittelman; Dr. E.A. Mirand (Ex Officio), has met with the principal investigator (Dr. Gerald P. Murphy) and Dr. Walter Korytnyk, and have reviewed the proposal herein contained. We feel that the request as written represents a legitimate core function for activities conducted at RPMI and respectfully request that it be considered as a supplement to the present core grant."}
{"text": "This project aims at the visualization via MRI of freezinginterfaces in ice-water mixtures. Specifically, the in-situvisualization of frost and of freezing in porous media will bepursued. MRI is the only solution possible in such systems thatare strongly refracting to light. Attendant MRI thermometrystudies will also be pursued."}
{"text": "This research is directed toward elucidation of the enzymatic mechanisms involved in the assimilatory reduction of nitrate to ammonia. The significance of this pathway is manifest in that the various oxidized inorganic forms of nitrogen provide the ultimate nitrogen source for all life. Nitrate assimilation is a two-step process: the two-electron reduction of nitrate to nitrite, followed by the six-electron reduction of nitrite to ammonia. The enzymes catalyzing these reaction in Neurospora crassa, namely nitrate reductase and nitrite reductase, are adaptively formed in the presence of either NO3 or NO2. Their synthesis is repressed by NH plus 4. Nitrate reductase is a soluble, sulfhydryl-containing molybdoflavohemoprotein, mediating the NADPH-dependent reduction of nitrate via the electron transfer sequence: NADPH yields ((-SH) yields FAD yields cytochrome b-557 yields (Mo)) yields NO3. The nitrite thus formed is stoichiometrically reduced to ammonia by nitrite reductase, also a soluble, sulfhydryl-containing flavo-protein, possessing non-heme iron/sulfur centers and the novel iron tetrahydroporphyrin, siroheme, as prosthetic groups. The suggested electron transfer sequence is: 3NAD (P) H yields ((-SH),FAD, (Fe.S) yields siroheme) yields NO2. The prime objective at this point involves elucidation of the electron transfer catalysis mediated by nitrite reductase through UV/visible spectrophotometric and EPR spectroscopic observations, kinetic studies, and chemical and physical analyses of the nitrite reductase protein and its complement of prosthetic groups. Integration of these results should provide for reliable conclusions regarding the important relationships between structure and function in this multi-electron transferring homodimeric enzyme. The subunit organization of the nitrate reductase and the form and function of its molybdenum cofactor in this organization is also under investigation."}
{"text": "The major objective of the proposed research is the development of an in vitro model for exocytosis. Preliminary investigations indicate that the ways in which Ca2 ion and ATP are involved in this cellular process can be determined from study of the interactions of these molecules with isolated secretion granules. Submillimolar concentrations of Ca2 ion induce total lysis of suspensions of secretion granules isolated from acinar cells of the rabbit parotid. I have identified two processes which precede lysis: 1) the Ca2 ion-specific activation of a granule-associated phospholipase A2 and 2) the large-scale rearrangement of granule membrane proteins (viewed as intramembranous particles in freeze fracture preparations) into interconnecting linear assays. I intend to evaluate the extent to which granule lysis in vitro correlates with exocytosis in vivo and to investigate the relationship of phospholipase activity to granule lysis in a series of comparative kinetic studies. Lysis will be determined by the extent of release of alpha-amylase, a secretory protein found in the granule content. Phospholipase activity will be measured by quantitating the hydrolyzed phospholipids resolved by thin layer chromatography. Membrane protein rearrangements induced by Ca2 ion will be characterized by electron microscopy of freeze fractured preparations. Provided the linear arrays can be stabilized, the molecular species involved will be identified by techniques employing covalent crosslinking and spin labeling."}
{"text": "Abdominal aortic aneurysms (AAA) occur commonly in the elderly population and are a major cause of morbidity and mortality. The mechanisms responsible for aneurysm formation are poorly understood, which has impeded identification of effective medical therapies for this disease. Mounting evidence from studies in human AAA, and in experimental animal models, suggests that oxidative stress plays a key role in the pathogenesis of AAA. While a number of enzymatic pathways are capable of inducing vascular oxidant stress and potentially contributing to AAA, we hypothesis that myeloperoxidase (MPO) is instrumental in this process. MPO is an enzyme expressed primarily in neutrophils (PMNs) and to a lesser extent in monocytes/macrophages, that catalyzes the formation of HOCl, a powerful oxidizing specie, and induces protein nitration. In addition, MPO can be taken up into the blood vessel wall, thus amplifying inflammation, oxidative stress and protease degradation. Using two distinct murine models (elastase-induced and angiotensin II infusion in hyperlipidemic mice), we present data showing that aortic MPO activity and chlorotyrosine expression (a marker of HOCl-mediated oxidative stress) are increased during experimental AAA formation. Our data also suggest a potentially novel role for angiotensin II to exacerbate HOCl-mediated oxidative stress in the blood vessel wall. Moreover, supplementation with taurine, a beta amino acid that reacts with and detoxifies HOCl, prevents AAA formation in these experimental models. To test our hypothesis, we propose three specific aims. In aim 1, we will perform selective immunodepletion and adoptive transfer of PMNs from control or MPO- deficient mice.to test the hypothesis that MPO expression in PMNs contributes to experimental AAA formation in the elastase model. Also, we will immnuodeplete PMNs to determine their role in AAA formation in the angiotensin II infusion model. In aim 2, we will test the hypothesis that genetic deficiency of MPO ameliorates AAA formation in the elastase-induced and angiotensin II infusion models. In aim 3, we will test the hypothesis that transgenic expression of human MPO augments experimental AAA formation. In aims 2 and 3, we will also determine whether angiotensin II upregulates expression and activity of MPO in leukocytes, and whether MPO modulates hypertension and atherosclerosis induced by angiotensin II infusion. Our studies will provide novel insight into mechanisms of AAA formation and linkages between key oxidant stress generating pathways in the pathogenesis of cardiovascular disease."}
{"text": "The overall objective of this project is to obtain a 10 year update and extension of the 1995 National Survey of Adolescents (NSA) by conducting a three wave, longitudinal survey of a new national household probability sample of 12-17 year-old adolescents. The original NSA was conducted via telephone with a national household probability sample of 4,023 male and female adolescents between the ages of 12 and 17. The methods to be used for the proposed project are deliberately modeled after the original NSA. The proposed project will involve a baseline survey of a new cohort of 4,000 12-17 year-olds using identical sample selection and interview procedures as that in the original NSA. The proposed project will also include two additional assessments of adolescent participants to be conducted one and two years after the baseline assessment. As was the case with the odginal NSA, a structured interview will be used to measure a broad range of exposure to witnessed and direct violence victimization (e.g., sexual assault, physical assault, and physically abuse punishment) occurring in family, school, and community settings. Several types of mental health problems will be measured, including PTSD, major depression, suicide ideation and attempts, substance use and abuse, and delinquency. The study will also obtain information from parents as well as adolescents regarding academic performance. The proposed project will provide invaluable comparisons about changes over the past decade in violence exposure, substance use, and related adjustment issues among American youth. Although modeled after the NSA, the proposed project departs from the original in several important respects. First, the assessment of violence exposure in the proposed study is broader and more specific; specifically, assessment of physically abusive punishment will be expanded, and there will be a more comprehensive focus on episodes of school, domestic, and community violence to permit greater description of adolescents' exposure to violence in each of these important settings. Second, the range of outcomes assessed will be diversified beyond mental health, substance abuse, and delinquency to include academic performance, an important developmental marker of child adjustment, as well as risky sexual behaviors. Third, the proposed survey methodology is longitudinal. Thus, specific aims of the proposed project are: 1)To obtain prevalence rates and descriptive data regarding adolescents' exposure to violence (both direct and witnessed) across a range of settings (school, home, community), including sexual assault, physical assault, physically abusive punishment, domestic violence, community violence and school violence; 2) To determine whether there have been changes among a nationally representative sample of 12-17 year olds over the 1995-2005 period with respect to the prevalence of exposure to violence, mental health problems, and risk or protective factors for a range of adverse social and mental health outcomes given exposure to violence;3) To examine the longitudinal trajectory of exposure to violence, development of mental health and substance use, abuse problems, risky sexual behaviors, delinquency and changes in academic performance;4) To test the hypothesis that the relationship between adverse family environment and the outcomes of delinquency and poor academic performance are substantially mediated by exposure to violence and/or violence-related mental health problems; and 5)To test the extent to which the relationships described above are moderated by gender, race, and ethnicity."}
{"text": "Translation of mRNA into protein is thought of as occuring in three stages: initiation, elongaton, and termination. During initiation, the mRNA to be translated and the initiator tRNA are aligned on the 70S ribosome so that peptide synthesis can begin. Three protein factors (IF1, IF2, and IF3), a molecule of GTP, mRNA and fmet-tRNA are required for initiation. In this research, complexes that correspond to intermediates in the initiation pathway will be formed in vitro and probed with hydroxyl radicals generated by both free Fe(II)-EDTA and Fe(II)-EDTA tethered to unique sites on the initiation factors. The sites of protection from hydroxyl radicals and specific cleavages of the rRNA, mRNA, and tRNA will be identified by primer extension. Together, the results of these probing experiments should provide (1) a comprehensive mapping of the RNA environment of each of these factors, (2) sufficient protein-RNA interaction constraints to orient each of the factors with respect to the other components of the initiation complexes to be studied, and (3) insight into the mechanism by which the initiation factors prepare the ribosome to begin protein synthesis."}
{"text": "Brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) play a crucial role in neuronal plasticity and survival. Increasing evidence suggests that deficient BDNF-TrkB signaling may mediate amyloid- (?)-induced neurotoxicity, synaptic dysfunction and memory deficits in Alzheimer's disease (AD). However, it has not been examined whether BDNF-TrkB dysfunction may also contribute to pathogenic mechanisms upstream to A? accumulation. Interestingly, our recent study demonstrated that the selective TrkB agonist 7,8-dihydroxyflavone not only restores impaired memory and TrkB signaling but also lowers A? levels by reducing the ?-secretase enzyme BACE1 in a mouse model of AD. In this R03 application, we will test our focused hypothesis that reduction in TrkB signaling, which is found in early AD or its harbinger mild cognitive impairment (MCI), may be responsible for inducing BACE1 elevation and consequently initiating and/or accelerating disease processes. Specifically, we will use (i) pharmacological (ANA-12, a selective and blood-brain barrier permeable TrkB antagonist) and (ii) genetic (TrkB haploinsufficiency) approaches to experimentally reproduce the impairment of TrkB signaling associated with MCI or incipient AD. In Aim 1-1, these approaches will be applied to young 5XFAD transgenic mice, which develop little or only faint amyloid pathology and are still normal in memory function. We will first compare the levels of BACE1 expression in ANA-12-treated 5XFAD or TrkB+/-.5XFAD bigenic mice (3-month old) with those of age-matched 5XFAD control mice. We will then explore the molecular mechanisms by which pharmacological and genetic reduction of TrkB signaling may cause BACE1 elevation (e.g., changes in transcription, translation, protein stability) and exacerbate AD-like phenotypes including memory deficits in these 5XFAD mice. In Aim 1-2, we will further test whether administration of ANA-12 or TrkB haploinsufficiency may be sufficient to upregulate BACE1 in wild-type mice. Given that A?42 may be important for increasing BACE1 expression in neurons around amyloid plaques during the progression of AD, it will be critical to determine a causal relationship between reduced TrkB signaling and BACE1 elevation in wild- type controls that completely lack toxic human A? species. If the data is positive, we will examine whether the same mechanisms as found in 5XFAD mice may mediate BACE1 elevation following impaired TrkB signaling in wild-type mice, regardless of the presence of human A?. Together, the proposed research is expected to address a key question whether deficient BDNF-TrkB signaling may not only be a mediator of memory impairments as a consequence of A? accumulation (as shown by previous studies) but also have a crucial role in initiating or accelerating the AD process via BACE1 elevation."}
{"text": "This proposal for a Clinical and Translational Science Center at the University of New Mexico Health Sciences Center (HSC) will catalyze the emergence of a transformative, novel, and integrated academic home for clinical and translational science at a flagship institution that serves the entire state of New Mexico and the Southwestern region ofthe U.S. This center will have the infrastructure and consolidated resources to: 1) synergize multi- and interdisciplinary clinical and translational research and researchers to catalyze the application of new knowledge and techniques to clinical practice at the front lines of patient care;2) recruit, train, and advance well-trained interdisciplinary investigators and research teams with strength in cultural sensitivity, health disparity, and biotechnology;3) create an incubator for innovative research and information technologies;and 4) expand existing partnerships between UNM Health Sciences Center (HSC) researchers, practicing clinicians, and communities to speed the development of research. Our vision of the CTSC links and focuses the efforts of our basic, clinical, and translational investigators, community clinicians, clinical practices, health care and research collaborators, and industry partners. Since New Mexico has a high proportion of ethnically diverse (i.e., Hispanic and Native American), rural, medically disenfranchised, and health-disparate populations, a CTSC in New Mexico can uniquely address these problems. The UNM CTSC will draw upon outstanding institutional commitment (at least $57,802,262 ) to achieve its goals. The UNM CTSC possesses authority over the recruiting, hiring, protected time, expectations, promotion and tenure, and evaluation of its faculty members. Evidence of the commitment and effectiveness of the UNM CTSC in these pursuits are apparent from the following achievements made during the planning process: (1) reorganization of the UNM HSC research mission under a new Vice President for Translational Research with authority over research education, compliance and research functions, (2) establishment of an institutionally-funded K12-like program, and recruitment of 5 junior faculty, (3) development of innovative new research education programs, (4) operation of a functionally expanded Participant and Clinical Interactive Resources component that is serving the needs of several community-based studies, including the landmark, new National Children's Study, (5) the redesign of our institutional approach to Biomedical Informatics and development of a regional clinical data warehouse that will be available to investigators using a $5.8 M investment, and (6) construction and renovation of new buildings for the CTSC. Additionally, UNM's commitment includes $14.5 M in new institutional funds committed to CTSC programs, and $8.5 M in matching funds to construct new space to create a physical home for the CTSC. Funding of this proposal will assure sustainability and growth of these transformations that is critically needed to support clinical and translational research at UNM. RELEVANCE (See instructions): The UNM Clinical and Translation Science Center will rapidly escalate transformative scientific discovery into improved human health outcome."}
{"text": "Ubiquitin (Ub) is an essential signal molecule regulating protein degradation, localization and other activities. Generally the C-terminus of the Ub molecule is covalently conjugated to lysine residues of protein substrates by the E1/E2/E3 reaction cascade, and the modification is reversed by the action of deubiquitinating enzymes (DUBs). PolyUb chains can be further assembled on the substrates by the linkage between lysine groups of the first Ub molecule and the C-terminus of the next. Our current proteomic analysis revealed that all seven lysine groups on the Ub molecule can be utilized for the formation of polyUb chains, including Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63. We propose that the formation of functionally distinct polyUb chains is a regulatory step for ubiquitination and deubiquitination. To examine the function of these polyUb linkages, we will first develop a mass spectrometry technology for quantifying the abundance of all seven polyUb linkages, and then study the catalytic specificity between DUBs and polyUb chain topology. Moreover, we will focus on defining the function of Lys11 polyUb chains. Our studies will lead to the development of general proteomic tools for polyUb chain topology and better understanding of the diversity and function of polyUb chains."}
{"text": "Hot flushes during the perimenopausal and post-menopausal periods pose a significant public health concern because they (i) are the primary reason that women seek medical care during the menopausal transition;(ii) often negatively impact the quality of life for women because they are associated with depression, sleep disturbances resulting in fatigue, irritability, and forgetfulness, as well as acute physical discomfort and negative effects on work;and (iii) are thought to be indicative of some impending, serious conditions. Despite the importance of hot flushes in a woman's life, little is known about the underlying mechanism or the risk factors for hot flushes. Although the most efficacious therapy for hot flushes is estrogen replacement therapy, due to the potential risks involved with hormone therapy, many women try to find alternative therapies, such as the selective serotonin and/or serotonin-norepinephrine reuptake inhibitors (SSRIs/SNRIs), clonidine, and gabapentine in spite of their lower efficacy compared to estrogen replacement therapy. Clinical and preclinical data indeed show that these neurotransmitters play critical roles in thermoregulation by changing the sensitivity of the thermostat and subsequently, affecting hot flushes. Although a subpopulation of perimenopausal women do respond to SSRIs/NRIs, the response is heterogenous (some individuals respond well some do not respond at all). The overall goal of this application is to explore why some women with hot flushes do not respond to SSRIs/SNRIs. Our hypothesis is that the heterogenouos response is due to single nucleotide polymorphisms of the serotonin transporter (SERT) and/or the norepinephrine transporter (NET) genes. Therefore, we propose the following specific aims: (1) Genotype a comprehensive set of SNPs within the SERT and NET genes in a sample of 800 perimenopausal women recruited during a previous funding period and 58% of whom experienced hot flushes and genotype these genes in a sample of 200 women being recruited and (2) Determine whether these SNPs are correlated with hot flushes. This proposal matches the R21 funding requirements in that it explores, for the first time, the relationship between SNPs of SERT and NET genes as predictors of hot flush relief. If the heterogeneous response to SSRI/SNRI therapies is due to these SNPs, then a customized, genotype-based therapy may be the ultimate solution to increase efficacy of this approach to treating bothersome hot flushes without hormones. PUBLIC HEALTH RELEVANCE: Although hot flushes during the perimenopausal period pose a significant public health concern. Little is known about the underlying mechanism of hot flushes. Many women try to find alternative therapies, to estrogen such as the selective serotonin and/or serotonin-norepinephrine reuptake inhibitors (SSRIs/SNRIs). However, the response to these is heterogenous and might be due to individual gene variations. This proposal will identify these variations and their relationship to hot flushes."}
{"text": "A limited number of properties separate bacterial pathogens from non-pathogenic species. One such property exhibited by many pathogens is the ability to enter and replicate within eukaryotic cells. Francisella tularensis (FT) is a Gram negative bacterial pathogen that invades and replicates within a myriad of host cell types including, but not limited to, macrophages, dendritic cells and alveolar epithelial cells. The focu of this project is to understand mechanisms by which pathogenic Francisella adapt to, and manipulate the host cell environment and the implications that these properties have on bacterial virulence. In the previous funding period we investigated the roles of specific proteins in FT intracellular growth. During these studies we found that a robust ATG5- independent autophagic response was mounted in FT Schu S4 infected cells and that the bacteria assimilated amino acids derived through autophagic degradation of host proteins. Autophagy is also an anti-microbial response against cytosolic pathogens. However, even chemically induced autophagy did nothing to control FT intracellular growth. Thus, FT Schu S4 simultaneously evades degradation via autophagy while scavenging nutrients that are produced by this process. In this continuing application we propose to determine the function of the pathogenicity island encoded protein PdpC in protecting FT from autophagy and to define the bacterial metabolic processes that make it possible for virulent FT strains to replicate within monocytes."}
{"text": "Phosphatidylinositol-4,5-bisphosphate (PIP2) modulates the actin cytoskeleton, cell migration, invasion and signaling by binding effectors that regulate cytoskeletal dynamics and receptor trafficking. Yet, the mechanism of how PIP2 is generated to regulate these processes is poorly understood. Phosphatidylinositol phosphate kinases (PIPKs) that synthesize PIP2 specifically associate with PIP2 effectors. PIP2 then modulates the proteins and enzymes associated with the PIPK. PIPKI? regulates growth factor stimulated directional cell migration, vesicular trafficking of adhesion receptors and invasion. Here, we show that PIPKI? and PIP2 are required for establishment of cell polarity required for directional cell migration and invasion. In addition, we have discovered a novel signaling nexus that may explain how PIP2 mechanistically regulates many key effectors in adhesion, migration and invasion. Hypothesis: PIPKI? controls key aspects of tumor cell migration and invasion by controlling the assembly of cytoskeletal components and the trafficking of adhesion receptors. Interactions between PIPKI?, the exocyst and IQGAP1 function to integrate and specify the PIP2 generation and signaling required for growth factor stimulated directional cell migration, adhesion and invasion. In vivo the PIPKIg signaling nexus plays a key role in tumor cell intravasation and extravasation, processes required for tumor cell metastasis. Aim 1. The role of PIPKI?i2, the exocyst, and ?1-integrin trafficking in directional cell migration will be investigated. Regulation of integrin trafficking by PIPKI? and the exocyst will be defined with an emphasis on regulation by PIP2, the protein-protein interactions and cell polarity pathways, roles of small G-proteins, and specificity toward integrins. We will determine if a PIPKI?i2 interaction with talin acts as a tether for targeting the PIPKI?i2/?1-integrin/exocyst complex during directional cell migration. Aim 2. Define the mechanism for PIPKI? and IQGAP1 regulation of directional migration. We will study the IQGAP1/PIPKI? interaction and define how signals modulate this nexus. We will reveal the mechanism for PIPKI? control of IQGAP1 association with membrane. We will determine if and how PIPKI? modulates the association or activity of known IQGAP1 interactors, such as small G-proteins and the exocyst. Aim 3. Define the role of the PIPKI?, IQGAP1 and exocyst in cell invasion. The role of PIPKI?, the exocyst and IQGAP1 in formation of invadopodia will be revealed. An emphasis will be on targeting of key proteins required for invasion, such as IQGAP1, the exocyst, ?1-integrin and MMPs to the invadopodia in a 3D matrix environment. Aim 4. Determine if PIPKI?, the exocyst and IQGAP1 are required for extravasation and metastasis. Highly metastatic human breast cell lines will be engineered to over or under express PIPKI?, IQGAP1 or Exo70. We will use a well-established mouse xenograft model to transplant engineered cells into the vasculature. The development and number of metastases will be used to quantify extravasation."}
{"text": "This proposal addresses itself to the study of brain mechanisms involved in visual pattern recognition and perception. We have discovered that it is possible to actually control the development of functional properties of single cells in the visual cortex of kittens by the simple device of controlling their visual experience. We have demonstrated that the \"shape\" of receptive fields in the visual cortex of controlled experience kittens is directly related to the shape of the visual stimuli they viewed during development. For some units the receptive field shape is a recognizable, though blurred, image of the stimulus! Recently, we have also shown that simple visuo-motor training, during development, powerfully affects somatosensory cortex cells and their polisensory properties in ways which, again, are very directly related to the nature of the experience. These findings form the bases for the proposed experiments. We intend to raise kittens in environments designed to engage specific aspects of visual perception such as texture, line detection and simple recognition tasks. We expect that the kittens will have cells whose functional characteristics will reflect specific aspects or features of the experience thus enabling us to relate function with mechanisms directly. We will continue to try and separate neuronal properties which are inborn from those that are experientially determined. Finally, with the help of computer and neuroanatomical methods we will increase the functional connectivity and trace the structure of networks that have been engendered by controlled experience."}
{"text": "The proposed studies constitute part of a larger effort to understand how the contraction of smooth muscle is regulated at the level of contractile proteins, actin and myosin. The focus of this proposal is to elucidate the mechanism by which the putative regulatory protein calponin interacts with the actin filament to modulate contraction. In particular, thee studies will attempt to determine if calponin regulates a well described state of smooth muscle contraction called a \"latch- state\". The latch-state allows smooth muscles to remain contracted for long periods with relatively low expenditure of chemical energy. The high-economy of smooth muscle contraction is essential for normal physiologic function. In spite of the central importance of this contractile state for normal function of smooth muscle, the molecular basis for the regulation of the latch-state is unknown. Our central hypothesis is that calponin slows the rate of cross-bridge dissociation from actin, and this leads to activation of unphosphorylated cross bridges via a thin filament-linked mechanism. To test this hypothesis we will measure 1) actin filament sliding velocity, 2) changes in the level of force exerted on regulated actin filaments by a field of immobilized myosin molecules, 3) the force, displacement (step size), and attachment time for single myosin molecules interact with single actin filaments, and 4) the rate of myosin dissociation from actin using stopped-flow techniques. These measurements will provide insights into the physiologic parameters of isometric force and unloaded shortening velocity that characterize the contractile state of intact smooth muscles. These assays, in conjunction with recent x-ray diffraction data and high resolution electron microscopic images of actin myosin, tropomyosin, and calponin allow us to formulate and test specific molecular models for how calponin might interact with actin, tropomyosin, and/or myosin. The proposed studies will begin to address the issue of how calponin might interact with actin, tropomyosin, and/or myosin. The proposed studies will begin to address the issue of how calponin-mediated regulation interacts with the now well established myosin phosphorylation regulatory system. A major goal of the proposed studies will be to elucidate the role of calponin in thin-filament linked regulation of unphosphorylated myosin (i.e. the latch-state)."}
{"text": "Treatment of PANC-1 cells with the CB1R agonist Win55,212-2 resulted in a time-dependent decrease in ligand-induced phosphorylation of the EGF receptor and downstream signaling. In contrast, inhibition of CB1R signaling by AM251 led to a significant increase in the expression of EGF ligands (heregulin and betacellulin) and EGF receptor (ErbB1). These results were established by quantitative PCR, Western immunoblotting and EGF/PDGF signaling PCR array. There was no increase in IGF-1 receptor expression by AM251, and inhibition of CB2R with AM630 did not affect expression of the EGF receptor or its ligands, indicating the specific nature of AM251 action in these cells. Flow cytometry analyses and selective labeling of cell surface proteins with biotin probe indicated an increase in EGF receptor at the plasma membrane of AM251-treated PANC-1 cells, which coincided with higher levels of EGF-dependent phosphorylation of EGF receptor and its downstream mediator, Akt, when compared to EGF alone. In addition to the potentiation in EGF signaling, AM251 elicited distinct cellular morphological changes, with the cells changing from a cuboidal shape to a rounded cell with elongated projections. The loss of E-cadherin in response to AM251 suggests that these cells may be undergoing an epithelial to mesenchymal (EMT) transition, a hallmark of invasiveness. Indeed, AM251 enhanced significantly the invasive potential of PANC-1 cells as assessed by Matrigel invasion chambers. Work is underway to systematically examine the signaling pathways and activities of multiple transcription factors that directly control the extent of gene expression and function of the EGF receptor and its ligands in response to AM251. These studies will be expanded to include the use of cell lines from other cancer cell types (e.g., colon, breast, ovaries) and tumor xenograft models in mice."}
{"text": "The bulk of information that has contributed to our understanding of mechanism of progesterone action originates from avian models. During recent years rabbit uteroglobin has been successfully utilized as a model to exploit progestin action in mammalian tissues, too. Previous studies in our laboratory have yielded many interesting features in the regulation of uteroglobin gene activity and its relation to progesterone receptor dynamics, such as inhibition (and potentiation) of progesterone action by simultaneously administered estradiol or tamoxifen; progesterone receptor consumption during progesterone action; multihormonal control of uteroglobin synthesis, and dissociation of receptor changes from the biologic response. The present proposal is a continuation of these studies and utilizes uteroglobin and its mRNA (measured by RIA and cDNA hybridization, respectively) as biological markers for progestin action. To ensure that the regulatory events studied are not peculiar to uteroglobin, other progesterone-induced uterine proteins are concomitantly evaluated. Since uteroglobin is under multihormonal control--progestins, androgens, and estrogens regulate uteroglobin synthesis--this model offers an excellent system for studies of steroidal interactions in the regulation of a specific endometrial gene product. One of the major specific aims is to clarify the mechanism of antiprogesterone action of estradiol in the uterus. Steroid action is mediated via cytosol receptor proteins, but the extent to which biological responsiveness, maintenance and termination of steroid action are related to receptor dynamics are poorly understood. In the case of progesterone, receptor consumption (disappearance of steroid binding site) during hormone action still complicates the above relationships. The second specific aim of this proposal is devoted to clarification of these issues with a special emphasis on progesterone receptor consumption its biological significance. It may well be that an ultimate answer cannot be achieved utilizing current receptor assay techniques based on the use of labeled steroids, and hence progesterone receptors will be purified, characterized, and an immunologic assay for their measurement will eventually be set up."}
{"text": "The balanced growth of a normal cell or the development of a virus in its host cell involves a complex pattern of gene expression in which normal individual genes, or groups of genes, are activated or repressed at appropriate times during the growth cycle. Initial control over this process is effected at the first step of the genetic pathway: the transcription of DNA sequences into RNA. For both bacterial cells and bacteriophages specific transcribing enzymes have been isolated which carry out accurate and regulated reading of genetic sequences in vitro. Since this kind of seective transciption has not yet been observed in vitro for eukaryotic transcribing systems, these prokaryotic transcribing systems serve as essential model systems with which to sudy the factors that govern the regulation of gene expression. We propose to continue our studies on the mechanism of selective transciption--that is, the recognition and utilization of specific promoter and terminator signals on DNA by RNA polymerases. The studies will focus on the transcription of DNA from bacteriophage T7 that is carried out by E.coli polymerase and by T7 phage specific RNA polymerase."}
{"text": "Sun exposure has been consistently implicated in the development of non-melanoma skin cancers, but its role in melanoma etiology remains controversial, and is complicated by poorly understood genetic and behavioral factors. Melanoma incidence continues to increase (it is now the 4th most common cancer among males in California). Efforts to reduce melanoma incidence through sun exposure avoidance have had limited success, underscoring our lack of understanding of the true mechanisms of melanogenesis. Melanoma epidemiology is based on retrospective studies: recall bias probably explains associations between sunburn (almost always self-reported) and melanoma. Moreover, sun burn represents only excessive exposure to solar radiation, while sun-induced DNA damage potentially occurs across all levels of intensity: there is no accepted method for directly measuring sun exposure in epidemiological studies. In order to better understand the etiology of melanoma (particularly the role of sun exposure), valid methods of assessing both current and historical sun exposure are needed. While genetic factors probably play some role in melanogenesis, no study has yet managed to distinguish genetic from sun exposure-related risks. We will address both these issues in a case-control study of identical twins (nested in an established population-based cohort of 52,000 twins in California) assessing the role of sun exposure in melanoma etiology in the absence of genetic effects. Historical sun exposure will be assessed by combining residential history with novel fine-layer interpolated solar radiation level using GIS: we have already shown this method significantly reduces misclassification and recall bias. We will measure the actual impact of solar radiation on the skin, measuring UV- induced epidermal damage in tumor specimens noting differences in risk with varying UV intensity and wavelength. These results have the potential to improve primary prevention of melanoma and other skin cancers, and to direct the research team's future work on gene-environment interactions relevant to melanogenesis. We will measure the actual impact of solar radiation on the skin, measuring ultra-violet (UV)-induced epidermal damage in tumor specimens noting differences in risk with varying UV intensity and wavelength, and estimating the risk of melanoma with lifetime and age- specific exposures to specific wavelengths of UV. These results have the potential to improve primary prevention of melanoma and other skin cancers by indicating the appropriate means of avoiding harmful UV exposure, and to direct the research team's future work on gene-environment interactions relevant to melanogenesis. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]"}
{"text": "DESCRIPTION (provided by investigator): We request funds to establish a large-scale (up to 100 liters) bacterial culture facility in Memphis, TN, to be located at St. Jude Children's Research Hospital (St. Jude). This facility will be available to serve a broad community of scientists in Memphis, including those at St. Jude, The University of Tennessee Health Sciences Center (U. Tennessee), St. Jude's partner in graduate education, and The University of Memphis (U. Memphis), a partner in many collaborative research projects. The purpose of this facility is to enable the large-scale production of proteins and other biomolecules for structural, biochemical and drug discovery studies. The proposed Fermentation Facility will impact a wide variety of NIH-funded biomedical research projects at the three institutions noted above, ranging from structural investigations into basic biological function and structure-based drug design, to the production of small molecules as intermediates for the synthesis of new therapeutics and high-throughput small-molecule screening. Overall, the Fermentation Facility will contribute to advancing research funded by more than one dozen R01, P01, U01, or R37 applications distributed between the three institutions. The principal contribution to ongoing research will be to enable large-scale production of biomolecule samples, especially proteins. In some cases, large-scale protein production is already under way but is performed in a highly time inefficient manner. Access to the proposed fermentor will allow scientists to focus much more of their time on biochemical and structural studies rather than on protein production. In other cases, production of certain proteins is currently out of reach due to low expression levels. Studies in this category will be made possible through access to the 100-liter fermentor. An additional benefit associated with creating this facility is the promotion of intra- and inter-institutional collaboration. While many of the participants here already collaborate, use of a shared Fermentation Facility will foster interactions between groups, especially at the level of graduate students and postdoctoral trainees who will actually perform fermentation experiments."}
{"text": "PROJECT SUMMARY The human skin barrier does not always function as intended; in fact, defects in the outer layer of human skin account for a significant number of health problems for people living in the United States. As one example, it is estimated that up to 90 million Americans suffer from some form of atopic dermatitis. Atopic dermatitis and other forms of severely dry skin, such as ichthyosis vulgaris, are associated with defects or mutations in profilaggrin and its processed fragment, filaggrin. Mutations in keratin 1 and keratin 10 proteins also account for a variety of inherited skin disorders manifested by red, dry, scaly skin. Together, profilaggrin and keratins 1 and 10 are critical proteins involved in the stratum corneum of human skin, and elucidating the biochemistry behind their function is important for improving our understanding of how the human skin barrier works. The hope is that advancing our knowledge of how protein structure dictates function in the stratum corneum will generate new methods for treatment of human skin diseases. This proposal aims to address a deficiency in our understanding of the atomic resolution structure of key epidermal proteins, namely profilaggrin and keratins 1 and 10. In particular, it is still unclear (1) what the precise mechanisms are for profilaggrin binding to target proteins; (2) is there correlation between structure and function of the profilaggrin B domain; (3) does profilaggrin interaction with target proteins direct specific events in terminal epidermal differentiation; (4) what are the molecular mechanisms behind keratin intermediate filament aggregation; and (5) can enhanced structural knowledge of key skin barrier proteins pave the way for newly designed topical therapeutics. Given the direct association of profilaggrin and keratins 1 and 10 with multiple human diseases, we believe focusing our studies on these medically important proteins will help us address these outstanding questions. In this project, we examine the biochemical and structural properties of key proteins involved in a functional human skin barrier. Our first aim uses x-ray crystallography and biochemical analysis techniques to determine the structural basis of binding between profilaggrin and one of its targets, annexin II. The second aim examines the x-ray crystal structure of profilaggrin in the absence of bound molecules and performs mutational analysis on key inter-EF-hand linker residues in order to understand the functional role of this protein region. The third aim examines the structural basis for keratin intermediate filament interaction with different parts of the profilaggrin molecule using x-ray crystallography and biochemical techniques. Accomplishing these aims will provide novel insight into the principle biochemistry and atomic resolution structure of key epidermal proteins involved in maintaining the integrity of the human skin barrier."}
{"text": "The correct functioning of the endogenous circadian clock enables organisms to anticipate daily environmental changes and temporally modify behavioral and physiological functions appropriately. All organisms maintain a large number of physiological variables (sleep-wake cycle, locomotor activity, temperature regulation, water/food intake and levels of hormones) under control of the circadian clock. The biological clock readjusts itself by synchronizing to the daily light-dark cycle. Disruption of these rhythms has drastic effects on human health, leading to insomnia, depression, coronary diseases, various neurodegenerative or metabolic disorders and cancer. Recent advances have revealed unexpected links between circadian regulators, chromatin remodeling and cellular metabolism. The HDAC activity of the NAD+dependent SIRT1 enzyme is regulated in a circadian manner and SIRT1 transduces signals originated by cellular metabolites to the circadian clock. We aim to gain new insights into the regulation of chromatin transitions that govern the expression pattern of circadian genes, by understanding the biochemical aspects of the interaction between two chromatin remodelers, SIRT1 and MLL1, a histone methyltransferase. Our preliminary results convincingly indicate that MLL1 is acetylated and it is directly regulated by SIRT1, leading to a global control of circadian gene expression. We will unravel yet unexplored molecular mechanisms in which these two critical epigenetic regulators coordinate their function. This line of research is innovative as it links specific metabolic pathways to the epigenetic control of chromatin remodeling."}
{"text": "Modern techniques in Molecular Biology including cDNA arrays and automated sequencing have made possible the identification of genes expressed by a particular type of cell. The most common strategy to identify novel cell-type specific genes is to sequence cDNA clones obtained from the reverse transcription of mRNA using oligo dT and reverse transcriptase (RT). Due to limitations of the technique, these cDNAs often consist of partial clones of the 3' region of the mRNA. Since this 3' region often consists of non-coding sequences, insights into the function of the protein encoded by the mRNA are not possible. Techniques such as \"random priming\", optimization of mRNA preparation and improvement of the enzymatic reactions used in cDNA synthesis can increase the yield of full-length product. Even these optimized procedures result in many truncated cDNAs. Furthermore, these procedures require large amounts of mRNA starting material. This laboratory is interested in the developmental regulation of stem cells, a rare population of cells that play a critical role in organogenesis. Genes expressed by these cells are among the most likely to escape identification in the EST database efforts. In this proposal, we propose to develop novel in vitro and in vivo methods to make cDNA from such cell populations. The Specific aims of this project follow: Specific Aim No. 1. To make long cDNA libraries from rare cell populations. Specific Aim No. 2. To develop a method to use random priming to clone the 5' end of the mRNA obtained from rare cells. Specific Aim No. 3. To develop a method to make cDNA in vivo."}
{"text": "In recent decades, the United States has experienced an unabated rise in the number of individuals diagnosed with diabetes;as a result 23 million people are currently living with type 2 diabetes. The pathogenesis of type 2 diabetes involves a complex interplay between insulin resistance and failure of pancreatic [unreadable] cell compensation. [unreadable] cell compensation occurs through improved [unreadable] cell function and [unreadable] cell mass expansion, which are both regulated by the critical pancreatic transcription factor PDXI. Preliminary data show that PDXI deficiency increases [unreadable] cell apoptosis during diet induced insulin resistance by increasing susceptibility to endoplasmic reticulum (ER) stress (M. Sachdeva, unpublished data, 2008). The ER of a [unreadable] cell processes one million insulin molecules per minute, and ER stress has been associated with type 2 diabetes in mice and humans. High throughput gene expression and promoter occupancy analyses suggest that PDXI regulates several genes involved in maintaining ER homeostasis, including EROlip and GRP170. PDXI regulation of EROlip and GRP170 is hypothesized to be important for insulin biosynthesis and [unreadable] cell survival. This hypothesis will be tested in the following specific aims: I. Determine the role of DX1 in insulin biosynthesis, II. Determine the role of PDXI in the regulation of ER homeostasis and p cell survival. Insulin secretion will be studied in mouse insulinoma cells with shRNA silenced EROlip and GRP170. The role of EROlip in insulin disulfide bond formation and the function of GRP170 as a [unreadable] cell chaperone will be analyzed. To determine the effects of EROlip and GRP170 deficiency on ER homeostasis, ER stress markers such as BiP and CHOP will be measured, and Annexin V and TUNEL staining will be used to quantify [unreadable] cell apoptosis. Overexpression of EROlip and GRP170 may partially rescue the PDXI deficiency phenotype suggesting that they are important mediators of the effects of PDXI on insulin secretion and [unreadable] cell survival. Relevance: Failure of [unreadable] cells to sufficiently secrete insulin and death of these insulin-producing cells results in diabetes. Studying the mechanism for PDXI regulation of insulin secretion and [unreadable] cell survival may lead to targets for treatment of type 2 diabetes."}
{"text": "Histone mRNA synthesis is being studied in a cell-free system derived from synchronized mouse myeloma cells. This system carries out many of the reactions of RNA processing as well as transcription. The histone mRNA in mouse cells is analyzed by its ability to hybridize with plasmid DNA coding for the sea urchin histone genes. Our evidence indicates that the primary transcript of the mouse histone genes is a large molecule containing sequences for more than on histone protein. This transcript is synthesized only in early S phase cells. Thus, the primary control is at the level of transcription."}
{"text": "Over the past decade the genetics of development and cancer have converged in the identification of signaling pathways that control embryo patterning and, when aberrantly regulated, lead to cancer. Typically, tumors form in tissues in which the pathways normally operate. Tumors arise due to oncogenic mutations in components of these pathways that lead to ligand-independent constitutive activity. A classic example is the canonical Wnt signaling pathway, an essential developmental pathway that is disrupted in several kinds of tumor. Mouse Wnt1 is a prototypical oncogene first identified when it was shown that activation of Wnt1 by integration of Mouse Mammary Tumor Virus led to cancer of the mammary gland in mice. Mutations in several components of the Wnt/beta-catenin pathway have identified both oncogenes and tumor suppressors in this pathway that lead, in particular, to colorectal cancer. Recently, we have shown that aberrant Wnt signaling may lead to cancers of the immune system, specifically T-cell lymphomas. A large-scale survival study has revealed that relatively late-onset lymphomas and thymomas arise in Tcf mutants older than 6 months of age, however when one copy of Wnt5a is removed, severe, early-onset lymphomas arise as early as 3 months of age. These results suggest that Wnt5a has tumor suppressor activity. Our current experiments are aimed at understanding the underlying mechanisms and suggest that Wnt5a normally antagonises the canonical pathway ie. Wnt/beta-catenin target genes such as the proto-oncogene c-myc. We have also initiated a microarray project to generate a transcriptional profile of tumors so that we can molecularly compare our lymphomas with clinically"}
{"text": "Methicillin-resistant S. aureus (MRSA) infections are a major cause of morbidity and mortality in veterans. MRSA is transmitted through direct or indirect contact in the healthcare setting. In acute care settings, we use Contact Precautions (health care workers [HCW] wear gowns and gloves for all patient contact) for patients colonized with MRSA to prevent transmission to other patients. Current Centers for Disease Control and Prevention (CDC) Isolation Guidelines suggest modifying Contact Precautions in long term care facilities (LTCF), but there is little evidence to guide how to modify them. The goal of this revised proposal is to determine the optimal modifications of Contact Precautions for LTCF in order reduce the risk of MRSA transmission and allow care in a home-like, patient-centered environment consistent the Community Living Centers Cultural Transformation. LTCFs provide multiple levels of care including rehabilitation, skilled nursing and maintenance care. In the PI's current VA Merit award, we demonstrated that MRSA transmission is four-fold higher in rehabilitation care than in other long term care. This suggests that the types of care delivered increase the risk of transmission. MRSA transmission to other residents is difficult to study. Recently we developed a novel surrogate measure of MRSA transmission, detection of MRSA on HCW gown and gloves during HCW-patient interaction. Using this new methodology, we will test two major hypotheses based on expert opinion recommendations from the CDC Isolation Guidelines. 1. Risk of MRSA transmission will vary by type of contact with the resident and each activity will have its own risk of transmission. Some activities such as those involving contact with secretions (e.g. draining wounds, ostomy care) will be of higher risk than others (e.g. vital signs, medication administration). 2. For any given type of contact, resident characteristics increases the risk of transmission (e.g. residents totally dependent upon healthcare personnel for healthcare and activities of daily living or residents whose secretions or drainage cannot be contained). Our aim is to estimate the frequency of and risk factors for MRSA transmission to protective gowns and gloves worn by HCW interacting with 400 MRSA colonized VA long term care residents in a multi-site observational study in VA Community Living Centers from four states and the District of Columbia."}
{"text": "This application's major objective is to test the efficacy of a potential microbicidal agent to prevent transmission of HIV to women. Such an effective agent is urgently needed to combat the predominant rise globally of new HIV infections in women. Based on promising preliminary results in vitro, the specific aim of this application is to test in vivo the efficacy of glycerol monolaurate (GML) to prevent intravaginal transmission of simian immunodeficiency virus (SIV) to rhesus macaques, a highly relevant nonhuman primate model of sexual mucosal transmission of HIV. Treated or control monkeys will be infected and biopsy and necropsy specimens will analyzed by immunohistochemical and in situ hybridization methods and viral loads in blood to assess the efficacy of GML in preventing transmission or altering viral replication in cervicovaginal and [unreadable] lymphatic tissues. [unreadable] [unreadable] [unreadable]"}
{"text": "Previously, we reported the alternative splicing of gc pre-mRNA as a new mechanism to downregulate surface gc protein expression and to produce a new bioactive molecule in the form of soluble gc proteins. Notably, we found that increased expression of sgc resulted in impaired generation and differentiation of thymic NKT cells, which critically depend on IL-15 signaling. Interestingly, generation of IL-2-dependent Foxp3 T regulatory cells or IL-7-dependent CD8SP thymocytes were not affected, indicating distinct sensitivity of different gc cytokine receptors to sgc proteins. Altogether, these results demonstrated a previously unappreciated role for sgc in downregulating surface gc expression and in dampening gc cytokine signaling in thymocytes. Realizing such impact of alternative splicing on cytokine receptor expression, we shifted our focus to other receptors of the gc cytokine family. Among others, we found soluble IL-7Ra proteins in serum of both human and mice suggesting a potential role for these protein products in controlling T cell immunity. In humans, soluble IL-7Ra has been previously described and it is known that they are produced by alternative splicing of the IL-7Ra pre-mRNA. In marked contrast, so far, soluble IL-7Ra proteins has not been reported in mice. In fact, there is no molecular evidence reported for an alternative IL-7Ra mRNA splice isoform in mice, so that soluble IL-7Ra has been considered not evolutionally conserved. However, here we found that mice do express soluble IL-7Ra. By establishing a sensitive ELISA assay to serum IL-7Ra proteins, we could detect soluble IL-7Ra proteins in serum of wildtype mice. In humans, soluble IL-7Ra is produced by alternative splicing that omits exon 6, which encodes the entire transmembrane region. However, we were unable to detect such alternative transcripts in mice, suggesting that the mechanisms to produce soluble IL-7Ra proteins differ between humans and mice. Cloning of IL-7Ra mRNA species from mouse T cells showed that alternative splicing of IL-7Ra pre-mRNA utilized a distinct mechanism from human T cells in that is use intron-retention, instead of exon exclusion, for alternative splicing. Thus, we identified the molecular basis of soluble IL-7Ra proteins in mice. Nonetheless, we do not exclude the possibility of membrane protein shedding to produce soluble IL-7Ra proteins, and we are also investigating other post-transcriptional mechanisms of IL-7Ra production. Whether soluble IL-7Ra proteins are involved in controlling T cell immunity and T cell differentiation is an important question that is currently under investigation. In parallel to these studies, we used fluorescent beads to quantify the absolute number of gc cytokine receptors on cell surface of T cell subsets with the aim to gain better understanding of the expression under steady state condition and during T cell activation/differentiation. We found that IL-7Ra proteins outnumbered gc molecules at a ratio of four to one on surface on resting naive CD4 T cells. On the other hand, other gc cytokine receptors such as IL-4Ra or IL-2Rb were expressed at significantly lower numbers than gc. These results indicated that the availability of gc proteins is limited for association with IL-7Ra, and consequently IL-7 signaling is curtailed on CD4 T cells. However, this is not the case of other gc cytokines, such as for IL-2, IL-15 and IL-4, where gc expression and availability are not limiting to initiate cytokine receptor signaling. To understand the quantitative effects of cytokine receptor availability and signaling, we have generated a series of new experimental models that include enforced expression of gc or IL-7Ra proteins on T cells. The downstream biological effects of altering gc availability is currently under investigation."}
{"text": "The purpose of this proposal is to develop an Implementation Science Resource Core to support the research efforts of several CHAART consortia, building upon existing infrastructure within these consortia. The current proposal would create the infrastructure to close the loop of the research cycle for NIAAA-funded research. ORCAAA (The Operations Research Collaboration for Alcohol Abuse and AIDS) would be a resource core using implementation science to inform decisions important to investigators, public health authorities, and clinician/patient dyads. In particular, ORCAAA will create 3 different PODS (Portals Of Decision Support) that will function as catalysts of collaboration, providing CHAART investigators with tailored decision support in order assure that research will be harnessed most effectively to improve population health. In particular the ORCAAA resource core will implement the following aims: Aim 1: Facilitate implementation of CHAART HIV/AIDS interventions targeting highest risk populations; Aim 2: Increase capacity for implementation science to impact CHAART study design; Aim 3: Increase capacity for evaluating effect of CHAART alcohol interventions on HIV progression"}
{"text": "ABSTRACT Therapeutic interventions that favorably impact the untoward natural history of heart failure (HF) either slow or reverse left ventricular (LV) remodeling. In some patients with HF with a reduced ejection fraction (HFrEF) reverse LV remodeling is associated with freedom from future clinical HF events (?myocardial recovery?), whereas in the great majority of patients the initial stabilization of LV structure/function is followed by progressive LV remodeling and untoward clinical outcomes (?myocardial remission?). Thus, although recovery of LV structure and function are associated with stabilization of the clinical course of HF, as well as reversal of many aspects of the HF phenotype, it is not associated with freedom from future HF events. Understanding the clinical and biological features of ?compensated? HF patients, who have normalized or partially normalized LV structure and function, but remain vulnerable to hemodynamic/neurohormonal stress, represents a major unmet need in the field of heart failure. The long term goal of this research initiative is to delineate the mechanisms responsible for the functional instability of hearts that have undergone reverse LV remodeling with recovery of LV function, and to develop new therapies that will address this unmet clinical need. To explore the biological basis for the clinical phenomenon of ?myocardial remission,? we have developed a clinically relevant pre-clinical model of ?reversible heart failure? that combines moderate aortic constriction (TAC) and distal LAD ligation (MI), which are the major comorbidities that cause HFrEF in industrialized nations. To ?reverse? the HF phenotype, the TAC + MI mice are hemodynamically unloaded by surgically removing the aortic constriction, resulting in the normalization of LV structure and function. Germane to the present proposal, when the de-banded TAC + MI mice (?HF-DB? mice) are exposed to a neurohormone stress, they develop increased LV hypertrophy and increased LV dysfunction, analogous to what is observed in HFrEF patients who develop functional instability following recovery of LV structure and function. Based on our preliminary observations that the autophagy-lysosome system is dysregulated during the development and recovery from HF, we prose to test the following three hypotheses: (1) autophagic flux is impaired during the development of HF and, although flux is relatively improved following hemodynamic unloading, flux remains ?inefficient? (Aim1); (2) autophagic flux is required for effective reverse LV remodeling (Aim 2); and (3) insufficient autophagic flux is responsible, at least in part, for the functional instability that develops in reverse LV remodeled hearts that are exposed to neurohormonal stress (Aim 3). Specific Aims 1-3 will provide definitive information with respect to the potential role of autophagy in the recovery of LV structure and LV function following hemodynamic unloading, as well as the functional instability of reverse LV remodeling in a pathophysiologically relevant model of reversible HF."}
{"text": "We plan to isolate and characterize as many enzymes of the pyrimidine-synthesis system of Crithidia fasciculata as is possible. We will use standard procedures of purification including ammonium sulfate precipitation, heat treatment, acid and alkali treatment, Sephadex and ion exchange chromatography and electrofocusing. All enzymes will be tested for inhibition by natural end products as well as metabolic analogs. Special attention will be given to those enzymes which differ in control from those of mammalian systems."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The SSRL BTP staff provides user hands-on training for the effective use of the SMB SAXS/D instrument. Users first receive a brief safety orientation specific to BL4-2 and updates on recent equipment changes. Users receive a short tutorial on Blu-ICE used for the majority of non-crystalline scattering and diffraction experiments. They also receive training on the use of MarParse, the data processing program for solution scattering developed recently by the BTP staff, as well as its GUI version MarParseDlg. Users are instructed on the basics in solution x-ray scattering data analysis, such as inspecting data for possible radiation-induced aggregation, subtracting the background, and calculating radii of gyration. The data processing program Fit2D, developed at the European Synchrotron Radiation Facility, as well as Primus from EMBL-Hamburg are also covered upon request. Introductory tutorials to the sophisticated solution scattering data interpretation software packages, developed by Dr. Svergun (EMBL-Hamburg), are covered. Users are advised on practical experimental aspects on experiment geometries, sample-handling equipment, and data collection strategies especially for minimizing radiation damage to help improve data quality. These are given to new user groups and have been found useful also for experienced users."}
{"text": "The specific molecular mechanisms and nuclear events involved in leading to the transcription of pathological growth factor and inflammatory genes under diabetic conditions are not fully resolved. Circulating monocytes in diabetic individuals would be continuously exposed to hyperglycemic conditions. Monocyte activation, adhesion, transmigration and foam cell formation are key events in the pathogenesis of atherosclerosis. There is evidence of increased leukocyte-endothelial interactions in diabetic animals and with monocytes from human diabetic subjects. However, the behavior of monocytes cultured under high glucose (HG) and diabetic conditions have not been well studied. Much less is known about in vivo transcription mechanisms leading to the regulation under diabetic conditions of inflammatory cytokine and chemokine genes such as tumor necrosis factor- alpha (TNF-alpha), monocyte chemoattractant protein-1 (MCP-1) and cyclooxygenase-2 (COX-2) that are implicated in monocyte activation and atherosclerosis. The hypothesis is that diabetic conditions lead to in vivo nuclear chromatin remodeling and key alterations in the nuclear transcriptome machinery. This induces the transcription of NF-kB-regulated inflammatory genes in monocyte/macrophages and leads to enhanced monocyte activation and adhesion. The Specific Aims are: 1. To determine whether HG conditions increase interactions between NF-kB p65 transcriptionally active subunit and key transcriptional coactivators with histone acetyl transferase activity in monocytes. 2. To examine in vivo nuclear transcription and novel chromatin remodeling mechanisms by which HG and advanced glycation end products (AGEs) lead to inflammatory gene transcription in monocytes. 3. To evaluate the functional relevance of NF-kB activation under HG and AGE treated conditions by adopting state-of-the-art RNA-interference techniques to silence NF-kB p65. The project is supported by our preliminary data that human monocytes under HG conditions, as well as monocytes from diabetic patients produce significant amounts of TNF-alpha and MCP-1 in an oxidant stress and NF-kB-dependent manner. Our new data also shows evidence of chromatin remodeling under HG conditions. This project adopts several innovative approaches including in vivo chromatin screening and gene silencing, and could lead to novel new therapies for diabetic complications. [unreadable] [unreadable]"}
{"text": "The histone genes of the sea urchin provide one of the best systems for the study of gene regulation in eukaryotic organisms. These genes represent as much as 0.3-0.5% of the sea urchin DNA as there are as many as 1000 copies of each histone gene in the genome. The genes are organized into a repeating unit of 6-7 kb, which contains the genes for the five histones interspersed with five spacer regions. The availability of gradient-enriched histone DNA as well as cloned histone DNA recombinants provides suitable nucleic acid hybridization probes to investigate both the nature of the genes and the presence of complementary transcripts. Our objectives are twofold: a) To learn more about the organization of the histone genes - the extent of length heterogeneity and where it exists, and the identity and organization of the genes coding for the sequence variants of H1, H2A, and H2B histones. b) To study the regulation of histone synthesis during development, including the basis of control of transcription of these genes. The first set of studies will employ the techniques of restriction enzyme mapping, Southern transfer hybridization, electron microscopic visualization of DNA heteroduplexes, and recombinant DNA techniques. The second objective will be pursued by the use of cell free translational systems and RNA-DNA hybridization to assay for content, rate of synthesis, and turnover times of each histone mRNA. An in vitro system will be used to assay the factors involved in the control of gene transcription. Our results should provide significant information on the maintenance, organization, evolution, and regulation of the histone genes."}
{"text": "Despite our understanding that consolidation of long-term memories (LTM) require new protein synthesis, the specific molecular substrates of translation are yet to be understood in detail. Recent studies have described abnormal protein synthesis in several neuropsychiatric disorders. Importantly, therapeutic potential of inhibition o translation in specific brain regions for treating Post-traumatic stress disorder (PTSD) have been described. Thus, better understanding of the molecular substrates of translation will help develop novel therapeutic treatments for disorders such as PTSD. The central hypothesis of this proposal derived from our own studies is that activation of translation of specific mRNAs in specific compartments of pre and postsynaptic neurons is required for establishing LTM. To test this hypothesis, we will identify molecular substrates of translation in specific neuronal compartments of pre and postsynaptic neurons by exploring the advantages of recently described tagging of polyribosomes (TRAP: Translating ribosome affinity purification) and well- characterized neural circuitry of gill-withdrawal reflex (GWR) of marine snail, Aplysia californica Specifically we will (1) isolate and characterize tagged polyribosomes immunoprecipitated from somatic and synaptic compartments of pre and post synaptic neurons of GWR that are exposed 5HT, a neurotransmitter important for learning in Aplysia and (2) characterize their function in memory storage by knockdown of specific candidates and electrophysiological analysis. We anticipate that these studies will provide the first ever description of molecular substrates of translation in specific pre and postsynaptic compartments of a neural circuitry and their temporal regulation during memory storage. Furthermore these studies will bring novel insights into dynamics of translation during LTM. These studies will have an important positive impact on our fundamental understanding of neural circuit function and memory storage and likely lead to identification of new targets for preventive and therapeutic interventions of memory disorders."}
{"text": "The cause of syphilis, Treponema pallidum, has never been cultivated in vitro. The inability to grow this spirochaete in large numbers free from tissue has prevented successful studies leading to the development of a useful vaccine, studies on the mechanism of disease production, and the solution of complex immunologic problems associated with this disease. We have shown that this organism is capable of obtaining energy from glycolytic pathways, terminal electron transport to 02 and coupled oxidative phosphorylation. We propose to obtain additional physiological information on its biosynthetic capabilities, transport problems, control mechanisms and lytic phenomena, with the aim of applying such information to the in vitro cultivation of this spirochaete."}
{"text": "Five patients with malignant melanomas (4 choroidal and l of the ciliary body) were treated with proton irradiation at the Harvard Cyclotron during the period from July l975 to January l976. The patients were given a tumor dose of 5000 to 6000 rads Cobalt 60 equivalent delivered in 5 fractions over a period of 8 to 9 days. All patients tolerated treatments quite well without adverse effects. Response of tumor to therapy could't be evaluated at completion of treatment since there was no evidence of any reaction at the tumor or the surrounding retina. No definite regression is yet observed in any patient but a change in the color of the tumor has been seen in the patient with the longest follow-up. No further growth of the tumor occurred in any patient. No complications were noted on the follow-up visits except minimal epilation (loss of few eyelashes) in one patient and a small erythema of the lid which disappeared gradually without any sequelae. An accurate method of aiming the proton beam within the eye has been developed. Four to five tantalum rings 2 millimeters in diameter are sutured to the sclera at the edges of the tumor, which are localized by indirect ophthalmoscopy and transillumination. The rings are used as markers for stereotactic radiography in order to align precisely the tumors with the proton beam. Monkeys irradiated two to three years ago have been followed with general eye examinations, color fundus pictures and fluorescein angiography. This should be continued for another two to three years in order to be sure that all delayed effects of irradiation have been observed. A new on-axis TV viewing system with a camera of higher sensitivity and resolution (greater than 500 TV lines at 4.5 ft. c) has been worked out and is expected to allow monitoring of eye movements continuously throughout all phases of preparation and treatment, eliminate parallax and improve monitoring at lower levels of illumination with reduced eye discomfort for the patient."}
{"text": "After decades of a protein-centric view of gene regulation, it has become clear that the control of gene expression by regulatory RNAs is equally important (1-2). New small and large noncoding RNA molecules continue to be discovered at a staggering rate in bacterial model organisms as well as in the transcriptomes of bacterial communities (3-5). Newly discovered structural and functional aspects of such RNAs have reached a degree of breadth that requires a meeting with a strong focus on bacterial RNA research to fully address the diversity of these new regulators of gene expression and bring together the scientists involved in these studies. Regulating with RNA in Bacteria will be the first conference dedicated to this topic and will be a premier forum for the presentation of cutting-edge advances and the latest perspectives in the areas of discovery, mechanisms and structure of bacterial riboregulators. Funds are requested to provide partial support for this meeting, which will be held on March 7-11, 2011, in Cancun, Mexico. The conference is sponsored by the American Society for Microbiology (ASM), which provides both management expertise and financial support. We have carefully chosen 31 invited speakers with high international visibility, known affability and effectiveness to maximize the breadth and timeliness of presented work. Our program will cover prominent and emerging roles of RNAs in regulatory networks, and on current understanding of mechanisms of action for diverse RNAs. One session will be dedicated to other molecules that impact sRNA function, including ribonucleases, which are both friend and foe of regulatory RNAs, as well as RNA-binding proteins. Another session will focus on structural analyses of RNAs, which have lagged behind protein-related work. Systems and synthetic approaches to studying and using riboregulators, as well as emerging mechanisms of RNA-based immunity against invading genetic elements will complete the program. In addition to the invited scientists from around the world, the program will include 20 speakers selected from submitted abstracts to provide flexibility in programming to include cutting-edge advances and to promote high visibility of rising young investigators. The main goal of the meeting is to bring together researchers that use different approaches to study different aspects of RNA regulation in divergent bacterial systems, thus facilitating cross- fertilization of ideas among investigators, postdoctoral fellows and graduate students studying a variety of bacteria. PUBLIC HEALTH RELEVANCE: Bacteria constitute the largest class of infectious agents, causing devastating diseases in humans, animals and plants in both the developed and developing world. Our understanding of control of bacterial gene expression, which forms the basis for successful prevention and treatment strategies, until recently has neglected the many roles that RNA plays in pathogens as well as commensal organisms. Recognizing the high pace of discovery in RNA research and its great potential for therapeutic interventions, this conference will foster a better understanding of how regulatory RNAs might be exploited as new drug targets in order to treat and prevent disease at a particularly critical time when the pipeline of conventional antibiotics is waning."}
{"text": "Epidemiological and clinical studies have established an association between periodontitis and Type 2 Diabetes Mellitus (T2DM)/obesity. However, the causality and mechanisms linking periodontitis to T2DM are not known. We have established animal models of induced periodontitis to study the causality underlying this association. Using Zucker Diabetic Fatty (ZDF) rats, we demonstrated that periodontitis accelerates the development of insulin resistance (IR) and T2DM in high fat (HF), but not low fat fed animals. Since certain bacterial lipopolysaccharides (LPS) from periodontal lesions and free fatty acids (FFAs) from HF diet promote inflammation through Toll-like-receptor 4 (TLR4), and inflammation in general promotes insulin resistance, we have recently explored the role of TLR4 in mediating periodontitis-enhanced IR using mice with TLR4 loss-of-function (LOF) mutation and wild-type (WT) controls. Our results demonstrate that insulin signaling is impaired in the livers of WT TLR4 vs. TLR4-/- animals with periodontitis and furthermore, the plasma glucose level improves in TLR4-/- animals with periodontitis. Insulin functions to maintain whole body glucose levels in a narrow ideal range by balancing production of glucose by the liver and glucose uptake by skeletal muscle and adipose tissue. We therefore hypothesize that: 1) periodontitis affects insulin target organs (liver, muscle and adipose tissue) by suppressing insulin signaling via TLR4, leading to an alteration in whole body glucose homeostasis, 2) LPS triggers hepatic IR, 3) periodontitis/LPS affects insulin secretion via a TLR- dependent mechanism by influencing beta-cell mass and/or glucose-stimulated insulin secretion. To test these hypotheses, we will 1) determine the effects of periodontitis/LPS on insulin target organs via TLR2 & TLR4 by a) identifying specific sites of inhibition of insulin signaling, and b) determining the expression of key molecules involved in hepatic glucose production (G6Pase, PEPCK) as well as peripheral glucose uptake (AS160) by skeletal muscle and adipose tissue using whole body TLR4 and 2&4 knockout mice, 2) determine the pathways involved in periodontitis-induced hepatic IR and altered glucose homeostasis via TLRs using TLR2&4 adoptive bone marrow chimeric mice, 3) identify mechanisms by which periodontitis influences pancreatic beta-cells by modulating beta-cell compensatory alterations (insulin secretion, beta-cell hyperplasia & apoptosis) via TLRs. The impact of the proposed studies will: 1) advance our understanding of how periodontitis directly influences distant organs/tissues involved in glucose homeostasis, and 2) identify for the first time, specific TLR-mediated effects of periodontitis on insulin sensitivity and glucose homeostasis. The results will impact future treatment modalities, especially for subjects who are consuming a high fat diet where inflammation/LPS resulting from periodontitis may play a central role in IR and diabetes."}
{"text": "Currently, failure to persist after transfer into cancer patients is a factor that limits the effectiveness of adoptive cell transfer of T cells genetically modified to express a tumor-reactive T cell receptor. The goal of this application is to modify th human TCR TIL 1383I (melanoma-reactive) by appending the cytoplasmic signaling domains of DAP10 to the end of the cytoplasmic domains of the - and - chains of the TCR. We believe that the TCR/DAP10 will directly activate the DAP10 pathway upon TCR engagement, and that these unique signals will enhance the survival of transferred T cells. Preliminary data: We have demonstrated in CD8+ T cells that signaling through the [naturally expressed] activation receptor complex NKG2D enhances memory development. Our data show that NKG2D signaling in TCR-transduced CD8+ T cells augments their anti-tumor potency and in vivo persistence. These data argue that signaling through NKG2D in CD8+ T cells is a viable approach to overcoming the limited survival of TCR- Td T cells. We will couple TCR ligation and activation of downstream NKG2D signaling. Because NKG2D is unable to signal by itself, we will use the signaling domain of DAP10, the adaptor molecule that mediates signaling downstream from NKG2D in CD8+ T cells. We will modify the human TCR TIL 1383I (tyrosinase- reactive) by appending the cytoplasmic signaling domains of DAP10 to the end of the TCR cytoplasmic domains of the - and - chains. Hypothesis: We hypothesize that the TCR/DAP10 will directly activate the DAP10 stimulatory pathway upon TCR engagement, and that these unique signals will enhance the survival of adoptively transferred T cells. Strategy: The retroviral vector containing TCR/DAP10 construct will then be transduced into human T cells. Human T cells expressing TCR/DAP10 will be examined for their signaling pathways, capacity to survive and ability to mediate the regression of established human cancer using a humanized model of melanoma. Immune deficient A2/NSG mice and human melanoma cell lines will be used for anti-tumor experiments. We will test our hypothesis through the following specific aims SA1. Develop TCR/DAP10 constructs to induce and dissect DAP10 signaling. SA2. Study the signaling pathways utilized by TCR/DAP10 and their cellular consequences in human T cells. SA3. Test T cells expressing TCR/DAP10 against human melanoma. The significance and innovative character of our strategy would be that these engineered T cells will have specificity for tyrosinase, and engagement of their TCR will activate the DAP10 costimulatory pathway as well as downstream TCR signaling."}
{"text": "The core resources include the computer hardware and support personnel required to supply the computational needs of the component projects. The core will also provide the administrative personnel for the visiting scientist program for the program project and for the organization of meetings."}
{"text": "In the first 3 quarters of FY 2010 , the FACS Core Lab provided service to 24 NCI labs or branches and 2 other institutes. From these labs, there were 68 principal investigators using the FACS Core Facility. These labs, with the number of principal investigators and the number of individuals using the FACS Core in parentheses, are: CCBB (5 principal investigators,18 individual users), Dermatology Branch (1,1), Genetics Branch (3,11), HAMB (1,2), LBMB (3,5), LCB (1,3), LCBG (7,21), LCMB (6,15), LCO (3,8), LCP (1,1), LEC (2,10), LHC (3,15), LICB (3,14), LM (3,6), LMB (4,13), LMP (5,11), LRBGE (1,3), LTIB (2,2), MBTL (3,6), Medical Oncology Branch (3,9), Pediatric Oncology Branch (1,2), Radiation Oncology Branch (1,3), Surgery Branch (3,4), NCI-Frederick (1,1), NHLBI (1,1), and NHGRI (1,1). From these labs, 186 scientists have used the FACS Core in the first 9 months of FY 2010 and of these, 60 were new to the Core lab this past year and received training from the core staff. 163 people used the flow cytometers and sorting was provided by the core lab staff to 84 users. Identifying and studying cancer stem cells is one of the major research areas of the FACS Core Lab users. A number of NCI labs are using flow cytometry and fluorescence-activated cell sorting to identify and sort the cancer stem cell by membrane antigen expression using monoclonal antibodies or with a functional assay involving active membrane substrate transport. Investigators in CCBB, MBTL, LEC, LHC, and LCBG are studying cancer stem cells from breast, ovarian, hepatic, thyroid, pancreatic, and lung carcinomas. The LSRII flow cytometer, the FACS Vantage cell sorter, and the new special order FACS Aria, because each is equipped with a UV laser, are frequently used for these assays. Transfection of cells with genes expressing fluorescent reporters is a technique used by the majority of the labs using the FACS Core. The FACS Core cytometers and cell sorters have been equipped with specific lasers to allow detection and sorting of cells labeled with any of the green, yellow, blue, red, and UV fluorescent proteins or with combinations of these fluorescent reporters. Sorted transfected cells are used to prepare protein, DNA, and RNA that can be used in Western blotting and microarrays. Sorted cells are also used to determine effects of siRNA, to look at signaling proteins, or may be further passaged to create stable cell lines. Fluorescent reporter proteins may also be linked to luciferase. Tumor cell lines have then been sorted based on their expression of green or red fluorescent protein to establish cell lines with high levels of luciferase. These cells have then been used to establish tumors in mice and to image metastasis. In addition to the 3 co-authored publications, 12 additional publications this year have included work done in the FACS Core and, of these, 7 acknowledged the assistance of the FACS Core staff. These publications were authored by investigators in CCBB, LBMB, LCBG, LHC, LICB, LMB, and ROB."}
{"text": "The distant objectives of this study are the evaluations of thermodynamic characteristics associated with selected nonhelical and aberrant structures of DNA, some that may provide specificity for the attraction of enzymes for replication, transcription, etc. Quantitative appreciation of the forces that induce different modes of dynamic behavior in DNA should help explain subtle effects on gene expression of small changes in sequence in noncoding regions, as well as the evolution of DNA sequences. The near-term goal is to measure the energetic costs of interrupting the helix and maintaining nonhelical loop structures, including the shear forces needed to separate opposing chains over short lengths in internal stretches of helix. Initially it is intended that the loop forming specimen be short lengths of oligo(A degrees T) N, where 50 greater than N greater than 10 bp, sandwiched between stable boundaries of (G degrees C) 40-80 degrees. The specimen will be synthesized as a recombinant in the Pst I site of pBR322 and excised for the physical studies. Thermodynamic characteristics are to be extracted from observations of the helix-nonhelix equilibrium monitored by high resolution optical melting at several wavelengths simultaneously by the digital-difference approximation method. Later studies will take up the question of loop formation in domains of different base compositions and sequence features. In a correlated study the thermodynamics associated with each of the ten unique stacking interactions will be evaluated from the stabilities of specific, well characterized domains producing the multitransitional melting curve of pBR322. Nearest neighbor frequencies, to which the ten stability constants are related, will be assigned to specific subtransitions that reveal themselves by changes in melting temperature when the domains responsible for the subtransitions are cut by restriction enzymes. In a separate study, the precise amounts and level of sequence variation will be determined for the 1399bp satellite I and 46bp satellite II repetitive sequences from the bovine genome. Sequence variations in these satellites will be determined from the increase in the range of temperature of the subtransition for the population of repetitive sequences over that of single cloned specimens."}
{"text": "High fat diet and enduring obesity pose a more dangerous threat in lupus patients than in general population. Obese lupus patients show much higher inflammation markers and risk of heart attack. The link between high fat western diet and accelerated atherosclerosis is clinically well established. Recent studies also show evidence that healthy eating patterns enhance immune function and reduce inflammation in obese patients. The inability of a large population to eat healthy signifies the need to explore medical interventions for diet-induced cardiovascular diseases in lupus patients. However the molecular and cellular mechanism of how high fat diet dysregulates the immune system is still unclear and thus hindering efficient therapy. B1 B cells reside in the peritoneal cavity and omentum, in direct contact with accumulated and inflamed visceral adipose tissue. B1 B cells are the source of natural IgM antibodies that protect from atherosclerosis through masking oxidized LDL, inhibiting uptake by macrophage and preventing foam cell formation. Dietary effect on B1 B cells that may lead to accelerated atherosclerosis has never been studied. Based on our preliminary study, this proposal aims to test the hypothesis that high fat western diet and chronic obesity may over-stimulate and disarm B1 B cells. Even worse, it may transform protective B1 B cells into self-attacking B cells. The result of this study will reveal new molecular and cellular mechanisms underlying diet-induced arthrosclerosis and provide new avenues for future design of B cell-targeted immune therapy. INNOVATION: This is a novel study where B1 B cells are for the first time investigated as a link between diet-induced obesity and atherosclerosis. The novel concept that high-fat-diet-stimulated B1 B cells may alter the balance of inflammatory and regulatory T cells will be tested using a novel animal model. A newly developed assay will replace traditional flow cytometry-based analysis of B1 B cells that will revolutionize B1 B cell study. Short-term high fat western diet might activate B1 B cells to produce atheroprotective IgM, cytokines and promote Treg cell differentiation. However chronic western diet consumption and obesity would switch protective B1 cell HYPOTHESIS: functions to pro-inflammatory response leading to the aggravation of atherosclerosis. SPECIFIC AIMS: Aim #1: Determine the extent to which diet-induced obesity dysregulates B1 B cell functions~ Aim #2: Determine how B1 cells from obese gld/ApoE-/- mice may affect Treg vs. Th17 balance. IMPACT: We propose to investigate an important yet unexplored immunologic component that may be the key intermediary between diet-induced obesity and heart disease in lupus patients. The result of this study will unravel unique aspects of B1 B lymphocytes in the abdominal cavity and omental milky spots and will provide a novel mechanism, as well as a non-static view of B cell-mediated protection and pathogenesis. The knowledge obtained from this study will be fundamental for the future design of B cell subpopulation-tailored risk-assessment, prevention and therapeutic intervention at different disease stages. PUBLIC HEALTH RELEVANCE: Obesity poses a severe life threat in lupus patients by escalating inflammation and increasing the risk of heart diseases. This proposal aims to test the hypothesis that chronic consumption of high fat western diet and consequently, the deposition of abdominal visceral fat may over- stimulate B1 B cells in the abdominal cavity and as a result, lose their atheroprotective immunologic functions. The result of ths study will provide a rationale for future design of B cell- targeted immune therapy to reduce inflammatory heart diseases in lupus patients."}
{"text": "Broad, long-term objectives and career goals: To acquire the knowledge and skills needed for independent patient-oriented research concentrated on improving the diagnosis and management of pulmonary malignancy through imaging and image analysis techniques. Health relatedness: About 1.2 million cases of invasive cancer are diagnosed annually in the United States. About 500,000 Americans die annually from cancer, and bronchogenic cancer is the leading cause of cancer deaths. Early diagnosis of pulmonary malignancy, comprised primarily of bronchogenic cancer and metastatic disease, is important for early treatment and patient survival. Early primary lung cancer and metastatic disease to the lung parenchyma commonly manifest as indeterminate nodules, meaning nodules that are too small to be characterized as benign or malignant by other methods, on diagnostic and, more recently, screening computer tomography (CT). Other than calcification, morphologic characteristics have not been shown to differentiate benign from malignant nodules. Change in volume over time has been used as a marker of malignancy; however, for small nodules in particular, subtle volume changes are difficult to detect and quantify consistently using current techniques. Specific aims: The immediate career goal is to obtain knowledge and research skills through a structured career development program in a mentored research environment. This entails multidisciplinary didactic training in digital imaging, image segmentation, image analysis, epidemiology and mechanisms of tumorigenesis, and clinical study design, and conducting the proposed research in a mentored environment. The overall aim of the proposed research is to facilitate the CT diagnosis of malignant nodules by the development of precise quantitative techniques for the measurement of pulmonary nodule volumes. Our central hypothesis is that early nodule growth can be detected on CT using computer-assisted techniques and be correlated with biologic indicators of preneoplasia and neoplasia. The proposed method will enable early recognition of nodule growth and, therefore, the early diagnosis of malignancy."}
{"text": "Self-control may be vital when individuals cut back or cease drinking. To regulate their alcohol consumption, individuals must resist temptation and restrain their desire to drink. Resisting the temptation to drink should require self-control. Recent research suggests that the exertion of self-control is costly, as it requires and consumes a limited resource (self- control strength) that may be critical to the success of self-control. Individuals who previously exerted self-control may have less self-control strength and therefore perform more poorly on tests of self-control than individuals who did not exert self- control. Self-control strength may be particularly important when individuals are regulating their alcohol intake. Specific Aim 1 will help establish whether self-control strength is consumed when heavy drinkers resist the temptation to drink. After sniffing alcohol in a cue exposure paradigm, heavy drinkers should perform more poorly on subsequent tests of self-control as compared with their performance after sniffing water when contrasted with the performance of light drinkers. Negative mood, frustration, or arousal should not mediate the decline in self-control Performance after sniffing alcohol. In addition, resisting a temptation should not influence performance on a task that does not require self-control. Comparisons between light and heavy drinkers after sniffing alcohol and water will help establish whether self-control strength is required for, and consumed in the process of resisting the temptation to drink. Specific Aim 2 will explore whether proximal and distal risk factors related to self-control may influence the risk of violating alcohol consumption limits. Any factor that reduces self-control capacity may increase the likelihood of drinking limit violation. To test the relationship between limit violations and self-control demands, heavy drinking participants who were tested in the cue exposure paradigm will call an interactive voice response (IVR) system daily for 3 months to report on their drinking behavior, self-control demands, and self-control strength. Participants who experience an increase in self-control demands or a reduction in self-control strength may be more likely to violate their drinking limit. Survival and multilevel analyses will be used to determine whether excessive drinking is related to changes in day-to-day levels of self-control strength, effort exerted to resist the temptation to drink during experimental chase of the study, and individual differences in trait self-control. The present study has the potential to elucidate the self-control processes needed to enhance the maintenance of limits on alcohol intake."}
{"text": "Although a number of studies have been conducted on the efficacy of various medications in the treatment of alcoholism, opiate antagonist compounds appear to show the most promise. There is increasing evidence that the endogenous opiate system is involved in the experience of alcohol effects and in the maintenance of alcohol consumption. Several opiate antagonist drugs which have reported utility in the treatment of alcoholism have different opiate receptor binding characteristics. While both naltrexone and nalmefene are predominantly mu opiate antagonists, nalmefene binds to the delta receptor with 2 times the affinity of naltrexone. Given this difference and preclinical reports of grater potency of delta antagonism on alcohol consumption, nalmefene may have more pronounced affects on acute alcohol reactivity in humans. While prospective pharmacologic treatment trials in alcoholism are the only definitive method for evaluating the utility of medications for the treatment of this condition, these trials are costly, time consuming, and put patients at risk for adverse events. Laboratory paradigms which examine the effect of medications on acute alcohol reactivity and consumption in alcoholics may be able to identify compounds which would be the most efficacious to employ in treatment trials. The proposed study will be a randomized placebo controlled comparison of the ability of two opiate antagonist drugs, naltrexone and nalmefene, to alter the reactivity of alcoholics and social drinkers to an acute administration of alcohol and to consumption of alcohol in a \"free choice\" limited access paradigm. Non-treatment seeking individuals with alcohol abuse or dependence (N-135) and social drinkers (N=135) will be randomly assigned to take placebo, naltrexone, or nalmefene for 7 days. On the eighth day each individual will receive their study medication in a laboratory setting and will ingest a fixed dose of the beverage of their choice followed by a limited access alcohol consumption period. Reactions to alcohol presentation (anticipatory), consumption (pharmacologic), and free choice ingestion (cognitive control and craving), will be measured utilizing subjective, cognitive, physiologic and biologic assessments. Subjects will be paid for their participation and alcoholic individuals will undergo a brief motivational counseling session at athe end of the protocol to educate them about the risks of heavy alcohol consumption and motivate them to seek treatment."}
{"text": "Factors and processes influencing entry into alcohol treatment programs will be investigated among population-based samples of alcoholics and first admissions to alcoholism treatment agencies. The research will be guided by a comprehensive model of factors predicting entry into an alcohol treatment program. The model includes state-of-the-art conceptualization of biopsychological factors thought to influence the severity and chronicity of alcohol abuse and dependence; a comprehensive assessment of the severity and chronicity of alcohol problems, including blood and urine tests; informal pressure by family and friends and formal pressure by employers and the legal system to seek alcohol treatment; health beliefs; and stage of change in addictive behavior. In addition, factors associated with unassisted or natural recoveries will be investigated among population-based alcoholics. Population-based alcoholics will be recruited from two prospective alcohol epidemiologic studies and from the household sample employed as controls for cases of coronary heart disease and lung cancer in Research Components 2 and 3. It is estimated that 795 individuals having a lifetime DSM-III diagnosis of alcohol abuse/dependence will be available for study; a subset of 398 will have had symptoms in the past year. Samples will be available for study; a subset of 398 will have had symptoms in the past year. Samples will be merged and post-stratified on sex, race, education, and heavy drinking. A representative samples of Erie County residents entering alcohol treatment programs for the first time(N-616) will be recruited using data on number of previous admissions to alcohol treatment, county of residence, and key study factors. These data are available from the NYS Office of Substance Abuse Service Information System for clients seen in all 50 alcohol treatment agencies in Erie County, making the recruitment of this sample feasible and cost-efficient. Analyses will employ simple and multivariate logistic regression and structural equation modelling techniques, where appropriate. A better understanding of alcohol treatment seeking behavior and unassisted recovery is critical to the improvement of current efforts to intervene more effectively and efficiently at earlier stages of alcohol abuse and dependence."}
{"text": "Endometrial epithelial cells undergo dramatic remodeling during the peri-implantation period. Such changes include alterations in cell-cell adhesions, in cell-matrix interactions, modified apical-basal polarity, and, in some species, cell-cell fusion. We have documented the expression of an mRNA encoding a transmembrane protein rbMDC9, a member of the ADAMs gene family with potential cell binding, cell-matrix interactions and fusogenic properties. RbMDC9 expression is up-regulated in rabbit endometrium during hormonal preparation for implantation, and expression is further augmented by blastocysts. Preliminary evidence suggests a similar up-regulation in mouse and human uteri. We will test the hypothesis that MDC9 in rabbits serve as an integrin-binding adhesion molecule between epithelial cells and, in doing so, also functions in the redistribution of junction and cytoskeletal proteins. Furthermore, we hypothesize that ADAMs family proteins participate in cell-matrix interactions and in the fusions between adjacent epithelial cells and between trophoblast and epithelial cells during implantation, and in the ectopic attachment and invasion of endometrial tissue during endometriosis. Aim of the project will be 1) to determine if domain specific-targeting and post-translational processing regulate the function of epithelial cell rbMDC9 in peri-implantation-stage endometrium, 2) to determine if rbMDC9 ligand interactions are required for uterine epithelial junction or cytoskeletal protein modifications during implantation, 3) to define the function of rbMDC9 in implantation-specific cell-cell adhesion and/or fusion processes, and determine whether its expression of processing are regulated by blastocysts in vitro, and 4) to determine if MDC9 is expressed in a cycle-specific pattern in endometrium of women with and without endometriosis, and to analyze its regulation and potential roles in the adhesion and invasion of ectopic endometrium in an experimental model of endometriosis."}
{"text": "(This application was written in response to the NIH Announcement for the Availability of Recovery Act Funds for Competitive Revision Applications (NOT-OD-09-058). The experiments outlined in this application were designed to expand the scope of the specific aims, research design, and methods of the parent grant (RO1 EB007357) entitled \"Image-guided Hydrodynamic Gene Delivery\". The objective of the parent grant is to develop an image-guided hydrodynamic gene delivery system for site-specific gene delivery to the liver. Since the start of the project last year, we have established the procedure using pigs as an animal model and demonstrated that the image-guided hydrodynamic gene delivery is highly effective and safe for liver gene delivery. In this competitive revision application we propose to expand the study by including the non-human primate as an additional animal model for further development. We plan to systematically assess the effectiveness of the image-guided hydrodynamic gene delivery in baboons in order to establish the hydrodynamic parameters that can be used for development of a computer program for gene delivery in humans. The specific aims are: (1) To study the effect of selected injection pressures and volumes of DNA solution on gene delivery efficiency to baboon liver, and (2) to assess the persistency of transgene expression and the long-term effect of hydrodynamic procedure on the transfected animals. In compliance to Acceleration of the Tempo of Scientific Research and/or Allow for Job Creation and Retention, We are going to hire two full time employees for the proposed study. In addition, the use of animals will also create job for those who are animal caring professionals. The reagents used in the proposed would also help in creating or retaining people who work in research reagent companies."}
{"text": "SUMMARY Glial cells play an essential role in defending brain health and managing neuronal stress and damage. Neurodegeneration triggers robust glial immune responses, including changes in cytoskeletal dynamics, glial cell migration, and increased phagocytic activity. Timely removal and degradation of degenerating axons and neuronal debris by glia confers neuroprotection in the brain. Despite the importance of glial responses to axon injury, we still know surprisingly little about how damaged neurons invoke immune reactions in glial cells. What signals are released from degenerating neurons? What prompts the release of these injury cues? Finally, how are these signals translated by glia to carry out efficient immune responses to damage? We are using the fruit fly Drosophila melanogaster as a tractable model to investigate the immune communication relays that exist between neurons and glial cells in vivo. The fly nervous system contains distinct glial subtypes that are molecularly and functionally similar to vertebrate glia. Moreover, well-established axotomy assays in the adult olfactory system and the adult wing reveal that Drosophila axons undergo a classic Wallerian degeneration (WD) program, which includes increased intra-axonal calcium waves, axon fragmentation, and subsequent clearance by phagocytic glia. Notably, our lab has recently shown that axon degeneration triggers activation of the insulin-like signaling (ILS) pathway in reactive ensheathing glia, which, in turn, elicits essential glial immune responses, including transcriptional upregulation of immune genes (e.g. the engulfment receptor Draper) and phagocytic activity. We hypothesize that neuropeptide-containing dense core vesicles (DCVs) are broadly released from severed axons to trigger immune responses in local glial cells. Here, we propose to use static and live confocal imaging, transcriptional profiling, and newly developed in vivo reporters to investigate how neuropeptide signaling between neurons and glia informs glial immune responses to nerve injury. Specifically, we will 1) monitor DCV dynamics and release in adult severed nerves, 2) utilize novel single transcript labeling methods to visualize local translation of immune mRNA transcripts in glial extensions at sites of injury, and 3) determine how neuropeptide signaling between discrete glial subtypes ensures that glial responses to degenerating axons are properly carried out. Together, these findings will offer exciting molecular and cellular insight into how neuropeptide signaling between neurons and glia govern immune responses in both acute and chronic degenerative conditions."}
{"text": "Pulmonary arterial hypertension (PAH) in its familial form (FPAH) is a heritable autosomal dominant disorder that, in the majority of patients, results in progressive right heart failure and death within five years of diagnosis. Bone morphogenetic protein receptor (BMPR) 2 haploinsufficiency occurs in over 70% of the FPAH patients, but intriguingly, only 20% of mutation carriers get clinical disease. This proposal aims to uncover the mechanism underlying the reduced penetrance of BMPR2 mutation in causing FPAH using patient-specific induced pluripotent stem cells derived endothelial cells (iPSC-ECs). Understanding the molecular mechanisms underlying the protective phenotype in those BMPR2 mutation carriers without FPAH could lead to novel therapeutic approaches for familial and non-familial forms of PAH as they all share reduced expression or function of BMPR2. These studies also hold the key to understanding penetrance in other genetic conditions related to PAH, such as the caveolin 1 (CAV1) mutation. Studies carried out during Dr. Gu's AHA postdoctoral fellowship utilized iPSC-ECs from three sets of FPAH patients and from their family members with the same BMPR2 mutation but without disease. Dr. Gu uncovered a compensatory p-p38 signaling pathway leading to preserved adhesion and survival of iPSC-ECs from the unaffected mutation carriers. The mechanism accounting for the preserved p-p38 signaling appears to differ among the families. The first aim (K99 phase) of Dr. Gu's proposed studies is to extend the findings described above, through gain and loss of function studies to pursue the mechanism of `carrier compensation'. She will also determine whether correction of the BMPR2 mutation by CRISPR/Cas9 technology restores BMP signaling pathways and EC functions in FPAH iPSC-EC from all three families. The second aim (K99 phase) will determine how the transcriptome and epigenome explain the protective phenotype in the unaffected mutation carriers. RNA-Seq was carried out on iPSC-ECs from all family members (n=11). Seventy-one differentially expressed genes were identified by comparing iPSC-ECs from controls and mutation carriers versus FPAH patients, and four genes of interest have been verified by qPCR. This aim is strengthened by a collaboration with Dr. Michael Snyder's lab, to help relate gene expression changes with alterations in histone marks identified by ChIP-Seq and polymorphisms called by whole genome sequencing. The third aim (R00 phase) will extend studies in Specific Aims 1 and 2 to novel mutations associated with FPAH, such as caveolin1. In the fourth aim (R00 phase), Dr. Gu will establish a high-throughput platform for personalized drug screening using iPSC-ECs as a continuous cell source. These studies will help Dr. Gu to launch a research program that optimizes the use of iPSC-derived vascular cells and integrative `omic' technologies to model vascular disease pathophysiology and to develop personalized treatments using either a bioinformatics' approach or high throughput screening to repurpose FDA approved drugs that activate the protective pathway."}
{"text": "Many geriatric patients are being treated for extended periods of time with neuroleptic and antidepressant drugs. The degree and type of therapeutic and side-effects of these drugs may be different in the elderly than in younger patients. Recent evidence suggests that many of the therapeutic and side-effects of these psychotropic drugs may be mediated by their interaction with neurotransmitter receptors in the brain. We are proposing to study the effects of older age on the changes in receptor function in the brain induced by chronic administration of neuroleptic and antidepressant drugs. We will use an animal model to study their effects in the rat brain. Four different age groups of rats ranging from young adult to senescent ages, will be chronically administered a neuroleptic or an antidepressant drug. Radio-ligand binding techniques will be used to assay dopaminergic, adrenergic, and cholinergic receptors in several regions of the rat brain both during, and after termination of chronic drug administration. Questions we will attempt to answer include: Do old-age rats develop quantitatively greater or lesser degrees of changes in receptor binding on these ligands during or after chronic drug treatment than younger animals? Is there a different pattern of changes in receptor binding in old rats than in young rats? The results of the proposed research may provide an animal and biochemical model to determine the differences in the therapeutic and toxic effects of neuroleptic and antidepressant drugs given to elderly patients; it may help provide an explanation for the much greater prevalence in the aged of one important side-effect of neuroleptic drugs, tardive dyskinesia."}
{"text": "ONE OF THE AIMS OF OUR LAB IS TO MAKE AN EQUIVALENT CIRCUIT MODEL OF THE MUDPUPPY, NECTURUS MACULUOSUS, GASTRIC EPITHELUM. MUCH OF OUR PRIOR WORK ON THE EQUIVALENT CIRCUIT HAS ALREADY BEEN COMPLETED (DEMAREST AND MACHEN, 1985). WE HAVE YET TO DETERMINE THE MAGNITUDE OF THE PARACELLULAR SHUNT. THE MUDPUPPY HAS SEVERAL ADVANTAGES FOR DOING ELECTROPHYSIOLOGICAL STUDIES, MAKING IT MUCH EASIER TO USE THAN THE STANDARD MAMMALIAN MODELS. THESE INCLUDE LARGE CELLS AND A GASTRIC MUCOSA THAT READILY LENDS ITSELF FOR USE IN A TRANSEPITHELIAL VOLTAGE CLAMP. THIS PROJECT IS CURRENTLY IN PROGRESS. THE ACID-SECRETING REGION OF THE STOMACH OF VERTEBRATES IS A COMPLEX EPITHELIUM, SINCE IT IS COMPOSED OF SURFACE EPITHELIAL CELLS AND GASTRIC GLANDS. IN THE GASTRIC GLANDS ARE CHIEF CELLS AND PARIETAL CELLS IN MAMMALS AND OXYNTIC CELLS IN OTHER VERTEBRATES. BECAUSE OF THE PRESENCE OF MULTIPLE CELL TYPES, THE STANDARD MACROSCOPIC TRANSEPITHELIAL VOLTAGE CLAMP TECHNIQUES CAN NOT BE USED TO DETERMINE THE SHUNT RESISTANCES OF THE DIFFERENT CELL TYPES. THE VIBRATING PROBE, HOWEVER, CAN BE SITUATED ABOVE OPENINGS OF THE GASTRIC GLANDS AND ABOVE THE SURFACE EPITHELIUM IN THE REGION OF EACH OF THESE CELL TYPES. USING PHARMACOLOGICAL TOOLS, THAT INCLUDE AMILORIDE, CIMETIDINE, AND ION REPLACEMENTS, WE ARE ABLE TO DISSECT THE CURRENTS AND RESISTANCES DUE TO THE PARACELLULAR AND TRANSCELLULAR PATHWAYS."}
{"text": "DESCRIPTION: This program for Ph.D. and postdoctoral students will train specialists who will be able to conduct preclinical research at levels ranging from the molecular to the behavioral on biological mechanisms underlying the development, maintenance, and elimination of alcoholism. Twenty members of the graduate faculty of the Oregon Health Sciences University will train postdoctoral research fellows and graduate students matriculating into programs in Behavioral Neuroscience or Neuroscience. Training will include firm curricular grounding in the basic sciences, specific training in the pharmacology of alcohol and other abused drugs, exposure to clinical and psychosocial aspects of human alcoholism, and extensive and continuous participation in basic research. The focus of our training opportunities is on biological processes involved in the etiology of problem drinking and alcoholism. Our general approach is interdisciplinary, emphasizing genetic, molecular, physiological, pharmacological and psychological/ behavioral processes. The research questions addressed by trainees fall into four general areas: (a) genetic bases for alcohol and drug responses, (b) learned and unlearned determinants of alcohol and drug reward, biological bases for self-administration of alcohol and drugs, and (d) stress and the biology of alcoholism and drug abuse. These areas reflect the research interests and expertise of a participating faculty using behavioral, systems-level, and cellular/molecular methods. Their shared biobehavioral perspective is consistent with growing evidence indicating that many forms of human alcoholism are best understood in terms of an interaction between genetic and environmental factors. Areas of faculty collaboration include: studies of genetic determinants of alcohol and drug responses; neuroendocrine and neuroactive steroid participation in alcohol's effects; studies of dopaminergic, gabaergic and glutaminergic systems involved in alcohol and drug effects; study of learned and unlearned determinants of responses to alcohol and other drugs, particularly their rewarding effects and self-administration; and studies of sensitivity, tolerance, and dependence/withdrawal phenomena for alcohol and all major classes of drugs of abuse."}
{"text": "Engagement of the T cell receptor (TCR) triggers a number of intracellular signaling events that lead to the differentiation and activation of T cells. Several studies have shown that SLP-76 plays a central role in T cell activation and T cell development, as mice deficient in SLP-76 lack mature T cells. SLP-76 has three functional domains: an acidic domain with three phosphorylatable tyrosines, a central proline-rich domain, and a C-terminal SH2 domain. Of these domains, mutation of the three N-terminal tyrosines results in the most profound defects in T cell development and function. It is hypothesized that the individual tyrosines of SLP-76 activate specific signaling cascades required for T cell development including thymocyte selection, and T cell differentiation and function. This hypothesis will be tested by 1) analyzing SLP-76 deficient Jurkat cells reconstituted with SLP-76 bearing mutations at one or multiple tyrosine phosphorylation sites, 2) analyzing SLP-76 knock-in mice expressing tyrosine mutations at particular tyrosine residues and 3) mating SLP-76 knock-in mice to T cell receptor transgenic mice to evaluate thymocyte selection and mechanisms of T cell tolerance. Understanding how T cells transmit signals to direct thymocyte development and mature T cell function is critical to understanding the mechanisms to drive several disease processes including autoimmune diseases such as arthritis. This work will be performed by Dr. Martha S. Jordan at the Abramson Institute at the University of Pennsylvania. Dr. Jordan's background in cellular immunology and current training in molecular immunology provides her with a distinct angle from which to approach questions of T cell development. This proposal will provide her with the tools and reagents required to study T cell function in vitro and in vivo, in normal and diseased states as an independent investigator at a research university. Full access to faculty and services provided by the University of Pennsylvania, especially those within the Abramson Institute, will ensure the success of this proposal. [unreadable] [unreadable] [unreadable]"}
{"text": "Core C: Data Management and Statistics Core Project Summary/Abstract The overarching goal of the Data Management and Statistics Core (DMSC) within the UCI ADRC is to provide data management support and statistical collaboration to all ADRC investigators at all phases of scientific projects. Data management support includes comprehensive data management and dissemination of data arising from the Uniform Data Set (UDS) and all ADRC projects. DMSC personnel are responsible for the development of National Alzheimer's Coordinating Center (NACC) approved collection forms, maintenance of the ADRC database, and the timely transfer of accurate data to the NACC. Strong statistical design and efficient data analyses are also crucial components to achieving the scientific goals set forth by the UCI ADRC. DMSC members work closely with ADRC investigators to refine scientific hypotheses and develop analytic plans that are both appropriate and efficient for meeting the scientific needs of each investigator. DMSC statisticians are key collaborators in conducting studies and interpreting and reporting results. While a large portion of effort from DMSC faculty is devoted to providing collaboration and service to ADRC investigators, the DMSC also emphasizes the development of independent research programs among members of the DMSC. These DMSC-specific research endeavors not only increase the intellectual contribution of the DMSC to the scientific community at large, but will also lead to improved methods for collecting, entering, and analyzing complex data in the study of AD as well as other neurodegenerative diseases."}
{"text": "The principle of ALARA (as low as reasonably achievable) has been developed to reduce the risk of induced malignancy from radiation exposure, In practice this requires a rigorous quality control process for clinical operations in radiology. Currently, a critical tool (a radiographic test phantom) needed for quality control is not commercially available for computed radiology and digital radiography. The application's long-term goal is to develop a new class of phantoms and objective system performance tools for use in digital radiographic systems. These devices will be used to address emerging needs in computed radiography and digital radiography. These needs include (1) quality control procedures for daily operations, (2) quality assurance methods to evaluate products arid perform acceptance testing, (3) quality assurance methods for optimizing system operating parameters, and (4) development tools for manufacturers to assess new hardware and software products. Because neonatal chest radiography is a technically challenging procedure, with profound radiation dose implications, a prototype neonatal chest phantom was successfully developed in Phase I and is the focal point for Phase II phantom development. The neonatal chest phantom will be developed to simulate the disease states of pneumothorax and hyaline membrane disease. An observer's ability to diagnose these two disease states will test system resolution and noise, respectively. An automated objective system performance tool will also be developed to measure quantifiable parameters, such as resolution (modulation transfer function), noise (noise power spectrum), and detector efficiency (detective quantum efficiency). Phase II has two Specific Aims: 1) Clinical Validation of the Neonatal Chest Phantom in Normal and Disease States; 2) Clinical Validation of the Objective System Performance Tool. Successful neonatal chest phantom design will be determined by the appearance and histogram analysis of images; the attenuation values of anatomic structures; and the diagnostic performances of radiologists for detection of disease states within the phantom. Successful objective system performance tool design will be determined by agreement with established methods of calculating quantitative parameters. The commercial applications are driven by the phantom's clinical significance, which is to assure optimal equipment performance and minimize x-ray exposure, thereby reducing the concomitant risk of induced malignancy to neonates."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The LTQ-Velos ion trap mass spectrometer has recently been modified to allow performing higher-energy collision-induced dissociation (HCD) within the first quadrupole (Q00) in the intermediate vacuum region of the instrument. The new instrument is called the LTQ-Velos-Pro. HCD is a collision-based fragmentation technique which uses electric potentials to drive ions through a collision gas, in this case air. This beam-like fragmentation technique produces fragmentation spectra that are qualitatively similar to fragmentation patterns observed in triple-quadrupole instruments. We investigate the utility of performing HCD on the LTQ-Velos Pro for proteomics. In particular, we compare HCD and resonant CID, using several different experimental configurations, comparing the methods in the same LC/MS run and between different runs. We also evaluate the utility of HCD in producing library spectra that are well-matched to triple-quadrupole data and are thus well-suited to providing a transition from discovery-based proteomics to a more targeted approach."}
{"text": "This application requests funds for travelling expenses of speakers and session chairmen participating in the 31st Gordon Research Conference on Cancer. The conference will be held at Colby-Sawyer College, New London, New Hampshire during the week of August 22-26, 1977. The pattern of the conference will follow the format utilized in the previous conference, which in general has been considered very successful. Admission will be limited to approximately 150 persons with participation of academic, clinical, industrial and governmental scientists. The topic of the conference will be \"Regulation from within and from without of the Tumor Cell Phenotype.\" The initial session will be devoted to the operation and definition of the tumor cell phenotype as delineated by in vitro and in vivo parameters, with later sessions exploring the various mechanisms and methods by which this phenotype can be controlled or reversed. The selection of the mechanisms to be discussed will obviously be limited to those which at present appear more promising. Whenever possible, in vitro approach will be correlated to in vivo examples or experimentation. It is conceivable that some of these methodologies could eventually be useful as adjunctives to other therapeutical approaches."}
{"text": "A murine model of sdisseminated histoplasmosis has been established in this laboratory to facilitate studies of immunoregulatory distrubances occurring in the disease. To foster these investigations, the proposal attempts to compare the agbsolute and relative numbers of the T cell subsets in the various lymphoid organs of normal and infected animals. Studies to determine whether these subsets retain their normal function during the course of infection will also be undertaken. Attempts will be made to clone a T cell capable of modulating the response to Histoplasma antigens in vitro and in vivo. The surface phenotype of such a cloned cell will be determined. The possible production of soluble factors by these cloned cells will be studied. Finally, attempts will be made to produce anti-idiotypic antibodies capable of blocking/modulating the effects of thse cloned cells both in vitro and in vivo."}
{"text": "The investigations described herein are designed to provide new information on the action(s) of pituitary somatotropin (GH) and/or prolactin upon the endocrine cells of the gastroenteropancreatic (GEP) system in normal and diabetic rats by means of radioimmunoassay (RIA), quantitative light microscopic immunocytochemistry and ultrastructural immunocytochemical analysis. Both qualitative and quantitative information will be obtained on the chronic effects in vivo of exogenous FH and PRL or endogenous GH-PRL mixtures produced by transplantable mammosomatotropic tumors upon the numbers, distribution, morphology and fucntion of the insulin, glucagon, somatostatin (SRIF), pancreatic polypeptide (PP), vasoactive intestinal polypeptide (VIP), and possibly gastrin cells of pancreatic islets and the glucagon-, and GIP-producing cells of the gastrointestinal mucosa. The results of these studies correlating tissue and serum hormone levels with quantitative measurements of immunocytochemically identified endocrine cell populations within the GEP system with the diabetic status of the animals should provide further information on interactions among the pituitary, gut and pancreatic endocrine cells and, in particular, should help to elucidate the role and/or fate of the non-B pancreatic islet cells and gut hormone cells in metabolic dysfunctions characterized by either hypersomatotropism (acromegaly or giantism) or hyperprolactinemia. Since large mammosomatotropic tumors promote both an increase in B cells and a decrease in D cells in islets of tumor rats, these studies may provide insight into the mechanisms underlying the development of carbohydrate intolerance and diabetes in the above conditions, and also they may well provide a useful model for future studies of pancreatic endocrine cell interactions in normal and pathological conditions (like maturity onset diabetes)."}
{"text": "This is a revised competing renewal of a project studying the synaptic organization of postmortem striatum in schizophrenic subjects (SZ) at the ultrastructural level. The striatum, which interacts with other brain areas to affect motor, cognitive and limbic behavior, is one of the regions affected in schizophrenia. The results of the studies in the last grant cycle indicated an increase in cortico-striatal type synapses in the caudate matrix and putamen patches, that was not caused by antipsychotic medication. The higher density of cortical-type synapses in the SZ cases than in controls suggests hyper-stimulation of striatal projection neurons. This could have several important and different downstream effects depending on the precise circuitry involved. The present application seeks to identify the specific striatal circuitry affected in SZ. SA#1) To test the hypothesis that limbic and prefrontal circuitry are perturbed at the level of the striatum, we will examine synaptic density in the subregions of the striatum that process these circuits. SA2 will examine synaptic density of striatonigral and striatopallidal neurons in the patch and matrix in select striatal territories determined in SA1. SA#2A) To test the hypothesis that striatopallidal matrix neurons in the caudate receive more excitatory inputs, the immunocytochemical localization of enkephalin, a marker of these neurons, will be performed; the number of synapses formed onto labeled spines will be compared between groups. SA#2B) Tests the hypotheses that striatonigral matrix neurons in the caudate receive more excitatory inputs, but that striatonigral neurons in the putamen patch receive normal or fewer numbers of synapses. The immunocytochemical localization of substance P, a marker of striatonigral neurons, will be performed; the number of synapses formed onto labeled spines will be compared between groups. SA#3) To test the hypothesis that typical vs atypical APDs have different effects on the patch and matrix compartment, we will treat rats chronically with APDs, process the tissue for calbindin immunocytochemistry to identify the patch and matrix and analyze EM samples obtained from each. In monkey tissue obtained from Dr. Lewis, we will examine the synaptic density (labeled with synaptophysin) within the patch and matrix compartments in chronic haldol treated animals and controls using light microscopy. The proposed experiments will: 1) distinguish between drug effects and disease related alterations in synaptic pathology; 2) will provide insight into the mechanisms of action of antipsychotic drugs; and 3) are an important initial step in identifying putative abnormal striatal circuitry that may underlie some of the psychopathology of schizophrenia. [unreadable] [unreadable]"}
{"text": "Children with sickle cell disease (SCD) are living longer with the advent of medical advances such as prophylactic penicillin, chronic transfusion, and hydroxyurea. Despite greater longevity in SCD, the period following the transition from pediatric to adult care is critical; youth aged 18-30 years are at high risk for mortality and have high rates of healthcare utilization, leading to high healthcare costs. As such, health care transition (HCT) programs have been created to prepare patients for adult-centered care and subsequently, improve health outcomes. However, very few programs have been evaluated for effectiveness in achieving optimal health outcomes in SCD. This paucity of program evaluation is attributed to a lack of identifiable predictors and outcomes. The aims of this proposal are to: 1) identify potential predictors of successful HCT through a survey of pediatric and adult SCD providers, 2) examine the associations between predictors of successful HCT and HCT outcomes in a cohort of youth with SCD (ages 16-20 years), and 3) identify and characterize trajectories of HCT over a 24-month period and determine predictors of these trajectories. The results of this project may have significant implications for the delivery of healthcare in SCD during the period of HCT. This study will contribute to the understanding of the predictors and outcomes of HCT among youth with SCD and the long-term goal of developing approaches to best evaluate HCT interventions and identify areas of improvement. This project will provide support for Dr. Porter to establish herself as an independent investigator with a research program in HCT in youth with SCD. The short-term goals of the project are to develop her skills in systematic assessment of HCT predictors and outcomes, assist her in learning and applying more advanced methodology to examine longitudinal HCT relationships, and expand her abilities to investigate, disseminate, and translate her work into effective interventions. The goals will be accomplished via mentorship by experts in the areas of SCD and HCT, participation in didactic activities, and the conduct of a research project focused on HCT in youth with SCD. (End of Abstract)"}
{"text": "The goal of this project is to quantify airway secretory activity in horses with recurrent airway obstruction (RAO), to investigate mechanisms underlying increased production and accumulation of airway secretions, and to assess the functional significance of this increase. Equine RAO mimics many of the changes observed in human diseases, such as chronic asthma, chronic bronchitis, and organic dust-induced airway disease, in which persistent mucus overproduction is an important component of airway obstruction. Immunochemical, morphometric, functional, and in vitro methods will be utilized to identify temporal alterations in the production, secretion, and intraepithelial storage of mucus/mucus-like material in disease-affected horses versus controls, and to assess the functional significance of any such alterations. In addition, mechanisms of increased secretion will be addressed by evaluating the effect of neutrophil elastase and endotoxin on airway secretory activity. It is my general hypothesis that equine RAO is associated with a persistent increase in mucus production that contributes to airway obstruction even during periods of apparent disease remission, that increased intraepithelial storage accompanies this persistent increase, and that increased concentrations of the inflammatory mediator neutrophil elastase or a hypersensitivity to this mediator contributes to increased secretory activity."}
{"text": "The long-term objective of the research described in this application is to develop new stereocontrolled methods for the synthesis of a variety of unsaturated heterocyclic systems. The vast majority of pharaceuticals used in medical practice are heterocycles. We believe by developing efficient synthetic entries to heterocyclic ring systems, we will allow promising pharacological leads in the future to be rapidly developed with potential therapeutic implications. Heterocyclization reactions are the focus of the studies we propose for the 04 to 07 project years. Cyclizations of this type are much less well developed than the corresponding preparation of carbocycles by polyene cyclizations. Our attention will continue to focus on heterocyclizations which are terminated by vinylsilanes. Since our introduction of these cyclization terminators in 1980, vinylsilanes have proven useful in a number of laboratories to terminate a wide variety of cyclization reactions. Because of the pivotal role that new initiators plan in expanding the horizons of heterocyclization chemistry, we also aim to develop new azacyclic and oxacyclic cyclization initiators. Tetrahydropridines related to the Parkinson's disease stimulaten, MPTP, will be prepared. The new antibiotic streptazolin will be prepared in enantioselective form, as will the powerful \"uteroevacuant\" diterpene zoapatanol. Intermediates and natural product targets will be broadly sdcrened for biological activity at the Biological Sciences Research Center of the Shell Development Co., and at NCI."}
{"text": "Immunosuppressants are absolutely essential for successful organ transplantation and are useful in the treatment of autoimmune disorders. Analyzing their mechanism of action can, as in the case of cyclosporine and calcineurin, yield important insights into basic features of T cell biology. We recently embarked upon an ambitious three-step project intended to use chemical biology approaches to identify unknown pathways involved in lymphocyte function. The over-arching rationale underlying the project was that compounds that inhibit T cell activation and work via unknown molecular mechanism (MMOA) could be developed into chemical probes that could be used to identify novel cellular targets, which would reveal currently-unknown aspects of basic T cell biology and might become the basis for new classes of immunosuppressant agents. The first step of the project- screening the NIH's Molecular Libraries Small Molecule Repository of ~375,000 compounds and identifying compounds with unknown MMOA- succeeded. We monitored lytic granule exocytosis using TALL-104 human cytotoxic T lymphocytes as a model, measuring externalization of LAMP-1/ CD107a using flow cytometry. Among hits with unknown MMOA was 2-N-[(2-methoxyphenyl)methyl]-4-N-[(4- propan-2-ylphenyl)methyl]thieno[3,2-d]pyrimidine-2,4-diamine, CID 49792547, which is the subject of this application. This compound inhibits lytic granule exocytosis with potency in the micromolar range, but does not work via any of the mechanisms we tested. It inhibits IL-2 production by Jurkat human leukemic T cells, confirming that has broad immunosuppressive activity. It is a drug-like molecule that is amenable to synthesis and the generation of diverse analogs. The project's intended second step was to generate analogs for structure-activity analysis, then use that information to design chemical probes to use in the third and final step, applying affinity-based and/or photo- crosslinking approaches to identify the unknown target that underlies the compound's activity. However, Chemistry Center support was withdrawn when the NIH MLPCN program ended. This application for an R03 is intended to allow us to continue to pursue the overall goals of the project by creating probes for target identification. We will create analogs of CID 49792547, then test their effects on lytic granule exocytosis, IL-2 secretion and toxicity. This information will allow us to identify the highest possible affinity analogs and will reveal sites that can be used to attach linkers which will allow the compound to be coupled to biotin for creation of an affinity matrix and to bifunctional photo-crosslinking/ click-chemistry groups that can be used to label target proteins. Future efforts will be directed towards target identification using probes developed in this application and in a companion application submitted previously focused on another compound."}
{"text": "Over the past 30 years there has been a dramatic increase in the number of harmful algal blooms (HAB's) in coastal waters throughout the world. As a result, there are more toxic algal species, more algal toxins and more geographic areas impacted than ever before. When these toxic species proliferate, they may cause massive kills of fish and shellfish, wildlife mortality, human illness and death. One of these marine organisms, pseudo-nitzchia produces a neurotoxin, Domoic Acid (DA). Most of what we know about the human health effects of Domoic Acid has been derived from a single documented outbreak in Montreal Canada in 1987. Persons who ate mussels with high levels of DA suffered serious medical illnesses, including seizures and coma, and 3 people died. Survivors were left with a profound memory disorder, Amnesic Shellfish poisoning (ASP). Based upon animal models, regulatory levels of DA in shellfish were established. Within the past 17 years, DA levels have been close to or exceeded these safety levels at razor clam harvesting beaches on Native American Reservations in the Pacific NW. Recent data indicate that this population is currently at risk for significant, but preventable, neurobehavioral impairment (ASP) from razor clam consumption, with memory problems ranging from the low average to amnesic range. There appears to be a dose- response relationship between exposure and memory problems and the base rate of persons meeting the criteria for severe memory impairment, or ASP is 4.3%. The purpose of this 5 year longitudinal cohort study is to extend these findings to establish their clinical and public health significance. A prospective longitudinal cohort design of 735 Native Americans (ages 6 months to 75 yrs) from three Tribes, with nested case-control study of identified cases of ASP will be implemented. The health impacts of chronic, low level exposures to DA over time will be determined as well as the exposure and host factors associated with DA neurotoxicity. A new model of ASP, to include the potential for delayed or latent toxicity, recovery, and recurrence will be tested with state-of-the-art procedures for assessing human exposure (mobile technology) and behavioral neurotoxicity in infants, children, adults and geriatric groups. Findings will have a significant clinical and public health impact. The diagnosis and prognosis for ASP will be defined and established safety levels for exposure will be re-evaluated to insure they are protective of persons with long term, repeated exposure to DA."}
{"text": "Chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia and remains incurable. The introduction of the CD20 antibody rituximab combined with chemotherapy (chemoimmunotherapy) was the first intervention that has been shown to impact survival of CLL patients. Building upon the success of antibody directed therapy in CLL, we have actively pursued development of similar therapies directed at alternative antigens expressed selectively on CLL cells. CD37 is a tetraspanin protein of uncertain function that is expressed predominately on mature B-cells. Our research group has worked with Trubion Pharmaceutics, Inc/Emergent Biosolutions to develop CD37 directed TRU-016, a small modular immune pharmaceutical similar to an antibody but lacking the CH1 domain. Prior pre-clinical work demonstrated TRU-016 to have potent direct and immune based killing of CLL cells and also in vivo activity in several lymphoma pre-clinical models. A first in man phase 1 clinical trial initiated with TRU-016 in previously treated CLL has demonstrated single agent activity and combination studies have now been initiated. During this early phase 1 development of TRU-016 we have continued pre-clinical work to discern its mechanism of action. Preliminary data described in this proposal demonstrate that TRU-016 ligation of the CD37 antigen causes recruitment and phosphorylation of SHP1 via a potential ITIM domain within the intracytplasmic domain. SHP1 activation leads to decreased PI3-Kinase activity and AKT phosphorylation concomitant with FOXO3A accumulation in the nucleus and induction of the pro-apoptotic gene BIM and CLL cell death. siRNA targeting either SHP1 or BIM antagonizes death induced by TRU-016. Interestingly, the C-terminal of the CD37 protein contains ITAM like motif with associated activation function. Deletion of this motif enhances TRU-016 induced cytotoxicity suggesting a potential negative regulatory role for the C-terminal in TRU-016 cytotoxic effect. Consistent with this, inhibition of pro-survival signaling by PI3K and BTK overcame the inhibitory effects of the C-terminal domain resulting in enhanced TRU-016 induced apoptosis. Moving forward, this proposal seeks to further characterize in detail the ITIM and ITAM function of CD37 activated by TRU-016, study the mechanisms of cross talk between the activation and inhibitory signaling relevant to TRU-016 response and pursue development of afucosylated TRU-016 (TRU-016GV) leading to rational combination strategies that will facilitate enhanced TRU-016 and TRU-016GV response. Knowledge gained from this proposal has potential to impact not only CLL but also lymphoma and acute lymphoblastic leukemia patients who may also derive benefit from TRU-016 or TRU-016GV. Additionally, mechanistic study of CD37 ITIM and ITAM dual signaling has relevance to general B-cell immunology. The senior investigative team formed to perform this project includes expertise to accomplish the proposed goals and support from a large internationally known leukemia program. Collectively, TRU-016 and its application to CLL therapy have potential to transform treatment of this disease."}
{"text": "Defects in the core human mismatch repair (MMR) genes are the cause of Lynch syndrome (LS/HNPCC), as well as 10-40% of sporadic colorectal, gastric, endometrial, ovarian and upper urinary tract tumors. MMR recognizes and repairs polymerase misincorporation errors, suppresses recombination between non-allelic partially homologous DNA sequences during double-stranded break (DSB) repair (homeologous recombination; HEOR), and functions as a lesion sensor in DNA damage signaling. Unrepaired errors in MMR-deficient cells lead to mutations (Mutator Phenotype) that drive tumorigenesis. Importantly, MMR- deficient tumors display resistance to several common chemotherapeutic drugs. The components of MMR from numerous organisms have been purified and MMR reactions have been reconstituted in vitro. Approximately a dozen fundamental biochemical properties have been detailed that together may account for the excision of a mismatch from DNA. Based on these studies several mechanisms for the MMR reaction have been proposed. Remarkably, the biochemical, genetic and structural studies performed to date have not fuly determined the function(s) of the core MMR components or resolved the validity of the proposed mechanisms for MMR. Moreover, the vast majority of biochemical studies have been performed with naked DNA that is significantly different from the cellular chromatin where MMR functions occur. We have developed three robust real-time single molecule measures capable of visualizing and interrogating the detailed function(s) of MMR components. Using these new methodologies we propose to resolve the mechanism of MMR on naked DNA and then determine the function(s) of MMR components on physiologically relevant chromatin for comparison. In this renewal application we propose the following Specific Aims: 1.) enumerate the precise mechanism of human MMR using single molecule analysis, 2.) determine the functions of the human MMR proteins on physiologically relevant chromatin, and 3.) examine the biophysical mechanism of homeologous recombination suppression by MMR. We will apply our results to determine the functional defects associated with missense mutation found in LS/HNPCC families. These studies should provide a visual and quantitative platform for understanding the detailed function(s) of core MMR components in repair, damage signaling, drug resistance and tumorigenesis"}
{"text": "When simian virus 40 (SV4O) is serially passaged at high multiplicity, a heterogeneous collection of naturally arising variants is generated. Some of these variant viruses become very abundant in later serial passages presumably due to their enhanced replication efficiency. Previous studies have shown that these abundant variants contain multiple copies of the SV40 replication origin, frequently as inverted repeats and in association with inserts of host DNA. These evolutionarily-selected origin regions will be cloned into a bacterial plasmid and then transfected into monkey cells constitutively producing T antigen (COS cells) in order to quantitate the effects of duplicated sequences, inverted repetitions, and insertions of host DNA on the replication efficiency of the SV40 origin region. In addition, the evolutionarily-optimized sequences will be further modified by in vitro mutagenesis in order to precisely define those features of the nucleotide sequence which affect replication. Clones from a monkey genomic library which hybridize to the variant-selected host DNA segments will also be analysed for functional activity. The proposed studies constitute a unique approach to understanding the functional aspects of a replication sequence and to understanding the regulation of replication. Both types of understanding have important implications for understanding oncogenesis and viral-host interactions."}
{"text": "Tougaloo College has a long history of producing graduates who have gone on to PhD program and to careers in research. The science division of the college has several programs such as the Minority Access to Research Careers program, Minority Biomedical Research Support program, the Alliance for Minority Progress program and the Jackson Heart Study Undergraduate Training Program that provide excellent research opportunities for our students. Between these programs we can provide research training experience for 25 students every year. However, we do not have enough faculty involved in research with whom the students can work. At present the students gain their experience off campus. We are convinced that our students will benefit even more if more of our faculty were involved in research. This EARDA FRESP/Phase II proposal will enable Tougaloo College to continue the task start under Phase I of increasing the number of faculty members involved in research. During the past three and a half years the PI Asoka Srinivasan's focus has been on underscoring the importance of faculty involvement in research, informing them of funding opportunities and improving t infrastructure in the preparation and submission of proposals. The objectives during Phase II will be to continue the efforts started under phase I and to focus more sharply the efforts to involve the faculty in research by funding 2 pilot research study awards per year and to formalize the establishment of the Office of Research Development. We expect at least 50 percent of the faculty receiving the pilot research study awards to be successful in obtaining research grants."}
{"text": "DESCRIPTION (Adapted from applicant's description): This proposal is requesting funds for a very promising and exciting new direction of research that may shed light on a controversial issue in the scientific community. The proposes to isolate and characterize a novel form of gonadotropin-releasing hormone (GnRH), that is lamprey-like, in the primate hypothalamus. She proposes to test the hypothesis that the primate brain has two hypothalamic GnRHs. This hypothesis is based on recent data from preliminary and collaborative studies that support her earlier claims of a novel ir-GnRI-I (lamprey GnRH-like) form in humans. The applicant proposes to isolate and characterize this novel form of GnRH in the hypothalamus of primates by screening the cDNA libraries to hypothalamus using oligonucleotide probes against lamprey GnRH as well as other related probes. She theorizes that an ancient form of GnRH (lamprey) as well as the more modem mammalian form of GnRH function as hypothalamic neurohormones stimulating gonadotropin release in the primate. In mammals, it has not been fully explained how one GnRH hormone differentially stimulates the release of two gonadotropins (FSH and LH). She is in a unique position to accomplish this proposed project because she has recently identified a full length cDNA encoding the lamprey GnRH-I and has an established collaboration with research in humans. The presence of multiple GnRHs in the hypothalamus could provide the answer to this question. Her specific aims include 1) screening and isolating cDNA clones encoding the precursor forms of brain GnRH(S) in primates and (2) screening genomic libraries using the newly identified cDNAs as probes to isolate the complete GnRH gene(s). She predicts that findings from these studies will contribute to our understanding of reproductive physiology and provide further information for developing medical therapies using GnRH analogs. A new hypothalamic GnRH in mammals could shed light on the formation of new GnRH analogs that may not have some of the side effects noted with the GnRH analogs currently used."}
{"text": "Tetrahydropterin forms the basic structure of a series of analogues which serve effectively as cofactors for hydroxylase enzymes. Most analogues are formed by placement of various side chains on the 6-carbon. We have recently acquired a compound which has phenyl group attached at this 6-position. The present investigation compared this compound with tetrahydrobiopterin for effective hydroxylation of phenylanine, tyrosine and tryptophan. The enzymes used were: phenylalanine hydroxylase purified from rat liver and tyrosine and tryptophan hydroxylase from rat brain. In addition we examined the ability of the reductase system to continually regenerate reduced 6-methylpterin. Our kinetic analysis showed that 6-phynylpterin is as effective as the natural cofactor (tetrahydrobiopterin) for the various hydroxylases. Also, 6-phenylpterin is incorporated effectively into the endogenous reductase system."}
{"text": "Cdk5 and nociception: In general nociceptive (painful) neural signals originate from sensory neurons in peripheral tissues and are transmitted to second-order neurons in the spinal cord, which then convey the message to specific nuclei in the brain for perception of pain. Nociception results from the activation of molecular and cellular mechanisms in damaged tissue, sensory neurons, and the spinal cord. Many cellular pathways have been implicated in nociceptive signaling, but their precise molecular mechanisms have not been clearly defined. Abnormalities in molecular pathways underlying nociceptive processes may result in chronic pain conditions. It follows that a detailed characterization of these pathways is necessary for developing effective strategies to treat pain. Signal transduction mechanisms activated by nociceptive stimuli have not been fully characterized. Recent advances, especially the cloning and characterization of the capsaicin receptor (also known as vanilloid receptor 1, VR1, TRPV1) remarkably improved our understanding of signal transduction in nociceptive neurons. TRPV1 is a member of the transient receptor potential (TRP) family. It is a polymodal ligand-gated ion channel that is expressed in small-diameter sensory neurons (C-fibers) and activated by heat, protons, anandamide, and leukotrienes. TRPV1 knockout mice show reduced thermal hyperalgesia following inflammation and reduced nociceptive responsiveness in a model of bone cancer pain. TRPV1 has been connected to a broad spectrum of physiological conditions and diseases such as thermal hyperalgesia, allodynia, inflammatory bowel disease, Crohn's disease, vulvodynia, osteoarthritis, pancreatitis, gastroesophageal reflux disease, bladder disease, cystitis, and asthma. There is mounting evidence that TRPV1 is subject to multiple interacting levels of control. Phosphorylation of TRPV1 is critical for its function in response to nociceptive stimuli. We previously described the expression and specific activity of Cdk5/p35 in TRPV1-positive cells of dorsal root ganglia (DRG) and trigeminal ganglia (TG) and the modulation of Cdk5 activity in response to peripheral inflammatory pain. We also found that p35 knockout mice, which have significantly reduced Cdk5 activity, behaviorally mimic the TRPV1 knockout pain phenotype. [unreadable] [unreadable] Cdk5, a neuron-specific, proline-directed serine/threonine kinase is activated by 2 noncyclin activators, p35 and/or p39. Cdk5 phosphorylates serine and threonine immediately upstream of a proline residue. In addition to an absolute requirement for proline in the +1 position, Cdk5 shows a marked preference for a basic residue in the +3 position and phosphorylates the consensus sequence (S/T)PX(K/H/R). Several neuronal and nonneuronal substrates are phosphorylated by Cdk5, and the list of new substrates is increasing. Earlier studies showed that Cdk5 knockout and p35/p39 double-knockout mice are embryonically lethal with neuronal migration defects, whereas p35 knockout mice show inverted neuronal layering with a concurrent attenuated response to noxious heat; conversely mice overexpressing p35 were hyperalgesic. [unreadable] [unreadable] Subsequently, we analyzed TRPV1 for potential phosphorylation by Cdk5. We report that Cdk5 can directly phosphorylate TRPV1 at threonine 407, and this in turn modulates agonist-induced calcium influx. We initially found that inhibiting Cdk5 activity resulted in attenuation of capsaicin-induced calcium influx in cultured DRG neurons, and this attenuation was reversible. These observations suggest that Cdk5-mediated phosphorylation of TRPV1 is important for capsaicin-mediated calcium influx through this receptor. Since germline Cdk5 knockout mice are embryonically lethal and p35 knockout mice present various neuronal disorders, we generated primary nociceptor-specific Cdk5 conditional knockout (Cdk5-CoKO) mice to identify the precise role of Cdk5 in primary afferent pain signaling. In the basal state, the conditional knockout mice showed significant hypoalgesia, confirming the direct role of normal Cdk5 activity in primary afferent. Collectively, our findings describe a novel molecular mechanism for the functional regulation of TRPV1 by Cdk5 and provide further insights into the role of Cdk5 in the pain pathway.[unreadable] [unreadable] Cdk5 and brain development: Analysis of functional roles of Cdk5 in postnatal brain development has been hampered because of perinatal lethality of Cdk5-/- mice and the compensated phenotype of p35-/- mice. To study the role of Cdk5 in postnatal development of the midbrain-hindbrain (MHB), we generated MHB-specific Cdk5 conditional knockout mice by crossing Cdk5 floxed mice with Wnt1-Cre mice. Wnt1-Cre-mediated Cdk5 conditional knockout (WCOKO) mice have lower body weight than controls, an ataxic gait, and early postnatal lethality. Histological analysis indicated a smaller cerebellum with defective migration of the Purkinje cells. A detailed analysis of WCOKO mice showed a complete lack of inward migration of the granule cells. In addition, we also identified a complete absence of the superior colliculus in Cdk5-/- mice and its abnormal development in WCOKO mice. These results indicate that Cdk5 plays important roles in mouse MHB development."}
{"text": "Protein-protein interactions are essential to almost all cellular processes. The ability to rationally manipulate these interactions would allow for the creation of new tools for studying cell biology and the creation of new protein therapeutics. The objective of this research is to develop and test computer protocols for protein interface design that include full backbone and side chain flexibility. We will test these protocols on two important problems in interface design: enhancing the binding affinity of naturally occurring interactions and changing the binding specificity of signaling proteins. To achieve this objective we will make use of a computer program, Rosetta Design, that we developed for simultaneously optimizing the amino acid sequence and backbone conformation of a target structure. Encouragingly, this protocol has been previously used to design a novel protein structure with atomic level accuracy. Several lines of evidence suggest that the accuracy of protein design simulations increase when more side chain conformations are considered during the simulation. To allow complete flexibility in side chain torsion angles we will use a Monte Carlo minimization procedure in which discrete rotamer substitutions are followed by gradient-based minimization of side chain torsion angles. We will test the flexible side chain model by designing functionally orthogonal binding interactions between two sets of model proteins: proteins from the ubiquitination pathway and proteins involved in G-protein signaling. We will test our model for protein design with a flexible backbone by designing protein extensions that enhance binding affinity by increasing the number of favorable contacts with the partner molecule. Specifically, we will incorporate additional residues off the N-terminus of the ubiquitin conjugating enzyme, UbcH7, that are predicted to enhance its binding affinity for its natural binding partner, the ubiquitin ligase E6AP. Binding affinities will be determined with various biophysical techniques and activity assays will be used to determine if the redesigned proteins bind as an active complex."}
{"text": "Understanding the mechanisms for the breakdown of self-tolerance in autoimmunity is essential for the development of strategies to prevent and treat autoimmune diseases. Abundant evidence has shown that defective apoptosis in the immune system is associated with the development of systemic autoimmune diseases in humans and mice. However, inhibition of apoptosis in lymphocytes alone is not sufficient to break immune tolerance, indicating that impaired apoptosis in other cell types plays a critical role in the breakdown of self-tolerance. Targeted inhibition of apoptosis in dendritic cells (DCs) has been shown to induce systemic autoimmune responses. Experiments are proposed to test the hypothesis that apoptosis in DCs is essential for limiting lymphocyte activation and preventing autoimmunity in the following specific aims: 1) to characterize the apoptosis pathways in DCs. Death receptor-mediated and mitochondrion-dependent apoptosis pathways will be characterized in DCs. Preliminary studies suggested that the lifespan of DC subsets in vivo was correlating to the molecular ratios between anti-apoptotic and pro-apoptotic bcl-2 family members. Experiments will be performed to further characterize the bcl-2-regulated mitochondrial apoptosis pathways in regulating DC apoptosis; 2) to test the hypothesis that defective apoptosis in DCs contributes to dysregulated lymphocyte activation. The lifespan of DCs can potentially influence immune responses by affecting the duration of DCs in stimulating lymphocytes. The potentials for apoptosis-deficient DCs in over- activating lymphocytes will be examined; and 3) to test the hypothesis that defective apoptosis in DCs contributes to the development of autoimmunity. Studies will be performed to examine whether DCs harboring apoptosis deficiency in the mitochondrial apoptosis pathways leads to the development of autoimmunity. Experiments are proposed to test the hypothesis that apoptosis regulates DC homeostasis and immunogenicity, and defective apoptosis in DCs contributes to the onset of autoimmunity. In the long term, the knowledge gained from these studies will be used to develop more specific and effective strategies to prevent the onset of autoimmunity by targeting apoptosis in DCs. [unreadable] [unreadable] [unreadable]"}
{"text": "A new electrophoretic method is proposed for efficiently separating membrane proteins while retaining function. Fluid, smooth, electrically neutral lipid bilayers are proposed as the electrophoretic medium. The proposed separation would maintain the membrane proteins in their natural type of environment to produce separated zones of membrane proteins, which are then displayed as functional arrays. The separation would support such applications as membrane proteomics, drug screening and development of cancer diagnostics. Membrane proteins play key roles in human health, since they direct cellular signaling, recognition and transport, yet the steps for isolating membrane proteins for research are expensive, laborintensive and inefficient. Today's methods for separating proteins rely on water solubility. The proposed work will test the feasibility of separating membrane proteins in lipid bilayers by investigating 3 essential properties. First, the fluidity and uniformity of the lipid bilayer will be studied on ultrasmooth polyacrylamide films, grown by atom-transfer radical polymerization. The polyacrylamide film provides a uniform hydrophilic boundary on the underside of the bilayer, an aqueous buffer will be used on the upper side. Second, the electrophoretic mobilities of 5 well characterized receptors will be studied: the 3 opioid receptors and 2 melanocortin receptors. All 5 are medically important G-protein coupled receptors. Their ligands are peptides, which will be singly labeled with rhodamine. Third, the function of these membrane proteins will be studied by single-molecule fluorescence spectroscopy of the rhodamine labeled ligands. The R21 research will critically test the idea of whether the electrophoretic separation of functional membrane proteins is feasible in supported lipid bilayers. If feasible, an R01 proposal would address separations of real mixtures of membrane proteins from cell lysates. The work could have enormous impact on drug discovery, allowing inexpensive screening that encompasses many receptors."}
{"text": "PROJECT SUMMARY Specific compositions of periodontal microbiota are associated with risk for the various forms of periodontitis. Serum antibody levels are considered as surrogate markers for the activity levels of periodontal microbiota, which represent the protein synthesis and inflammatory potential of the microbiota. The chronic trickling of periodontal microbiota into the bloodstream also elicits a low-grade systemic inflammation which has been suggested to increase the risk for several extra-oral diseases, such as atherosclerosis, rheumatoid arthritis, metabolic disorders, and neurodegenerative diseases, including cognitive impairment and Alzheimer's disease. Furthermore, our recent study using data from the Third National Health and Nutrition Examination Survey (NHANES III) indicated that specific compositions of periodontal microbiota, even without inducing clinically significant periodontitis, may have a significant impact on the risk for human disease and mortality, and that Porphyromonas gingivalis (P gingivalis) play a key role in the dysbiotic periodontal microbiota. In this proposal, we will collaborate with experts from related disciplines to test our hypothesis that the activity levels of specific periodontal microbiota/P gingivalis are associated with the occurrence of age-related macular degeneration (AMD). We will first use partial least squares (PLS) regression to identify specific patterns of 21 serum immunoglobulin G (IgG) levels for periodontal microbiota which are associated with the risk for AMD in a case-control study from a representative sample of the US population, the NHANES III. PLS is an important statistical method in bioinformatics and used to discover variables (i.e. specific patterns of IgG levels) that are not directly observed but are rather inferred from other observed variables (i.e. 21 individual IgG levels). Since our hypothesis is novel and no previous study in the topic has been done, it is possible that the observed associations between specific IgG patterns and AMD will be confounded in spite of adjusting for confounding variables. To further verify the associations, we will use complement factor H (CFH) genetic polymorphisms as an instrumental variable in our Mendelian randomization study. CFH genetic polymorphisms are a well-established risk factor for AMD. The Mendelian randomization approach is a novel epidemiologic study design that incorporates genetic information into traditional epidemiologic methods and addresses exposure-outcome relationships without many of the typical biases that impact the validity of traditional epidemiologic approaches. The Mendelian randomization approach is especially useful when a randomized trial is impossible. This project will serve as our first step toward our long-term objectives of understanding how the phenotypes (such as serological markers) and genotypes/strains (microbiome) of oral microbiota, together with the human genome and nutrition and other known risk factors, affect eye health. Since periodontitis is one of the most prevalent diseases in humans and AMD is the leading cause of blindness among elders, understanding the associations between periodontal microbiota and AMD may lead to new therapeutic and preventative strategies for AMD."}
{"text": "We are developing an artificial bone marrow for ex vivo culture of hematopoietic stem cells (HSCs). This platform has significant scientific value for testing hypotheses regarding the cascade of external signals responsible for directing HSC fate decisions within the bone marrow. An artificial marrow would also have significant clinical value for therapeutic expansion of HSCs or for study of the etiology and treatment of hematopoietic pathologies. However complicating this effort is limited information regarding the regulatory role played by the continuum of sub-niches that exist in close spatial order across the marrow responsible for maintaining hematopoietic homeostasis. We have recently described microfabrication approaches to generate an engineered bone marrow (EBM) containing overlapping patterns of marrow-inspired cellular, biophysical, and biomolecular cues to begin to examine the coordinated impact of these signals on HSC quiescence vs. activation. However, the small scale that makes the EBM advantageous introduces concerns regarding the unknown heterogeneity of a stem cell's response to these cues. While not surprising HSCs may exhibit a range of responses to a niche signal, effects may be magnified in multi-cue environments. We therefore propose to demonstrate a label-free approach to temporally track and quantify the heterogeneity of single HSC fate decisions in response to multiplexed EBM niche signals. To do this, we will combine photonic crystal enhanced microscopy (PCEM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) to create an integrated detection instrument able to trace single HSC fate decisions. We hypothesize that individually profiling these decisions across a continuum of biomaterial sub-niches will allow us to better predict niche regulation of HSC fate than more traditional metrics that report ensemble averages. Aim 1 will integrate SIMS and PCEM to determine the heterogeneity of HSC response to biophysical cues demonstrated to have an effect on populations of HSCs. Aim 2 will employ the PCEM/SIMS-based biosensor to examine HSC fate decisions within multi-cell colonies containing HSCs and supportive niche cells. Combining this novel biosensor with traditional functional assays will allow us, for the firt time, to determine the heterogeneity of HSC response to engineered niche signals. It also offers a framework to rapidly assess the impact of multiple signals using a minimal number of cells in order to identify hierarchies and/or synergies between these cues. Such data will bring new richness to our understanding of how HSCs integrate niche signals as well as identify critical design elements of an engineered bone marrow. By quantifying the level of heterogeneity in these fate decisions, we will also establish where along the continuum between single HSC and ensemble averages future investigations must focus."}
{"text": "Human rotaviruses are an important cause of severe diarrhea in young children throughout the world. Rotavirus infections are especially devastating in developing nations because of the high mortality rate associated with gastroenteritis. By the age of 3, the incidence of rotavirus infections decreases markedly, but outbreaks of rotavirus-induced diarrhea have been reported among the elderly, the acutely ill, and the immunocompromised. Other mammals and birds can develop rotavirus diarrhea as well, making this disease one of veterinary interest and economic concern. Effective means of preventing this disease are obviously needed. The rotavirus genome consists of 11 segments of double-stranded RNA. Most of the gene segments appear to be moncistronic and together they encode at least 12 viral polypeptides (VP). The genome is enclosed in a core which is surrounded by a double- shelled capsid. VP3 and VP7 are the major outer shell proteins. VP3 is activated by protease cleavage and is associated with host range, hemagglutination, virulence, and both homotypic and heterotypic neutralization. The glycoprotein VP7 is the primary determinant of serotype specific neutralization. This proposal focuses on defining and characterizing those genetically-determined features of VP3 that are involved in gastrointestinal infection. To accomplish this, we plan to: 1) determine the nucleotide sequence encoding the homotypic and heterotypic neutralizing domains on VP3, 2) compare the gene 4 sequences of various murine rotavirus stains with human or simian strains in an effort to clarify the genetic basis of host range, 3) design and execute a series of genetic experiments to determine whether rotavirus RNA is \"infectious,\" 4) develop a strategy to clone full length gene 4 cDNA, and 5) use full length cDNA clones of rotavirus gene 4 in a suitable expression vector to produce a biologically active VP3."}
{"text": "Dietitian support only."}
{"text": "The purpose of this project is to add approximately 22,500 person years for the detection of cancer and other major medical illnesses among the 5,464 women who have previously been enrolled in the DESAD Project. This continued surveillance will enable us to accomplish five specific aims: to determine the magnitude of the excess of squamous neoplasia in exposed over unexposed women and to determine if the increased risk continues over the next five years, to determine if the risk of adenocarcinoma increases during the next five years, to determine the rates of serious medical illnesses in the DES exposed women and to compare these rates with the control groups and published age specific incidence rates for the same diseases, to determine the frequency of abdominal and gynecologic surgery among DES exposed women and to compare these rates with the unexposed controls and the rates available from the National Center for Health Statistics, and to maintain contact with the DESAD Project exposed cohort at minimal expense. These aims will be accomplished by soliciting information by the method of mailed questionnaire. Each participant will be questioned annually concerning the diagnosis of medical diseases or the performance of surgical procedures. Previous experience of the DESAD cohort has indicated that in excess of 90 percent of the women previously enrolled in the project can be expected to comply with the requests for information. The sample size considerations are explained in detail in the project and indicate that it will be possible to detect, with a power of .9, a two-fold difference in the rates of many common medical diseases and surgical procedures at the p=.05 level."}
{"text": "The primary objective of this study is to compare the long-term safety of a hydrofluroalbane propellent (non-ozone depleting) with that of the standard chloroflurocarbon based propellant used in metered-dose inhalers. The secondary objective of this study is to evaluate the safety and efficacy of salbutamol sulfate in those subjects who were randomized to receive Ventolin (trade name for salbutamol) in a previous study. This study has been completed."}
{"text": "Ovulation is essential for successful reproduction. The ovulatory luteinizing hormone (LH) surge stimulates periovulatory follicles to produce prostaglandin E2 (PGE2). Blockade of PGE2 production causes ovulation failure. LH-stimulated neovascularization of the follicle is also essential for ovulation as inhibition of follicular angiogenesis prevents ovulation. This proposal puts for the overall hypothesis that PGE2 mediates the ability of the ovulatory LH surge to regulate angiogenesis within the ovulatory follicle. Angiogenesis is regulated via vascular growth factors. In Aim 1, we will determine if LH acts at follicular granulosa cells to regulate production of vascular growth factors in a PGE2-dependent manner. Monkey ovaries with multiple follicles will be obtained before and after administration of an ovulatory dose of the LH-like hormone hCG; some monkeys will also receive concomitant administration of a prostaglandin (PG) synthesis inhibitor. Granulosa cells, follicular fluid, and whole ovaries will be assessed for vascular growth factor mRNAs by qPCR and proteins by western blotting, ELISA, and immunofiuorescent detection on tissue sections. In vitro studies will be performed to determine if PGE2 acts directiy at granulosa cells to regulate vascular growth factor mRNA and proteins. Vascular growth factors act at endothelial cells to promote the formation of new blood vessels. In Aim 2, we will determine if PGE2 and vascular growth factors act at endothelial cells of the follicle to stimulate angiogenesis. Using a novel proliferating population of monkey ovarian endothelial cells, we will determine if PGE2 stimulates endothelial cell proliferation, migration, and formation of new capillaries. Similarly, vascular growth factors produced by granulosa cells, granulosa cell-conditioned media, and coculture with granulosa cells will be used to determine if vascular growth factors stimulate endothelial cell functions necessary for angiogenesis. Finally, monkey ovaries obtained after treatment in vivo with hCG, hCG+PG synthesis inhibitor, or hCG+PG synthesis inhibitor+PGE2 will be assessed histologically for neovascularization of the ovulatory follicle. The proposed studies will likely demonstrate that PGE2 and vascular growth factors act directly at endothelial cells to promote the neovascularization of the primate ovulatory follicle. Modulation of follicular PGE2 levels or PGE2 receptor activity may provide effective treatments for infertility or suggest targets for the development of novel contraceptives."}
{"text": "The effects of illness and nutritional status on various areas of the immune system are important considerations. This is especially true in attempting to define the effects of aging on immune function. Vitamin E apparently has little effect on immune function in vivo or in vitro. Burn trauma results in an overall depletion of circulating T cells, especially CD8+ cells. The receptor for IL-2, an activation marker, is induced not only by antigen and mitogen activation but by endotoxin. This can be seen frequently in older individuals. The T cell antigen receptor is under the control of at least 4 genes, which all are equally transcribed by an activation signal. Studies on genes involved in neurologic disorders have demonstrated that abnormal superoxide dismutase and tubulin gene transcription are not a features of Alzheimer's disease. Also, there are no common viral infections associated with this disorder."}
{"text": "Marrow myeloid cells can be separated into classes which exhibit different self-renewal capabilities carrying morphological features with respect to maturation and differing immunophenotype. The greater the self-renewal potential, the less frequent are these cells. Fluorescent activated cell sorting (FACS) has been used to develop populations of early myeloid cells which are enriched with the phenotype CD34+CD38-Lin-Thy-1 +(lo)Rho(lo). These cells have been shown to have high proliferative potential. Some AML cells show negativity for the Thy-1+lo phenotype. The t(15; 17) AML subset has been shown to be negative for CD34+CD33- cells. This data has suggested to some that the good prognosis AML subsets do not penetrate into the early myeloid compartment, whereas the poor prognostic subsets may contain cells with the immunophenotype CD34+CD38-CD33-Thy-1(lo). We will test this hypothesis by using high speed FACS cell sorting to develop populations of cells from AML patients which have the CD34+CD38- Lin-Thy+1(lo)Rho(lo) phenotype and then test this by FISH and PCR for evidence of AML cells."}
{"text": "The long-term objective of this grant is to analyze the central neural circuits that regulate autonomic functions with an emphasis on the cardiovascular system. Neuroanatomical techniques will be used to identify and chemically characterize specific CNS neural circuits which affect the sympathetic supply to the heart and adrenal gland. Much of the work described here will utilize the viral transneuronal tracing method-a technique which depends upon the use of an attenuated pig herpes virus to produce transneuronal infections that spread within functionally related chains of central neurons. The first project will analyze the descending projections arising from the amygdala-an important brain region thought to be responsible for emotionally triggered cardiovascular changes. The second project deals with the preoptic region and its potential role in sympathetic control. The third project explores the sympathetic control systems represented within the suprachiasmatic hypothalamic nucleus the CNS pacemaker that provides circadian modulation of some sympathetic functions. The fourth project focuses on the descending cell groups. The fifth project investigates the efferent projections of the periaqueductal gray matter in reference to sympathetic control; this area of the brain has been implicated in programming the fight-or-flight response. These studies will be useful for understanding the brain circuits responsible for sustaining and modulating basic life functions, such as blood pressure and cardiac regulation. In addition, they may provide insights into the underlying CNS mechanisms of certain clinical conditions, such as stress-induced cardiovascular changes and essential hypertension."}
{"text": "The overall goal of this research is to evaluate relevant issues confronting the integration of research findings into clinical practice, and to outline the perceived benefits of an automated, multi-use computer program for the assessment and treatment of alcohol abuse, and associated outcome and program evaluation functions. This multi-site, statewide (NM) randomized trial utilizes an automated computer-based assessment, treatment, and follow-up evaluation program to examine two empirical questions. First, does the program effectively disseminate the use of clinical (brief) interventions known to be efficacious for the treatment of alcohol and/or drug abuse across a variety of treatment settings? Second, how useful do treatment providers find an automated intervention and data management program to be, and what, if any, issues arise with respect to the diffusion of such an innovation?"}
{"text": "The growing HIV pandemic and its increasing burden on women highlights the urgent need for novel safe and effective preventative strategies such as topical microbicides. We hypothesize that a truly safe and effective microbicide will require sustained, local delivery of a combination of drugs that target different steps in the HIV life cycle and a delivery system that addresses the challenges related to adherence. A safe microbicide should not interfere with host defenses nor increase the risk of selecting for drug resistant viruses. Achieving this goal requires more stringent pre-clinical and clinical evaluation of microbicides, focusing on their interactions with the genital tract mucosal environment. The goal of this iterative Program is to develop a combination microbicide delivered via intravaginal ring (IVR) to prevent the sexual transmission of HIV. This will be accomplished either through IVR formulation of a dual-active single agent, a pyrimidinedione, which is both a potent reverse transcriptase and entry inhibitor, or by combining two distinct antiretroviral (ARV) drugs. We will address the importance of the biological synergy between HIV and HSV by also combining acyclovir with ARV drugs to provide local sustained suppression of HSV. The safety of the microbicides and formulations will be explored using novel assays designed to assess their impact on the epithelial barrier and mucosal immunity. Exploratory clinical studies to evaluate the genital tract mucosal environment of Rwandan women will be conducted as defining the healthy genital tract environment will provide crucial information for establishing clinical biomarkers of microbicide safety. Pilot studies to examine the safety and tolerability of three different polyurethane rings will be conducted to identify the optimal composition for formulation of microbicide rings. Results of this study will guide the IVR formulation of our lead drugs, which will be advanced for further safety and regulatory studies including macaque PK and safety studies. A pre-Phase I clinical study is proposed to evaluate PK and safety of our lead IVR formulated microbicide and to determine if the released drug retains its antiviral activity using a spiking strategy. RELEVANCE: These studies will provide crucial information for advancement of safe and effective IVR combination microbicides to prevent HIV and to suppress HSV. These studies will also address critical gaps in microbicide science focusing on the development of sustained delivery systems and safety biomarkers. PROJECT 1: Efficacy &Safety of Multitargeted Combination Microbicides to Prevent HIV &HSV (Herold, B) PROJECT 1 DESCRIPTION (provided by applicant): Topical microbicides, a female-controlled strategy to prevent HIV, are crucial in stemming the pandemic. An optimal microbicide should protect against infection without disrupting the mucosal environment or its mediators of host defense. This project focuses on the preclinical evaluation of combination microbicides with the goal of identifying safe and effective prevention strategies. Combinations that target at least two steps in the HIV life cycle will be prioritized, thus providing protection against circulating resistant viruses and reducing the risk that the drugs will select for resistant variants. This will be accomplished through the dual activities of a single antiretroviral drug (ARV), a pyrimidinedione, which is a potent reverse transcriptase inhibitor and also blocks HIV entry, or by combining two different ARV microbicides. The biological synergy between HIV and HSV will be addressed by evaluating the possibility of delivering acyclovir in combination with the ARV microbicides to provide local sustained suppression of HSV replication. The rationale for focusing on HSV suppression reflects the pre-existing high prevalence of HSV-2 (60-90%) in the developing world and the overwhelming biological and epidemiological evidence demonstrating that subclinical HSV recurrences, which are quite common, increase the risk for HIV acquisition. Critical gaps in microbicide development are the lack of biomarkers predictive of safety and efficacy. This project will address these gaps by developing and expanding novel assays, which focus on the interactions between microbicides and the vaginal environment, using primary cells, explant cultures, and a dual chamber culture system and murine models. The proposed safety models focus on the impact of sustained drug delivery on epithelial integrity and genital tract mucosal immunity. The biological significance of any observed changes will be assessed by examining changes in the ability of HIV to traverse the epithelial barrier in the dual culture model and changes in the susceptibility to genital herpes in the mouse. Results obtained from these studies will provide crucial information for the advancement of novel safe and effective combination microbicides."}
{"text": "A number of self-management procedures will be systematically implemented in representative middle school classes (5th grade through 8th). The basic self-management procedures to be used in the project are self-evaluation, self-determined work requirements, self-recording, and self-scheduling. In addition, a self-control science course for middle school students is being developed. The effects of the self-management procedures on classroom academic performance will be evaluated by both within group designs and comparisons with peer control subject. Student attitudes towards school will be carefully monitored using multiple administrations of likert scales. Finally, the Peers Harris Children's Self-concept Scale. The Self-esteem Invetory, and the Intellectual Achievement Responsiblity Questionaire will be given to participating students and peer controls pre and post treatment. The scores on these scales will be analyzed to determine if the self-management procedures have any desirable effects on student self-concepts."}
{"text": "Flow disturbances in model vascular conduits and anastomoses will be studied under conditions of steady and pulsatile flow, including clinically encountered pulse configurations. The flow field will be analysed using dye injection, hydrogen bubble generation, and laser doppler anemometry. Emphasis will be placed on determining the formation and behavior of the boundary layer and on the origin and structure of flow separation and secondary flow. The introduction into the separated flow of fluid which has been subject to prolonged contact with the conduit wall will be determined. This will provide information on the role of contact-activated blood in downstream anastomotic hyperplasia. The contribution of fluid which has been subjected to high shear prior to entering the downstream secondary and separated flows will also be determined. This will provide information on the role of rheologic platelet activation in downstream anastomotic hyperplasia. The work will also have important implications with regard to the role of fluid dynamics in atherogenesis. Downstream anastomotic hyperplasia will be studied in paired prosthetic grafts in dog carotid arteries. The structure and progress of hyperplasia with time will be compared using dacron as the control with umbilical vein, PTFE, and autogenous vein as the test materials. An additional group of paired grafts to be studied will involve covering the downstream end of the test dacron graft with an onlay vein patch to provide a source of endothelial cells in an attempt to retard hyperplasia. Quantitative histologic analysis of the area of hyperplasia will be performed using digital computer integration of the projected microscopic image. Light, scanning, and transmission electron microscopy will be used to determine the cellular and subcellular structure of hyperplasia and neointima. These studies will provide a valuable basis for comparing commonly used prosthetic graft materials. Clinically relevant information will be gained concerning the fate of downstream anastomotic hyperplasia, the period of greatest risk of graft failure, the timing of antiplatelet aggregant therapy and of post implant angiography and angiodilation."}
{"text": "The research described in this proposal will provide access to tetrodotoxin, a potent neurotoxin widely used in pharmacological studies as a probe for sodium channels. This highly complex molecule will be assembled in optically pure form in a concise manner using a novel synthetic strategy. Furthermore, the proposed synthetic route is flexible enough to permit preparation of natural congeners of tetrodotoxin as well as its nonnatural analogs. The structure-activity relationship study of these derivatives, which cannot be accessed by chemical modification of tetrodotoxin itself, will make possible a better understanding of the structure and the mechanism of action of the sodium channel."}
{"text": "The programs of the SERCEB have immediate and projected requirements for monoclonal antibodies (mAbs) for studies of pathogenesis, immune response, vaccine development, therapeutics and diagnostics. These reagents and bio-recognition elements will be vital to the success of proposed research projects. The nature and source of the monoclonal antibodies required will depend on the objective of the research program, ranging from murine monoclonal antibodies for immunohistochemistry and detection to human or humanized mouse antibodies for development of therapeutics. Broad expertise exists within the institutions of the SERCEB in mAb development, testing and use. The spectrum of expertise in mAb development includes human, mouse, humanized mouse, rhesus, and phage display human and mouse F abs and ScFv. Finally, the antibodies will be deliverables that may have immediate application in therapeutics and diagnostics. The proposed Core C is a distributed core that uses established labs at several institutions to provide each of the needed services. The Core will provide all of the biorecognition elements needed in the SERCEB and will develop them into new assays and biosensors. The core has four Specific Aims. Aim 1. Generate human mAbs to poxvirus neutralization targets and anthrax protective antigen. Aim 2. Generate high-quality mAb reagents for SERCEB research programs. Aim 3. Develop biosensors for diagnostics or detection of select agents using novel mAbs available in the SERCEB Aim 4. To develop a database and repository for mAbs within the SERCEB that can be shared across institutions, to facilitate the other scientific programs."}
{"text": "We are studying the development and function of mucosal M cells and their role in immune surveillance. Our goal is to define the genes and mechanisms involved in the development and function of mucosal M cells. By identifying the critical steps and mechanisms in M cell biology, we will begin to establish their specific role in the mucosal immune response and its ability to mediate mucosal tolerance and the balance with commensal microbes. Our Working Hypothesis in these studies is that specific TNF Superfamily and TNF Receptor Superfamily genes along with coordinated expression of Jagged-1 mediate cellular interactions that specify M cell development and function. We will study two main steps in M cell development, defined by our studies on CD137- deficient mice. The first step is the commitment of M cell lineage progenitors from stem cells, which is dependent in part on ligands for the lymphotoxin receptors and the TNFa receptors. Expression of Jagged-1 by the established M cells may also inhibit generation of M cells from adjacent enterocytes. The second step, functional maturation of M cells, appears to be dependent on interactions between M cells and basolateral pocket B lymphocytes. Here, CD137 (TNFRSF9) and its ligand CD137L, may be an important signaling pair in this interaction. Two specific aims examine these components of our hypothesis: (1) How is M cell lineage commitment and development regulated by Jagged-1/Notch interactions? (2) What are the specific CD137/CD137L cellular interactions regulating M cell basolateral pocket formation and M cell functional development? Regulation of the steady state numbers of M cells in the intestinal mucosa is a dynamic process, and the process depends on an active interplay between crypt stem cells, intestinal epithelium, and lymphocyte subpopulations. This process works in parallel among Peyer's patch, Isolated Lymphoid Follicles, and Villus M cells, and will be shaped by intestinal infection and inflammation (e.g., in Inflammatory Bowel Disease). Thus, a feedback loop exists where M cell transcytosis of lumenal microbes induces mucosal immune activation, which in turn drives production of new M cells. Our studies will provide important details on both the positive and negative regulators of this process. PUBLIC HEALTH RELEVANCE: M cells are a specialized subset of epithelial cells overlying mucosal lymphoid tissues such as intestinal Peyer's patches; they have a unique selective particle transcytosis capability that enables particles as large as a few microns in diameter to cross the epithelial barrier. Thus, M cells play a central role in host-pathogen interactions. Curiously, while the host mucosal immune system relies on this transcytosis function to detect pathogens and induce protective secretory IgA immunity, many pathogenic viruses and bacteria also hijack M cell transcytosis to invade. This paradoxical dual role of M cells in infection and immunity becomes even more complex as infection- and autoimmune disease- induced inflammatory cytokines affect M cell differentiation. Regulation of the steady state numbers of M cells in the intestinal mucosa is a dynamic process, and the process depends on an active interplay between crypt stem cells, intestinal epithelium, and lymphocyte subpopulations. This process works in parallel among Peyer's patch, Isolated Lymphoid Follicles, and Villus M cells, and will be shaped by intestinal infection and inflammation (e.g., in Inflammatory Bowel Disease). Thus, a feedback loop exists where M cell transcytosis of lumenal microbes induces mucosal immune activation, which in turn drives production of new M cells. Our studies have identified a set of genes that appear to regulate M cell lineage commitment (Jagged-1, Notch) and functional development (CD137, CD137L) and interactions between M cells and associated B lymphocytes. The proposed project will provide important details on the role of these genes in both the positive and negative regulation of M cell development."}
{"text": "Time-resolved absorption and fluorescence spectroscopy are used to study the dynamics of protein structural changes subsequent to rapid mixing or excitation with short laser pulses. Kinetic models are used to fit and interpret the measured data. Our recent efforts have focussed on understanding the folding of the villin subdomain and the assembly of amylin. The 35-residue villin subdomain consists of three helices which interact to form a hydrophobic core. Amylin is an extremely hydrophobic 37-residue peptide which is coexpressed with insulin and fibrilized in the islet cells of diabetic patients.[unreadable] [unreadable] To probe the folding mechanism of villin we previously carried out structural and physical-chemical studies of molecules with point mutations and other sequence modifications. These studies have included determination of the structures of some of the folded states by X-ray diffraction carried out in collaboration with Dr. Thang Chiu and David Davies in the LMB/NIDDK. Conservative mutations had no significant effects on the folding rates, suggesting that the transition state for folding of villin is close to the denatured ensemble of conformations. On the other hand, the K24Nle and K29Nle mutations both stabilize the folded structure and accelerate folding by factors of ~2.5, and the double mutant, with a folding time of ~ 700 ns, ranks as the fastest folding protein studied to this time.[unreadable] [unreadable] To further explore the folding of villin we are engineering a variant which includes a pair of probes which undergo fluorescence resonance energy transfer. We have replaced the N-terminal leucine by a cysteine to which the dye Alexa-350 can be attached and the C-teminal phenylalanine at position 35 with naphthylalanine. In the native state these two residues are only about 1 nm apart so the naphthylalanine (donor) is more than 90% quenched by the Alexa-350. In the unfolded state this distance increases to ~4.0 nm and the donor is only ~40% quenched. This pair thus provides a probe of the global unfolding of villin and of the average end-to-end distance in the unfolded state. At this point we know that the folding of this doubly-labelled variant is very similar to that of the variant which we have previously studied. More detailed equilibrium and kinetic characterization is in progress.[unreadable] [unreadable] We have also begun to study the dynamics of the unfolded states of human and rat amylin. The rat sequence, which contains three prolines at positions 25, 28 and 29, does not assemble into fibrils. To do so, we make use of the triplet quenching method developed in our laboratory (Lapidus et al 2000). The C-terminal tyrosine (Y37)is replaced by tryptophan, the triplet excited state of which is quenched by an internal disulfide formed between residues C2 and C7 in both sequences. By measuring loop formation dynamics as a function of the concentration of guanidinium chloride, which solubilizes the these hydrophobic peptides, and temperature we will be able to observe the effects of intramolecular interactions on their chain dynamics. These studies may provide insight into the mechanism of assembly of human amylin into fibrils."}
{"text": "Population-based surveys estimate that the prevalence of methamphetamine (meth) use is 20 times higher among men who have sex with men (MSM) compared to the general population. Meth-associated sexual risk behavior is also a driving force in the MSM HIV epidemic: meth use is consistently associated with high-risk sexual behavior and sexually transmitted diseases, including HIV. Despite these alarming data, relatively few interventions have been tested among meth-using MSM, and no studies have tested the efficacy of pharmacologic interventions in reducing meth use in this population. Pilot studies demonstrate that aripiprazole (Abilify), an FDA-approved, well-tolerated antipsychotic and partial dopamine agonist, reduces the effects of meth in humans, decreases meth craving, and exhibits an excellent safety profile. Partial agonists - - ligands with target receptor affinity but low intrinsic activity - - have already been shown to be effective in treating opiate and nicotine dependence. In response to the compelling evidence supporting aripiprazole and the partial agonist approach, we propose conducting a randomized, double-blind, placebo- controlled trial of intermediate size (60 participants) and length (90 days of follow-up) to assess the efficacy of aripiprazole in reducing meth use among meth-dependent, sexually active MSM. Our specific aims are: 1) To test the hypothesis that aripiprazole 20 mg daily will reduce meth use significantly more than placebo among meth-dependent MSM, as determined by the proportion of meth-negative urines and by self- report of meth use in the aripiprazole versus placebo group. 2) To measure the acceptability of aripiprazole and placebo among meth-dependent MSM, by determining (via electronic pill caps and self-report) medication adherence to aripiprazole and placebo. 3) To measure the safety and tolerability of aripiprazole and placebo among meth-dependent MSM, as determined by the number of adverse clinical events in the aripiprazole and placebo arms. All participants will receive HIV risk-reduction counseling and brief substance use counseling. If promising, we anticipate that study results will be used to design a phase III study to determine if aripiprazole's effects on reducing meth use lead to significant reductions in meth-associated sexual risk and, if the trial sample size is appropriate, HIV incidence. [unreadable] [unreadable] [unreadable]"}
{"text": "This proposal describes a 5 year career development plan for Dr. Pallavi Gopal to serve as a transition to successful independent physician-scientist. Dr. Gopal completed her clinical training in Anatomic Pathology and Neuropathology at the University of Pennsylvania (Penn), and is now developing an independent research and training program that will allow her to gain expertise in spatial and temporal dynamics of mRNA transport under physiological conditions and in disease. This proposal brings together diverse resources in RNA metabolism, molecular neuroscience and neuropathology and will provide superb training for Dr. Gopal to develop into an independent physician-scientist. Research will be performed under the mentorship of Dr. Erika Holzbaur, an internationally recognized expert in microtubule-based motors and real-time axon transport dynamics. This grant will provide protected time for Dr. Gopal to gain expertise in neuronal cytoskeletal, organelle, and RNA-protein dynamics through formal coursework, scientific seminars and meetings. The collaborative environment at Penn will foster utilization of novel techniques to conduct the proposed project and will provide Dr. Gopal with the training required to proceed towards a successful academic career. Amyotrophic lateral sclerosis (ALS) and front temporal lobar degeneration (FTLD) exist on two ends of a clinic pathologic spectrum but share clinical, genetic, and pathologic features. Ubiquitinated cytoplasmic inclusions composed of Tran's active response DNA-binding protein of 43 kDa (TDP-43) are a shared feature of sporadic ALS and the most common form of FTLD; there is concomitant loss of normal nuclear TDP-43 expression. Moreover, the discovery of disease-linked mutations in TDP-43 and other RNA processing proteins highlights altered RNA metabolism as a common pathogenic mechanism of neurodegeneration. However, our knowledge of how TDP-43 mislocalization disrupts its nuclear and cytoplasmic RNA processing functions and/or mediates toxicity in the cytoplasm is still incomplete. The research plan will utilize innovative approaches with real-time imaging techniques in primary neurons to test two main hypotheses: that loss of nuclear TDP-43 results in reduced dynamic flux of TDP-43 target mRNA and proteins in axons and that cytoplasmic redistribution of TDP-43 under pathological conditions results in mislocalization of TDP-43- associated mRNA. The specific aims are to: 1) Determine whether loss of nuclear TDP-43 function reduces axonal trafficking of synaptic proteins and organelle turn over and 2) Determine how (A) loss of cytoplasmic TDP-43 RNA binding function and (B) stress-induced cytoplasmic TDP-43 aggregation affect localization and trafficking of mRNA in axons and dendrites. These studies will provide temporal and spatial resolution of individual RNA transcripts in neurons in order to gain a clearer understanding of altered RNA metabolism in ALS/FTLD pathogenesis."}
{"text": "We have studied the susceptibility of the chicken globin gene in erythrocyte nuclei and the vitellogenin gene in liver nuclei to DNAse I pancreatic digestion. Whereas the globin gene was preferentially digested in erythrocyte nuclei, we were unable to show preferential digestion of the vitellogenin gene in liver nuclei isolated from roosters that were actively synthesizing vitellogenin mRNA."}
{"text": "This modified grant proposal develops methods to address the complexity of modern Pharmacokinetics/Pharmacodynamics (PKPD) population studies focusing on computational PKPD and modeling of complex PKPD systems. AIM 1, Statistical Estimation Methods for Population PK/PD Data, develops improved estimation methods for population PKPD models, and general methods to incorporate patients compliance behavior and uncertain dosing history into PKPD models. AIM 2, Modeling System- PKPD, proposes two specific projects modeling system level PKPD across multiple organs, in particular for Cocaine/Nicotine PKPD and addiction dynamics modeling, and drug absorption physiological modeling. AIM 3, Population Stochastic Differential Equations (SDE), investigates novel approaches to population model building that are based on the use of stochastic differential equation, in particular investigating methodology to include covariates into population PKPD modelsm, and apply SDE to drug-absortion modeling."}
{"text": "The ultimate goal of this proposal is to develop gene therapy for the treatment of hereditary kidney diseases. The carbonic anhydrase (CA) II deficient mouse is an ideal model for kidney-specific gene therapy since it manifests renal tubular acidosis and effects of gene therapy can be easily monitored. CA II is an enzyme present in cells of the kidney and other organs such as the brain and bones, and is important for acid-base homeostasis at both cellular and whole body levels. The investigators have developed a novel approach by infusing the CA II gene, driven by a viral promoter, into the urinary space of the kidney of the CA II deficient mice using liposomes as carriers, and shown a transient (3 week) correction of renal tubular acidosis. During this grant period, new strategies will be tested to improve the efficacy and duration of gene expression using this gene therapy model. Specifically, the goals will be: 1) to add polycations, polymers of positive-charged organic compounds such as basic amino acids, to the liposome delivery system in order to enhance the transfection efficiency in vivo. These polycations may serve as signals for the therapeutic gene to be delivered to the nucleus; 2) to replace the viral promoter of the therapeutic gene with the CA II promoter in order to produce a cell-specific, regulatable, and long-term gene expression in mice; 3) to evaluate the safety of the liposome/polycation-mediated gene therapy in mice; and 4) to determine whether gene therapy in CA II deficient mice will convert the phenotypical changes associated with CA II deficiency, i.e., up-regulation of CA IV, and depletion of intercalated cells, which are responsible for acid secretion in the kidney. It is hoped that results obtained from the proposed study will lead to an optimum gene therapy suitable for future clinical trials on a broad range of renal diseases in humans."}
{"text": "The Gordon Research Conference (GRC) on Cell-Cell Fusion will provide scientists from a wide range of disciplines with the newest research findings on the functions of cell fusion in development in a number of model organisms and in a variety of diseases. The emphasis will be on emerging universal mechanisms and future applications to cancer and stem cell research. This research conference will be the third Cell-Cell Fusion GRC and it is part of over 150 conferences that will be organized in 2011 by the GRC, an organization internationally known for the cutting edge nature of its meetings. Attendance at the meeting has grown steadily since its inception five years ago, and in 2011 the organizers expect to have 140 participants. The 2011 GRC on Cell-Cell Fusion will be held August 7-12, 2011 at the University of New England in Biddeford, ME. The program for the meeting described in this application has been designed around the theme of \"Cell Fusion in Sex, Life, Development, and Disease\". The organizer will deal mostly on the fundamental process of cell fusion during health and disease as well as the relationship between cell fusion and the larger fields of membrane fusion and fission during exocytosis, endocytosis, viral entry, and cytokinesis. The cell fusion community is entering a particularly fascinating phase as the fundamental principles of fusion emerging from theoretical analyses and studies on viral entry and intracellular trafficking start to be applied to understanding the functions and mechanisms of cell-cell fusion in development as well as disease. Important processes such as membrane fusion and fission, egg-sperm fusion, formation of bones, muscles, eye lens, stem cells, placenta, rare genetic diseases, and the invasion of cells by pathogens will be covered in eight sessions. This progress is being helped by classic and new methodology, including high throughput screening, developmental genetics in model organisms (e.g., fungi, flies, worms, and mice), and single-molecule biophysical methods. Exciting developments will be covered by the invited speakers, along with the latest progress on the basic mechanisms and applications of cell membrane fusion in vitro and in vivo. In addition there will be one round table discussion on myoblast fusion, a hotly debated topic that will encourage wide participation and scholarly organized discussions. The first Gordon-Kenan Research Seminar (GRS) on Cell-Cell fusion will be held August 6-7, 2011. This new GRS is organized by graduate students and postdoctoral fellows, and the activities during the GRS will be oriented to junior investigators and are intended to: (1) provide them with the basic background on mechanisms involved in membrane fusion in health and disease necessary to maximize their understanding of the science which will be discussed in the subsequent conference, (2) receive feedback on their ongoing research projects from experts in the field, and (3) facilitate their interaction with senior members of this scientific community to promote networking between junior and more senior researchers in the field. It is fully anticipate that the scientific discussions, research talks, poster sessions, and other informal interactions among the participants of this conference will contribute to advancing our understanding of novel molecular mechanisms involved in cell fusion during normal development as well as in human diseases, and will set the basis for the development of multidisciplinary projects aimed at discovering new treatments for these disorders. NARRATIVE: The Cell-Cell Fusion Gordon Research Conference (GRC) together with the first sister Gordon-Kenan Research Seminar (GRS) will bring leaders in diverse scientific disciplines together with graduate students and postdoctoral fellows constituting the future generation of researchers in the emerging field of cell fusion. The scientific presentations, discussions, round table discussions, as well as workshops during the GRC and GRS conferences on cell-cell fusion are tailored to expand our knowledge on the mechanisms by which membranes fuse in model organisms, and may contribute to our understanding of the first events in sexual development, the formation of many organs, cancer, stem cell biology, and rare diseases. The exciting, friendly, and multidisciplinary atmosphere that has traditionally characterized the Cell-Cell Fusion GRC will provide the ideal setting for the intellectual development and implementation of groundbreaking approaches to treat some cancers, as well as use stem cells to address fundamental biological questions and diseases."}
{"text": "Physical activity is crucial in maintaining health. Understanding how physical activity impacts pelvic floor disorders is important: half of American women have urinary incontinence or pelvic organ prolapse and one in nine undergoes surgery for these conditions. Studies to date find both protective and harmful effects of physical activity on pelvic floor disorders. The investigators, a multi-disciplinary team of urogynecologists, exercise scientists and statistician, believe this discrepancy is in large part due to inadequate ascertainment of both physical activity data and pelvic floor outcome measures. The primary aims of this study, addressed in two carefully designed case-control studies, are to determine, after adjusting for confounders, whether 1) moderate to severe stress urinary incontinence, defined by a validated questionnaire, and 2) pelvic organ prolapse, defined by structured pelvic examination, are associated with increased or decreased a) current leisure activity and b) total lifetime activity (leisure, household, and occupational). Participants will include 650 women, largely from primary care clinics: 175 with moderate to severe stress urinary incontinence, 175 with pelvic organ prolapse and 300 controls with neither condition frequency matched for age, body mass index and site. A secondary aim of this study is to establish, from the population screened for these studies, a registry of approximately 1,500 women with neither prolapse nor urinary incontinence; this will provide a foundation for future studies on the incidence of these conditions. Physical activity will be measured using validated questionnaires designed to assess current and cumulative lifetime activity in women; activity items will be supplemented with additional loading and impact physical activities. Lifetime physical activity amongst women with and without each condition will be compared using both MET hours/week (the most widely used and traditionally accepted method) and strenuous activity hours/week. PUBLIC HEALTH RELEVANCE: Pelvic floor disorders including urinary leakage and pelvic organ prolapse (dropping of pelvic organs such as the uterus into or out of the vagina) are very common. Limited data to date suggest that physical activity may be both helpful and harmful in terms of its effect on the pelvic floor. We will study the long-term effects of different levels of activity on pelvic floor disorders. Given the health benefits of activity, we believe that women should be encouraged to be active unless there is scientific evidence to the contrary."}
{"text": "Ocular immune privilege is maintained in part by the induction of systemic regulatory T cells mediated by events that are initiated after the introduction of antigen into the Anterior Chamber. This potent (ocular) response specifically preempts the systemic induction of tissue-damaging cell-mediated immunity, some complement fixing antibodies and atopic antibodies. Accordingly, we suggest that regulatory T cells induced by intracameral antigen could be used to selectively regulate systemic autoreactive T cells. Most investigations on the induction of regulatory T cells in autoimmunity do not discriminate between the regulation of the induction of an autoimmune response and the regulation of T cells that effect autoimmunity during on-going disease. Preliminary studies by our laboratories have demonstrated that the injection of myelin basic protein or myelin oligodendrocyte glycoprotein into an anterior chamber of mice immunized to these myelin proteins induces the production of antigen-specific regulatory T cells that suppress delayed- type hypersensitivity and/or experimental allergic encephalomyelitis (EAE) effected by an encephalitogenic T cell clone. Therefore we propose that the injection of myelin proteins into an anterior chamber of mice induces T cells that specifically regulate the induction of EAE by EAE effector T cells and may modulate on-going disease. We will test this hypothesis by raising antigen-specific splenic regulatory T cells by the injection of myelin oligodendrocyte glycoprotein into an anterior chamber and (1) Test the hypothesis that antigen-specific regulatory T cells that effect and/or induce the suppression of an encephalitogenic T cells are produced (but not necessarily activated) during an immune response to the autoantigen. This Aim will also test the hypothesis that inactive regulatory T cells are produced during an immune response to autoantigen and how these cells may be activated in vivo to prevent autoimmunity and/or eliminate an on-going autoimmune response;(2) Determine the mechanism(s) of cell-mediated suppression of EAE induced by encephalitogenic effector T cells. We will test the hypothesis that the suppression of encephalitogenic effector T cells by antigen-specific, regulatory T cells is mediated by cytokines that induce the regulatory T cells or effect suppression. The results of this study could aid our understanding of the systemic basis for the ocular regulation of autoimmunity and develop procedures for the modulation of active autoimmune disease."}
{"text": "The objectives of this undertaking are to originate new epidemiologic approaches to the study of cancer causation in man and animals, to develop sources of data related to specific epidemiologic problems, and to stimulate epidemiologic research in other health agencies. Opportunities for research in cancer epidemiology are based on questions arising from clinical or laboratory observations, on unusual groupings of cancer cases in the population, and on study of the characteristics of groups of persons prior to the onset of a specific type of cancer as compared with similar persons who have not developed the disease."}
{"text": "Dr. Diane Elliot and associates are doing a study entitled \"School-Based Drug Use Prevention for Girl Athletes\". Subjects will be girls involved in a sports team at school. The purpose of this study is to 1) determine student's knowledge about the effects of weight loss/physique altering drug use, and unhealthy exercise and nutrition habits; 2) measure drug use, 3) gain information about nutrition and exercise habits among female students-athletes; and 4) understand factors that influence drug use and unhealthy eating and exercise habits."}
{"text": "Completed CRC study. Used CDMAS only."}
{"text": "This proposal is to assess the behavioral predictors of weight gain and the development of overweight and obesity in adolescence and early adulthood in the National Longitudinal Study of Adolescent Health (Add Health). In cross-sectional studies researchers have observed a strong association between dieting and being overweight or obese. The cross-sectional association between dieting and weight status may be due to overweight individuals being more likely than their normal weight peers to go on diets to lose weight. Since most dieting efforts are not successful, or at least not successfully maintained, overweight individuals may remain overweight despite, not because of, their dieting attempts. Only prospective analyses can help to determine whether dieting leads to great weight gain or is simply an ineffective strategy to lose and maintain weight. Add Health is a school-based study of adolescents from 80 high schools and 52 middle schools from the U.S. This sample is representative of U.S. schools with respect to region of country, urban/city, school type, ethnicity, and school size. We seek to analyze data from Wave I (1995-1996), Wave II (1996), and Wave III (2001-2002) to assess whether weight change between 1995 and 2001 is predicted by weight change goals (e.g., desire to lose, maintain, or gain weight) or weight control practices in 1995 and 1996. Specifically we will assess whether the development of overweight and obesity is predicted by dieting, using diet pills, self-inducing vomiting, and/or using laxatives to lose weight. In addition, we will investigate whether adolescents who report using exercise to maintain their weight will gain less weight than their peers not using exercise as a weight control behavior. We will also investigate the weight change patterns of adolescents who report that they are trying to gain weight. We will assess whether self-reported use of dieting, exercise, or weight lifting to gain weight or build muscle predicts weight change over a six-year period. We will assess whether these associations exist and whether they vary by gender and/or race/ethnic group. This study will have good power to detect moderate associations (e.g., relative risk >= 1.6). Add Health is the only large prospective study of adolescents with enough males and non-white subjects to assess whether predictors vary by or are modified by gender and/or race/ethnicity, therefore our results should make an important contribution to the field."}
{"text": "The majority of adolescents struggling to avoid substance use during early recovery are also coping with many other issues (e.g., peer relationships) for which they require consistent support. We propose to develop an online relapse prevention program for adolescents called Staying Off Substances (SOS). Specific content in the program will be recommended to counselors based on their client's a) age, b) current motivation level, and, when available, c) reported needs and strengths on the Comprehensive Health Assessment for Teens (CHAT(R)), a Web-enabled interactive adolescent self-report assessment for use in treatment settings. Whereas current online programs for adolescents are geared for primary prevention, CHAT(R): SOS will be an intervention for use in clinical populations. Counselors will assign the client's user name to access privileges for that content and/or other content per their clinical judgment. We expect that CHAT(R): SOS will ultimately have six modules corresponding with the CHAT(R) subscales (i.e., Psychological Health, Family Relationships, Peer Relationships, Tobacco Use, Alcohol Use, Drug Use). CHAT(R): SOS will include opportunities to reinforce the skills and philosophies that adolescents learn in treatment through online interactive exercises, writing activities, and access to a supportive, online community. This resource will be available to them during treatment and post-treatment to help them navigate challenges in early recovery. Adolescents will receive tailored recommendations and an individualized user experience because CHAT(R): SOS will utilize an innovative content management and logic solution called Ektron. CHAT(R): SOS will also provide counselors with guidelines on how to use this program with their clients (e.g., in sessions, during aftercare) and custom user reports. To demonstrate the feasibility of the program concept, the Phase I study focused on the Peer Relationships domain. All feasibility criteria were met and findings indicated: (1) Proposed content was based on highly positive findings from concept mapping, as well as usability and acceptance testing with clients and experts. Ratings of potential effectiveness and appeal were high. (2) Usability testing indicated that the program was usable by the target audience, the skills and outcomes were relevant to the target audience, and the program was regarded as potentially very helpful to the treatment process. We surpassed feasibility benchmarks for both clients and experts. (3) Our technical/design team a) produced a demonstration program that was perceived by key stakeholders as highly usable and engaging multimedia program and b) determined the necessary technologies to produce the complete program in Phase II. In Phase II we will develop the program then test its efficacy in a field trial. The field trial will examine the primary hypotheses that, relative to the control condition (CHAT(R) only), CHAT(R): SOS will be associated with significantly higher motivation, higher self-efficacy, more relapse coping, and lower substance use. PUBLIC HEALTH RELEVANCE: Adolescent substance abuse is a significant public health issue. Unfortunately, the 2005 federal Treatment Episode Data Set indicated that less than 150,000 adolescents received treatment. Typically, 75% of those who receive treatment relapse within the following year. To address the need for cost-effective assessment, Inflexxion, Inc. developed the Comprehensive Health Assessment for Teens (CHAT(R)), a Web-enabled interactive self report assessment for use in treatment settings. We propose developing a supplemental online relapse prevention program called CHAT(R): Staying Off Substances (SOS). CHAT: SOS will use an adolescent's 1) age, 2) motivational level, and, when available, 3) responses to the CHAT(R) assessment to recommend Web-based relapse prevention content to address the adolescent's individual needs. This resource will be available to adolescents during treatment and post-treatment to help them navigate challenges in early recovery."}
{"text": "This project addresses the NHLBI RC2 GO application entitled \"Characterizing Differentiated Heart, Lung, and Blood Cells Derived by Reprogramming Human Embryonic and Induced Pluripotent Stem Cells.\" Emerging technologies to generate induced pluripotent stem cells (iPSCs) by reprogramming human somatic cells promises to revolutionize biomedical research and clinical medicine. Through in vitro culture methods, iPSCs can be differentiated into numerous cells types derived from all three germ layers. This raises the possibility that patient-derived iPSCs can be used to create relevant tissues for the study of many human disorders. In addition, iPSCs may provide starting material to manufacture transplantable cells for transfusion and regenerative therapies. However, the field is in its infancy and many core questions must be solved in order to realize these exciting long-term prospects. This proposal seeks to advance the use of iPSCs for the study of normal and pathological hematopoiesis. Multiple investigators with broad areas of expertise in hematopoiesis, embryonic stem cell/iPSC biology, chromatin biology, clinical hematology, bioinformatics, cell banking and bioethics/regulatory affairs will work together to pursue the following global issues 1) Mechanisms by which hematopoietic developmental potential might vary between different normal iPSC clones;2) The extent to which iPSC-derived hematopoietic precursors resemble normal ones with respect to cellular phenotypes, gene expression and epigenetic signatures;3) Whether hematopoietic disease phenotypes can be recapitulated by in vitro manipulation of patient-derived iPSCs. We will execute these studies using novel methods to create and culture iPSCs and state-of-the art tools to analyze and manipulate their resident genomes. Pursuit of these problems will serve as a framework in which to develop a facile infrastructure where investigators at our large pediatric institution can create, analyze, bank and distribute iPSCs from any patient of interest. If successful, this work will help to accelerate practical applications of iPSCs for the study and treatment of human diseases. This work will be based at Children's Hospital of Philadelphia with subcontracts to The Pennsylvania State University (State College, PA) and The Coriell Institute for Medical Research (Camden, NJ). The project will create 6 new jobs, thereby stimulating the economy in three different regions of the Northeastern United States. PUBLIC HEALTH RELEVANCE: Efforts to better understand blood production from patient-derived induced pluripotent stem cells (iPSCs) will enhance our understanding of blood disorders and generate new therapeutic approaches. Additionally, this work could create new general paradigms for studying the genesis of many normal tissues and their associated diseases."}
{"text": "Legionella pneumophila is a gram-negative bacteria that utilizes a type IV secretion system to alter its intracellular trafficking in host macrophages. Internalized Legionella containing vacuoles (LCV) immediately avoid transport to lysosomes and within 15 minutes of internalization, begin remodeling their phagosomal compartment with ER-derived host cell vesicles. Dot/icm mutants are efficiently transported to and fuse with lysosomes suggesting that the type IV secretion apparatus is required for altered LCV trafficking and remodeling. Identification of factors utilized by Legionella to alter trafficking and remodel its intracellular vacuole may provide new targets for therapeutic intervention. Furthermore, understanding how ER-derived vesicles are recruited to and fuse with a membrane-bound compartment originating from the plasma membrane may provide insight into novel host cell functions. Confocal microscopy, coimmunprecipitation studies, 2D-gel electrophoresis and in vitro budding and fusion reactions will be used to identify host and/or Legionella derived SNARE-like molecules that play a role in ER-derived vesicle fusion with LCV."}
{"text": "General rationale and organization of the Cores The main potential technical barriers to efficient execution of the biology are (1) the quality control and execution of the 3-D culture system, and (2) the requirement for expertise and ready access to state of the art confocal microscopy. To make these essential technologies readily available to everybody, and to coordinate the efforts of the various groups, we propose two core facilities, one to coordinate the 3-D culture methodology (3-D Core), and another to manage the confocal microscope (Confocal Core). Although the 3- D Cell Culture and Microscope Cores will be described separately, they will be located in adjacent labs and are designed to work together to standardize data collection procedures and provide a seamless transition from the 3-D culture phase to the confocal analysis phase of each study."}
{"text": "The focus of this proposal is on the mechanism of host lysis by bacteriophage. Building on recent progress, it is proposed to investigate the biochemical and genetic properties of holins, small phage-encoded membrane proteins that act as the timers of phage infections. Holins have the remarkable ability to accumulate during the phage infective cycle without harming the cell, then suddenly triggering to permeabilize the membrane. This terminates the infection and activates muralytic enzymes called endolysins, or lysozymes, resulting in degradation of the cell wall, leading to bursting of the cell and release of the progeny virions. The work is aimed at determining how these proteins can form holes in membranes, and how the scheduling of the hole-forming event is programmed into the sequence of the holin. Fundamental issues of lipid-protein and protein-protein interactions in membranes will be addressed, including an investigation of how integral membrane domains of some lysis proteins actually are able to exit the membrane upon physiological cues. [unreadable] [unreadable] The holin-endolysin mode is completely general for all phages except those with very small genomes. However, single-stranded DMA and RNA phages, limited to 3 - 10 genes for their entire genome complement, accomplish host lysis by expressing single genes. In two of these cases, recent progress has shown that the phage lysis protein causes lysis by inhibiting different enzymes in the murein precursor biosynthetic pathway. It is proposed to investigate the molecular basis by which these \"protein antibiotics\" effect inhibition of these conserved enzymes. Other small single-stranded RNA phages effect lysis by an unknown mechanism, the elucidation of which is another goal of this project. [unreadable] [unreadable] Public health implications: These studies are critical to our understanding of how bacterial viruses, or phages, kill their prey and effect dispersal of their progeny. This may have direct practical benefits because there is a growing consensus that phages, as natural antibacterial agents, will become an important tool in combating bacterial pathogens, which are increasingly resistant to available antibiotics. In addition, the research may reveal new modes for design of chemical antibiotics. [unreadable] [unreadable]"}
{"text": "Despite advances in resuscitation of trauma victims, a large number of such patients subsequently die of sepsis and multiple organ failure. Improvement in survival of such victims should, however, be possible. Our hypothesis is that the generation of toxic substances and the various metabolic and microcirculatory alterations produced after severe injury are not adequately corrected by resuscitation with conventional fluids alone, and that such alterations eventually lead to organ failure. Although numerous attempts at overcoming these problems have been made, including the use of hypertonic solutions, substrates, inotropes and other agents, there has not been a systematic investigation of the specific needs and interdependance between the heart, liver, and kidney following low flow states. Low flow produces an ischemic environment resulting in depletion of tissue energy stores, cellular electrolyte imbalance, generation of toxic substances, cell swelling and eventually microcirculatory and organ failure. Our plans are to determine the specific needs of the organs which are not met with volume resuscitation alone, and to then correct those abnormalities by pharmacologic means. Organ blood flow and function following injury may be better maintained by providing osmotic and oncotic agents along with metabolic support and agents that improve microcirculatory flow and inhibit the generation of toxic substances. We plan to develop a pharmacologic solution (i.e., composite of pharmacologic agents which will act synergistically) which, together with appropriate volume replacement, will maintain cell and organ function and host defense mechanisms after injury better than conventional solutions alone, and thus prevent subsequent multiple systems failure. Such a solution (osmolarity approximately 600 mOsm) will initially contain dextrose, albumin and electrolytes. To this solution we will systematically add dopamine, diltiazem, imidazole and ATP-MgC12 individually and in various combinations. In this manner, we will evaluate the action of each of the above agents individually and their interaction with each other. Such an approach should yield an optimal combination to produce synergistic beneficial effects on cell and organ function following injury, and produce resistance to a subsequent septic challenge. If these studies uncover a lack of improvement in any cell, organ or system function, the constituents of the pharmacologic solution will be modified to specifically address that problem. To accomplish these goals we will utilize a rat hemorrhage model."}
{"text": "While tremendous advances in pharmacotherapy for adults have been achieved in recent years, expanding the benefits of these new therapies to infants and children remains a significant challenge. Important developmental changes in pharmacokinetics, pharmacodynamics, disease presentation and progression all impede direct translation of adult therapeutics into pediatrics. The discipline of pediatric clinical pharmacolog provides necessary training in developmental physiology to leverage existing knowledge and guide rational therapeutics for infants and children. However, the current pool of pharmacologists and pediatric sub- specialists with formal training in clinical pharmacology is both small and aging. This T32 Fellowship Program led by the Department of Pediatrics at the University of California, San Diego (UCSD), entitled Developing Pediatric Clinical Pharmacologists for the Advancement of Therapeutics (DPCPAT), will provide cutting-edge research training and mentorship in pediatric clinical pharmacology, enabling our trainees to emerge as leaders among the ranks of pediatric academic investigators. The biomedical research community at UCSD has a long- established reputation of excellence, and now ranks in the top 5 in NIH research funding of all US institutions. Notably, the last 5-10 years have witnessed an impressive academic expansion within the UCSD Department of Pediatrics and its research mission, coupled with the solidification of the synergistic relationship among Rady Children's Hospital San Diego (RCHSD) and the UCSD Schools of Medicine and Pharmacy. We will provide ample evidence that the timing for creation of the DPCPAT training program at UCSD has never been better, and that our research training environment and faculty are truly world class by any measure. The overarching goal of the DPCPAT program will be to prepare Pediatric Physician-Scientists for the lifelong challenges of a sustained, independent and highly productive research. Dr. David Brenner, Dean and Vice Chancellor for Health Sciences, Dr. Jack Dixon, Associate Vice Chancellor for Scientific Affairs, Dr. Gabriel Haddad, Chair of the Department of Pediatrics and Dr. James McKerrow, Dean of the School of Pharmacy, have provided letters of UCSD institutional commitment to our proposed DPCPAT, confirming the central importance of this Program to the academic mission of the UCSD School of Medicine. The key objectives of our DPCAT are as follows: (1) To increase the number Pediatric clinical pharmacologists and Clinician-Scientists engaged in clinical pharmacology research as applied to child health; (2) to attract outstanding young pediatric clinicians to UCSD and to expand their translational capacity through clinical pharmacology methods; (3) to facilitate career development of DPCAT trainees under the guidance of world class, established investigator-faculty mentors; and (4) to cultivate the early careers of women and minority investigators in pediatric therapeutics. With our impressive group of mentors with outstanding training track records, UCSD DPCPAT Fellows will be poised to be tomorrow's leaders in pediatric clinical pharmacology."}
{"text": "The main objective of this proposal is to analyze the molecular mechanisms by which retinoic acid inhibits mouse skin tumor promoter 12-)-tetradecanoylphorbol-13-acetate (TPA) induction of epidermal ornithine decarboxylase (ODC), a biochemical change associated with skin tumor promotion by TPA. We first demonstrated that the mechanism by which certain retinoids inhibit skin tumor promotion is due to their ability to inhabit it the induction of ODC by TPA. However, the nature of retinoic acid inhibition of ODC induction by TPA remained undefined. Recently, we found that retinoic acid inhibits TPA-caused synthesis of ODC protein. Retinoic acid may inhibit the synthesis of ODC protein at the transcriptional and/or translational levels. In a preliminary experiment, we found retinoic acid inhibits TPA-increased level of ODC RNA. Thus, we hypothesize that TPA-induce ODC activity (peaks at 6 hr and approximately 200-fold above control) is the result of increased synthesis of ODC protein at the transcriptional level and retinoic acid inhibits ODC induction by inhibiting TPA-increased ODC messenger. Specifically, we plan to investigate: 1) a correlation among TPA-induced epidermal ODC activity, ODC protein, rate of ODC synthesis, and ODC mRNA levels, 2) whether retinoic acid inhibition of TPA-induced ODC activity is proportional to the inhibition of ODC protein and ODC mRNA levels, 3) the mechanism of inhibition by retinoic acid of ODC messenger. We will determine whether TPA causes amplification of ODC gene(s) and retinoic acid inhibits this process. Experiments will be performed with CD-1 mice. Total RNA from skin will be isolated by urea extraction and CsC1 gradient centrifugation. Poly(A)+-contining mRNA will be fractionated by affinity chromatography on an agarose poly(U) column. ODC mRNA will be quantitated by solution hybridization using [32p]-labeled single stranded probe and Dot-blot analysis using radiolabeled cDNA probe. Size of ODC mRNA transcripts will be determined by Northern blotting. Mouse skin genomic DNA will be analyzed by Southern blotting. We will analyze both hybridizable and translatable ODC mRNA; translatable ODC mRNA will be determined by translation in a reticulocyte lysate cell-free syftem. Amount of ODC protein will be analyzed by the radioimmunoassay using [3H] difluoromethylornithine-labeled ODC. These data will permit an evaluation of the role of transcription in the regulation of ODC. These data will permit an evaluation of the role of transcription in the regulation of ODC induction by TPA and whether retinoic acid inhibits TPA induction of ODC at the transcriptional level."}
{"text": "Integrins are critical components of the immune system through their activities in directing lymphocyte development, movement and activation. The function of the b2-integrin CD11c in vivo is largely unexplored. Our data show that CD11c is induced on primary and memory CD8 T cells responding to infection. Moreover, in the absence of CD11c, the CD8 T cell response is severely diminished indicating an integral costimulatory role for CD11c in driving CD8 T cell activation. The studies proposed here will test the hypothesis that CD11c controls CD8 T cell response by promoting T cell- DC interactions early after activation. Three specific aims are proposed to analyze the mechanisms of CD11c costimulation: Specific Aim 1. To determine the role of CD11c in the early CD8 T cell response to infection. Using bacterial and viral infections in conjunction with CD11c-deficient CD8 T cells, the role of CD11c in promoting proliferation, differentiation and survival of CD8 T cells will be tested. Specific Aim 2. To analyze the role of CD11c expressed by CD8 T cells in determining the anatomy of the immune response to infection. Our preliminary results using in situ MHC class I tetramer staining identified a series of stepwise events that drive CD8 T cell activation in response to infection with Listeria monocytogenes. In situ analysis using confocal microscopy will be performed to assess the role of CD11c in activation and movement of responding endogenous and adoptively transferred CD8 T cells. Specific Aim 3. To analyze the role of CD11c in development and function of dendritic cells. The high level expression of CD11c by DC strongly suggests an important role for CD11c in DC function. However, the analysis of CD11c-deficient DC is complicated due to the lack of a reliable marker for DC identification. We have produced transgenic mice expressing a fluorescent reporter protein under control of the CD11c promoter to circumvent this issue. These mice, when rendered CD11c-deficient, will provide the means to track and analyze CD11c-/- DC under conditions of homeostasis or infection. In sum, these studies will provide the first analysis of the role of CD11c in CD8 T cell and DC activation. PUBLIC HEALTH RELEVENCE: This proposal is focused on understanding the role of a specialized adhesion molecule, the integrin CD11c, in CD8 T cell activation in response to a bacterial infection. CD8 T cells are cytotoxic lymphocytes involved in killing of infected cells and play a central role in protection against intracellular bacterial infections. Therefore, this research has substantial impact on understanding immunity to infection and holds implications for design of effective vaccines."}
{"text": "Hospice is a key model of home care for seriously ill individuals and those with dementia represent the fastest growing group of hospice users. Hospices support patients and caregivers through interdisciplinary care with the goal of enabling individuals to remain at home at the end of life. Living in the community in the last phase of life for those with dementia has increased in the last two decades. Although site of death for decedents with dementia in both 2000 and 2015 was predominantly a nursing home, the proportion dying at home in the community almost doubled from 15% in 2000 to 26% in 2016. Community residential alternatives to long term care in nursing homes, such as assisted living facilities, are becoming more prevalent and are considered a positive phenomenon for promoting independence and improving quality of life for those with dementia. Yet research on end of life care for those with dementia is primarily focused on the nursing home setting and little is known about characteristics of those with dementia who remain in the community and the types of supportive services (e.g., personal care, medication assistance, meal preparation, transportation) that they receive. Also unknown is the extent to which hospice is added as an extra layer of support for those with dementia and their families who remain in the community. To address these gaps in knowledge, we propose a supplement to our geriatric palliative care focused Claude D. Pepper Older Adult Independent Center. We will employ the Medicare Current Beneficiary Survey, a nationally representative panel survey of Medicare beneficiaries linked to Medicare administrative data and regional characteristics, which provides a unique opportunity to examine those with dementia living in a range of settings including private residences, assisted living facilities, retirement homes, continuing care communities, and nursing homes, with a range of supportive services (e.g., personal care, medication assistance, meal preparation, transportation). We will determine patient, family, and regional characteristics associated with residential setting and associated with hospice enrollment across residential settings for those with dementia. We will also describe the trajectory of transitions across residential settings at the end of life for those with dementia. The proposed research will lay the foundation for a program of research regarding hospice use in the context of dementia and across residential care settings leading to a more comprehensive picture of the consequences of the increase in home care for those with dementia at the end of life."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This event is held on demand during the summer months, if some students cannot attend the NBCR summer institute due to schedule conflict.."}
{"text": "This K award will allow me to develop as an independent investigator, focusing on the role that paternal factors play in the risk of schizophrenia in offspring, specifically paternal reproductive health. The proposed program includes research and training in the translation of findings from (1) observational studies into basic experiments through collaborative efforts using animal models and (2) from experimental data into testable hypotheses using epidemiology. The training activities build on my prior experience in psychiatric epidemiology and toxicology, including coursework and research training in neuroscience and experimental animal studies. The skills that I acquire will be directly applicable to the study of many kinds of exposures relevant to schizophrenia. The research program described in this application is based on new findings that prenatal and pre-conceptual exposure that affect paternal reproductive health, including advanced paternal age and exposure to lead (Pb), a known toxin may increase susceptibility to schizophrenia and other psychotic disorders in offspring. Lead exposure results in reductions in fertility in both men and women, and is associated with behavioral abnormalities in animal models of paternal exposure. Two phases of research are proposed that will involve parallel career development activities in both training and research: (I) studies of behavioral phenotypes congruent with schizophrenia using mouse models of paternal lead exposure and advanced paternal age and (II) epidemiologic studies of paternal Pb exposure and SSD. At the conclusion of this award I will design of new collaborative programs, incorporating lifecourse epidemiology and basic research with the goal of submitting an R01 application."}
{"text": "The long-term goal of our work is to understand the mechanism of transcriptional regulation by steroid hormone receptors and their dysregulation during disease. We seek to reveal how specific coactivators and corepressors translate the hormonal signals into promoter-dependent transcriptional outputs. Our work centers on the mechanism of gene activation by the glucocorticoid receptor (GR). GR harbors two major transcriptional activation functions (AF1 and AF2) that are differentially utilized in individual promoter and cellular contexts. In contrast to the C-terminal AF2 function, little is known regarding the coregulators that interact with, and mediate the functions of the N-terminal AF1. This region plays key roles in important therapeutic effects of glucocorticoids such as the induction of thymocyte apoptosis. We recently generated specific mutations in AF1 that pinpoint the critical residues responsible for function. We have used them in complementary approaches to discover AF1 coregulators. In a genetic approach, we isolated two extragenic suppressor yeast strains where the function of deficient AF1 mutants is restored. The genes responsible are likely to encode conserved factors intimately involved in AF1 function. Through an independent functional approach, we identified the 150kDa subunit of the DRIP/TRAP/Mediator complex as a protein that interacts with a transcriptionally competent AF1. In Aim 1, we propose to define the nature and structural basis for the interaction of DRIP 150 with GR AF1. We plan to map the minimal regions necessary in each protein and identify key residues through random and targeted mutagenesis coupled with screening in yeast. We will explore the structural basis of the interaction through a biophysical characterization of the complex. In Aim 2, we will ascertain the functional consequences of AF1 binding to DRIP 150 and define the effector regions of DRIP 150 involved in transcriptional activation. Using multiple approaches, we will test the hypothesis that AF1 recruitment of the DRIP complex determines whether GR activation of a given promoter requires participation of AF1. In Aim 3, we propose to identify novel conserved AF1 coregulators by cloning the genes responsible for the extragenic suppression in the yeast strains we have isolated. We will then identify mammalian counterparts and begin to ascertain their role in AF1 function using some of the assays outlined in Aims 1 and 2."}
{"text": "The pathogenesis of asthma results from interactions between the innate (Project 1) and adaptive (Project 3) immune systems. A subset of immune cells, regulatory CD4+ T cells, play an important role in suppression of both innate and adaptive responses. Although there are indications that regulatory T cells participate in asthma, much remains to be learned before therapeutic interventions can be entertained. The goal of the first part of this Project is to better define the role of complement regulator (CD46) activated human T cells, which appear to represent a unique adaptive regulatory T cell subset. In Aim 1, we will determine if complement activation plays an important role in asthma, as well as providing a physiologic in vivo stimulus for these adaptive regulatory T cells. We will ask if genetic deficiency in various complement components (beginning with C3) modulates the Sendai virus-induced acute infection and the chronic asthma phenotype in mice (as described in Project 1). We will also determine whether markers of altered complement activation occur in situ in human patients with asthma. In Aim 2, we will look for the presence of T cells with the CD46 adaptive regulatory T cell phenotype in human patients with asthma. We will also determine if human CD46 adaptive regulatory T cells inhibit the function of pathogenic cells in asthma (Th2 cells and B cells). If so, this would argue for further studies into the use of these adaptive regulatory cells for the treatment of asthma. In the second part of this Project, we will complement the above studies on adaptive regulatory T cells and define the role of natural regulatory T cells in a mouse model of asthma. By moving to this model, we can utilize a newly described marker Foxp3-GFP for natural regulatory T cells to facilitate studies of natural regulatory T cell specificity. In summary, our goal is to further address the role of the complement system in asthma and whether adaptive or natural regulatory T cells participate in controlling the severity of asthma. Both avenues represent attractive therapeutic targets for the treatment of asthma. Asthma is a growing problem in the United States, but current therapies are expensive, chronic, and, unfortunately not curative. By exploring the body's innate immune system and natural regulatory mechanisms that inhibit excessive pathology, our hope is that curative therapies can eventually be generated not requiring global immunosuppression."}
{"text": "M. tuberculosis is the world's leading cause of death from a single infectious agent and the leading cause of death in AIDS patients. The emergence of multidrug resistant tuberculosis poses a major threat to the public health, giving new urgency to research aimed at combating this ancient scourge. Moreover, multidrug resistant strains of M. tuberculosis (MDRTB) are a potential weapon of bioterrorism, and such strains have been classified as NIAID/CDC Category C Bioterrorism Agents. Studies proposed in this grant application build upon advances made in collaborative efforts between the Horwitz laboratory at UCLA and the Griffith laboratory at the U. of Wisconsin over the past two years to develop novel antimicrobials against M. tuberculosis for treatment of drug resistant organisms. During the past several years, the Horwitz and Griffith laboratories have laid the groundwork for the development of a new antimicrobial strategy against M. tuberculosis - targeting M. tuberculosis glutamine synthetase (GS). Thus, Horwitz et al. demonstrated that M. tuberculosis GS is a promising antimicrobial target, and that the high production of this enzyme is correlated with pathogenicity in mycobacteria and with the presence of a poly-L-glutamate/glutamine structure in the cell wall of pathogenic mycobacteria. Horwitz et al. showed further that inhibition of GS with L-methionine-SR-sulfoximine (MSO) inhibits M. tuberculosis growth in cell-free culture, in human macrophages, and in vivo in guinea pigs challenged by aerosol with M. tuberculosis. In combination with ascorbate, MSO is almost as potent as isoniazid, the leading antituberculous drug. Importantly, working in a collaboration during the past year, the Horwitz and Griffith laboratories have identified analogs of MSO that are highly potent against M. tuberculosis in vitro but lack certain drawbacks of MSO. This application has two major goals: 1) Develop novel MSO analogs that are better drug candidates than MSO because they are a) more selective for glutamine synthetase (GS) vs gamma-glutamylcysteine synthetase (gamma -GCS); and/or b) less well taken up into the brain where MSO exerts its major toxicity in sensitive species; and/or c) even more selective for M. tuberculosis GS vs. mammalian GS, and test the toxicity of the analogs in mice. 2) Test the novel MSO analogs for their capacity to inhibit M. tuberculosis growth in broth culture, in human macrophages, and in guinea pigs."}
{"text": "The Core Support Grant is in support of the organizational structure of the Cancer Center of Hawaii. The grant supports leadership personnel and key investigators in the Center's five departments of Basic Sciences, Clinical Sciences, Epidemiology, Data Resources and Cancer Control. The grant also supports shared resources which consists of computer facility, animal facility, tumor cell bank and a carcinogen biohazard laboratory. Epidemiological research is being conducted at the Center in the multi-ethnic population of Hawaii. Case control studies on diet and breast cancer, trace metals in prostate, role of vitamin A in lung cancer and the mechanism of action of nitrosoamines in gastric cancer are just a few of the many projects funded at the Center."}
{"text": "Catecholamine regulation of vasopressin in hypertension. The major aim of this proposal is to examine the role of catecholamine regulation of vasopressin in the supraoptic nucleus on the development and maintenance of hypertension. Secondary aims are the establishment of normal parameters of CA regulation of vasopressin and the identification of afferent nuclei and pathways to the supraoptic nucleus. The methodologies to be used will be specific and regional catecholamine depletions, immunocytochemical analyses, biochemical assays, and modulations of fluid content of the cardiovascular system. These experiments will be carried out on normotensive and hypertensive rats."}
{"text": "The concept of the neurovascular unit states that the pathophysiology of stroke, brain injury, and degeneration cannot be investigated as a purely neuronal phenomenon. Instead, interactions between cells from all neuronal, glial, and vascular compartments must be considered. The first cycle of our P01 program was focused on dissecting acute mechansims of disease in the neurovascular unit. In this renewal, we now propose to look at how the neurovascular unit undergoes repair and recovery. Project 1 (Lo/Xing: Gliovascular regulation of the microglial switch) examines how endothelium and astrocytes differentially regulate the activation of microglia. Microglia can span the range from good to bad. We will test the hypothesis that signaling at the gliovascular interface regulates the microglial switch as the neurovascular unit transitions from initial injury into repair. Project 2 (Arai/Huang: Mechanisms of recovery after white matter injury) examines how oligovascular signaling between brain endothelium and oligodendrocyte precursor cells mediate remodeling in diseased white matter. This allows us to extend the neurovascular unit concept for understanding how white matter recovers and reconnects. Project 3 (Ayata/van Leyen: Targeting ROCK for neurovascular recovery after stroke) will ask how the rho-kinase system may be a central signaling system for specific neuronal and vascular recovery endpoints during stroke recovery. This project is also our attempt at translation since it provides a potential therapeutic target for promoting neurovascular unit repair. Direct collaborations exist between all projects. All studies are supported by three cores. Core A (Boas/Sakadzic) provides powerful tools for in vivo and cellular imaging. Core B (Whalen) supports neuro-behavioral assays for assessing recovery in animal models across all projects. Core C (Lo) is our program coordination core that provides infrastructure for all collaborations We are excited about this next phase of our highly integrated P01 that dissects new mechanisms of cell-cell signaling in endothelial, glial and neuronal systems as the neurovascular unit transitions from initial injury into recovery after stroke, brain injury and degeneration."}
{"text": "Elevation of body temperature to between 41.8 and 42.4 degrees centigrade is now being used both alone and together with chemotherapeutic agents to treat patients with cancer. The mechanisms by which hyperthermia is cytotoxic are poorly understood as are the ways in which elevated temperature affects cell killing by chemotherapeutic agents. We propose to study the interaction between hyperthermia and selected chemotherapeutic drugs utilizing both in vitro and in vivo systems. Our general approach will be to test for synergistic cytotoxicity by measuring in vitro colony survival in chinese hamster ovary cells and by measuring the in vitro colony survival of RT9 astrocytoma cells after in vivo treatments. We then plan to study the mechanism behind the observed increased cytotoxicity by examining such things as altered drug uptake and enzyme inactivation. We will also study the scheduling of the drugs with heat in order to maximize cell kill. These studies should suggest clinically useful combinations of drugs with heat as well as provide basic cellular pharmacologic data. This work is already underway and we have discovered an interesting relationship between methotrexate and adriamycin cytotoxicity when each is combined with heat. In addition we have found that the rate of heating to peak treatment temperature is a critical determinant of the cell kill achieved. Our ultimate aim is to use these studies as starting points in the selection of drugs and hyperthermia schedules for clinical use."}
{"text": "In order to understand the role of the Xenopus forkhead protein (Xfkh3) in the differentiation of presomitic mesoderm, I have tried to overexpress and misexpress this protein by injecting synthetic mRNA in the animal pole region of both blastomeres of 2 cell stage embryos. 0.25, 0.5 and 1 ng of mRNA was injected. At stage 8, animal caps were dissected and cultured until sibling embryos reached tailbud stage in the presence or absence of activin. Although misexpression of the message by itself does not induce any muscle in the animal caps, still, muscle induction by activin is not inhibited. Further analysis of these results is currently under progress. By whole mount in situ hybridization, I have localized the distribution of Xenopus nonmuscle myosin heavy chain B (Bhatia-Dey et al., Proc. Natl. Acad. Sci. USA 90, 2856, 1993) using a probe that comprises part of the rod and the entire 3' UTR. The transcript is expressed ubiquitously in blastula and gastrula stage embryos, however, at stage 21, the transcripts begin to localize in the anterior part of the embryo, especially in the developing eye and fully differentiated anterior somites. At early tailbud stage, the transcripts become more concentrated in differentiated somites and in the eye. No expression is detected in the presomitic mesoderm. In swimming tadpoles, the transcripts are localized in differentiated somites, the eye and the branchial arches. Using a probe from a similar region of Xenopus nonmuscle myosin heavy chain A, no localized transcripts were detected throughout embryogenesis. However, Northern analysis, using the same probe, detected 2 transcripts during development. A 7.5 kb transcript is detected throughout embryogenesis from unfertilized eggs to swimming tadpoles and in a number of adult tissues. Another 8.3 kb transcript is first detected around stage 15/16, peaks between stage 25 to 30, gradually decreases in swimming tadpoles and is no longer detected in stage 47 embryos. Preliminary analysis suggests that the larger transcript arises due to differential polyadenylation."}
{"text": "Project Summary This study aims to prove the concept and feasibility that TendonCure, extracellular vesicles that contain signaling molecules (i.e. exosomes), secreted by mesenchymal stem cells (i.e. tendon stem/progenitor cells [TSPCs] or adipose-derived stem cells [ADSCs]) from individual patients (donors) cultured on a novel scaffold exert therapeutic efficacy on tendinopathy when injected into the diseased tendon of the same donor. Tendinopathy is a common chronic tendon disorder that affects 30-50% of individuals over 60 years old. It is characterized by pain, swelling, loss of function, and impaired performance. There is currently no cure for tendinopathy. Spontaneous repair or treatment typically leads to scar formation, resulting in a weakened tissue with reduced function and mechanical properties that may ultimately rupture with further use. Our previous and preliminary studies show that MSCs grown on the TendonCure scaffold are modulated towards tenocyte differentiation with enhanced tenogenesis-related gene expression. Furthermore, TSPCs cultured with exogenous TendonCure-T or TendonCure-A, TendonCure derived from TSPCs or ADSCs, respectively, exhibited tenogenesis-related gene expression profile changes, with increased expression of tenogenic markers and decreased expression of gene markers for the adipo- and chondrogenic lineages. Importantly, local injection of TendonCure showed therapeutic efficacy in an overuse-induced supraspinatus tendinopathy rat model in vivo. While the results are promising, the suggested efficacy of TendonCure was based on statistically significant results in TendonCure from MSCs of a young to middle-aged group. However, whether TendonCure derived from MSCs of aged individuals exerts such therapeutic effects is unknown. We therefore hypothesize that TendonCure from MSCs of aged individuals exerts therapeutic efficacy on tendinopathy. TendonCure-T and TendonCure-A will be harvested from TSPCs and ADSCs, respectively, which are isolated from aged patients at 65-80 y.o. and cultured on TendonCure scaffolds. Control exosomes will be derived from TSPCs and ADSCs from the same patients as the TendonCure groups and grown in regular 2D culture. Adult nude rats subjected to decline treadmill at two weeks (with expected mild tendinopathy) will be injected weekly in the supraspinatus tendons with TendonCure or control exosomes, or placebo (phosphate buffered saline), or kept for normal cage activity (control). Four weeks after injection, animals will be evaluated for efficacy on pain behaviors, and supraspinatus tendons will be dissected for efficacy evaluation with assays for histology and mechanical properties testing. Upon successful completion of Phase I and II studies, we will carry out clinical trials focusing on common sites of tendinopathy. TendonCure will be marketed as a biologic for treating tendinopathy and other tendon disorders. 1"}
{"text": "Oxidative stress is thought to be a major contributor to the development of retinal degenerative disorders such as macular degeneration, retinopathy of prematurity and diabetic retinopathy. In order to understand the mechanism(s) of oxidative damage to the retina, we have investigated the gene expression in the retina of rats exposed to intense visible light. The expression of heme oxygenase-1 (HO-1), a marker for oxidative stress, in the retina is highly increased following light exposure. The expression of HO-1 mRNA in the retina in response to acute intense light was investigated as a function of age in dim cyclic light reared and dark reared rats. The HO-1 mRNA in the retina of treated rats was analyzed by Northern blotting. The intense light exposure markedly increased the HO-1 mRNA expression in the retina irrespective of the rearing environment. However, the response was more pronounced in the dark reared rats. The HO-1 induction increased with age in both dim cyclic light reared and dark reared rats. The increase in HO-1 mRNA is accompanied by apoptotic photoreceptor cell loss. DMTU, an antioxidant, was highly effective in blocking HO-1 induction and photoreceptor cell loss resulting from the intense light exposure. The age or rearing light conditions did not influence the DMTU effect. Thus, age, light history, and antioxidant status are important factors affecting the retinal gene expression in response to stress. Activin, a member of transforming growth factor-beta superfamily, is known to be expressed in the vertebrate retina. We have identified the first invertebrate activin gene from Drosophila. A genomic clone representing 102 F region of Drosophila chromosome 4 was found to encode a putative activin b. The predicted protein sequence showed a multibasic protease site that would generate a mature 113 amino acid C-terminal peptide having more than 60% similarity to vertebrate activin bB. A TGF-b family signature as well as 9 cysteine residues conserved in the vertebrate activins were also present in the mature peptide sequence. The gene was expressed in embryo, larva and adult stages of Drosophila."}
{"text": "Microbial communities associated with the human body, in particular the gastrointestinal tract, play crucial roles in health and disease. The objective of this proposal is to understand how specific patterns in gut microbial succession are related to health and disease, and specifically to neonatal necrotizing enterocolitis (NEC). Although available evidence suggests that intestinal microbes contribute to the pathogenesis of NEC, the details of this relationship remain poorly understood. At present, relatively little is known about the gut colonization process in premature newborns, and about differences between this process in premature infants with and without NEC. We propose complementary high-throughput phylogenetic and metagenomic analyses to study microbial colonization of the premature infant gut during the first three weeks after birth. Our proposed work will elucidate, at high resolution, the population structure of microbial communities that develop during colonization of the premature infant gut and examine the roles of early colonists, gastrointestinal tract-mediated selection, immigration, the effects of mobile elements on genomic variation and microbial survival, and examine how these processes relate to onset of NEC. We will use next-generation sequencing to resolve species- and population-level community succession patterns during the critical initial period of gut colonization in babies that do and do not go on to develop NEC. We will profile community development using high-throughput 16S rRNA tag sequencing of stool samples collected daily during the first three weeks after birth. We will then carry out deep metagenomic sequencing of microbial DNA from half of these fecal time series samples to reconstruct genomes for coexisting bacterial, archaeal, phage, and plasmid populations. This will allow us to track species membership, community structure, metabolic potential, and population-level genetic heterogeneity. We will use these data to test the extent to which initial consortia predict succeeding community diversity [Aim 1], the importance of in situ diversification mediated by phage, insertion elements, and plasmids vs. immigration in determining population structure and metabolic potential [Aim 2], and to define ecological trajectories that correlate with health and disease [Aim 3]. Our preliminary metagenomic data conclusively demonstrate that the proposed approach can be used to reconstruct near-complete genomes of coexisting organisms from premature infant gut fecal samples with sufficient population depth to analyze population heterogeneity and dynamics. Improved understanding of the colonization process in the premature infant gut could translate to improved outcomes for premature babies by suggesting more effective strategies for disease prevention and treatment. More broadly, this research will uncover aspects of ecosystem colonization dynamics that have implications for other aspects of human health and the environment. !"}
{"text": "The overall goal of the Duke University - University of North Carolina at Chapel Hill (UNC) Training Program in the Genome Sciences and the Hemoglobinopathies is to train a cohort of both domestic and foreign postdoctoral fellows to be outstanding investigators of the basis for and treatment of the hemoglobinopathies, using genomics and related large-scale and high-throughput approaches in the genome sciences. Whether each trainee's chosen area of focus is in genomics, proteomics or computational biology and whether one's future research is basic, translational or clinical, each fellow will be expected to gain a broad and comprehensive understanding of the genome sciences as applied to ongoing research projects in the hemoglobinopathies. Training towards this goal will be achieved through: advanced coursework;extensive rotations in genomics, proteomics, and computational laboratories;a weekly colloquium involving researchers in the genome sciences;research in laboratories with active programs in hemoglobinopathy research;and co-mentoring by training faculty with expertise both in the genome sciences and in the hemoglobinopathies in order to develop a full appreciation for the multidisciplinary nature of this training program. The training program will be directed jointly by the Director of the Duke Institute for Genome Sciences &Policy and by the Director of the Duke-UNC Comprehensive Sickle Cell Center. Outstanding fellows will be identified from among a strong pool of applicants to existing postdoctoral training opportunities at both Duke and UNC in hematology, transfusion medicine, medical genomics, vascular disease and the genome sciences;from applicants to individual mentors at Duke and UNC;and from applicants identified by an international advisory group consisting of colleagues and collaborators in Tanzania and Thailand. Research opportunities will be provided by a group of more than 25 Duke and UNC faculty members with expertise in the genome sciences and in the hemoglobinopathies. We will develop a group of domestic and foreign colleagues who are dedicated to advance our understanding and treatment of the hemoglobinopathies. This network of investigators will form the basis for an ongoing series of international collaborations to reduce the pain and burden of these disorders."}
{"text": "This research concerns quality of life among younger women (premenopausal) diagnosed with, and treated for, early stage breast cancer. There is evidence that breast cancer leads to more distress among younger than older patients, and many possible reasons have been suggested for this difference. Study 1 (descriptive study) will examine this issue, assessing the levels of concerns expressed by younger breast cancer patients with regard to a range of issues suggested by various sources as important. We will also assess factors that we view as vulnerability/resilience variables (personality qualities of optimism and investment in body image as a source of self-esteem, contextual qualities of social support/social integration), to determine how the latter variables render individuals vulnerable to higher levels of various concerns. Finally, we will assess mediating variables (coping, perceptions of partner reactions to the surgery) and levels of quality of life (affective, social, and psychosexual disturbance) to determine the extent to which the concerns reported are linked to the distress and dysfunction that are the ultimate outcomes of interest, and to examine the role of coping differences as pathways in this chain. We will also collect open-ended data to ensure that we have identified all meaningful sources of concern among these patients. Subjects will be equivalently represented on extent of surgery (mastectomy vs lumpectomy), and 3 periods of time since surgery (3, 6, and 12 months). We will also recruit as close to equal numbers as we can from the 3 ethnic groups represented in large numbers in this population (Black, non-Hispanic White, and Hispanic). Study 2 (in a new sample) will implement and test the effectiveness of an intervention designed to meet the special needs and concerns of younger breast cancer patients. This intervention is based on findings from the literature of breast cancer, on previous intervention studies of breast cancer patients, on the experiences of this research team in intervention research using other populations, and on the results of Study 1. The once-a-week, 10-week intervention will target participants' sense of confidence about remaining cancer free, their coping responses (increasing acceptance and reframing of the experience, and decreasing avoidance coping), their utilization of social support, and their self- image, in a group-therapy format. The control group will be an education/standard of care group, in which subjects learn of the benefits of implementing some of the changes as described above in their orientation, but will not undergo the group experience in solidifying and acting on this information. Treatment efficacy will be assessed at 3, 6, and 12 months post-surgery, in terms of all the quality-of-life outcome variables listed above. This study will also have the benefit of addressing the generality of the treatment effects across ethnic/cultural boundaries."}
{"text": "The purpose of this study was to determine the feasability and utility of adenoviral mediated suicide gene therapy in women with recurrent ovarian cancer. A phase I trial was conducted in women with recurrent intraperitoneal ovarian cancer. Adenovirus encoding Herpes simplex virus-thymidine kinase (AdHSF-TK) was administered intraperitoneally through a percutaneous Tenckhoff catheter; the dose of AdHSV-TK was escalated in cohorts of patients between 1 x 10(9) pfu. Two days later, patients were treated with intravenous ganciclovir (GCV) at a dose of 5 mg/kg BID for a total of 14 days. Patients were monitored at regular intervals for eight weeks post-treatment for evidence of clinical toxicity and response. Serum samples and peritoneal aspirates were obtained at various time points to assess for evidence of gene transfer and immunologic response. Fourteen patients with persistent/recurrent ovarian cancer were treated per study specifications. Most patients had been extensively treated with a variety of chemotherapy regimens. Transient and easily ameliorated vector-associated fever was experienced by 4/14(29%) of treated patients. Other possible vector-associated constitutional (fatigue, insomnia) symptoms, abdominal pain, and gastrointestinal (nausea, diarrhea) symptoms were experienced by 6 of 14 (43%) treated patients. No other vector specific side effects were noted. Six patients experienced catheter-associated complications (infection-5, partial bowel obstruction-1). One patient expired due to a pulmonary embolism three weeks post therapy."}
{"text": "Computed Tomography (CT) has had an enormous impact on medicine since its introduction, and many physicians consider CT (along with MRI) to be the most important recent technological innovation in medicine. Further technical improvements would have important clinical benefit, but the system design in use in current CT systems, even the most advanced clinical scanners, is not able to achieve the combination of capabilities that is needed. We recently proposed a radically different CT system design, Inverse Geometry CT (IGCT) that promises to deliver wide volumetric coverage in a single rapid scan with no \"cone-beam\" artifacts, high spatial and temporal resolution, improved dose efficiency, and reduced radiation dose to the patient. Our preliminary results provide strong evidence that these goals can be achieved The goal of this proposal is to perform research leading to and including designing and constructing a fullscale prototype IGCT system capable of animal and human scanning, to quantitate its performance, and to perform pilot in-vivo studies. The research involves a collaboration between Stanford University and GE's Global Research (GEGR) Center, building on the pioneering work on IGCT performed at Stanford and the important advances and unique capabilities of the team at GEGR. The groups will collaboratively optimize the system design, perfect calibration and reconstruction algorithms, and perform detailed evaluations. The Stanford group, led by the PI, will be responsible for defining the clinical requirements and performing the animal and human studies. The GEGR group, led by Bruno De Man, will be responsible for detailed system design and construction. While the significance and potential impact of this research are very large, the scope and risk preclude it from being performed by industry alone and public support is required. At the same time, the impact of the requested budget is amplified by existing funding and ongoing research at both Stanford and GEGR, and by a commitment of 1$M from GE Healthcare to fund construction of the gantry based system. We believe that important research and clinical application would be possible with CT systems capable of much wider volumetric coverage in short scan times, requiring lower radiation dose than present systems, and delivering uncompromised image quality and temporal resolution. CT technology currently in use is not up to this task, and that a new approach is needed. We believe, and our preliminary studies show, that our IGCT approach will be able to open this new era in CT scanning. [unreadable] [unreadable] [unreadable]"}
{"text": "Methods for the synthesis of N-hydroxy-pyrroles and -imidazoles will be extended and the chemistry of these substances will be explored with a view of preparing new heterocyclic ring systems related to known antineoplastic agents. It is hoped that these will function as pyrimidine and purine antagonists. Substituents will be introduced to confer desirable properties to some of these molecules: alkylating groups, groups that increase lipid solubility, or groups that permit the molecules to cross the blood-brain barrier and thus make them more target-specific. Similar studies will be carried out with derivatives of pyrido(1,2-b)indazoles."}
{"text": "Considerable research suggests that major depression (MD) is common in women with bulimia nervosa (BN). Yet, despite more than two decades of research, the precise relationship between these forms of psychopathology remains unknown. This project aims to examine the depressive symptoms and cognitive correlates of MD, BN, and comorbid MD and BN. It is hypothesized that the depression experienced by individuals with comorbid MD and BN will differ from that experienced by individuals with \"pure\" MD and that evidence for a depressive subtype of BN (i.e., depressive bulimia) will be illuminated. Approximately 100 women meeting one of four diagnostic criteria (MD, BN, comorbid MD and BN, and asymptomatic control) will be recruited for the study. Proposed methods include diagnostic interviews, self-report questionnaires, and information processing tasks designed to assess: (1) depressive and bulimic symptoms, (2) depressogenic cognitive biases, (3) dysfunctional attitudes regarding needs for achievement and approval, and (4) dysfunctional attitudes and cognitive biases regarding weight, shape, and food. The results of this project are expected to illuminate important symptomatic and cognitive distinctions between MD and depressive bulimia. Such findings will have significant implications for the classification of MD and BN and will inform treatment protocols for both forms of psychopathology."}
{"text": "Ionizing radiation is widely utilized in cancer therapy. While some cancers have been treated successfully, an unacceptable fraction of treatments still fail. The ensuing recurrences and secondary tumors are often more aggressive than the original, possibly because of treatment-induced mutations. These mutations are not well defined due to the varied types of DNA damage ionizing radiation, the randomness of the damage, and its relative infrequency in the genome. Recent advances in nucleic acid chemistry make it possible to synthesize many of the types of damage detected in DNA subjected to ionizing radiation. These modified nucleotides can be incorporated into specific sites in cloned genes by standard molecular biology techniques. The proposed work will utilize this combined approach to place types of DNA damage, characteristic of ionizing radiation, at designated sites in a cloned DNA fragment. We will then determine how such damage affects eukaryotic gene expression and gene integrity. DNA damage will be positioned in synthetic oligonucleotides corresponding to transcription factor binding sites. A gel shift assay will be utilized to determine the binding and stability of transcription complexes containing either normal or damaged DNA elements. Damage will also be incorporated into specific sites in a synthetic DNA template and transcribed in vitro with a nuclear extract. The effect of specific types of DNA damage of gene transcription and fidelity will be determined. Related experiments will also determine if transcription transforms two opposed single-strand breaks into a more lethal double-strand break. Finally, we will measure the relative stability of duplex oligonucleotides containing base damage sites using the competitive mobility shift assay and determine whether a stability correlates with the polymerase by-pass or mutagenicity results obtained from the in vitro transcription studies."}
{"text": "A single injection of cocaine (24 hr prior to injection of carbamazepine) caused a trend (p=0.06) toward increased plasma levels of carbamazepine (single injection). More studies are required to test the effects of chronic cocaine treatment on carbamazepine plasma levels. These data suggest that carbamazepine treatment of humans for cocaine withdrawal may require close monitoring of carbamazepine blood levels to prevent potential toxic effects of this drug. Studies were started to investigate the gamma-aminobutyric acid (GABA-A) receptor system in amygdala-kindled rats that were made contingent tolerant to the anticonvulsant effects of diazepam. A study parallel to this one using contingent tolerance to carbamazepine was reported in last year's project report (Z01 MH 02459-06 BPB). Thus far, only the alpha-4 subunit of the GABA-A receptor has been measured. Results were qualitatively the same as for tolerance to carbamazepine. Kindling increased mRNA for the alpha-4 subunit selectively in the dentate gyrus of the hippocampus, and this increase was significantly less in rats tolerant to diazepam. However, the increase in rats tolerant to diazepam was greater than that observed in carbamazepine-tolerant rats."}
{"text": "Adolescence is a critical neurodevelopmental period that is associated with dramatic increases in rates of substance use. Identifying predictors of substance use and its effects on child and adolescent development is critically important, as substance-related decrements incurred during ongoing maturation could have long- lasting effects on brain functioning and behavioral, health, and psychological outcomes. This Research Project Site application from the University of Michigan and University of Florida is in response to RFA-DA-15-015, as part of the ABCD-USA Consortium (9/13) to prospectively determine the neurodevelopmental and behavioral effects of substance use on children and adolescents. A representative community sample of 975 9-10 year olds will be recruited as part of this application, contributing to the sample of 11,111 to be collected from 11 total sites across the ABCD-USA Consortium. All participants will undergo a comprehensive baseline assessment, including state-of-the-art brain imaging, comprehensive neuropsychological testing, and extensive assessment of substance use patterns and mental health functioning. These comprehensive assessments will occur at 2-year follow-up intervals, with intermediate assessments of functioning and substance use at 6- month intervals. The brain, behavioral, psychological, social, genetic, and environmental data collected during the course of this project will elucidate: 1) the effects of substance use patterns on the adolescent brain; 2) the effects of substance use on behavioral and health outcomes; 3) the bidirectional relationship between psychopathology and substance use patterns; 4) the effects of individual genetic, behavioral, neurobiological, and environmental differences on risk profiles and substance use outcomes; and 5) the gateway interactions between use of different substances. Elements Unique to this Site: This hub's Research Project application leverages site-specific expertise to address two aims focused on the identification of risk and resilience factors for adolescent substance use. Using baseline data categorized into distinct domains (Demographic, Cognitive, Mental Health, Personality, Life Stressors, Family History/Genetics, Environmental, and Brain), we will use cutting-edge, multi-stage analytic methods involving data reduction within each domain (e.g., latent variable analyses), identification of etiologically-distinct subgroups (e.g., community detection), and the construction of integrated multi-modal predictive models (e.g., regularized regression). This approach will delineate subgroups characterized by distinct profiles of risk and resilience. This approach is essential for informing outcomes of substance use during adolescence, which will ultimately inform the development of more efficacious interventions and clarify the toxic effects of exposure on adolescent brain, health, and cognition."}
{"text": "The purpose of this Task Order is to characterize the plasma pharmacokinetics (PK) and bioavailability of small molecule candidate anticancer compounds in mice as outlined below and optionally, provide analytical support for the quantification of such anticancer agents in plasma and/or tumor samples to be provided by NCI. Rapid turn-around is required. There is a base period and options under this task order."}
{"text": "The purpose of this five-year Institutional National Research Service Award is to provide funding for five predoctoral trainees and four postdoctoral trainees per year to prepare them for academic-research careers in the Speech and Hearing Sciences. Because of the multidisciplinary nature of the scientific substrates underlying Communication Sciences and Disorders, the 15 participating faculty for this training grant are drawn from five different departments in three colleges. Each faculty member has agreed to serve as a research preceptor to one or more trainee supported from this grant during a five-year period of time. Faculty members were chosen for participation on this training grant on the basis of the nature of their ongoing research activities, research funding, and experience in supervising doctoral and postdoctoral students in the speech and hearing sciences. Predoctoral and postdoctoral trainee-are selected on the basis of outstanding credentials that reflect a bonafide potential for productive research careers in the Speech and Hearing Sciences. Funding is requested to provide trainees selected for this training grant up to a maximum of three years of support at either the predoctoral or postdoctoral level."}
{"text": "AFC HDPTP Project Summary A disaster, by definition, is an unexpected destructive event that brings harm and damage to a community. While the community members often feel powerless to control the disaster, they can take steps to prepare for their response if it occurs. AFC proposes to use funds from the NIEHS Worker Training Program?s Hazmat Disaster Preparedness Training Program (HDPTP) to provide preparedness training to public safety responders in the southeast US and to Native American tribes throughout the country. The proposed project will continue AFC?s ten-year history with of delivering quality training focused on safety for first responders to a disaster, whether natural or man-made. This project will target public safety (fire, law, EMS, EMA) responders with training that helps them recognize hazards, assess response actions, and bring some order to the chaos of the disaster scene. Disasters often become mass casualty incidents (MCI) and responders may need triage their limited resources to do the most good. Also, unfortunately, these MCI events may be ?man-made? acts of terror in which the perpetrator of violence is still at the scene. AFC proposes to bring Hostile Event Response training that teaches enhanced situational awareness and survival as well as life-saving tactical ?stop-the- bleed? care in an active shooter or improvised explosive device event. AFC projects to bring a total of 70 classes to 1,400 trainees in 11,200 contact hours. It will be split between both the public safety and Native American target populations."}
{"text": "The objective of the project is to determine if cells other than early embryo cells can clone the mammalian blastocyst and participate in embryo development. The mouse will be used as the experimental animal. Cells for transfer will be obtained from late stage embryos, normal adult tissues, and neoplastic tissue. The effect on the cells and blastocysts will be determined by (1) recording microscopic changes in the transferred and blastocyst cells during in vitro development of the blastocyst, (2) following autoradiographically the fate of the cells in the embryo, and (3) placing the recipient blastocysts into foster mothers and studying the offspring for signs of an effect from the transferred cells. BIBLIOGRAPHIC REFERENCE: Brinster, R.L., Can teratocarcinoma cells colonize the mouse embryo? In: Teratomas and Differentiation, (Eds. M. I. Sherman and D. Solter), Academic Press, Inc., New York, pp. 51-58, 1975. Brinster, R.L., Participation of teratocarcinoma cells in mouse embryo development. In: Proceedings of the Meeting on Embryonic & Fetal Antigens. Edited by Dr. J. H. Coggin, Jr., 1976. In Press."}
{"text": "Retrotransposons comprise almost half of the human genome and substantial fractions of other metazoan genomes. Nonetheless, the mechanisms and localization of retrotransposon assembly are poorly understood. Ty3 is a long terminal repeat retrotransposon in budding yeast containing GAG3 and POL3 ORFs encoding structural and catalytic proteins, respectively. Because Ty3 exists in a simple eukaryote that facilitates molecular, genetic, biochemical, and even cytological approaches, it provides a useful model for understanding retrotransposon mechanics. In the current funding period, the assembling Ty3 VLP was characterized by density sedimentation, RNA protection, transmission EM and atomic force microscopy performed on Ty3 VLPs blocked at specific stages and also on mature VLPs. In addition, sixty Ala substitution mutants were analyzed to dissect the functions of Gag3 (capsid, spacer, and nucleocapsid) subdomains. These studies showed that the amino-terminal domain is critical for assembly and nuclear pore association of VLPs and that the nucleocapsid domain is important for targeting Ty3 RNA and Gag3 protein to the P body for assembly. A set of Ty3 RNA variants was developed and used to show that the Ty3 5'and 3'untranslated regions (UTRs) and POL3 contain independent cis-acting P-body targeting activities, but that only the UTR mediates packaging of Ty3 genomic RNA. The finding that the RNA processing body (P body) previously described as a site where nontranslating RNAs are sequestered or degraded, is the site of Ty3 assembly was particularly significant as assembly sites have not been characterized for retrotransposons and how retroviruses preassemble RNA and protein at a perinuclear site is not well understood, but appears to involve packaging signals. In Aim A of the proposed work, we will use mass spectroscopy to identify host proteins which are associated with Ty3 RNA and structural protein. Our collection Ty3 wt and variant RNAs tagged with the MS2- binding site coupled with a novel high affinity TAP-tagged MS2 binding protein and our antibodies against Ty3 proteins will be used to isolate proteins directly involved with Ty3 components. Strains with attenuated functions of identified genes will be tested for defects in Ty3 VLP assembly and proteins identified will be investigated in Aims B-D. A key step in assembly occurs when the mRNA transitions from translation to packaging. Based on the retrovirus model, this is associated with structural rearrangements in the UTRs of the RNA which are facilitated by nucleocapsid binding. In Aim B we will define at higher resolution the packaging site of Ty3 RNA and its interaction with the reverse transcription primer initiator tRNAMet. Previous work in the Darlix laboratory, with whom we collaborated, showed that initiator tRNAMet has a bipartite primer binding site in Ty3 5'and 3'UTRs. Retroviruses have dimeric genomes and we have shown that Ty3 is similar. Darlix proposed a novel model for Ty3 genomic RNA dimerization mediated by two itRNAMet molecules annealing at the 5'ends. We will specifically test whether the PBS, and by implication, the putative dimerization interface, is required for packaging of Ty3 RNA into VLPs. If so, that would suggest that initiator tRNAMet, which has unique roles in translation initiation, and priming Ty3 reverse transcription, is a central player in the key transition from translation to packaging. SELEX and in vitro RNA-protein binding assays will be used to identify important nucleocapsid binding contacts in the packaging site of the Ty3 UTR which could mediate primer annealing and dimerization. In Aim C we will test our hypothesis that the Ty3 UTR structure antagonizes translation initiation and imposes requirements for specialized translation helicases. In addition, we propose that reduced numbers of translating ribosomes downstream of the frameshift site attenuates 60S subunit delivery to the 5'end of the RNA and explains the sensitivity of Ty3 and other retrotransposons to imbalances in translation initiation factors and 60S ribosomal subunits. This hypothesis will be tested using host mutants and variant Ty3 RNAs. We will test whether translation is ultimately repressed by Gag3 binding in cis to the Ty3 UTR or POL3. In Aim D we will characterize the contribution of P bodies to Ty3 assembly. We hypothesize that P-body proteins cooperate with Gag3 to repress Ty3 translation, allow the concentration of assembly factors, and may promote unwinding of the RNA to allow packaging. Nonetheless, isolation of an unusual mutant which does not form large P body clusters suggests that P bodies might repress transposition at a post-assembly stage. Factors identified in genetic and mass spectroscopy screens will be tested for direct interactions with Ty3 components and for their roles in clustering P body proteins and assembling Ty3 viruslike particles. In summary, Ty3 is a well-characterized retrotransposon in a model eukaryote. It offers a unique opportunity to understand the retrotransposon particle assembly process and how P bodies might chaperone this process."}
{"text": "The Environmental Protection Agency calls arsenic the most prevalent environmental toxin and carcinogen in the United States (://www.atsdr.cdc.gov/cercla/07list.html). Arsenic causes cardiovascular and peripheral vascular diseases, neurological disorders, diabetes mellitus and various forms of cancer such as skin and bladder cancer. Arsenic is biomethylated by the liver enzyme As (III) S-adenosylmethionine (SAM) methyltransferase (AS3MT) to mono- and dimethylated species. Because the trivalent products methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)) are more toxic than inorganic arsenite, they have been proposed to be associated with arsenic carcinogenesis and other diseases in humans. Individuals with AS3MT polymorphisms produce increased amounts of methylated species. How methylation contributes to disease depends on the mechanism of human AS3MT and differences between wild type and polymorphic enzymes. The uncertainty over the consequences of methylation makes it imperative to understand how this enzyme works. The overall goal of this study is elucidation of the structure and function of hAS3MT and its polymorphic forms."}
{"text": "Approximately 100,000 of the over one million people in the United States infected with HIV are women, 80% of whom are in their childbearing years. Consequently, AIDS has already become one of the leading causes of death in children between the ages of 1 and 4 in the United States. Maternal and infant HIV infection is occurring disproportionately among lower socioeconomic populations and minority women. Poverty, drug use, severe stress, and family disorganization characterize the ecological environment of these infants. As a result, these infants, already at risk for developmental delays because of the impact of the HIV infection, are at even greater risk for delays in their developmental trajectory. The quality of parental caregiving has been found to be a major environmental determinate of a normal child's social, physical, and mental development. Within the context of HIV-infected families, the quality of caregiving is critical. Yet, providing care to a seropositive child creates further strains on the family, which is already compromised by the burden of illness in the biological mother. Inadequate caregiving may also be caused by other lifestyle characteristics of the mother and family. The quality of parenting in these at-risk infants may influence the progress of the child's illness. The field of psychoneurology has demonstrated that stressful caregiving environments may create physiological responses in the child, specifically the immune system, at a time when the child is highly vulnerable to such changes. The overall goal of this longitudinal study of HIV seropositive infants born to HIV-infected women is to identify child, parent, family and community factors that influence the developmental trajectory of these children. Special emphasis will be placed on whether quality of parental caregiving plays a mediating role in developmental outcomes and, with HIV-infected infants, in the progression of the disease. This prospective longitudinal study will be done at the Duke Infectious Diseases Clinic where medical services and supportive care are currently provided in a comprehensive team approach. Infants sero-positive for HIV will be enrolled in the study when the infant first enters the Pediatric Infectious Disease program, generally about 24 months of age, and followed until the child is at least 12 months old, with a subsample followed to 18 and 24 months as feasible within the 3 year time frame. Date will be collected using multiple methods (interview, questionnaires, observation, and assessment) with multiple informants (parents, health care staff, research team) over time. Data collection methods assess selected infant, caregiver, family, and community characteristics, the quality of parental caregiving, and disease progression and developmental outcomes in the child. The ultimate goal is to identify factors which place infants born to HIV seropositive women at developmental or health risk so as to identify appropriate interventions to help parents or other primary parental caregivers cope with HIV and more adequately meet the child's developmental needs."}
{"text": "We recently discovered a thrombogenic mechanism for pregnancy loss in the antiphospholipid antibody syndrome that antiphospholipid antibodies displace the placental anticoagulant protein, annexin-V, from the anionic phospholipid surfaces of trophoblasts and accelerate coagulation reactions at that site. The core hypotheses of this grant are: a) Annexin-V serves a thromboregulatory role at the anatomic site where maternal blood and placental villi interface by shielding anionic phospholipids (which markedly accelerate coagulation enzyme reactions) from participation in coagulation reactions and; b) antiphospholipid antibodies competitively displace annexin-V from that phospholipid surface, disrupting the annexin-V \"shield\", thereby increasing the exposure of the thrombogenic anionic phospholipid. This process leads to thrombosis in the maternal circulation at the sites of interface with fetal trophoblasts and to ultimately to pregnancy complications and losses and determine their clinical efficacy. Our specific aims are: 1) To determine the mechanism(s) by which antiphospholipid antibodies and cofactors reduce the quantity of annexin-V on trophoblast apical membrane and accelerate coagulation reactions and 2) To develop tests for the reduction of annexin-V binding to phospholipids and the inhibition of annexin-V anticoagulant activity. This innovative project opens a new path toward elucidating the mechanism of pregnancy loss in the antiphospholipid syndrome and is likely to lead to improved diagnosis and treatment."}
{"text": "During the last year, the project has focused on the chemokine system in cancer. Chemokines and chemokine receptors can have roles in cancer by bringing white cells to the cancer and through direct effects on the cancer cells themselves. Although white cells can combat cancer by killing cancer cells, white cell products can also serve to enhance cancer cell growth, which is thought to be a mechanism whereby inflammation can promote cancer. Cancer cells that express chemokine receptors can also receive growth and/or survival signals from chemokines. In analyzing samples from human cancers that typically arise in association with inflammation, we have found that many of these cancers express the chemokine CXCL16. We concentrated on prostate cancer and using immunohistochemistry and immunofluorescence with confocal microscopy, we found that the cancer cells express both CXCL16 and CXCR6, and that high expression was associated with poor prognostic features. We also found expression by tumor-associated lymphocytes, and we hypothesize that both by bringing lymphocytes to the cancer and by directly stimulating cell growth, CXCL16 may be promoting prostate cancer. Another aspect of the project has explored using a second chemokine receptor, CXCR4, which is expressed on many cancers and is thought to be associated with aggressive cancer, in order to image cancers by positron emission tomography (PET). We have synthesized a radiolabeled CXCR4 antagonist, (64)Cu-AMD3100, that could potentially be used to identify CXCR4-expressing cancers and to correlate CXCR4 expression with prognosis and responses to therapy, thereby providing additional clinical information that might guide effective cancer therapy in individual patients."}
{"text": ". Repetitive use of hand tools, in particular those that compress the palm of the hand and are held with a pinch or precision grip may be a large part of the high incidence of carpal tunnel syndrome (CTS) that is overwhelming the industrial work place. This proposal is to conduct preliminary investigations into the relationship of carpal tunnel pressure (CTP) to the design of handles used on squeegees and related hand tools such as pliers and screwdrivers. The purpose of this investigation will be to determine the feasibility of preventing CTS using the squeegee handle as a hand tool model and redesigning the standard shape. In Phase I we will investigate how handle shape and size affects CTP to determine the feasibility of designing an ergonomically sound squeegee handle. If the results of this investigation suggest an ergonomically sound squeegee handle design will prevent CTS then in Phase II we will use the results of our study to design and test prototype squeegee handles and develop the biomechanics to properly use these ergonomic handles. The screen printing industry is actively seeking a solution to the rampant CTS problem."}
{"text": "The research outlined in this proposal deals with several aspects of biologically active compounds produced by microorganisms. The main emphasis is on the biosynthesis of a number of antibiotics. The methods to be used include feeding experiments with precursors labeled with radioactive isotopes, but also to a large extent the use of 13C-labeled compounds in conjunction with 13C Fourier Transform NMR. Antibiotics to be investigated are pyrrolnitrin, chlorothricin, boromycin, granticin, the naphthocyclinones, dihydrophenylalanine, ketomycin and anticapsin. In addition some structural work is proposed which involves the isolation and structure elucidation of some new naphthoquinone-type compounds found in the cultures of the granaticin-producing organism. Also within this project, some pilot studies will be carried out to explore possibilities of biotransformation of antitumor antibiotics. Also in collaboration with some synthetic organic chemists engaged in total synthesis of natural products and analogs we will evaluate their synthetic compounds for biological activity and/or help them establish identity of the synthetic and the natural material."}
{"text": "This application is for the continuation of studies to design and develop inhibitors of O6-alkylguanine-DNA alkyltransferase (AGT) as chemotherapeutic agents. The presence of AGT is a major factor in the resistance of tumor cells to killing by drugs that form adduct at the O6- position of guanine. The aim of these studies is to extend the effectiveness and the range of tumors that can be treated with these drugs. Studies during the first period of support have led to Phase I trials with O6-benzylguanine (BG) but further work is needed to optimize the use of this agent and to develop compounds with better therapeutic properties in order to improve the discrimination between tumors and normal tissue. There are 5 laboratory programs and 3 cores. Dr. A.E. Pegg will serve as the PI and the Leader of Program 1 which will carry out studies on the interaction of possible AGT inhibitors with the control and mutant AGT proteins and design improved inhibitors including compounds able to inactivate BG-resistant AGTs. Dr. M.E. Dolan will head Program 2 in which detailed metabolic studies of novel AGT inhibitors will be carried out. Specific compounds include:metabolism-resistant 8-substituted BG derivatives; compounds with ester linkages that are activated as AGT inhibitors by esterases; and benzyl pyrimidine derivatives which are very potent but rapidly cleared. The use of regional therapy with AGT inhibitors using intraarterial delivery for CNS parenchymal tumors and intrathecal administration for leptomengeal neoplasms and the selection of the most promising compounds for targeting the inhibitor to brain tumors will be studied in Program 3 led by Dr. H.S. Friedman. Program 4 led by Dr. S.C. Schold will focus on the development of 9-substituted BG derivatives including the nucleoside and deoxynucleoside which appear to offer significant advantages over BG in terms of systemic availability, penetration of the blood-brain barrier and localized metabolic activation. Program 5 led by Dr. S.L Gerson will investigate methods for optimizing tumor cytotoxicity after AGT inactivation. He will study the extent of AGT inactivation needed to achieve maximal inactivation, methods to overcome resistance to BG-therapy of colon and breast tumors, and procedures including gene transfer to minimize bone marrow toxicity of the combination of AGT inhibitors and BCNU. Routine evaluation of compounds both for AGT inactivation and for the ability ot sensitize tumor cells in culture to BCNU will be carried out in Core B led by Dr. Pegg. Promising compounds will be examined for in vivo pharmacokinetics, tumor AGT inactivation and sensitization of tumor xenografts in nude mice in Core C led by Dr. Dolan."}
{"text": "= No large-scale study within the United States has examined any aspect of the epidemiology of intracranial meningiomas, a lesion which accounts for up to 25% of all primary intracranial neoplasms. We propose to conduct a population-based case/control study of meningioma within the states of Connecticut, Massachusetts, North Carolina, and Texas as well as the San Francisco Bay area under the mechanism of an integrated R01 (Note that identical applications are submitted for all five sites). Cases will be selected using rapid case ascertainment and will include all male and female individuals diagnosed with a histologically confirmed intra-cranial meningioma between the ages of 20 and 79 years from 7/1/2006 through 6/30/2010 in the above listed regions. Control subjects will be identified via random-digit dial methods and matched to the cases by sex, ethnicity, geographic location, and five-year age category. Study subjects will be administered a telephone questionnaire to collect information on the two primary categories of risk, exposure to ionizing radiation and hormones as well as additional risk factors such as family history of meningioma and other tumors, cell-phone utilization, and head trauma as well as questions on outcome and quality of life. Parraffin-embedded tumor tissue blocks will be obtained for case subjects to allow for 1) a uniform histological review, and 2) immunohistochemical testing for estrogen, progesterone, androgen and MIB-1 receptors. Blood or buccal specimens will be collected from all study subjects for testing of DNA polymorphisms in DNA repair and cell cycle genes. This study represents the first concentrated effort to examine environmental and genetic risk factors for meningioma."}
{"text": "This interagency agreement has the following scope: Clinical research on HIV and AIDS associated complications and co-infections with emphasis on optimizing therapeutic approaches to the treatment of HIV and related co-infections an co-morbidities in resource poor developing countries, including phase I-4 therapeutic clinical trials Clinical research on HIV prevention strategies ranging from novel pre-exposure prevention modalities such as Prevention of Mother to Child Transmission (PMTCT) and between discordant couples, treatment of sexually transmitted diseases, post exposure prophylaxis, and microbicides"}
{"text": "The overall goal of this project is to directly test how cholinergic input from the nucleus of the horizontal limb of the diagonal band of Broca (HDB) alters olfactory bulb (OB) glomerular circuits and how this modulation ultimately affects odor coding, perception, and learning. We will use of both wide-field and 2-photon in vivo and in vitro calcium imaging in mice expressing genetically encoded indicators of neuronal activity in defined OB cell types, to test the novel overarching hypothesis that synaptically-released acetylcholine bidirectionally modulates mitral/tufted cell odor responses as a function of the prevailing odor intensity. We further test the hypothesis that this modulation is due to opposing muscarinic and nicotinic receptor actions on inhibitory periglomerular cells that differentially regulate the strength of presynaptic inhibition of olfactory sensory neuron input to the glomeruli. Using imaging and well characterized olfactory-mediated behaviors, we will also investigate how this cholinergic modulation of the glomerular odor representation affects odor perception and learning. Specifically, we test the hypotheses that HDB-evoked acetylcholine release in the OB: (1) enhances olfactory sensitivity, (2) dishabituates odor responses adapted at the peripheral olfactory sensory neuron level and (3) is critical for associative olfactory learning. By investigating receptor type-specific cholinergic modulation in morphologically- and physiologically-distinct neuron types at both the population and single cell levels, the experiments of this proposal will for the first time elucidate how cholinergic modulation of neural odor responses in the OB impacts olfactory perception and learning behaviorally. The overall findings will dramatically advance our understanding of how acetylcholine modulates sensory representation, odor coding, perception and behavior."}
{"text": "The contractor shall serve as the coordinating center for the Epidemiologic Studies of Human Retroviruses in Volunteer Blood Donors program. The objectives are as follows: 1) determine the prevalence of retrovirus seropositivity in first time blood donors: 2) determine the rate of retrovirus seroconversion in repeat blood donors as a measure of incidence of infection: 3) ascertain risk factors for antibody-positive donors: 4) characterize the blood population by geographic location, age, sex, race/ethnicity, and donation history to permit analysis on prevalence, incidence, and risk factors: 5) identify recipients of retrovirus-positive blood units and conduct clinical and laboratory follow-up of these recipients to determine the transmissibility of these agents via transfusion; and 6) establish blood specimen repositories for long-term storage of specimens from study donors and recipients for future testing."}
{"text": "This project investigates the role of mitochondrial integrity during the development of TBI pathology and periods of functional recovery. Treatment paradigms involving cyclosporin A (CsA) are used to test the hypothesis that a triad of injury pathology, including plasmalemmal microporation, cytoskeletal proteolysis and mitochondrial degeneration, can be mediated through direct effects on mitochondrial membrane integrity. We will investigate the effects of blocking the mitochondrial permeability transition pore complex (MPT) with CsA both prior to and following TBI. Given that our original observations show that CsA administration offers protection against axonal injury pathology in TBI, we will now test the hypotheses that somato-dendritic domains within injured neurons also exhibit the same triad of pathologies and that these domains can also be protected with CsA intervention. Our hypotheses will be tested the same triad of pathologies and that these domains can also be protected with CsA intervention. Our hypotheses will be tested in two well known models of TBI, impact acceleration and combined neuroexcitation + deafferentation lesion. Using these models we can directly compare similarities and differences in both diffuse and focal injury paradigms. We will use immunohistochemistry (IHC), electron microscopic permeability tracer methods, assays for mitochondrial oxidative enzyme function and mRNA expression, and behavioral endpoints to measure effects of CsA on TBI pathology. In addition, we will explore the pharmacokinetics of both intrathecal and intravenous administration of CsA, assaying effects of administration time and dose on the above outcome measures, as well as determining the degree of drug permeability through the blood brain barrier. Finally, we will begin to address alternative mechanisms of action for CsA in TBI, first testing its potential influence on calcineurin mediated pathways. To do this we will administer a calcineurin specific inhibitor, FK506, in treatment paradigms shown to efficacious with CsA and determine its effect on structural outcome measures. The value of our proposed studies is twofold. First, they clearly define cellular mechanisms of TBI pathology and recovery associated with metabolic function. Perhaps more importantly, these studies, combined with the related human studies in this program project proposal, will establish a solid knowledge base supporting potential CsA therapeutic efficacy in TBI patients."}
{"text": "Patients with high level of eosinophils in their blood frequently die because of heart disease. Using ultrasound, we are able to detect abnormalities of the heart long before heart symptoms occur. It is hoped that early treatment of this problem will prevent or delay heart abnormalities."}
{"text": "Rodent pests have eaten our food and transmitted diseases to us for millennia. There are an estimated 300 million introduced Norway or brown rats in the U.S. that cause $27 billion of economic losses annually. Historically the strategy has been to kill rat pests through a variety of methods but primarily by using poison baits that are anticoagulant rodenticides. In addition to all of the problems associated with using poison bait, e.g. poisoning nontarget animals/pets, children under the age of 6, and contaminating the environment, poison does not address the problem. A non-lethal strategy that has significant potential to manage rodent pest number is fertility control. But thus far effective control of free-ranging wildlife, such as small, nocturnal rodents, has not been achieved because distribution to dose rats must be via an oral route. The industrial chemical 4-vinylcyclohexene diepoxide (VCD) accelerates the natural process of atresia leading to depletion of rat ovarian follicles causing ovarian failure and permanent sterility. Follicle depletion occurs when VCD is given in repeated intraperitoneal daily doses and on average, complete elimination of primordial/primary follicles and premature ovarian failure occur by day 58 following the onset of dosing and in mice causes infertility. To date VCD induced follicle depletion has been achieved by intraperitoneal administration. However to develop and commercialize a product to cause wild rat infertility it must be given orally in a bait. This Phase I application aims to test the following hypothesis in three specific aims. Oral administration of VCD to rats will lead to complete depletion of ovarian follicle populations, resulting in infertility demonstrating feasibility for development of a fertility control bait for rats. Aim 1. Determine the dose needed to deplete primordial ovarian follicles in rats by oral exposures to VCD. Aim 2. Determine the time course over which VCD completely depletes ovarian follicle populations. Aim 3. Determine the pharmacokinetics of VCD excretion. Results from Phase I experiments will define the dose and duration of VCD exposure necessary to cause complete ovarian follicle depletion. These results will enable us to obtain sufficient information to progress to a Phase II application to develop an oral bait and baiting protocol to manage wildlife rodent populations. Rats are being targeted in these studies, with the understanding that rats are more resistant to VCD than mice. Thus, success with rats, can easily be translated to mice. Furthermore, rats are the primary target pest species based on the enormous economic and significant public health risks they pose both by their contamination and destruction of our food supplies and the dangerous rodenticide control approaches currently being used to manage rat populations. PHS 398/2590 (Rev. 11/07) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: Management of wild rats is critical to minimize agriculture and property damage and the spread of infectious diseases. Lethal approaches introduce poison into the environment and consequently are being targeted for removal from over the counter availability. The goal of this project is to provide data to support the feasibility of using the industrial chemical 4-vinylcyclohexene diepoxide (VCD) to ultimately develop an environmentally neutral permanent rodent fertility control bait to manage wild rat populations"}
{"text": "Major disasters occur with regularity throughout the world, causing widespread destruction and death and leaving stunned survivors in their wake. Relatively little is known of the extent of lasting psychic impairment produced by these events or what can be done to ameliorate it. One such disaster took place at Buffalo Creek, West Virginia, on February 26, 1972, when a dam collapsed, unleashing 150 million gallons of water and coal waste into the valley. A group of approximately 650 survivors are suing the coal company for compensation for physical and mental pain and suffering and the destruction of their former community life. Our research team is in the unique position of having access to all medical and school records, psychiatric interviews and psychological tests, obtained on this group of litigants. We propose to quantify the psychiatric data and the familial life-stress produced by the disaster, making them amenable to statistical analysis and hypothesis testing. These will be supplemented with symptom data on non-litigants and on members of another similar community that has not had such a disaster. A follow-up study is also planned for two successive years after the case is settled, to determine its effect on the community. Independent random samples of 50 litigant and 25 non-litigant families each year will be selected for this purpose."}
{"text": "This SBIR Phase II proposal is a plan to further develop a novel therapeutic approach that will save the lives of patients with sepsis. Despite the increased understanding of the complex pathophysiology of sepsis, severe sepsis still results in significant morbidity and mortality. As such, there is an urgent unmet medical need for an effective novel therapy for septic patients. The global market potential for sepsis treatment is estimated at over $30 billion annually. Thus, successful development of a new anti-sepsis therapy will not only have a positive impact on health care, but also will have significant commercial benefits. A balanced inflammatory response is an essential element of a successful host defense after injury. However, excessive production of proinflammatory cytokines may cause further tissue injury. Macrophages/Kupffer cells play important roles in producing proinflammatory cytokines in sepsis. The nervous system reflexively regulates the inflammatory response in real time. We have demonstrated that the release of the sympathetic neurotransmitter, norepinephrine (NE), from the gut is increased in sepsis, and that NE potentiates endotoxin-induced TNF-( upregulation via the A subtype of (2-adrenoceptors (i.e., (2A-AR) expressed on the surface of Kupffer cells. Pre-treatment with a specific (2A-AR antagonist, 2-[(4,5-dihydro-1H-imidazol-2-yl) methyl]-2,3-dihydro-1-methyl-1H-isoindole maleate (BRL- 44408 maleate), downregulates TNF-(, attenuates tissue injury, and improves survival in a rat model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). However, it remained unknown whether the delayed administration of BRL-44408 maleate (which is more clinically relevant) reduces sepsis-induced mortality as well. Accordingly, the primary objective of our completed Phase I project was to determine the effect of delayed administration of BRL-44408 maleate on sepsis-induced inflammation, organ injury, and mortality. We have clearly shown that administration of BRL-44408 maleate at 5 h after CLP (i.e., at the early stage of sepsis) is protective in experimental animals. These results have established the technical merit and feasibility of the proposed Phase II project. We therefore continue to hypothesize that the administration of the small molecule drug candidate BRL-44408 maleate in established sepsis attenuates tissue injury and improves survival. In this Phase II proposal, we will perform detailed toxicological evaluation and pharmacokinetic characterization, and determine the optimal protective dose(s) and time- course of BRL-44408 maleate in sepsis in the rat. In order to advance the technology to clinical trials, the efficacy of BRL-44408 maleate will be tested in a rabbit model of sepsis. These proposed studies should provide important preclinical data that will help us filing an IND application to the FDA to initiate clinical trials in order to obtain commercial utilization of BRL-44408 maleate as a safe and effective therapy for sepsis. PUBLIC HEALTH RELEVANCE: Sepsis is one of the leading causes of death in intensive care units. Over 210,000 people succumb to this overwhelming infection in the United States annually. A recent epidemiologic study estimated that more than 750,000 people develop sepsis each year at a cost of $16.7 billion nationally. Given the intensive and prolonged care necessary to treat patients with sepsis, the economic burden is profound. Thus, there is an urgent unmet medical need for an effective novel therapy for patients with sepsis."}
{"text": "Background Endocrine malignancies (including thyroid, adrenal, parathyroid, and pancreatic neuroendocrine tumors) are among the fastest growing cancer diagnoses in the United States, but it is difficult to distinguish benign from malignant tumors by routine clinical, laboratory, and imaging studies. So, even patients who have seemingly benign endocrine tumors often choose to undergo surgery to get a definitive diagnosis in the hopes of ruling out cancer. Most patients with endocrine cancers have a relatively good prognosis. However, anywhere from 10% to 40% (depending on tumor type) have aggressive disease which often cannot be reliably determined at the time of initial treatment. Prognostic markers which can reliably risk stratify patients with high risk of recurrence and death would help determine which patients should receive aggressive initial treatment and close follow up. Furthermore, prognostic markers may also help identify which patients are likely to respond to standard therapy and which patients do not respond to standard therapy if a distinct molecular phenotype is identified. Summary We have made progress with our pan-genomic (mRNA and microRNA expression, copy number changes, and DNA-methylation) analysis of endocrine neoplasms to identify candidate diagnostic and prognostic markers for endocrine malignancies (thyroid, adrenal, neuroendocrine pancreas), and to understand the dysregulated genes/pathways in endocrine cancers. Our analysis shows key changes in mRNA/protein expression and microRNA expression levels, and DNA-methylation status that serve as excellent diagnostic markers. These studies have been expanded to include the analysis of serum and urine samples to detect altered levels of microRNAs, proteins and DNA-methylation changes found in the tumor samples. In this analysis we found some microRNAs (downregulated in cancer) to be actively secreted in exosomes based on both in vitro and first in human venous sampling studies. The integrated analysis of our genomic data, especially in thyroid cancer and adrenal cancers, have also uncovered possible novel pathways which may have a role in thyroid and adrenal cancer initiation and progression which we are confirming using functional genomics studies."}
{"text": "SHARED RESOURCE CORE CRUMP PRECLINICAL IMAGING TECHNOLOGY CENTER ABSTRACT: The Crump Preclinical Imaging Technology Center will provide comprehensive support for the in vivo imaging studies carried out in the NSBCC projects. Under the direction of Core Director Dr. Michael Phelps, the Imaging Center is comprised of two other faculty, Dr. Arion Hadjioannou and Dr. Jason Lee, and one full-time staff member. All faculty members have extensive experience and recognition in preclinical molecular imaging. The Imaging Center will provide all radio-labeled probes, perform all microPET and CT studies, and data analysis. In addition, they will provide support and training to investigators, including animal preparation and monitoring, quality control and the strictest safety measures. The goal of the Shared Resource Core is to provide advanced molecular imaging support to assess the kinetics of tumor metabolism and therapeutic outcome in response to therapeutic intervention. These experiments will consist of highly-controlled, serially- acquired PET and CT scans for both functional and anatomical assessment, respectively. Since the PET radio- labeled probe used is also widely available in the clinic, the preclinical imaging data acquired by the Shared Resource Core can be translated as biomarkers for evaluation of treatment response in patients."}
{"text": "This is an application to renew a grant to study RNA synthesis in minus-strand RNA viruses. Nonsegmented negative-strand (NNS) RNA viruses include some of the most significant human pathogens that are a major ongoing threat to US public health. To combat those agents, we need a combination of antiviral drugs and vaccines. Our long-term objective is to understand the mechanisms by which the replication machinery of vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, functions. VSV is the ideal choice for such studies because it is the only NNS RNA virus for which robust transcription can be reconstituted in vitro from purified recombinant components. The catalytic core of the RNA synthesis machinery is a 241 kDa large polymerase protein (L) that contains an RNA dependent RNA polymerase (RdRP), a polyribonucleotidyltransferase (PRNTase) that caps the mRNA, and a dual specificity mRNA cap methyltransferase (MTase). During mRNA synthesis, those activities are coordinated so that the nascent mRNA is capped, methylated and polyadenylated. Although L contains all the enzymatic activities for RNA synthesis, it requires a 29 kDa phosphoprotein (P) that bridges interactions between L and the nucleocapsid protein (N) that completely coats the genomic RNA template. In the last grant period, we developed in vitro assays to separately study each of the steps of mRNA cap addition independent of ongoing transcription. Those assays, combined with a powerful reverse genetic system allow mechanistic analysis of lethal mutations in the viral RNA synthesis machinery. We have used those assays to provide a map of where the different enzymatic activities for each step of mRNA cap addition are localized within L. Those studies lead us to the hypothesis that L contains independent functional domains whose activities are coordinated by the assembly of L into the RNA synthesis machine of the L-P complex with the N-RNA template. A major gap to understanding the mechanisms by which the RNA synthesis machinery of NNS RNA viruses function is the absence of structural information for L. During the next funding period, we will use electron microscopy, X-ray crystallography and in vitro assays of polymerase function to provide unique structural and functional insights into the RNA synthesis machinery of VSV. We will: (i) determine the functional organization of the VSV polymerase complex; (ii) determine the three dimensional structure of the VSV polymerase, and (iii) probe the relationship between the mRNA capping and RNA synthesis activities of L. The successful completion of this study will provide a structure of the polymerase of an NNS RNA virus as well as new mechanistic insights into the function of this RNA synthesis machine that may help in the rational design of antiviral therapeutics and candidate vaccines."}
{"text": "The Bypass Angioplasty Revascularization Investigation (BARI) is a randomized clinical trial to compare the relative efficacy of an initial strategy of PTCA vs. CABG in patients with multivessel coronary disease and severe angina or ischemia. Treatment strategies will be compared to respect to clinical outcome and economic impact. The trial was initiated in 1987 and investigators from 14 primary sites and 5 satellites have randomized 1829 patients. Of these, 43% are elderly, 27% are female, and 6% are black. BARI patients are at high risk for cardiac events; 55% presented with a history of myocardial infarction, and 69% with unstable angina or non Q-wave MI. At baseline, triple-vessel disease was present in 41% and 73% had three or more significant lesions (greater than or equal to 50% stenosis). As of July 1993, the first patient randomized will have been followed for 5 years and the last patient will have been followed for 2 years. In addition to the randomized trial, BARI contains a registry comprised of 2013 eligible-not-randomized patients and a sample of 422 patients deemed ineligible based on their angiogram. For both randomized and registry patients, we propose to complete and report 5-year follow-up results and extend follow-up to 10 years. In randomized patients, treatment strategies will be compared with respect to mortality, myocardial infarction, need for repeat procedures and hospitalizations, functional status, radionuclide ejection fraction and quality of life. Periprocedural outcome will also be reported. Similar analyses will be reported for both registry groups. To accomplish 10-year follow-up, we will use the same methods of data collection and management, and continue the same lines of communication between the clinical sites, central laboratories and the NHLBI that have served BARI successfully to this point. The clinical sites will rely on the excellent long-term relationships established with their patients and referring physicians. Ten-year follow-up results will provide the long- term cost of PTCA vs. CABG, not only in terms of dollar expenditures but in terms of quality of life, morbidity and longevity. The results of BARI will have an important impact on the future practice of coronary revascularization and thus affect hundreds of thousands of patients who are expected to undergo these procedures."}
{"text": "Ethnic minorities tend to face more stressors than the majority population and to have excess risk of many health problems. These disparities are strikingly evident for Puerto Ricans living on the mainland, especially for older Puerto Ricans who face psychosocial stressors related to drastically changed social and physical environments. The limited research on Puerto Ricans has provided evidence of much poorer physical health and greater disability', including cardiovascular risk, compared to other Hispanic groups. The effects of psychosocial factors on heart health risk are not well understood for older Puerto Ricans, as research has primarily focused on other Latinos and has been mainly cross-sectional. Theoretically, it is important to understand the mechanisms of mediating factors and the longitudinal causal pathways through which psychosocial factors influence cardiovascular risk. It is therefore critical to identify and explore the interactions between personal, social and environmental factors which contribute to cardiovascular risk for Puerto Ricans. The longterm goal of this work is to contribute to understanding psychological, social and environmental contexts of cardiovascular risk of older Puerto Ricans. The objective of this proposal is to examine in our study population how stressors, personal resources, social networks, and perceptions and characteristics ofthe built environment relate to cardiovascular risk indicators over time. We approach the analysis of these factors using a multi-level approach and drawing on the large scale cohort longitudinal data set with 1350 participants and three points in time. The aims ofthe projects are 1) to assess associations between depressive symptomatology, psychosocial stress and CVD risk over a period of 5 years; 2) to assess the characteristics of social networks and their effects on cardiovascular risk, and 3) to assess the characteristics ofthe local environment and its impact on heart health and cardiovascular risk. This project is innovative in its integration of analj/sis at the individual, social and environmental levels which should yield a richer data set and a more thorough analysis. The work vrill contribute significantiy to gaining a fuller view ofthe context of psychosocial stress and how it influences the cardiovascular health of older Puerto Ricans. We add an interdisciplinaty social science perspective to grounding and contextualizing findings from our proposed work but also those ofthe other projects in this program. Results will allow the development of more effective strategies and programs to reduce health disparities in this high risk population."}
{"text": "Work planned for the coming year will concentrate more heavily on studies of the ultrastructural correlates of ion and fluid transport and macrom lecule secretion in epithelial cells which are possible candidates for the type of cell affected by the genetic defect in Cystic Fibrosis. Attempts at identifying an abnormal cell or cell organelle in patients with CF will entail cytochemical, fine structural, electrophysiologic and certain biochemical analyses of human eccrine sweat glands & possibly specimens of nasal or tracheobronchial epithelium from control & CF subjects. Alterations in these tissues maintained in explant under various experimental manipulations will be assessed along with the effect of so-called factors thought to be present in body fluids of CF patients. Efforts will continue in collaboration with Dr. W.H.J. Douglas to culture epithelial cells from sweat glands of normal and CF cubjects & investigate abnormalities in the cultured cells from the patients again employing fine structural, cytochemical, electrophysiologic, ion flux & biochemical measurements. Investigation of the presence of amiloride channels from passive Na ion transport in the human sweat gland & respiratory epithelium will be initiated. These studies, in collaboration with Dr. E.G. Cragoe of Merck Sharp & Dohme, will include radioautographic demonstration of binding of C14 and H3 labeled derivatives of this diuretic drug to specific epithelial cells. Fluorescence microscopy with various fluorescent derivatives & electron spin resonance measurements with a spin labeled derivative will be utilized for localization of amiloride channels in various epithelial cells. Freeze-fracture studies now in progress will be pursued to characterize further the nature of cell junctions between various cell types in the sweat glands and respiratory epithelium & characterize intramembranous particles in plasma membranes of these cells. Investigations of the pathogenesis of mega inclusions in cells of beige mice with a genetic defect analogous to the Chediak-Higashi syndrome will continue with emphasis on the pathogenesis of inclusions in the proximal nephron, gastric epithelium and culture fibroblasts. Development & application of cytochemical methods for localizing & characterizing complex carbohydrates of immunocytochemical methods for visualization of carbonic anhydrase & other antigens will continue."}
{"text": "Neurocognitive impairments in HIV-infected individuals, collectively known as HIV-Associated Neurocognitive Impairments (or HAND) remains a significant problem in the era of Combined Antiretroviral Therapy (CART). In many HIV-infected individuals there is evidence of accelerated aging, including aberrant processing of amyloid precursor protein (APP). These disruptions seem to result in accumulations of pathogenic forms of amyloid- () in brain and are thus likely to also decrease the formation of soluble APP? (sAPP?), an important neurotrophic peptide. Our preliminary data suggest that accumulations of sphingolipids and complex glycolipids in intracellular compartments accelerates A formation by enhancing the activity of - and ?- secretases (that process APP to A), and by perturbing the intracellular trafficking / clearance of A. Previously we have documented accumulations of multiple sphingolipid species in HIV-infected individuals. These combined findings prompted us to determine if the accumulations of sphingolipid products in endosomes, lysosomes and/or autophagosomes are associated with aberrant APP processing, increased A deposition and decreased sAPP? in the setting of HIV-infection. In this application we propose a comprehensive approach to address this question, using human brain tissues, cellular/molecular approaches, and transgenic model systems to determine if increased brain levels of these lipid metabolites shifts APP processing to a more amyloidogenic (A) and less trophic (sAPP?) pheotype and if interventions that target sphingolipid metabolism can reverse these effects."}
{"text": "Xiphophorus fishes have been used as a vertebrate experimental model for fundamental biomedical research for over 60 years. This R24 proposal outlines both resource and research components designed to maintain and enhance the Xiphophorus Genetic Stock Center (XGSC) that has operated within the United States since the 1930's making it one of the oldest live animal resources centers worldwide and a national scientific treasure. The XGSC houses over 60 pedigreed genetic lines representing 25 Xiphophorus species in over 1,400 aquaria. The XGSC has provided pedigreed fish and materials to researchers in over 30 laboratories representing 11 countries. The traditional strength of the Xiphophorus experimental model involves the non-biased assessment of genetic inheritance patterns associated with complex phenotypes within intact animals. For example, genetic control of tumor susceptibility has been documented in both pure Xiphophorus strains and select interspecies hybrids, for a variety of spontaneous and induced neoplasms including several genetically distinct melanomas. Our preliminary studies represent a first global comparison of the relative abundance of species-specific allele expression in two vertebrate species and Fl hybrids. In this initial study we were able to identify substantial changes in gene expression levels for parental alleles in the interspecies hybrid genetic background. We also found a new class of genetic interactions that lead to substantially suppressed gene expression in Fl hybrids relative to either parent. These initial experiments, employing RNAseq to follow allele specific gene expression in Fl interspecies hybrids, serve as examples of the Xiphophorus model may be used to uncover new knowledge regarding the mechanics of gene interactions, the genetic basis of developmental programs, complex behaviors, and the molecular processes underlying disease initiation and progression. If the goals of this proposal are realized, mechanistic dissection of Xiphophorus interspecies hybrid melanoma models may be approached and the results gleaned will have translational significance to tumorigenesis in humans."}
{"text": "This application is for a Patient-Oriented Research Career Development Award (K23) for Dr. Judith Tsui, an Assistant Professor in General Internal Medicine at Boston University School of Medicine, who is establishing herself as an investigator in hepatitis C virus (HCV) related outcomes. The research plan both extends her prior work in HCV, and allows her to grow in a new direction as a pain researcher focusing on substance users. The award will provide Dr. Tsui with the support necessary to accomplish the following goals: 1) develop expertise in the etiology of chronic pain in substance users with chronic viral infections like HCV and HIV; 2) publish research that will lead to improved treatments for pain among substance users; and 3) transition into an independently funded researcher by obtaining an R01. The comprehensive training plan includes formal coursework, as well as hands-on training in pain measurement and experimental pain models. Dr. Tsui's primary mentor will be Dr. Jeffrey Samet, an established investigator in the fields of substance use and chronic viral infections. She will receive additional mentoring from Dr. Jianren Mao, a leading translational pain researcher, and Dr. Robert Edwards, an accomplished junior investigator and clinical psychologist. Pain is a major problem among substance users, and a better understanding of its etiology is needed to improve clinical care. There is limited research to determine how viral infections such as HCV and HIV impact risk for developing chronic pain and painful conditions. Dr. Tsui will explore the novel hypotheses that HCV is associated with: 1) hypersensitivity to pain under experimental conditions and 2) clinical pain. To explore these hypotheses, Dr. Tsui will conduct an experimental, cross-sectional study of HCV mono- infected, HIV/HCV co-infected, and uninfected opioid addicts that compares experimental pain responses and self-reported clinical pain. She will assemble a cohort of 120 opioid dependent patients on buprenorphine or methadone: 40 HCV+/HIV-, 40 HCV+/HIV+ and 40 HCV-/HIV-controls. Participants will undergo the following experimental testing: 1) the cold-pressor test to assess cold pain tolerance 2) mechanical stimulus to test mechanical pain thresholds and 3) repetitive pinprick to assess for temporal summation. The study will also collect data on pain and pain-related variables using validated scales such as the Brief Pain Inventory, as well as measurements of pro- and anti-inflammatory cytokines (TNF-1, IL-2, IL-6, IL-2, IL-10 and IL-4) from peripheral blood samples. She will perform appropriate statistical testing to assess whether opioid addicts who are HCV infected (HCV+ alone or HCV+/HIV+) have different experimental pain responses (lower pain tolerance/thresholds and more temporal summation) and a higher prevalence of self-reported chronic pain, and to explore whether inflammatory cytokines mediate found associations between HCV and pain. This research will contribute to our understanding of the causes of pain among substance users, and will shape future studies to test new approaches (possibly including anti-HCV treatment) to prevent and treat pain in this vulnerable patient population. PUBLIC HEALTH RELEVANCE: Pain is a common problem among current and former substance users, many of whom are infected with HIV and HCV. A better understanding of pain etiology is needed in order to effectively prevent and treat chronic pain among patients with substance use problems. This research addresses the simple (yet relatively unexplored) question: how do chronic viral infections contribute to patients' vulnerability to pain? In addition, it begins to explore potential mechanisms for pain hypersensitivity in the setting of HCV by investigating the role of inflammatory cytokines in shaping pain responses. The results of this research may directly benefit patients with a history of substance use and pain by 1) providing an explanation for their pain and 2) leading to improved treatments for pain, including anti-HCV therapy or anti-cytokine medications (such as pentoxyphylline). In addition, it will inform clinicians and policy makers who must consider the long-term consequences of HCV infection when weighing costs/benefits of treatment and interventions to prevent transmission."}
{"text": "We have analyzed the development of B cells in Terminal Deoxynucleotide Transferase (TdT)knockout mice. Two aspects have been studied: 1) the effect on the immune response to phosphocholine (PC) in mice immunized with Proteus morganii; and 2) the effect on B1 B cell development. The immune response of normal mice to P. morganii is dominated by B cells expressing a M603 idiotype. Thus, the V1 H chain of these antibodies has a somatic mutation at the V:D junction in which the germline encoded Asp at position 96 has been changed to an Asn. It has been claimed that anti-PC antibodies of other idiotypes are not capable of binding to the PC-epitope on P. morganii. However, in TdT knockout mice immunized with this bacterial antigen only germline encoded T15-idiotype anti-PC antibodies are produced. This data demonstrates that TdT is responsible for the Asp to Asn change that occurs in the V1 gene of normal mice and it also shows that the response to P. morganii is not restricted to the M603-idiotype. Published work on TdT knockout mice shows that loss of TdT has very little effect on the animals ability to respond to a wide variety of antigens. This was surprising in that TdT isresponsible for generating diversity in the 3rd hypervariable region of the antibody molecule and this region of the antibody often defines the specificity of the antibody molecule. We found that TdT knockout mice have a higher level of B1 B cell development than normal mice. This increase in CD5+ B cells is accompanied by a 3 fold increase in the number of peritoneal B cells that bind to phosphatidyl choline (PtC). In normal mice up to 50% of the PtC binding cells display the VH-12 idiotype. However, in TdT knockout mice the VH-12 PtC-specific B cellsrepresent a much lower fraction of the PtC pool. These data demonstrate that TdT plays a role in controlling the homeostasis of the B1 B cell pool, and it also affects the H-chain makeup andantigenic specificity of this pool of B cells."}
{"text": "Terminally differentiated ventricular muscle cells virtually cease to proliferate, and subsequent cardiac growth occurs almost exclusively by cell enlargement. Hence, failure to increase cell number preludes cardiac regeneration as a means to reconstitute functional mass after infarction or other insult. This irreversible growth arrest poses a therapeutic challenge, especially in the elderly, where the includes of infarction and heart failure is high. Using an array of recombinant viruses to direct the expression of adenoviral EIA proteins, the applicant has recently demonstrated evidence for dual pocket protein-and p300-dependent pathways that govern the cell cycle in cardiac muscle. Drawing on these developments the present application proposes to study in depth the endogenous occupant of the \"pocket\", the transcription factor known as E2F; modulators of pocket protein function in the heart, known as cyclins and clyclin-dependent protein kinases (Cdks); and the molecular relationship between the pocket protein-and p300-dependent mechanisms. Specific Aims of the present project are: (1) to establish molecular mechanisms for the phenotype effects of E2F ( activation of the cell cycle, and repression of tissue-specific genes) in cardiac muscle cells; (2) to clarify what role is played by endogenous regulators of free E2F (G1 cyclins, Cdks, and Cdk inhibitors) in cardiac muscle cells; (3) To determine the epistatic relationship between E2F and the p300 pathway in cardiac muscle cells; and (4) To investigate the function of E2F in adult cardiac muscle in vivo, by direct injection of e2F virus and by cardiac-restricted homologous recombination of the pocket protein gene, Rb."}
{"text": "This proposal is directed toward the synthesis of cembranoid natural products, a class of compounds whose chemistry and biological activity have been little studied. The specific objectives, isolobophytolide, anisomelic acid, asperdiol and peunicin are cytotoxic and may exhibit antimicrobial, antiprotazoal, and cardiosvascular activity. A major objective is the development of reliable synthetic routes to this widespread class of macro carbocyclic natural products. Of particular interest is the assemblage of multifunctional chiral intermediates suitable for cyclization to the characteristic 14-membered cembranoid macrocycle. Biological testing of synthetic intermediates will be arranged with the appropriate N.I.H. division."}
{"text": "Neurological complications occur commonly in HIV infection, with over 40% of individuals being at risk for developing HIV-related neurocognitive impairment (NCI), with the numbers increasing in the era of effective antiretroviral therapy (1). Factors that underlie the development of NCI include neuronal dysfunction due to enhanced production of proinflammatory cytokines (e.g., TNF-? and IL-1?) and other inflammatory mediators that are secreted by HIV-infected cells in the brain (2,3). Contributing to such responses is impairment metabolism mediated by nicotinamide adenine dinucleotide (NAD), which is essential for normal mitochondrial function and the production of energy substrates in cells. In this grant, we will analyze potential effects of exposure to cigarette smoke (CS) and nicotine as co-factors for increasing the risk of NCI in HIV infected individuals. Cigarette smoking has been shown to alter a number of innate and adaptive immune mechanisms and can elicit cellular oxidative stress responses that can promote injury. Most marketed brands of cigarettes contain varying amounts of nicotine. In smokers, CS causes a leukocytosis and, in HIV-infected individuals, smoking is associated with higher plasma HIV viral loads and an increased mortality. It has been estimated that over 40% of HIV+ individuals are cigarette smokers (6), a number that is twice the estimated prevalence of smoking among adults in the general population (7). Nicotine, via activation of nicotinic acetylcholine receptors (nAChR), promotes cigarette smoking through its addictive properties. By activating these receptors, nicotine has been also shown to suppress immune activation and proinflammatory responses through direct effects on immune cells as well as through pathways that are mediated by activation of nicotine receptors (8-10). However, due to effects of HIV protein and with chronic nicotine exposure and upregulation of nAChR, proinflammatory responses have been observed to increase, thereby potentially contributing to CNS damage that occurs in the context of HIV infection. In previous studies in adult Lewis rats, we demonstrated that CS exposure can result in marked inflammatory and oxidative stress responses in the brains of the exposed animals (11). We have subsequently demonstrated that such effects can be also observed in HIV-1 transgenic rats, with more prominent responses developing than what are observed in corresponding wild-type animals. We also provide evidence that such responses can be driven by nicotine that is present in the CS. Activation of nAChR can also regulate cellular levels of NAD and activation of associated metabolic pathways. The potential effects of such activation in HIV infection and the generation of chronic inflammation in brain has not been previously studied. The proposed studies will be performed in vivo utilizing the transgenic rat model as well as in vitro utilizing primary astrocyte cultures that express the same HIV genes as the transgenic rat in vivo."}
{"text": "DESCRIPTION: What is proposed is to continue and expand a collaboration between an outstanding peptide and organic synthesis laboratory in Latvia and an outstanding cytoskeleton laboratory in Boston. Potential actin or polyphosphoinositide lipid (PPI)-binding peptides will be synthesized based on the sequences of various proteins identified by genetic or biochemical data. The structures of some peptides will be calculated by molecular modeling in Riga and their biochemical effects on lipids, actin binding proteins, and intact cells will be studied in Boston. These studies will be expanded to include fluorescent peptides shown to retain the PPI-binding properties of unlabeled analogs. One of these peptides exhibits the unusual property of crossing the plasma membrane of living cells. This peptide has potential applications as a research tool to identify or disrupt PPI localization in vivo or clinically as a carrier of pharmaceutical agents into specific cell types. These possibilities will be tested using derivatives synthesized in Riga and shown in Boston to form gels at very low concentration by assembling into rodlike micelles. These peptide gels are ideal materials to be studied by rheologic methods and have potential for application in drug delivery or antigen presentation."}
{"text": "In rural South Africa, only two-thirds of HIV+ pregnant women seeking antenatal care at community health centers took full advantage of available prevention of mother-to-child transmission (PMTCT) services in 2010 (SADOH). While engagement of male partners has been encouraged as a potential means of increasing PMTCT uptake, men have been reluctant to accompany their wives/partners to antenatal care. Recent studies generally support male involvement in promoting PMTCT, but the nature and impact of that involvement is unclear and untested. It is also clear that factors such as stigma, disclosure and intimate partner violence pose significant barriers to PMTCT uptake and retention in care, suggesting that male involvement may be necessary, but not sufficient to accomplish the WHO goal of <5% infant HIV incidence. Additional measures may be needed to increase participation by HIV positive pregnant women in PMTCT. In 2011, Mpumalanga Province had the highest rates of HIV in the country (36.7%) and rates of infant HIV incidence in rural clinics ranged up to 50%. Rates of PMTCT uptake in the Province have been among the lowest in South Africa (69%). This application proposes to expand on a successful PEPFAR- supported, PMTCT couples intervention pilot study conducted in Mpumalanga Province, (Vikela Umndeni: Protect Your Family) to include a more representative population of HIV positive pregnant women and their partners, the primary objective being to determine whether male partner involvement plus a behavioral intervention would significantly reduce infant HIV incidence by increasing levels of adherence to ARV/ PMTCT protocols, including breastfeeding and family planning, during the antenatal and post-natal periods. The proposed study will enroll two cohorts of HIV positive pregnant women recruited from 12 randomly assigned Community Health Centers (6 experimental, 6 control): a) Women attending without their male partners (n = 720), followed by b) Women attending with their male partners (n = 720 couples), to determine whether the influence of male participation itself or combined with a behavioral PMTCT intervention can significantly reduce infant HIV infection ante-, peri- and post-natally. It is our intention to significantly increase PMTCT participation from current levels (69%) in Mpumalanga Province to 90-95% through engaging women and couples in a unique, controlled, six session ante- and post-natal risk-reducing/PMTCT promotion intervention addressing the barriers to PMTCT (e.g., stigma, disclosure, intimate partner violence, communication, infant feeding practices, safer conception) that prevent women and men from taking full advantage of the treatment opportunities available to them and their infants. Based upon the encouraging preliminary results from our pilot study, successful CHC adoption of the Vikela Umndeni: Protect Your Family program could have major public health policy implications for containing the epidemic among the most vulnerable populations in rural South Africa: HIV+ pregnant women and their infants."}
{"text": "Mammographic screening leads to many women being diagnosed with ductal carcinoma in situ [DCIS], yet we cannot accurately predict which lesions will undergo malignant progression to invasive ductal carcinomas [IDC] or effectively block this transition. Studies of human breast biopsies have implicated in this process cysteine cathepsins V/L2 and B in tumor cells and macrophages and cathepsins F, K and L in myoepithelial cells/[myo]fibroblasts. Aberrant signal transduction, for example through p21-activated kinase 1 [PAK1], may contribute to increased pericellular proteolysis. Our working hypothesis is that the transition from pre-invasive DCIS to invasive carcinomas and the rapid progression of some DCIS lesions are mediated through alterations in proteolytic pathways in DCIS cells and DCIS-associated cells, and that dysregulated PAK1 contributes to the induction of these aberrant proteolytic pathways. To test this hypothesis, we will recapitulate the transition from pre-invasive DCIS to invasive carcinoma using in vitro and in vivo progression models that we have designated MAME for mammary architecture microenvironment engineering. In these models, we will use isogenic MCF10 cell lines [AT1, DCIS1 and CA1d] and two human DCIS cell lines [SUM-102 and SUM-225]. Our specific aims are to: 1. Modulate expression and activity of cysteine cathepsin V or B in the isogenic and SUM DCIS cell lines, both by direct targeting and through intervention at the level of PAK1, and determine using the in vitro MAME model whether the invasive phenotype is altered; 2. Determine using the in vitro MAME model whether the invasive phenotype can be altered by co-culturing modified cells from Aim 1 with myoepithelial cells, [myo]fibroblasts or both cell types, using wild-type cells and ones in which expression and activity of cysteine cathepsin F, K or L have been modulated; 3. Determine using the in vivo MAME model whether the malignant phenotype of xenografts of modified cells from Aim 1 can be altered by simultaneous implantation of myoepithelial cells, [myo]fibroblasts or both cell types, using wild-type cells and ones in which expression and activity of cysteine cathepsin F, K or L have been modulated; and 4. Screen [via our Hu/Mu ProtIn chip] the in vivo MAME model for proteolytic pathways that may contribute to the transition from DCIS to IDC and use the in vitro MAME model to define functional changes with libraries of reagents from the Center on Proteolytic Pathways. Validating, in the context both of the tumor and its microenvironment, proteases key to progression of DCIS to IDC, and kinase pathways that regulate them, should identify potential targets for therapeutic intervention as well as biomarkers to distinguish DCIS lesions that will rapidly progress to IDC. PUBLIC HEALTH RELEVANCE: Proteases and kinases are the subject of intensive efforts by the pharmaceutical industry to develop new treatment strategies for human diseases, including cancer. Our proposed studies will discover and validate protease pathways that are active in the tumor microenvironment and that mediate the transition to a full-blown malignancy, and kinase pathways that regulate these protease pathways. We anticipate that our studies will identify biomarkers to distinguish premalignant lesions that will progress to invasive cancers and define targets that will abrogate that progression. [unreadable] [unreadable] [unreadable]"}
{"text": "The immune system plays a critical role in the pathogenesis of pain triggered by injury or illness. A precise understanding of the mechanisms through which particular immune mediators contribute to sensitization of nociceptive neuronal pathways will be essential for the development of more efficacious treatment strategies. The complement system is a principal component of innate immunity. This proposal focuses on two highly active split products of the complement system, C3a and C5a. Increased production of C3a and C5a has been reported in various pathological states associated with pain, including arthritis, pancreatitis, inflammatory bowel disease, burns and surgical trauma. Blocking the synthesis of C3a and C5a or antagonizing their receptors produces analgesic effects in animal models of inflammatory, neuropathic and postoperative pain. Moreover, direct administration of the complement fragments increases nociceptive sensitivity to heat and mechanical stimuli in animal models. In spite of this strong evidence implicating the complement system in the development of pain hypersensitivity, the underlying mechanisms are not understood. The goal of this project is to investigate mechanisms that are responsible for the enhanced pain sensitivity produced by the generation of C3a and C5a in the affected tissues. We hypothesize that C3a and C5a receptors are expressed and functionally coupled with TRPV1 in a subset of primary nociceptors; activation of these receptors sensitize nociceptors via protein kinase C-dependent modulation of the TRPV1 channel, which is known to function as a molecular integrator of pain producing stimuli. We will use a multidisciplinary approach involving immunohistochemistry, single-cell RT-PCR, [Ca2+]i imaging, patch-clamp analysis, single-fiber recordings and measurement of nociceptive behavior to test our central hypothesis. In Aim 1, we will characterize the expression and subcellular distribution of C3a and C5a receptors (C3aR and C5aR, respectively) and TRPV1 in various classes of sensory neurons identified by specific molecular markers. We will also use Ca2+ imaging and patch-clamp recordings to test functional coupling of TRPV1 with C3aR and C5aR. In Aim 2, we will investigate intracellular signaling cascades that link the activation of C3aR and C5aR with TRPV1 sensitization. In Aim 3, we will use single-fiber recordings and behavioral studies to examine the role of PKC- dependent modulation of TRPV1 downstream of C3aR and C5aR activation in regulating nociceptor excitability as well as heat and chemical sensitization of nociceptors. The proposed studies will help to characterize the novel roles of C3a and C5a receptors in the regulation of nociceptor function, and may lead to the development of new therapeutic strategies targeting the complement system to alleviate pain. PUBLIC HEALTH RELEVANCE: Pain management remains one of the most serious public health problems. The proposed studies will help to better understand how the complement system contributes to the pathogenesis of pain caused by surgical trauma or illnesses such as arthritis, pancreatitis and inflammatory bowel syndrome, and may lead to the development of new therapeutic strategies targeting the complement system to alleviate pain."}
{"text": "Focal neuropathies of the hand are among the most common diseases treated by neurologists. The two most common are median neuropathy at the wrist (carpal tunnel syndrome) and ulnar neuropathy at the elbow. The current electrophysiological methods of assessment of focal neuropathies can be painful, and include nerve conduction studies and electromyography. Electrical impedance myography (EIM) is a new technique that can be used to assess neuromuscular disease. We have had success in using EIM in small muscles in animals, and limited experience using EIM in small muscles in human studies. We plan to refine these techniques in this proposal, while studying focal neuropathies of the hand. The long-term goal of this proposal is to make EIM a valuable tool for the assessment of neuromuscular disease. Establishing standards for use in small muscles will complement the known utility of EIM in larger muscles and help make it such a tool. We have three specific aims for this study. First, we aim to establish a range of normality for EIM for the three hand muscles that are tested most commonly in standard neurophysiologic assessment: the abductor digiti minimi, first dorsal interosseous, and abductor pollicis brevis. Second, we will evaluate EIM parameters in a group of patients with median or ulnar neuropathy, predicting that EIM parameters recorded from affected muscles in patients with median and ulnar neuropathy will differ from normal subjects due to the underlying architectural alterations in the muscle caused by neurogenic injury. Third, in order to determine the ability of EIM to judge lesion severity, we seek to determine the correlation between EIM parameters with standard electromyography (EMG) parameters in patients with focal neuropathy. Relevance to public health: Focal neuropathies of the hand, which include median neuropathy at the wrist (carpal tunnel syndrome) and ulnar neuropathy at the elbow, occur in approximately 10% of people. The standard neurophysiologic assessment of these problems includes electromyography and nerve conduction studies, both of which may be poorly tolerated. We are developing electrical impedance myography as a painless, noninvasive, quantifiable technique to assess focal neuropathies of the hand, and neuromuscular diseases in general. [unreadable] [unreadable] [unreadable]"}
{"text": "Type I collagen is a fibrous protein which is commonly used in the human body for structural and functional integrity. Mutations in the alpha-1 and alpha-2 chains of this triple-helical protein induce the disease Osteogenesis Imperfecta (OI). OI is expressed in people by a number of phenotypes ranging from a disease process that is lethal in utero to one that is only detected through gene analysis. We are using the techniques of molecular dynamics and free energy perturbation to predict the phenotypic severity that result from single point mutations of type I collagen. In particular, we are focusing on the following three issues in our investigation: 1) validation of the AMBER molecular mechanics force field for studying fibrous proteins; 2) determination of protein and water-protein structural changes induced by single point mutations of type I collagen; 3) determination of changes in stability between native collagen fragments and collagen fragments in which a single point mutation has occurred. We have completed the validation of the AMBER molecular mechanics force field for both molecular dynamics and free energy perturbations of collagen-like peptides. We have determined that solvent provides an overall network of stability to the collagen molecule, but does not provide the inherent stability to the triple helix. We have begun a collaboration with Professor Ron Raines at the University of Wisconsin to determine what molecular interactions/effects (e.g., hydrogen bonding, induction effects) contribute to the stability of the triple helix."}
{"text": "Project 4: Abstract Spliced X box-binding protein-1 (sXBP1) is prominent endoplasmic reticulum (ER) stress response factor of the Unfolded Protein Response (UPR) of the ER. The highly developed ER and protein processing and trafficking organelles of the pancreatic acinar cell are consistent with its specialized role as the site of synthesis and trafficking of large quantities of digestive enzymes. XBP1 is specifically necessary for development of exocrine pancreatic tissue and we have published and present further preliminary data here that inhibition of its expression promotes organellar disorders and cellular failure of the pancreatic acinar cell; and that increases in its expression are associated with protection from organellar disorders and cellular failure with stressors that cause pancreatitis. We find that XBP1s inhibition correlates with activation of additional ER- originating signals associated with inflammation and cell death, i.e., the PKR-like endoplasmic reticulum kinase (PERK) and C/EBP homologous protein (CHOP), i.e., pathways that promote the latter responses. Little is known about the regulation of XBP1s in exocrine pancreas. Our preliminary data suggest roles for calcium metabolism, ATP and a functionally pleiotropic ER transmembrane protein, Bax inhibitor-1 (BI-1) in regulating sXBP1. On the basis of these observations, we propose a series of hypotheses: that pancreatic acinar cell sXBP1 expression is subject to regulation by multiple factors including cellular Ca2+ responses; BI-1 expression; and mitochondrial ATP production. In response to stress, sufficient sXBP1 responses are required to feed back to restore control over many of these same factors to maintain cellular homeostasis. In contrast, sXBP1 deficiency exacerbates ER stress and leads to over-activation of the pathologic arm of the UPR (PERK/eIF2 /CHOP), disturbances of protein trafficking, and pancreatitis. We will pursue 3 aims in this project. Aim 1 will investigate the regulation of sXBP1 expression; Aim 2 is to determine the effects of sXBP1 expression insufficiency on UPR (PERK/eIF2 /CHOP), disturbances of protein trafficking, and pancreatitis; and the final aim is devoted to demonstrating that increasing sXBP1 expression enhances protective responses in the exocrine pancreas. Because of our initial findings that sXBP1 has significant effects on the endo-lysosomal/trafficking systems investigated in other projects in this program, it is highly integrated into the Program Project. Because we will show that sXBP1 can attenuate trafficking disorders and protect against pancreatitis, the project is highly translational."}
{"text": "Defects in human left right (LR) axis formation cause birth defects of the heart, vasculature, lungs, and gastrointestinal tract that are life-threatening and costly to the healthcare system. To understand how laterality defects develop, it is essential to understand the molecular mechanisms by which LR asymmetry is established. My long-term goal is to understand the mechanism of how LR asymmetry is established during early embryogenesis. My previous work including collaboration with my colleagues in this field has revealed important steps in this process in the mouse model. However, there are still gaps in our understanding of the entire process, such as the mechanisms by which the node structure and rotating node cilia develop, and how LR polarity in the node is transferred to the left LPM. Here I will address these questions by investigating roles of definitive endoderm in LR determination. To date, no report has been focused on roles of endoderm in LR asymmetry. But several series of evidence suggest importance of endoderm in this process. Our hypothesis is that endoderm cells regulate LR determination in two ways: regulating node formation and/or function, and transferring LR signal from the node to the LPM. This hypothesis will be addressed by analyzing Sox17 mutants because Sox17 is the only gene that specifically expressed in the endoderm and primary defects of the mutants are specific to the definitive endoderm. Aim 1 will study the nature of the initial LR determination defects occurring in Sox17 mutants by characterizing the node and cilia formation, in addition to the development of nodal flow. Aim 2 will examine the possible defects occurring in the process of transferring LR signals from the node to the LPM regarding the gap junctional transport and intracellular Ca2+ signaling. In addition to chemical blockers, the roles of gap junctions will be tested by a sophisticated transgenic approach using a dominant negative form of connexin that can be temporally and/or spatially controlled. Conditional mutants of Sox17 will be studied to identify the importance of endoderm cells in the signal transfer from the node to the LPM, being separated from the initial abnormalities in the node. We have found that the Nodal gene continues to be expressed in migrating endoderm in the Sox17 mutants, which could be the cause of endoderm defects in Sox17 mutants that affect LR determination. In Aim 3, I will test this possibility by transgenic techniques. I will rescue Sox17 mutants by expressing Nodal antagonists in endoderm and the Sox17 mutant phenotype will be reproduced by ectopically expressing the Nodal gene in the endoderm of wild type embryos. The 20 yrs of experience that the PI possesses in this model system has enabled us to design and optimize novel tools and techniques that are ideal for addressing the questions I have put forth in this proposal. The proposed experiments will reveal novel roles of endoderm cells in establishment of LR asymmetry in mammals. This work may ultimately provide insight into the mechanisms underlying human birth defects due to problems in laterality. PUBLIC HEALTH RELEVANCE: The proposed experiments will reveal novel roles of endoderm cells in establishment of LR asymmetry in mammals. This work may ultimately provide insight into the mechanisms underlying human birth defects due to problems in laterality."}
{"text": "Prediction of RNA secondary structure is important in many areas of molecular biology. For example, in phylogenetic studies, prediction of the secondary structure of \"16S-like\" rRNA is an important piece of information used in determining the correct alignment of sequences. The \"well-defined\" regions of the multiply aligned sequences are then used to make phylogenetic inferences. Secondary structures can be used in part to explain translational controls in mRNA, and replication controls in single-stranded RNA viruses. RNA structure also plays an important role in the regulation of retroviruses and cellular messenger RNAs. Secondary structure modeling can be used as a first step to the more intricate process of three-dimensional modeling. This could include the modeling of ribosomal RNA or catalytic RNA's such as group I introns. The folding patterns of individual RNA molecules are most often predicted using energy minimization approaches based on dynamic programming algo rithms. The optimization of the most modern versions of these algorithms on highly parallel computers will allow the application of these methods to biological systems previously too large for these calculations. Although the RNA folding problem is ill-conditioned, this drawback can be mitigated by the use of special \"energy dot plots\" that show the superposition of all possible foldings in the vicinity of a global energy minimum. Multiple predicted foldings indicate either the inability of the present modeling procedure to yield an unambiguous answer, or the actual presence of multiple structures (or both). We are now focusing on the MFOLD program, a more recent version of the program that incorporates these advances. Several cycles of optimization have been performed on MFOLD. First, the existing SGI version was simply ported to the Paragon. As expected this version was not particularly efficient since it required large global data arrays and frequent communication. In a second step, it was determined that the computationally intensive part of the code lies in the calculation of multiply branched loops. After optimizing this part of the computation the code performed relatively well, but was limited to relatively small molecules because of its replication of the stored energy values on each processor. Finally, a distributed memory version has been implemented that allows molecules of at least 20,000 bases to be analyzed. This appears to be both the fastest implementation, as well as the most memory efficient, currently available. In the current year, we proceeded with the porting of the parallel code from the paragon to the CRAY T3E. This meant a translation of the parallel communication calls from the nx library to the MPI library. Moreover, this port involved many declaration changes, and other formatting modifications needed to compile the code on the T3E. At this point in time, we have completed nearly 95% of the port. There remains a small error in the traceback routine. In the near future, we will complete the testing of the code, and add in a couple of communications optimizations that should improve performance. In the next year we will make the MFOLD code publicly available through the NBCR as an additional transparent supercomputing service."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Objective: To convert human pluripotent stem and somatic cells into hematopoietic stem cells (HSCs) and self renewing cardiac progenitor cells. The Midwest Progenitor Cell Consortium is a collaboration between the UW-Madison and the University of Minnesota, which will serve as a research hub within the NHLBI's Progenitor Cell Biology Consortium. The goal is to develop new strategies to convert human pluripotent stem and somatic cells into hematopoietic stem cells (HSCs) and self renewing cardiac progenitor cells through identification of genetic program leading to formation of pre-HSCs and cardiac progenitors and activation of self-renewal program in lineage restricted cells. This project uses WNPRC Stem Cell Resources."}
{"text": "Pulmonary fibrosis is the leading cause of death in systemic sclerosis (SSc). The NIH/NHLBI-sponsored Scleroderma Lung Study (SLS) was a 13-center, double-blind, randomized controlled trial designed to evaluate the effectiveness and safety of oral cyclophosphamide (CYC;less than or equal to 2 mg/kg/d) versus placebo as a 1- year treatment for patients with active, symptomatic scleroderma-related interstitial lung disease (SSc-ILD). The goal of this hyper-accelerated award application is to evaluate stored biologic samples from the SLS in order to define the pathophysiologic mechanisms that underlie SSc-ILD, to predict the presence and activity of SSc-ILD, to predict patient subsets that will benefit the most from cytotoxic therapy, and to aid in the design of future clinical studies in which other biologic agents, with other mechanisms of action, can be evaluated for their ability to improve clinical responses beyond those observed with CYC alone. Analysis of the BAL fluid, cell pellet mRNA, and plasma samples will be carried out with a focus on candidate biomarkers representative of the inflammatory, proliferative and obliterative vascular, and fibrotic/tissue matrix components of SSc-ILD. The unique strengths of this proposal are the near complete set of biologic samples which include material from the plasma as well as the primary target organ (lung);the rich data set from the SLS in which there are both extensive baseline features that define the presence and extent of disease and multiple positive outcomes in response to treatment;the identification of distinct pathogenic components upon which to focus our biologic analysis. By the completion of this study, we hope to have gained important new insight into the biology of SSc-ILD, to be able to use plasma and/or BAL samples to identify patients with potentially responsive lung and skin disease, and to provide important insight to the SLS Investigators as they plan future therapeutic clinical trials. Furthermore, we will garner new information as to the biology and pathogenesis of SSc-ILD. Relevance to Public Health: Successful completion of this study will further our understanding of scleroderma lung disease. It will also allow us to develop biologic markers and predictors of disease progression and to determine which patients would benefit most from treatment with cytotoxic therapy."}
{"text": "A study of the macroscopic properties of thermo-sensitive poly(DIOMMA) revealed a reversed temperature dependence in water versus alcohols. Gels made of the cross-linked analog also showed the same reversed trend between both solvents. We have studied the role of solvent in molecular processes of the polymer aggregations (PA) and the volume phase transitions (VPT) of gels in water or alcohols. We have completed extensive temperature dependence studies of 1H NMR spectra and some relaxation times for poly(DIOMMA) and its cross-linked analog (systems A and B) at both 360 and 500 MHz. Based on these molecular level data, we can deduce the relative strength and nature of the polymer-polymer, polymer-solvent and solvent-solvent interactions. Volumes of gel bead systems (these were created by cross-linking poly(NIPA), poly)NNPA), poly(NIPMA) and poly(NNPMA) with water) shrink at temperatures above the phase transition temperature (ph. tr.T). Spectral changes in 1H NMR (360 MHz) have recently been analyzed from 3 to 65 *C. These data have revealed details of differences in the molecular processes of the VPT among [unreadable]these gel systems. Spectra at temperatures below ph. tr. T (swollen state) consist of several peaks representing various chemical groups of the polymer. The linewidths of all peaks in the spectra suddenly broaden just above the ph. tr. T. These broadenings increase with rising temperature and, at the same time, a broad peak emerges near the HOD peak. Eventually all peaks coalesce to this new broad peak, except in the poly(NNPMA) system where the linewidths of all peaks become rather narrow again at high temperatures. Determination of the ph. tr. T. is usually defined as the onset of volume contractionexpansion measured by certain macroscopic methods. However, the IH NMR spectra indicate that significant spectral changes of the polymer component certainly begin within a narrow temperature range around the ph. tr. T., though gradual changes still continue for 10 to 30* C above the ph. tr. T. These results suggest that molecular processes in the VPT occur rather gradually by manifesting various conformational changes in the polymer chain with changing temperature. Our recent measurements of the temperature dependence of density of various gels demonstrated that onset of volume contractionexpansion corresponds to losing or gaining solvent molecules to or from the bulk liquid phase. Therefore, broadening of NMR linewidths in the collapsed state can be attributed to close proximity of various chemical groups caused by the loss of water and thus increasing the nuclear magnetic dipolar interactions between protons."}
{"text": "Structural abnormalities of the amygdala/hippocampal complex are a consistent finding in schizophrenia. In animal models, neonatal damage to the amygdala/hippocampal complex results in adult onset dopamine (DA) dysregulation, another key feature of this illness. Maintaining the integrity of amygdala/hippocampal circuits therefore appears critical to later DA function. We have previously shown that the extended amygdala, a major output region of the amygala and other temporal lobe structures, has broad inputs to the dopamine neurons. This pathway is thus a potential route by which amygdala-hippocampal abnormalities may eventually lead to DA dysregulation. The proposed studies will examine how the amygdala and hippocampus can influence the midbrain DA system through the extended amygdala. The fact that temporal lobe injury results in DA dysregulation only later in development suggests that plastic changes eventually influence DA output. Our preliminary results show that B lymphocyte 2 protein (bcl-2), which protects cells from excitotoxic damage and also has neurotrophic effects, is highly concentrated in specific subregions of the adult primate temporal lobe. Our preliminary results show high concentrations of Bcl-2 positive cells in the extended amygdala, and in subregions of the amygdala and hippocampus associated with schizophrenia. The presence of bcl-2 in specific circuits may help to identify excitatory pathways most susceptible to plastic changes and/or excitotoxic stress in adult animals. The proposed studies will identify temporal lobe circuits that influence DA through the extended amygdala. Specifically we will: 1) identify direct amygdaloid and hippocampal inputs to the extended amygdala-DA pathway, 2) identify indirect hippocampal pathways through the amygdala that influence the extended amygdala, 3) determine whether specific amygdaloid and hippocampal input/output paths contain Bcl-2 immunoreactive cells, 4) determine the extent to which hippocampal inputs overlap amygdala subregions that project to the extended amygdala, and the extent to which this input overlaps inhibitory interneurons and bcl-2-containing cells."}
{"text": "Cultured human melanocytes provide an excellent model for the study of neural crest development. Discrete growth and differentiation signals can be delivered to homogeneous cell populations and the effects of these signals monitored by changes in morphology, growth rate, differentiated functions and gene expression. In this way, an in vitro model of melanocyte differentiation can be defined and ultimately correlated with characteristic developmental abnormalities of the pigmentary system. The goal of these studies is to critically examine the effect of local tissue environment and exogenous factors suspected to impact on human melanocyte differentiation during development utilizing a sophisticated tissue culture system. We will use known and potential modulators of melanocyte growth and differentiation to examine the effect of these factors on the expression of specific genes and protein products potentially involved in the differentiated function of melanocytes. The effect of nerve growth factor, laminin, fibronectin and vitamins A and D, and medium conditioned by cultured keratinocytes on melanocyte morphology (dendricity), proliferative capacity, melanin synthesis and the expression of the receptor for nerve growth factor will be determined using the appropriate techniques (immunofluoresence, immunobloting, northern blotting). We will subsequently use phorbol esters, known to affect melanocyte morphology, to examine the induction of genes potentially involved in melanocyte growth and differentiation. We will ultimately attempt to transfect the human nerve growth receptor gene into cultured human melanocytes in an effort to explore the tissue specific expression of this gene. In this way, we hope to construct a model of normal human melanocyte differentiation in vitro."}
{"text": "The initial aim of this research is to determine whether persistently elevated luteinizing hormone (LH) in circulation is causally related to excessive follicular development and superovulation in the cyclic hamster. The ultimate aim is to determine the usefulness of the elevated LH system as an animal model for the study of cystic follicles. Elevation of the circulating LH levels will be accomplished by sc insertion of Alza mini-pumps containing ovine LH S 22 (less than 0.5% contamination with FSH). Preliminary studies have shown that elevation of serum LH concentrations (using 400 ug LH minipumps) beingnning on day 1 of the cycle (estrus) culminates in superovulation 4 days later. Further elevation of the circulating LH (using 1000 ug LH minipumps) blocks ovulation and cystic follicles persist in the ovary. Perhaps, the elevated serum LH levels and cystic follicles in the hamster may indeed mimic a condition found in some women who have elevated serum LH and polycystic ovaries. In order to substantiate the hypothesis that elevated LH (in the presence of normal serum concentrations of FSH) induces follicular development and to 'rule out' the possibility that FSH contamination of the LH is the active component in superovulation, several preparations of highly pure LH (less than 0.05 contamination with FSH) will be tested for the ability to induce follicular development. To provide some universality to the discovery that elevated LH enhances follicular development, I will attempt to induce superovulation in the rat, mouse and hamster. Since the recruitment of follicles by the persistently elevated LH (0.5% contamination with FSH) may be due to FSH contamination, several experiments are designed using hypophysectomized hamsters implanted with 400 and 1000 ug LH minipumps to assess whether LH (and less thna 0.5% FSH) can stimulate follicular development. In addition, hypophysectomized and cyclic hamsters will be implanted with 2 and 5 ug ovine FSH minipumps (the doses of FSH contamination in 400 and 1000 ug LH minipumps, respectively) in order to ascertain any effect of these small doses of FSH on follicular dynamics. The result should provide some insight into whether the LH-minipump system is a useful model for the study of the effects of persistently elevated circulating LH on follicular development and cystic follicles."}
{"text": "The Cold Spring Harbor Laboratory proposes to continue its highly successful summer course in Advanced Bacterial Genetics. This course has its roots in the original Cold Spring Harbor Bacteriophage Course initiated by Dr. Max Delbruck in 1945. The course will be offered for three weeks in June from 1993-1997. This course prepares the participant to enter directly into research that makes use of advanced and/or specialized techniques and concepts in molecular genetics. Participants gain extensive first-hand experience with materials and systems that have led to recent advances in prokaryotic molecular genetics. A solid grounding in classical genetic analysis supports training in contemporary uses of transposon mutagenesis, operon and gene fusion technology, molecular cloning, site-specific mutagenesis and polymerase chain reaction as applied to diverse problems in bacterial and bacteriophage genetics. This course, which is unique in providing instruction in both genetics and molecular biology, gives participants the wherewithal required to perform analogous experiments in their home laboratories. The course instructors are chosen on the basis of their contributions to and knowledge of the field of bacterial genetics. Daily discussion periods cover the basis of the methodology used and fine points of experimental design and analysis. The course instructors invite guest lecturers to give up-to-the-minute reports on current research. The lecturers have all made significant contributions to their fields, so that direct contact with the lecturers is a valuable experience, especially for the younger course participants. Because the Laboratory is open at all hours, and because of the informal atmosphere, spontaneous discussions between participants, instructors, lecturers and laboratory staff are constant occurrences. Course participants range from graduate students to senior investigators. They are chosen by the course instructors from a pool of highly-qualified and deserving applicants. Because of the short duration of the course, senior scientists can easily attend to experience a period of intense training in an environment that is remote from other demands on their time and attention."}
{"text": "Continuing a systematic study of the inotropic effect of changes in extracellular potassium concentration, including: clear demonstration of voltage-independence of these inotropic effects over as large a range of potassium concentrations as will allow adequate voltage control during voltage clamp; quantitative analyses of the effect of increased and decreased extracellular potassium on the voltage-tension relationship, examination of potassium's inotropic effects under experimental conditions, including potassium-excess and sodium lack contractures, as well as activation induced by voltage clamp depolarizations. Continuing mathematical modelling of activation-related calcium transport, with particular regard to its interraction with potassium."}
{"text": "Acne is a multifactorial disease affecting ~50 million people in the US. Three main factors contribute to the pathogenesis of acne;1) P. acnes growth 2) P. acnes-induced inflammation and 3) overproduction of sebum, all targets for therapeutic intervention. Treatments, such as antibiotics and benzylperoxide (BPO) are directed at the bacterium P. acnes and retinoids target sebum control. Several antimicrobial and retinoids also possess anti-inflammatory activity. However, to date a treatment directed at all three components of acne has not been identified. Signum's isoprenylcysteine (IPC) analogs represent a novel class of topical therapeutics that potentially targets all three contributors of acne pathology. Our preliminary results demonstrate IPC analogs inhibit P. acnes growth. In addition, when topically applied, using an in vivo irritant-dermatitis inflammatory model, our results demonstrate IPC analogs have strong anti-inflammatory activity inhibiting neutrophil infiltration, edema and erythema of the skin. Preliminary results support a mechanism involving the reduction in keratinocyte inflammatory mediator production. We have extended these observations to in vitro and in vivo P. acnes-induced inflammation models, demonstrating IPC inhibition of several pro-inflammatory cytokines. Lastly, IPC analogs have also been shown to modulate GPCR signaling, suggesting a potential role in regulating sebum control through inhibition of the GPCR melanocortin 5 receptor (MC5R) (critical for sebaceous gland lipogenesis). Based on our previous work, we hypothesize by screening our library of IPC compounds we will be able to identify IPC chemotypes with plieotropic effects to be developed as a novel therapeutic for acne. Structure-activity relationship of these results will lead to proof of principle by identifying, designing and testing candidate compound(s) having the highest combined potency towards each pathological factor, which will be subsequently developed in a Phase II application. PUBLIC HEALTH RELEVANCE: Acne is the most common disorder of the human skin and affects up to 80% of individuals in their lives. Three main factors contributing to the pathogenesis of acne are 1) P. acnes growth 2) P. acnes-induced inflammation and 3) overproduction of sebum all of which are potential targets for therapeutic intervention. To date a treatment directed at all three components of acne has not been identified. Successful development of this novel class of topical therapeutics will be the first that potentially target all three contributors of acne pathology."}
{"text": "The long term objective of this proposal is to develop and identify compounds which are effective in treating AIDS and other HIV- related diseases. The studies described in this proposal will aid in the collaborative efforts of this drug discovery group to develop and identify compounds which specifically inhibit HIV enzymes, either reverse transcriptase (RT), endonuclease, or a protease which is thought to be virus specified. The specific aims of this laboratory will be to: 1) collaborate to better characterize biochemical events following HIV infection in cultured cells, both T-lymphocytes and monocytes/macrophages; 2) furnish immunoaffinity purified RT to the group; 3) immunoaffinity purify the HIV endonuclease, characterize its substrate and develop an endonuclease assay; 4) evaluate anti RT, endonuclease, and protease compounds for cytotoxicity and anti-HIV activity in cultured cells, both T-lymphocytes and monocytes/macrophage; 5) using HIV infected cultured cells, collaborate with other investigators to determine the mode of action of anti-HIV compounds; 6) study selection of virus resistant to anti-HIV compounds; and 7) study the interaction of new compounds with other current or new compounds in inhibiting HIV in cultured cells."}
{"text": "Plasmacytoid dendritic cells (pDC), a distinct type of bone marrow-derived cell, play an important role in linking innate and adaptive immune responses. pDC are pivotal in the first line of defense against viral infections through recognitio of viruses by toll-like receptors as well as their ability to produce large amounts of type I interferons (IFN). In preliminary studies, we have discovered a novel population of resident pDC in the cornea. In vivo pDC depletion in our preliminary studies demonstrate that pDC are the major source of IFN-a in the cornea and play a protective role in the host defense in herpes simplex keratitis (HSK). Thus our results suggest that pDC are key participants in fighting viral keratitis, while preserving vision. Identifying specific functions of pDC in HSK and understanding the critical pathways of pDC and T cell migration in the cornea may provide new molecular targets for pharmacological intervention through immunotherapy. However, defining organ- and cell-specific molecular migratory mechanisms is critical, in order to inhibit cell subsets driving disease, without affecting leukocytes required for protective immunity. To address these questions, we have developed a new multiphoton intravital microscopy (MP-IVM) model to study pDC in intact corneas of living mice. This imaging approach uses transgenic mice, with fluorescent pDC, and fluorescent viruses, and will visualize pDC and viruses at subcellular resolution in living animals, allowing us to study their interaction with surrounding cells. Based on preliminary work and that up-regulation of organ-specific combination of vascular adhesion molecules and chemokines regulate pDC and T cell recruitment to the cornea and subsequent migration of pDC from the cornea in inflammation and infection. We further hypothesize that pDC are protective to the cornea in HSK through local corneal IFN-? and TNF-? production and migrate to distinct areas of the dLN after activation in HSK, where they mediate differentiation pathways of CD4+ T cells to T regulatory or T helper (Th)17 cells through IFN-? and IL-6 production. pDC will be studied to investigate the traffic signals that guide them to normal and inflamed corneas and will be used to address the following two specific aims: 1.) To characterize corneal pDC and dissect the molecular mechanisms that mediate their recruitment and egress during inflammation; 2.) To characterize corneal pDC and dissect the molecular mechanisms that mediate their recruitment and egress during inflammation. Identification of these critical pathways of cell migration to and from the cornea will provide new and highly specific molecular targets for pharmacological intervention in inflammatory, infectious, alloimmune and autoimmune diseases. Few effective anti-inflammatory drugs have emerged over the last decades in the ophthalmic field and an urgent need for new drugs exists. HSK is a leading cause of blindness. Effective therapy for HSK would significantly reduce visual impairment, increase productivity, and reduce the burden of treating HSK."}
{"text": "A combination of various pancreatic, morphologic and functional assessments, along with gut hormone release, serves more accurately to gauge severity of disease, and these criteria may then be used to trace, in a longitudinal fashion, the course of the disease and to assess the efficacy of various treatment options. In addition, on the basis of the observation that chronic pancreatitis is biochemically similar to Type I diabetes mellitus, the investigators will compare the complications of these two diseases."}
{"text": "Over 1,000,000 Americans and 15 million individuals worldwide are infected with HlV. These numbers have been projected to be in the range of 40 - 100 million by the year 2000. Chemotherapeutic suppression of viral replication provides the greatest likelihood of beneficial intervention in the natural history of disease progression, at least for the near future. Three nucleoside analogs (zidovudine [AZT], dideoxyinosine [ddI], and dideoxycytosine [ddC]) have been approved for use in the United States. In certain patient populations these can prolong life and reduce morbidity; nevertheless, much room for improvement remains. In addition, herpesviruses (HSV, VZV, and CMV) represent the most prevalent and significant opportunistic viral complications of HIV disease. The identification of effective drug regimens for both HIV and the herpesviruses must contend with a number of hurdles. One of the most important of these is drug resistance. The objectives of this proposal are to characterize the expression, the genetic basis, the mechanisms, and approaches to treat antiviral drug resistance. Specific aims include 1) the investigation of the nature and significance of nucleoside drug resistance, 2) the confirmation or denial of the hypothesis that higher plasma levels of non-nucleoside reverse transcriptase inhibitors can effectively suppress drug resistant virus, 3) the determination whether the clinical failure of the tat antagonist Ro 24-7429 is attributable to TNFalpha and whether inhibitors of TNFalpha can synergize tat antagonist activity, 4) to examine drug resistance with protease inhibitors, 5) to determine whether HIV gene therapies, like antisense oligonucleotides and ribozymes, can select for escape mutants (resistance), and 6) to develop compounds that effectively bypass drug resistance caused by acquired or innate kinase deficiencies of herpesviruses like HSV and CMV. These studies involving both specimens from clinical investigations and more basic molecular biologic and biochemical approaches are intended to elucidate new approaches to treat HIV and opportunistic herpesvirus infections more effectively."}
{"text": "Characterization of complications arising from cardiovascular invasive treatment procedures were analyzed using CDMAS."}
{"text": "Hepatitis C virus (HCV) infection is a major public health problem, putting infected individuals (~180 million worldwide) at risk of developing cirrhosis, hepatocellular carcinoma, and liver failure. Chronic hepatitis C is the leading cause for liver transplantation. Current standard interferon-based therapy, a costly and time-consuming process, has only a ~50% cure rate, and no anti-HCV drugs have yet been approved for hepatitis C therapy. Despite the promise shown by HCV-specific proteases and polymerases as drug targets, the rapid emergence of viral resistance indicates that additional targets and combinations of antivirals will be necessary for effective treatment. We propose to isolate genetic suppressor elements (GSEs) from a library comprising a fragmented HCV genome which could be used both as potent anti-HCV therapeutic agents and as probes to identify and validate new targets for further drug screening. GSEs are nucleic acid or protein/peptide molecules derived from a gene or genome that act as transdominant inhibitors of a particular biological function through a variety of mechanisms, which includes binding to and blocking essential interaction surfaces for protein activity. In order to identify GSEs from within the HCV genome that exert inhibitory activity against HCV, a novel function-based selection system will be developed and implemented. Briefly, a fragmented HCV genome will be delivered to a hepatoma derivative cell line that is sensitive to a cytopathic effect exerted by HCV. The cells containing the HCV-derived genetic fragments will be subjected to a cytopathic challenge by exogenously administered cell culture-derived HCV (HCVcc) infectious particles, and cells surviving this HCV challenge will be enriched in GSEs that exert an inhibitory effect against HCV. Iterative application of this selection procedure will result in the identification of highly potent GSEs that protect cells against HCV infection and cytotoxicity. Preliminary studies will evaluate the degree of the protective effect conferred by the identified GSEs against HCV-mediated cytotoxicity, and the stage in the HCV life cycle at which the anti-HCV effect of the GSEs is exerted. Future studies originating from the identified GSEs are expected to reveal the molecular basis of the anti-HCV GSE activity, thus providing new leads for anti-HCV drug development. It is expected that the GSEs identified from the proposed research, and molecular mimetics derived therefrom, will inhibit HCV infection/propagation through diverse mechanisms, including the inhibition of virus-host cell interactions, thus contributing to the urgent quest for new and effective HCV antivirals on multiple fronts. PUBLIC HEALTH RELEVANCE: Infection by hepatitis C virus is a serious global health problem that causes numerous debilitating liver conditions. The current treatment regime for hepatitis C is time-consuming, expensive, and often ineffective, creating an urgent need for new and effective drugs. Novel anti-hepatitis C genetic suppressor elements isolated from this research will serve both as hepatitis C drugs, and as keys to open doors to new avenues of research in hepatitis C antiviral development."}
{"text": "Investigate activation and deactivation of insulin action on hepatic glucose production and peripheral glucose utilization. There are two separate mechanisms which may account for changes in activation kinetics, namely, changes in regional blood flow in response to insulin and transit of insulin from the circulation to the intersttial space."}
{"text": "Summary The cilium is a widely distributed cell surface organelle that functions as a signaling hub for the vertebrate cell. Ciliary defects leads to a wide range of human diseases collectively referred to as ciliopathies. Multiple ciliopathies, including nephronophthisis, show renal fibrosis. However, the role of cilia in renal fibrosis has not been studied extensively. In this project, we use Arl13b, a gene essential for cilia biogenesis, as an entry point to dissect the role of cilia in renal fibrosis. We generated conditional Arl13b knockout mice. Our results show that deletion of Arl13b in renal epithelial cells leads to renal fibrosis and cysts. Moreover, multiple signaling pathways, including the Wnt, HH and Hippo pathway, are mis-regulated in the mutant kidney. This project focuses on dissecting tissue specific functions of cilia and communications between different cell types in renal fibrosis through both candidate and unbiased approaches. Aim 1 is focused on signaling in renal epithelial cells regulated by ciliary defects and the relationship between different pathways. Aim 2 studies targets in interstitial cells that are affected by ciliary defects and the functional significance of interstitial cilia. Through this study, we will elucidate how ciliary defects in epithelial cells triggers a signaling cascade that eventually lead to renal fibrosis and whether and how cilia in interstitial cells modify this response. These results not only will provide insight to the mechanism of renal fibrosis in ciliopathies, but will also be informative to fibrosis in general."}
{"text": "A recently discovered human epilepsy gene, LGI1 (leucine-rich glioma-inactivated; mutated to cause human autosomal dominant lateral temporal lobe epilepsy or ADLTE) encodes a protein secreted at glutamate synapses during postnatal glutamate synapse development. Consequently, we hypothesize that LGI1 might promote epilepsy through a novel mechanism, by regulating postnatal glutamate synapse maturation. We propose ADLTE mutant LGI1 acts as a dominant negative to inhibit native LGI1 function and arrest maturation. To directly address our hypothesis and contrast the functional effects of epilepsy-associated mutant LGI1 with those of excess wild-type LGI1 on native neural circuits, we created transgenic mice using bacterial artificial chromosomes (BAC) carrying a large 226 kb fragment of mouse genomic DNA encoding the full-length LGI1 gene. The ADLTE 835delC mutation introduced a premature translational stop codon to generate a truncated LGI1 protein. The full-length gene BAC transgenic approach is important to maintain native patterns of gene expression and transcript splicing in order to assess the genes effects on the glutamate synapse development process in vivo. LGI1 is heavily expressed presynaptically at medial entorhinal cortex perforant pathway glutamate synapses innervating dentate granule neurons (MPP- dentate) and also separately in a synapse targeting thalamus. Our overall goal is to define the cellular basis for human ADLT epilepsy. We specifically test whether excess LGI1 magnifies and mutant LGI1 blocks maturation of the following glutamate synapse properties during the postnatal periods when it becomes expressed and throughout adulthood in dentate gyrus and thalamus. We examine: 1) presynaptic glutamate release down-regulation and role of Kv1.1 K+ channel activity; 2) postsynaptic NR2B NMDA receptor current and synaptic plasticity down-regulation, PSD95-Src complex formation, and the role of Src kinase activity; and 3) dendrite branch (e.g., dentate) and axon input (e.g., thalamus) pruning. This murine model of human ADLT epilepsy could link a novel human epilepsy gene to the arrest of glutamate synapse maturation and potentially defining a cellular basis for human seizure disorders beyond ADLTE. PUBLIC HEALTH RELEVANCE: This project seeks to identify the cellular basis of the inherited human epilepsy disorder caused by mutations in LGI1, a secreted synaptic protein. The results should provide new insights into the cellular and molecular basis of human epilepsy, and help identify new potential therapeutic targets to treat this common brain disorder. [unreadable] [unreadable] [unreadable] [unreadable]"}
{"text": "Continuous wave EPR spectroscopy is being used to characterize the iron-binding domain of a synthetic model (PMA) of the antitumor antibiotic, bleomycin. The EPR spectra of two very similar low-spin ferric complexes have been differentiated and assigned as a methoxide and hydroperoxide form of Fe(III) PMA."}
{"text": "The U.S. appears to have worse health than people in a number of European countries. Because health reflects an accumulation of processes over a lifetime, we will study how stressful events during the working years may affect pre-retirement health and ultimately longevity. The broad aims of this application are to find whether in panel data the health gap between the U.S. and Europe increases during the working years, but not during the post-retirement years, and to explain these broad facts by differences in the stress induced in the labor market and by differences in the ameliorating effects of economic and social policy (including health insurance). Our analysis will use data on 15 countries, which provides a wealth of variation in institutions in time as well as in space. The principal outcomes will be fourfold. First, this study will clarify at what point in the life-cycle health differences emerge and chart their path thereafter. Second, this study will propose a formal economic and biological model linking indicators of physiological functioning to other health and economic outcomes clarifying some of the concepts measured in the literature and guide empirical work on the topic. Third, this study will provide micro as well as cross-country empirical evidence on the relationship between economic stressful events and physiological dysregulation using biomarkers but also reported health measures. Fourth, because of its cross-country design, this study will exploit differences in social policy across countries and over time to see estimate how these policies contribute to the international differences in health through the effect of physiological dysregulation on health. PUBLIC HEALTH RELEVANCE: The U.S. has fallen behind in terms of life expectancy in the pre-retirement years and large differences in health have emerged between the U.S. and Europe. This project aims to test the hypothesis that differences in physiological dysregulation due to a more stressful working life in the U.S. are responsible for those differences. This project will use a large array of longitudinal datasets in 15 countries to link health outcomes in the pre-retirement years to life histories and exploit differences in social policy across countries."}
{"text": "Squamous cell bronchogenic carcinomas continue to be chemically induced at preselected endobronchial sites in dogs. These preparations allow serial testing during the evolution (transitional continuum) of lung cancers like those which occur in humans. Material from patients and a hamster model will be utilized in adjunctive fashion. We aim to: 1) Make the existing cancer models more cost effective; 2) Delineate relationships between polyploidy and stages of respiratory epithelial carcinogenesis; 3) Develop methods for in vivo preparation and propagation of pre-neoplastic and cancer cells; 4) Determine the role of DNA methylation during lung carcinogenesis; 5) Explore factors of genetic control upon pulmonary carcinogenesis. Toward cost effectiveness we shall use endobronchial sustained release implants (SRI) of carcinogens and heterotopical autogenous segmental tracheo-bronchial transplants treated with carcinogen. Using image analysis and FACS separation techniques, we shall study ploidy serially during carcinogenesis and work toward improved cell culture methodology. Transfer of tumor cells to nude mice shall be used for cancer cell propagation; importantly adjunctive, in vivo autogenous tracheal segment grafts treated with carcinogen shall be used to propagate autogenous bronchial cancer cells. Pre-neoplastic cell populations shall be harvested from autologous tracheo-bronchial grafts and endobronchial sites undergoing bronchial carcinogenesis. Lung cancer specimens from humans, dogs, and hamsters shall be used to test the hypothesis that undermethylation is a key factor in bronchial carcinogenesis. The effect of the methylation inhibitor 5-azacytidine upon the continuum of bronchiocarcinogenesis will be tested in dogs and hamsters. Using karyotyping in correlation with assessment of total DNA and DNA methylation measurements, the biologic and genetic differences between more and less carcinogen susceptible hosts will be explored. This project involves collaboration between surgeons, cytogeneticists and molecular biologists seeking better to understand lung cancer. It utilized specimens from three species - man, dog, hamsters and proposed specifically to use the canine model for studies not possible with humans or hamsters."}
{"text": "This proposal concerns the roles of Axin and the related gene Axin2 in regulating the canonical Wnt signal transduction pathway during mammalian embryogenesis. The Wnts are a family of secreted factors that play important roles in cell proliferation, patterning and differentiation during development. Axin is a critical component of a protein complex that controls intracellular signaling downstream of Wnts, by regulating the levels of [_-catenin, a transcriptional co-activator and a key effector in the pathway. Axin2 is believed to have a similar function, although it is not as well characterized. Mutations in both genes in humans have shown that they are tumor suppressors, consistent with their roles in negatively regulating the Wnt pathway. The analysis of mice with mutations in Axin and Axin2 have revealed that these genes are important for early axial patterning, as well as for craniofacial and brain development. Craniofacial development is a complex process that involves interactions between the surface ectoderm, endoderrn, mesoderm and the neural crest, and it is highly susceptible to genetic and environmental perturbations, as craniofacial abnormalities are among the most common birth defects in humans. The Wnt signaling pathway has been implicated in eraniofacial development through several mouse mutations. In this proposal, the hypothesis will be tested that Axin and Axin2 are required to negatively regulate the response to certain Wnts by the cranial neural crest cells that form much of the face, and that mutations in Axin and Axin2 lead to inappropriate activation of the Wnt pathway. This will be addressed by analysis of mutant mice, as well as through the conditional manipulation of the Wnt pathway in the cranial neural crest, using transgenic approaches. To understand how Axin and Axin 2 cooperate during development, it is important to define the extent to which they are functionally redundant, and this issue will be investigated through a gene replacement strategy in mice. Finally, to test in vivo the importance of specific domains of Axin that are believed to mediate its functional interactions with other components of the pathway, several novel mutant alleles of Axin will be generated, and examined for their effects on mouse embryogenesis."}
{"text": "Critically ill patients with acute lung injury and acute respiratory distress syndrome (ALI/ARDS) who receive mechanical ventilation can suffer from severe sleep disruption despite continuous sedative infusions. Sleep disruption, in turn, may activate the sympathetic nervous system and cause elevation of circulating inflammatory cytokines, which, in turn, may play a causative role in delirium and post-traumatic stress disorder through consolidation of unpleasant memories during awakenings from sleep. Currently, there is very little understanding of the inter-relationship between critical illness, sleep, and neuropsychological well-being, due to the lack of intervention-based trials that improve sleep during critical illness. The central purpose of this application is to study the short-term effects of sedation with sympatholysis (central 12 adrenergic agent) on sleep and inflammation in critically ill patients with ALI/ARDS. Sedation with sympatholysis will be achieved by a novel sleep-promoting agent with central 12 adrenergic properties. We will undertake sleep studies and measure circulating inflammatory cytokines that modulate sleep in patients with ALI/ARDS randomized to receive two different sedation strategies: central 12 adrenergic sedative-analgesic (dexmedetomidine) versus a conventional sedation strategy (midazolam and fentanyl) in a randomized, double blind, cross-over study. Specific Aim 1: To assess the short-term effect of an 12 adrenergic agent on sleep quality in critically ill patients with ALI/ARDS. Specific Aim 2: To assess the short-term effect of an 12 adrenergic agent on sleep-modulating inflammatory cytokines in critically ill patients with ALI/ARDS. Specific aim 3: To determine the effect of 12 adrenergic agent on the in-vitro production of sleep-modulating inflammatory cytokines by peripheral blood mononuclear cells of patients with ALI/ARDS. Collectively, our study will identify the best sleep assessment tool in patients with ALI/ARDS and whether sleep disruption in such patients can be minimized. In the long-term, this program of research will identify sedation practices that are least associated with adverse short- and long-term consequences of critical illness, and thereby ultimately help improve quality of life of patients surviving critical illness. PUBLIC HEALTH RELEVANCE: During life support for respiratory failure, patients frequently awaken from sleep despite sedatives, and upon surviving may suffer from flashback of traumatic experiences that were memorized during awakenings from sleep (called 'PTSD'). There is little understanding of how such patients' sleep should be measured or improved. We will find the best sleep measurement tool and administer a new medication that improves sleep by soothing inflammation which may, in the future, improve the quality of life of patients."}
{"text": "Early onset generalized dystonia is a common but poorly understood movement disorder, affecting young people, with little meaningful intervention. Clinical evidence has strongly implicated abnormal dopaminergic transmission in the basal ganglia as a major etiology of dystonia. Two recent findings by our colleagues now allow the investigation of the molecular and cellular basis of early onset dystonia: 1) a strong candidate gene DYTl has been identified on human chromosome 9, and 2) expression of the DYTl message in the brain is restricted neuroanatomically to the substantia nigra, cerebellum and hippocampus. The DYTl gene product, torsinA, is a member of a novel family of human genes (torsin-related proteins) of unknown function. TorsinA has sequence homology with functional domains of the large Clp ATPase/HSP100 gene family. Prototype members of this family are characterized by ATP-binding and formation of functional oligomeric complexes. This project is designed to use an efficient expression system to test hypothesis of torsinA function modeled from its homology to the Clp/Hsp family, including protein stability in stress conditions. The regulation of torsinA gene expression, subcellular distribution of the protein will be determined in neuronal cell lines and primary neuron cultures. The role of mutant torsinA as a dominant negative will be measured directly in functional studies of neuronal function, such as stress response, oxidative phosphorylation and dopamine release. The expression system will be designed to differentiate different torsin members, and mutant from wild type torsinA and allow efficient delivery into neurons in different experimental systems. This expression system is designed as a 'bridge' technology, to link the new discoveries of the genetics of the torsins to the cell biology and function of these proteins in the normal and diseased state."}
{"text": "Project Summary/Abstract The emergence and reemergence of pathogenic viruses represent continuous infectious disease threats to public health. Among these, the paramyxoviruses, which include many important human and animal pathogens, also include two excellent examples of emerged, zoonotic viral pathogens of importance: the henipaviruses; Nipah virus (NiV) and Hendra virus (HeV). NiV and HeV have a uniquely broad host tropism capable of infecting at least 18 animal species across 6 orders of mammals. NiV and HeV can also cause a systemic and often fatal respiratory and/or neurological disease in 11 mammalian species including humans. These henipaviruses remain significant biothreats to humans and economically important livestock in Australia and throughout South East Asia. In addition, there are no vaccines or therapeutics approved for human use. The henipaviruses are single-stranded, negative sense, enveloped RNA viruses and possess two membrane anchored glycoproteins involved in virus entry, one mediates host cell receptor attachment (G glycoprotein) and the other is a Class I fusion (F) glycoprotein which facilitates virion and host cell membrane fusion. The viral G and F glycoproteins are the major antigenic targets of neutralizing antibodies and are the main focus of active vaccine strategies. We have been characterizing the non-pathogenic species of henipavirus, Cedar virus (CedPV). Using recombinant viral glycoprotein mediated cell fusion assays, we have determined that CedPV- mediated fusion tropism is similar to NiV and HeV, however, the ephrin receptor tropism of CedPV is remarkably broad and fusion is triggered by ephrin-B1 and -B2 as well as the glycophosphatidylinositol- anchored A subtype ephrins-A1, -A2, and -A5. The fusion tropism activity by each of these ephrins is also correlated with CedPV G binding, and using a recently developed reverse genetics system for preparing recombinant CedPV (rCedPV) we have confirmed the fusion tropism findings with rCedPV infection studies. The rCedPV platform now offers a new virological system that can be used in a variety of applications to study henipaviruses safely under BSL-2 containment which will be broadly used by the CETR Research Projects and Cores. But of additional importance, it is an authentic henipavirus system that is attenuated in comparison to NiV and HeV, and can be genetically manipulated to serve as a live-attenuated universal henipavirus vaccine platform, and one likely capable of inducting a long-lasting and balanced immune response. Using this new system our objectives here will be to develop rCedPV chimeric viruses encoding the F and G glycoproteins of NiV or HeV and characterize and explore their potential a novel henipavirus vaccine. Specifically, we will: 1) Construct and rescue rCedPV-NiV/HeV viruses; 2) Characterize the chimeric viruses in cell-based infection and tropism studies; 3) Analyze the immune responses and outcomes in both immunocompetent and immunodeficient mice following infection with rCedPVs; 4) Test the protective abilities of rCedPVs by vaccination in animal subjects, in response to NiV and HeV challenge."}
{"text": "[unreadable] The Center for Clinical Epidemiology and Biostatistics (CCEB), the Gastroenterology Division (GD), both of the University of Pennsylvania (Penn) School of Medicine (SOM), and Penn's Leonard Davis Institute (LDI), in collaboration with the Division of Gastroenterology, Hepatology, and Nutrition (DGHN) within the SOM's Department of Pediatrics, seek the continuation of a highly successful research training program for clinically oriented investigators in Gastrointestinal Clinical Epidemiology. The overarching objective of this program is to train individuals who have completed their clinical training to be rigorous and independent academic clinical investigators in the broad field of gastroenterology. [unreadable] This training program consists of (1) a core curriculum of required courses in clinical epidemiology and health services research methodology and biostatistics; (2) a series of seminars in gastroenterology combining faculty expertise from the CCEB, LDI, and GD; (3) extensive independent readings and individualized tutorials; (4) a required course in gastroenterology epidemiology; (5) elective courses in advanced methods for epidemiology, health services research, and biostatistics; (6) a series of seminars and workshops designed to improve trainees' skills in presentations and manuscript/grant writing; (7) instruction in the responsible conduct of research and regulatory affairs; and (8) the development/completion of a clinical research project in gastrointestinal diseases, under supervision of a mentoring team comprised of at least one methodological, one clinical, and one biostatistical preceptor. The program is designed to achieve: (1) indepth knowledge of the research techniques appropriate for clinical epidemiology and clinically-oriented health services research investigation; (2) intensive, supervised research experiences; and (3) integration and cohesiveness among faculty and fellows through participation in CCEB, GD, and LDI lectures, seminars, and journal clubs. Fellows are candidates for a Master of Science in Clinical Epidemiology (MSCE) degree. When feasible, candidates are encouraged to seek a Ph.D. degree. [unreadable] The strengths of the ongoing Gastroenterology Clinical Epidemiology Training Program are a long history of successful NIH T32 research training programs in the CCEB, GD, and LDI; comprehensive and rigorous courses, seminars, and research programs for trainees; and a rich history of collaborative links among the programs. In addition, there are excellent basic science laboratories in the GD (supported by R01 grant funded investigators and an NIH/NIDDK Center for Digestive and Liver Diseases) as well as throughout the SOM, several large databases and tissue banks, and a deep commitment of all SOM Chairpersons and Program Directors to the successful implementation of this training program. The existence of a K30 grant (Clinical Research Curriculum Award), diverse faculety expertise in the CCEB, GD, DGHN, and LDI, and recent successes of trainees provide a strong rationale for the continuation of this successful program [unreadable] [unreadable]"}
{"text": "We are concerned with the intracellular expression of the murine thymus-leukemia antigens in normal cells and tumor cells together with changes that follow modulation of surface antigen by specific antibody. The work focuses on mitochondria because they are relatively easy to isolate and have a characteristic morphology. TL antigens are demonstrated by immunoelectron microscopy. We are also characterizing plasma membrane differences on subsets of murine T cells by electrophoretic methods after iodination of intact cells."}
{"text": "In 2004, the Centers for Disease Control reported that AIDS was the leading cause of death for African- American women between the ages of 25 to 34 and African-American men between the ages of 35 to 44 in the United States. Researchers have explained these ethnic/racial disparities in terms of delays in accessing care and difficulties in adhering to medication regimens. These findings suggest that culturally appropriate interventions developed to reduce HIV/AIDS stigma could help improve treatment adherence and in turn, improve health outcomes for African-Americans living with HIV/AIDS. It is the career aspiration of Dr. Rao, applicant for this Patient Oriented Research Career Development Award (K23), to help lessen the burden of stigma associated with HIV/AIDS in vulnerable populations. In this proposal, Dr. Rao outlines a career development plan that will lead to a line of work aimed at making available an effective, culturally- appropriate intervention to reduce self stigma for African-Americans living with HIV/AIDS. Through coursework and mentorship, Dr. Rao will train in culturally-specific models of health beliefs and advanced qualitative and quantitative research methodologies. She will implement a research project that will adapt a self stigma reduction intervention for African-Americans living with HIV. In addition, she will adapt and validate an outcome measure of self stigma for use in a future clinical trial. These adaptations will lead to a pilot test and then directly to a full-scale clinical trial of the intervention. Drs. David Cella and Patrick Corrigan, senior investigators with extensive experience in behavioral interventions and patient reported outcomes measurement, will serve as co-sponsors for this project. The clinical site for this project, the outpatient HIV Clinic at Northwestern Memorial Hospital, serves a patient population with much ethnic/racial and gender diversity, and its patients enthusiastically participate in clinical research. During the award period, Dr. Rao will be based at Evanston Northwestern Healthcare and have faculty appointments at Northwestern University. Her location at these institutions, with their outstanding reputation in research and education, will provide her with an outstanding environment, rich with the research resources she will need to become an independent investigator. Dr. Rao will be uniquely positioned to carry out this work aimed at reducing HIV stigma, and subsequently improving health outcomes, for African-Americans living with HIV."}
{"text": "We face an epidemic of hospitalizations in heart failure (HF), which are often unrelated to HF. We hypothesize that extensive efforts have not successfully reduced hospitalizations in HF because management standards rely on disease-centric clinical guidelines, applied within provider-centric systems of care. Care models often overlook that the HF syndrome arises within a complex and poorly understood multi-morbidity context and how comorbid conditions present according to the type of HF (preserved vs. reduced EF) is not known. Further, as a chronic disease that coexists with multiple other conditions, HF causes self-management difficulties in elderly patients who may lack the needed support. Understanding the epidemiology of coexisting conditions and the determinants of successful self-management is fundamental to design new practice models and reduce hospitalizations in HF. The central goal of our revised application is to respond to the urgent need for new approaches by fulfilling 3 objectives: firstly, understand the complex epidemiology of coexisting conditions in HF, secondly, identify patient-centric attributes conducive to successful self-management by drawing upon the Chronic Care Model, thirdly, develop models that integrate cardiovascular characteristics, coexisting conditions and determinants of self-care to be used at the point of care to engage community support for at risk patients. Our 3 Specific Aims, designed to address these objectives, align with the priorities of the Department of Health and Human Services, outlined in their report Multiple Chronic Conditions: A Strategic Framework. Aim 1 will study the epidemiology of coexisting conditions in HF in a community cohort of persons with HF within the Rochester Epidemiology Project. We will assess the prevalence of coexisting conditions (comorbid diseases, geriatric syndromes) and determine which clusters of conditions most impact hospitalizations. We will assess the emergence and progression of coexisting conditions after HF diagnosis and their association with hospitalizations, according to the type of HF (preserved vs. reduced EF). Aim 2 will evaluate the role of patient-centric factors (social support and self-management) on hospitalizations in HF guided by the Chronic Care Model in a prospective cohort of patients living with HF. Aim 3 will develop and evaluate prediction models to identify patients with HF at high risk for hospitalizations. In doing so, we will leverage the unique Health Information Exchange (HIE) capabilities and community partnerships established by the Office of the National Coordinator for Health Information Technology-Funded Southeast Minnesota Beacon Program and assess the feasibility of adapting an existing HIE architecture to implement such models at the point of care, identify patients at high risk for hospitalizations and alert community care coordinators when their patients have been hospitalized to enable proactive community support. By executing these aims, we will understand how the epidemiology of coexisting conditions and how patient-centric factors contribute to the burden of hospitalizations in HF. We will develop risk prediction models, which integrate cardiovascular characteristics, coexisting conditions and patient-centric factors to optimize tools for interventions to reduce hospitalizations in HF."}
{"text": "The introduction of the PCR technique has provided a powerful tool to analyze the quality of complete responses in low grade follicular lymphomas. Complete remissions confirmed by PCR (\"molecular complete remissions\") rarely occur with traditional chemotherapy regimens for low grade lymphomas. Preliminary studies conducted in our Institution have shown that a new strategy consisting of 3 alternating non-cross resistant chemotherapy combinations, is capable of inducing a high rate of molecular complete remissions in these disorders. The primary objective of project #1 is to use the molecular response as a surrogate endpoint to compare this new strategy against less intensive strategies. All low grade follicular lymphoma tissues will be phenotyped and genotyped. The peripheral blood and bone marrows will be examined by the PCR technique to detect bcl-2 or JH rearranged sequences before and' after therapy. Patients with Ann Arbor stage I-III will then be randomized to either the novel intensive alternating therapy or to standard management with total nodal radiation. Those with stage IV presentations will be randomized to either the same intensive alternating therapy or a new and effective but less toxic regimen consisting of Fludarabine, Mitoxantrone and Dexamethasone. Patients will be followed periodically with the standard clinical restaging techniques as well as with peripheral blood and bone marrow PCR. The primary endpoint of these trials will be the percentage of cases who achieve a molecular complete response at the end of 18 months. A secondary goal will be to more firmly establish the value of the PCR as a means of assessing response by correlating it with the clinical outcome. Rigorous evaluation of these therapeutic alternatives demands a large patient population with close and uniform disease monitoring as well as a cohesive group of investigators."}
{"text": "The proposed research will continue investigations on: Alterations in the metabolic pool at the site of both aqueous humor and cerebrospinal fluid production in groups of unoperated animals and in animals in which bilateral superior cervical sympathetic ganglionectomy will be performed. In addition, a third group of sham-operated animals will be used. That supersensitivity to catecholamines takes place in sympathectomized animals has been shown previously in this and other laboratories. Ocular fluid dynamics in all animals will be determined by repeated weekly testing of ocular pressure (IOP) (with the Mackay-Marg tonometer), as well as measurement of pupil size. A base-line of measurements will have been made for a period of about two weeks prior to the performance of any surgery. Again, prior to surgery, this base-line will be followed by a period of testing of ocular responses to phenoxybenzamine (PBA), norepinephrine (NE) and prostaglandins (PGs). At the termination of a study, the animals will be anesthetized, the eyes cannulated and outflow resistance and IOP measured. The animals will then be sacrificed and tissues and fluids removed. The metabolism and/or concentrations of cyclic adenylic acid (cyclic-AMP) and the PGs will be of particular interest. We shall, in other words, look closely at the metabolic and physiological nature of ocular and non-ocular tissues in animals which would be made catecholamine-supersensitive (via the above surgical procedure) at sites of sympathetic innervation as compared with sham-operated and unoperated animals. BIBLIOGRAPHIC REFERNCES: Waitzman, M.B. and Law, M.L., \"Changes in Prostaglandin Concentration in Blood Subjected to Repetitive Freezing and Thawing\", Prostaglandins 10:949-962, 1975. Jackson R.T., Waitzman, M.B., Pickford, L. and Nathanson, S.E., \"Prostaglandins in Human Middle Ear Effusions\", Prostaglandins 10:365-371, 1975."}
{"text": "Current and recent work generally has focused on understanding and improving cognitive and behavioral phenotypes and applying these in analyses of whole genome association data. In Dickinson et al (2011) we extended earlier analyses of cognitive structure in schizophrenia cases and controls (Dickinson et al., 2006; Genderson, Dickinson et al., 2007). To achieve greater cognitive phenotype reliability and avoid redundant statistical comparisons, we used exploratory and confirmatory factor analyses separately in the CBDB samples (schizophrenia n=496, unaffected siblings n= 504, and controls n=823) and identified six positively correlated cognitive factors (for verbal memory, visual memory, n-back working memory, processing speed, card sorting, and span working memory). Data also supported a higher-order factor reflecting general cognitive ability, so-called g. We found that this structure was reasonably consistent across schizophrenia cases and controls, as had been shown previously (Dickinson et al., 2006), and extended this finding to include unaffected siblings. These findings guided construction of cognitive composite scores, which are in wide use in CBDB data analyses. One application of these composite scores has been to explore genome-wide associations in the CBDB samples. One exciting finding has been presented at genetics and psychiatry conferences and will be submitted for publication in the near future. We have identified an exciting and novel genome-wide significant association between our global cognitive composite and a sodium channel gene polymorphism that explains substantial variance in the cognitive impairment in our sample of people with schizophrenia and in their unaffected siblings an association that would not have been detected without the cognitive data aggregation strategy and composite scores developed by the Neuropsychology Lab. To gauge better which cognitive variables are best suited for genetics analysis, separate work has taken different approaches to estimate the heritability of these variables. Recently, a novel technique (GCTA) has been developed that permits estimates of heritability based on genome-wide genotype data from samples of unrelated people. The technique correlates pair-wise distances between genotypes for all possible pairs from a sample, with pair-wise differences in performance on a trait of interest (in our case, cognitive variables). We will present our first results contrasting GCTA methods with more traditional family-based methods at an upcoming conference and are beginning work on an associated manuscript. Wallwork et al. (2011) describes our efforts to determine a more empirically sound dimensional structure for patient data from the Positive and Negative Syndrome Scale (PANSS). The 30-item scale was developed with three syndrome scores, but previous analyses suggest that the scale really captures 5 or more dimensions of schizophrenia-associated symptomatology. We used existing literature to build a consensus five-dimension model of PANSS data then tested variations of this model using confirmatory factor analysis in the CBDB schizophrenia data and in an independent data set from Japan, supporting construction of new PANSS composite scores for positive, negative, depressive, agitated, and concrete/disorganized symptoms. Analogous lines of work are examining the dimensional structure of typical and abnormal personality in the CBDB data. We have elaborated and are refining five-dimension models for the Tri-dimensional Personality Questionnaire (TPQ) and for the SCID-II Personality Questionnaire (SCID-II). The TPQ targets personality more in the non-clinical range. The SCID-II is used to assess maladaptive personality disorder symptomatology. All of this work has been presented at scientific conferences and a paper on the SCID-II analyses is nearing completion. We design cognitive batteries for and collect, score and manage data from pharmacological trials seeking evidence of treatment-related changes in cognitive performance in schizophrenia patients and healthy controls. Recently, we have helped to develop the assessment and analysis strategy for a protocol examining an experimental agent (LX6171) and have worked on data analyses of information from tolcapone and modafinil treatment trials. We have completed an update of earlier meta-analyses of cognitive impairment in schizophrenia (Dickinson et al., 2007), showing that the cognitive impairment seen in people with schizophrenia has been consistent in magnitude and pattern over the past 30 years, and across different geographic regions around the world (i.e., North America, Europe and Asia). An early version of this work was presented at a conference and is accepted for publication as a chapter in an edited collection (Dickinson et al., in press), and a journal manuscript describing the final analysis is in preparation. Illness heterogeneity is a major challenge to the development of improved treatments in schizophrenia. As described in Cole et al. (2012), we have utilized latent class growth analysis (LCGA) in an effort to derive illness subtypes based on pre-morbid academic and social adjustment. We found evidence supporting three developmental trajectory subtypes: good/stable, insidious onset, and poor/deteriorating. These classes differed significantly in terms of age of onset, processing speed, and functioning after onset. The finding illustrates a potentially powerful methodology to attack heterogeneity challenges in schizophrenia research. Higher degrees of intra-individual variability (IIV) across neuropsychological tests have been recently linked to risk for schizophrenia, other clinical disorders, and normal aging. Cole et al (2011) is the first report showing decreasing IIV across cognitive domains in people with schizophrenia, their siblings and controls, respectively. Ongoing work is examining whether IIV might be useful as an intermediate phenotype. We have a large sample of unaffected siblings in the CBDB dataset, who, on average, share half their genetic code with an ill sibling, but do not share confounds, such as medication history and low motivation. In Wisner et al. (2011) we further characterized this sample, showing how sex and psychopathology history associate with sibling cognitive performance."}
{"text": "The mechanism of action and regulation of the alpha-ketoglutarate dioxygenase reaction are being investigated through the study of thymine 7-hydroxylase, pyrimidine deoxyribonucleoside 2'-hydroxylase and prolyl hydroxylase. Streptomyces antibioticus and the simple eukaryotes Neurospora crassa and Rhodotorula glutinis are serving as sources for enzyme purification. The intact cells of the above mentioned organisms, as well as of mammals, are being studied, especially in regard to the factors which regulate the activities of the alpha-ketoglutarate dioxygenases. The objective of this proposal extends to the elucidation of the regulation and physiological functions of the oxidative and reductive pathways of pyrimidine catabolism and to gaining some insight into whether prolyl hydroxylase itself has a regulatory role."}
{"text": "The overall objective of the proposed research is to determine the mechanism for increased ACTH secretion during pregnancy. This increase is necessary for normal maternal and fetal homeostasis, and occurs without symptoms associated with increased steroid action, such as hypertension. We have hypothesized that action of corticosteroids at mineralocorticoid receptors (MR) leads to reduced MR action in pregnancy, and that progesterone mediates this effect as a competitive antagonist at MR. The studies done thus far have supported this hypothesis, showing changes in hippocampal MR consistent with reduced activation and the presence of antagonist activity, during progesterone treatment or pregnancy. The proposed studies will extend these studies to test the following hypotheses: 1) Progesterone treatment in vivo blocks MR effects on 5HT1A expression in hippocampal neurons, resulting in increased plasma ACTH levels. The magnitude of the effects on both 5HT1A expression and ACTH is related to the levels of cortisol relative to progesterone. 2) Progesterone treatment in vivo has selective effects on neurons in hippocampal regions expressing mineralocorticoid receptors (MR) but not progesterone receptors (PR) (including CA1 and dentate gyrus). 3) The increase in ACTH caused by pregnancy or progesterone treatment is caused by an increase in serotonin effects in the brain; 4) The interaction between cortisol and progesterone in hippocampal cells in vitro involves competition at MR, producing opposing effects on 5HT1A mRNA and on the hyperpolarization response to 5HT (serotonin); and 5) Progesterone inhibits activation of MR, resulting in reduced MR binding to specific response elements, including those in the 5HT1A promoter. The first 3 aims will be studied in experiments in sheep. The sheep will be studied either after chronic progesterone treatment, ovariectomy or during pregnancy; MR binding and 5HT1A mRNA, as well as MR, GR and PR mRNA will be measured in hippocampus, hypothalamus and medulla. To test whether changes in 5HT1A occur in the same types of cells in hippocmapus as express MR (but not PR), mRNA will also be determined by in situ hybridization. To test the role of 5HT1A during pregnancy, the ability of a 5HT1A agonist to inhibit the ACTH levels in pregnancy and in progesterone-treated ewes will also be determined. The final 2 aims will be tested in cultures of hippocampal neurons. The effect of progesterone on MR binding, 5HT1A mRNA and electrical responses to a 5HT agonst will be tested; single cell PCR will also be used to determine if cells contain MR, but not PR. The action of progesterone at MR will also be tested by using MR-selective antagonist and agonist, and GR-PR antagonist. Finally, the ability of progesterone to inhibit MR activation will be tested in gel-shift and super-shift assays using specific response elements in the 5HT1A promoter, including a nGRE and the SP-1 site."}
{"text": "Understanding the genetic and environmental mechanisms that underlie the development of reading ability and disability has important implications for literacy education and the prevention of learning disabilities in reading. To that end, the goal of the proposed competing continuation of HD38075 is to conduct a systematic developmental genetic examination of reading comprehension. The Western Reserve Reading Project (HD38075 \"Environmental Influences on Reading: A Twin Study\") includes a representative sample of 350 same-sex twin pairs born between 1996 and 1998 who, by the proposed start date, will have been assessed longitudinally across four measurement occasions from kindergarten through third grade on a broad battery of measures of reading skills, oral language skills, cognitive skills, mathematics, and family environmental factors. The proposed continuation will extend testing via three additional annual home visits spanning middle childhood to early adolescence, with a focus on emerging reading comprehension skills. We propose to examine the univariate genetic and environmental influences upon reading comprehension, the covariance between reading comprehension and oral language skills, decoding skills, and other related skills, as well as the relationship between proximal and distal measures of the home and school environments that are associated with reading comprehension and related skills. In doing so, the proposed research will offer a unique opportunity to examine the genetic and environmental etiology of the longitudinal development of reading comprehension and related skills during the critical transition from when children are \"learning to read\" to when they are \"reading to learn.\" Extending the unique resources of the Western Reserve Reading Project is an important opportunity for a highly cost-effective and powerful genetically-sensitive investigation of the etiology of reading ability within its various developmental, cognitive, behavioral, and environmental contexts. Developing a more informative model of how genes and environments work together to affect the development of reading comprehension has important implications fostering reading development as well as to detect, ameliorate and prevent reading problems."}
{"text": "The mechanism of cell division is studied, with special reference to cell cleavage in sea urchin eggs. A variety of experimental approaches are to be applied toward a deeper understanding of events which immediately precede the onset of cleavage furrowing in sea urchin eggs, because during this period the responsible mechanisms are established and begin to be expressed. In particular, the ultrastructure of the surface and of the cytoplasmic cortex will be examined and correlated with mechanical properties of the eggs which are explored by various physical determinations. In addition, living cells at critical times will be manipulated by physically damaging selected cell parts with a simple ultraviolet microbeam device."}
{"text": "The supplementation of vitamin D (vitD) during lactation has been poorly investigated: An expert panel convened by the CDC released a report stating that there are insufficient data to evaluate vitD supplementation requirements during lactation. A CDC study revealed a serious public health matter: 42.4% of African American women in their childbearing years exhibited hypovitaminosis D. Using new setpoints for hypovitaminosis D now renders >90% African American women affected. Nationally, there has been a steady and significant rise in nutritional rickets in breastfed (BF) infants, mainly in the African American population. Health organizations have recommended universal oral vitD supplementation of BF infants to diminish the risk of vitD deficiency in this group. Prolonged supplementation with 25X's the current DRI [10,000 lU/day) for up to 5 mos was shown to be safe in adult men and nonlactating women. Recent data suggests that a minimum of 6,400 lU/day vitDS is essential to prevent or reverse infantile hypovitD, particularly in darker pigmented BF infants. Due to compliance/dosing issues with infants, it is hypothesized that supplementation with higher dose maternal vitD will be safe and as effective as infant oral supplementation in the prevention of vitD deficiency in BF infants and will ameliorate vitD deficiency in lactating women, including women with darker pigmentation. The Aim of this proposal is to determine the effectiveness and safety of maternal and infant vitD supplementation (as a function of ethnicity and latitude) in the prevention of hypovitD in the BF dyad. Mothers from two study sites at different latitudes will be randomized to receive 1 of 3 treatment regimens of vitD3. Mothers, lactating or nonlactating controls will be randomized to either Group A: Standard tx (400 IU D3/d), Group B; 2,400 IU D3/day, or Group C: 6,400 IU D3/day. Infants of mothers randomized to Group A will receive 200 IU D3/d (recommended practice) and infants of mothers assigned to Groups B or C will receive placebo. By measuring an array of indicators, calcium homeostasis and skeletal remodeling in the postpartum mother and the breastfeeding infant will be monitored. Through this study, the prevalence of vitD deficiency in the BF dyad and the utility of maternal therapeutic intervention with vitD3 will be assessed. Lay Description: The Aim of this proposal is to determine the effectiveness and safety of maternal and infant vitamin D supplementation as a function of ethnicity and latitude in the prevention of vitamin D deficiency in the breastfeeding mother-infant pair. The findings of this study will generate important new information for health care professionals and policy makers with regard to vitamin D requirements and the potential benefit to both mother and infant. [unreadable] [unreadable] [unreadable]"}
{"text": "Over the past five to ten years, magnetic resonance imaging (MRI) has become an important tool for medical diagnostic applications. Newer techniques are leading to the use of higher static magnetic fields; more rapidly switched gradient fields and higher radiofrequency (RF) magnetic fields. Although use of the increasingly stronger electromagnetic fields is causing concern about patient safety, to date, only simplified homogeneous spherical, cylindrical and disc models have been used to obtain induced current density distributions and rates of energy absorption (specific absorption rates or SARs). Since this knowledge is vitally important, particularly for the critical regions of the body, we will adapt the anatomically based modeling techniques that we have successfully used for electromagnetic dosimetry. We propose to use the previously tested highly efficient impedance method and its new generalization for higher frequencies to calculate internal induced current densities produced by gradient magnetic fields, and SARs for RF magnetic fields, of realistic polarizations and variations typical of present and planned MRI systems. We also plan to use the newly developed modified finite-difference time-domain method, which has previously been used successfully for a wide variety of RF electromagnetic exposure conditions. For the various calculations we will use the new high- resolution model of a human volunteer based on MRI scans (with resolutions of 1.875-3 mm) and its scaled versions where different scaling factors alphax, alphay, and alphaz on the order of 0.6-1.0 will be taken to obtain models of different heights and weights representative of human adults and children. This latter step is needed since induced current densities and SARs are strongly dependent on the size and shape of the body. The current densities and SARs thus calculated will be compared with the safety guidelines suggested by USFDA and NRPB (U.K.)."}
{"text": "The career development plan described in this competing renewal application will allow the applicant to continue the development and expansion of his research career into new areas of investigation that complement current areas of expertise. The applicant has extensive training and experience in behavioral neuropharmacology and substance abuse with primary expertise in the neuropharmacology of CMS stimulants. During the previous funding period, the applicant developed in vivo microdialysis techniques in conscious monkeys trained to self-administer cocaine and related stimulants in order to determine neurochemical changes associated with the behavioral and reinforcing properties of abused stimulants. In addition, the development of positron emission tomography (PET) imaging techniques in conscious monkeys expanded further the applicant's ability to characterize and quantify the CMS effects of abused stimulants. More recent efforts have focused on the development of functional magnetic resonance imaging (fMRI) in conscious monkeys to document with high temporal and spatial resolution drug-induced changes in CMS activity. A major focus of the research plan will involve the pharmacological manipulation of monoamine and glutamate systems to assess potential changes in sensitivity to the CNS effects of cocaine. The studies proposed will provide an innovative preclinical model to identify useful pharmacotherapies for stimulant abuse. In addition to research activities, the applicant will commit effort to educational activities related to biomedical research and substance abuse. The host institution, Emory University, has a well-established program related to neuroscience and substance abuse research that is ideally suited to the career development plan. The University will provide the necessary resources and adequate time to ensure the development of the applicant as a productive, independent research scientist. The long-term commitment and support provided by continuation of the Independent Scientist Award will provide the opportunity to develop and integrate new skills and expertise into a unique multidisciplinary research program in substance abuse. [unreadable] [unreadable] [unreadable]"}
{"text": "QUALITY OF DYING AND DEATH IN PERSONS WITH AIDS aims are to l) use qualitative interviews and draft questionnaire to develop and select items for an instrument administered to loved ones and health care providers after a patient's death from AIDS that can be used in evaluation of the quality of dying and death; 2) assess the psychometric properties (measurement model, internal consistency, discriminate validity, and convergent validity) and respondent burden of the instrument; 3) compare physician\\casework evaluations with that of family and friends. Up to 3 loved ones and 3 health care workers will be interviewed following the deaths of up to 40 persons with AIDS to elicit domains and items that evaluate the quality of dying of death of the person with AIDS and to pilot existing questions. Respondents will be asked to describe last 7 days of person's life in detail. Content of final instrument to be determined by psychometric evaluation of existing items, new domains and items, and comparability among loved ones and health care workers."}
{"text": "This proposal describes the development of a Data Coordinating Center (DCC) at the University of Chicago. Progress in defining the pathogenesis of inflammatory bowel disease (IBD) through genetic approaches will be accelerated greatly through the establishment of an IBD Genetics Consortium. This proposal outlines a data coordinating center (DCC) at the University of Chicago. The overall objective of the IBD Genetics Consortium will be the identification of genes or genomic regions that are associated with increased risk of IBD and with specific phenotypic manifestations. Specific Aim I. To identify and implement IBD genetics projects which can most efficiently be completed through a Consortium structure. The specific objectives of the Consortium will be established through the Steering and Planning Committee and will reflect the nature of the studies proposed through individual Genetic Research Center (GRC) applications. It is expected that the DCC will play a key role in the planning, development and implementation of Consortium objectives. Potential joint issues include: mega-analysis of completed genomewide searches, stratified analysis of genome wide data based on established associations, support of positional gene identification efforts, candidate gene studies, and acquisition of data on clinical covariates, such as tobacco use, age of onset, and disease location. Specific Aim II. To perform the functions of a data coordinating center and genetic analysis center. Functional specifications of the DCC are described. In particular, careful development of a study protocol and Manual of Procedures will be the initial task of the Consortium, to be developed by the DCC with iterative input from the Steering and Planning Committee and individual GRCs. The use of both existing and novel methods of genetic analysis is proposed. Prior experiences in collaborative genomewide screens have been critical in advancing understanding of the challenges associated with pooling genomewide linkage data. These key concepts have been incorporated into the proposed mega-analysis. Methods to reduce genetic heterogeneity are proposed through stratified analyses based on established gene associations, locus-locus and gene-environment interactions. Specific Aim III. To collect common patient specimens and facilitate common genotyping requirements. The creation and maintenance of a DNA and cell line repository at the Coriell Institute containing appropriate patient and control samples is proposed. Provisions for common genotyping are described, if so requested by the Steering Committee or individual GRCs."}
{"text": "Long term objectives: To continue to offer a Master's degree in International Research Bioethics of the highest quality to developing country academics, researchers, health professionals, and members of non government organizations from countries in the Asia Pacific Region with the poorest match of ethics capability and research activity (with a special focus on China, Vietnam and Thailand, where increasing amounts of NIH- supported research are being conducted, and where we have existing institutional linkages). To thus foster the development of national capacity for the ethical design, review and analysis of both clinical and public health research in countries in the Asia Pacific Region, with a special focus on China, Vietnam and Thailand. Specific aims: i) Through the Master of International Research Bioethics, provide a comprehensive program consisting of 12 core units that encompass bioethics, research bioethics in an international setting, research methodology in international health, relevant law and practicums;ii) Continue to offer, evaluate, refine and improve the existing Master of International Research Bioethics;iii) Select a cohort of participants from the above-mentioned priority countries who have demonstrated capacity to benefit from the program and to take up leadership positions in research bioethics, including the provision of education in research ethics, in ethical review and in providing guidance to their institutions, governments and other relevant bodies in their home countries, to undertake the Master of International Research Bioethics;iv) Continue to provide ongoing support to participants subsequent to the program."}
{"text": "The proposed Administrative Core under the direction of Dr. Ellner (PI) and Mr. David Hom (Director) will take overall responsibility of management (fiscal, regulatory, operational), coordinating (operational, logistics), and oversight of all TBRU activities. The Administrative Core will assist proposed sites with study development (biostatistics, protocol, operating procedures), implementation and performance monitoring. The Core will also communicate and work with NIH program staff to monitor study progress and achieve proposed milestones. Lastly, the Administrative Core will work to leverage expertise across other TBRU programs with collaborative projects proposed by TBRU investigators. The Core will also operate a Data Management Center (DMC) to coordinate approved protocol and unit-wide scientific activities in concert with scientific decisions of the TBRU. The DMC will oversee the collection, storage, quality control, and evaluation of all study data. It will provide support and coordination for the statistical and data processing functions for proposed studies at all sites - clinical, animal, and translational. A detailed Management Plan will outline general activities, leadership structure, typical work-flow, chain of responsibility, review/approval processes, review committees and management activities across the TBRU members. Dr. Ellner has overall responsibility for the scientific, administrative, and fiscal activities of the TBRU and will provide vertical integration by directly interacting with leadership at each site. He will be ultimately responsible for scientific conduct and management of the TBRU."}
{"text": "Prostate cancer (PCa) is the second leading cause of cancer deaths in men in the U.S. with yearly treatment costs estimated to be near $8 billion. The increasing amount of research on bioactives and functional foods has fueled the development and marketing of foods and dietary supplements for prostate health, including both soy and tomato phytochemicals, and men may consume combinations of these products. However, it can't be assumed that combinations of supplements and functional foods will be more effective, and it is important to identify interactions that could alter the efficacy or safety of dietary interventions. The long range goal of the proposed research is to quantitatively define and understand the mechanisms by which consumption of soy or tomato products may reduce the risk of PCa. The objective of this application is to investigate the effects of soy germ and/or tomato powder containing diets on the progression of PCa in a transgenic adenocarcinoma of mouse prostate (TRAMP) model. In parallel, we will investigate the bioavailability of tomato carotenoids and soy isoflavones when tomato and soy germ are consumed together. To answer the questions raised in this proposal, diets containing 2% soy germ and/or 10% tomato powder will be fed to TRAMP mice prior to puberty and throughout progression of carcinogenesis. The specific aims for this proposal are 1) To investigate the effects of tomato and/or soy germ containing diets on progression of prostate carcinogenesis and identify their anticarcinogenic mechanisms of action in TRAMP mice 2) To measure bioavailability of tomato carotenoids and soy germ isoflavones in TRAMP mice. It is hypothesized that consumption of soy germ or tomato will reduce the incidence of poorly-differentiated carcinoma, and the combination of the two will not be as effective as when consumed individually. Potential anticarcinogenic mechanisms of the dietary interventions will be investigated through cancer pathway findway PCR arrays and immunohistochemistry. It is hypothesized that an interaction between soy germ and tomato alters bioactive bioavailability. Carotenoid bioaccumulation, serum lycopene metabolites, and urinary isoflavones will be measured to determine if combined consumption alters bioavailability of these bioactives and if the alteration correlates with how soy germ and/or tomato powder modulate prostate carcinogenesis. The proposed research is a unique approach to understand the efficacy of dietary interventions for PCa prevention. We will use whole foods rather than isolated bioactives and investigate a combination of foods to identify potential interactions. The research will benefit both consumers of supplements and the scientific community by providing a better understanding of the safety and efficacy of dietary interventions as alternative strategies for PCa prevention."}
{"text": "The increased availability and reduced cost of small desk-top computer has created increased interest in automating data acquisition and process or experiment control in areas where such things were not feasible before due to cost or complexity. With these changes also came a large increase in the use of the IEEE-488 GPIB by instrument makers. By combining the two, sophisticated instrumentation and data acquisition systems can be assembled quickly and inexpensively. The BEIB is continuing to develop the expertise to provide guidance and assistance to requirements where this approach provides the optimum solution. This capability is further asisted by the BEIB-SERP specifying the IEEE-488 interface on new equiment acquisitions whenever possible."}
{"text": "The affective behaviors symptomatic of the depressed adult are variously expressed in the parent role. How they are expressed by the mother and experienced and responded to by the child are the focus of this project. To be considered, also, is the fact that the child's affective expression, although a response to maternal interaction. The naturalistic laboratory paradigm (MH-02144) is the source of data. Measures of affect include: (a) a minute-by- minute rating of emotions and moods of mother and child, (b) a detailed accounting of physical touch and affection, and (c) an experimental intervention in which depressed affect is elicited from the mother in response to a standard stimulus and the subsequent mother-child interaction is recorded. Analyses are at various stages for these several sets of affective data. Among the findings are: The overall exposure of children to expressed negative mood or emotion of the mother is 17%, 11%, and 6% interaction time with unipolar, bipolar, and normal mothers, respectively. These mean percentages far underestimate the variability related to maternal diagnoses. There are extremes in the depressed groups in which 80 to 90% of the minutes are coded in negative affect. High frequencies on negative affect in the children (the outliers) tend also to be in the offspring of depressed mothers. Children of the unipolar mothers, more than other children, become involved in the mothers's sadness. In such interactions the young child's empathy for the mother and his/her caregiving responses appear to be exploited. Affect and touch were investigated in the interchange of mothers and their 2 to 3 year olds. Touch is initiated more often by the mother (75% of the occurrences) than the child. The major functions of mothers; touch is to monitor and regulate child behavior and to give affection. The young child's initiations of touch are predominantly to be physically close to (in contact with, leaning on) the mother"}
{"text": "Human growth hormone has proven to be an extremely important and valuable therapeutic whose full potential has yet to be realized. Recombinant DNA technology has increased the supply of this drug through commercial production in bacteria, but the extremely high cost of this product has put it out of reach from many patients. A cheaper and more abundant source of this therapeutic would be its production in the milk of transgenic animals. In phase I of this proposal we intend to test the feasibility of this approach by producing human growth hormone in milk of transgenic mice."}
{"text": "Physical activity among youth has declined over several decades. Low levels of physical activity have been associated with increased risk of both obesity and certain types of cancers. It is imperative that this trend be halted and/or reversed. Current methods attempting to increase physical activity among youth do not appear to be effective at promoting increased physical activity among youth, suggesting that innovative approaches are needed. Barriers, such as lack of time and interest, are commonly cited reasons youth do not engage in more physical activity. Self efficacy, a critical mediator of behavior change and a common target of interventions, affects the likelihood one will attempt to overcome barriers. Procedures to increase self efficacy for physical activity among youth need to be developed, tested, and theoretically explicated. Information processing techniques have been used to increase cognitive effort, and, thereby, impact information storage and retrieval. Thus, interventions to effect change in physical activity among youth would appear to benefit from including an emphasis on physical activity self efficacy to overcome barriers in a manner likely to enhance information processing. Computerized programs offer interesting possibilities for systematically manipulating characteristics of materials (e.g., visual and verbal information channels) that would affect information processing. Dual Code Theory predicts that information presented in a manner that utilizes visual and verbal processing channels simultaneously will result in more information processing and, therefore, learning. The studies in this proposal examine the effect of presentation format and processing channel on both information processing and physical activity self efficacy. Computerized multi media programs will be developed using Dual Code Theory and administered to 11-15 year old youth. In Study 1, the effect of presentation format on information processing and physical activity self efficacy will be examined. In Study 2, a factorial design will be used to more closely examine the effect of processing channel (i.e., visual vs. verbal) on information processing and physical activity self efficacy in computerized programs. The significance of these studies is that they will contribute to our understanding of the role of presentation format and processing channel in computerized multi media programs promoting increased physical activity self efficacy among youth."}
{"text": "Presently, several clinical Phase III clinical trials are ongoing at the University of Puerto Rico and affiliated Institutions for the treatment of our cancer patients. We are acutely aware of the need for an early clinical trial program at our Institution. The goal of this pilot project is to establish a program of early phase clinical trials at the University of Puerto Rico. To establish this program, we are starting a collaboration with Dr. Daniel Sullivan of the H. Lee Moffitt Cancer Center (HLMCC). This collaboration will emphasize the development of Phase I and Phase III clinical trials, targeting our Puerto Rican population. In addition, we propose to develop basic science infrastructure that will support investigator-initiated studies. Via this collaboration, the HLMCC, will have access to a unique population of patients that have a higher prevalence of specific malignancies in Puerto Rico, such as, esophageal, cervical, penile, anal, breast, and liver cancers. We propose to achieve these goals through the following three specific aims. In specific aim 1, we will evaluate the facilities of UPR and PRCC to conduct early Phase I and Phase II clinical trials. We will identify the availability of the infrastructure to perform pharmacokinetic, pharmacodynamic, and molecular analyses. In specific aim 2; we will delineate a plan to establish an early phase clinical trials program within the PRCC. Clinical and basic investigators from the UPR and HLMCC will join efforts in the development of specific clinical trials. Finally, in specific aim 3; we will initiate clinical trials that are based on basic research data."}
{"text": "Studies performed in our laboratory implicate the Porphyromonas gingivalis HtpG stress protein, the prokaryotic homologue of Hsp90, in the etiology of periodontal disease. We have reported that elevated levels of anti-Hsp90 antibodies, concomitant with P. gingivalis colonization, are associated with periodontal health. Transcription of HtpG message was also found to be upregulated 7-10-fold in P. gingivalis obtained from diseased subgingival plaque. There is a precedence for Hsp90 homologues contributing to pathogenicity of other microorganisms. Immunity to a single Hsp90 epitope of Candida albicans has been demonstrated to confer protection against systemic candidiasis. Studies performed by our laboratory have revealed that P. gingivalis HtpG has a significant degree of homology with human Hsp90, but remains clearly distinct from other HtpG proteins due to its unique C-terminal region. We have found that HtpG is localized to P. gingivalis membranes and extracellular vesicles, and that it cross-reacts with other prokaryotic and eukaryotic Hsp90 homologues. Our findings suggest that HtpG is readily accessible to participate in host cellular invasion processes, as well as to interfere with normal host cell functions one P. gingivalis enters the host cytoplasmic compartment. Transfection of KB cells with the P. gingivalis htpG gene stimulates IL-8 production by these cells. This application proposes to extend our investigations into the role that molecular mimicry by HtpG plays in the pathogenicity of P. gingivalis. Previous studies of other pathogenic microorganisms which appear to use the Hsp90 homologue as a virulence factor have been purely descriptive. Our application is unique in that while will propose to evaluate the role of HtpG in adherence and invasion mechanisms, we also propose to elucidate novel pathogenic mechanism(s) by which microorganisms such as P. gingivalis utilize molecular mimicry to disrupt normal eukaryotic cell function(s). Since the most clearly defined eukaryotic Hsp90-mediated mechanisms involved signal transduction pathways, these will be the primary foci of our investigations. The hypothesis to be tested in this study is: 1) HtpG plays a role in adherence and invasion of host cells; and 2) once internalized, signal transduction mechanisms mediated by Hsp90/TRAP1 within eukaryotic cells are disrupted by the HtpG of P. gingivalis through molecular mimicry. This leads to disruption of normal inflammatory cytokine responses to microbial invasion by P. gingivalis and other oral microorganisms."}
{"text": "The initial aim of this project was to examine the relations between two assessment methodologies. The CBCL (Child Behavior Checklist) is a quantitative tool which scores behavior along a continuum within each of eight behavioral syndromes: Attention Problems, Aggressive Behavior, Delinquent Behavior, Conduct Problems, Oppositional Behavior, Withdrawn Behavior, Somatic Complaints, and Thought Problems. The DSM- IV ( Diagnostic and Statistical Manual) is a categorical system of conceptualizing emotional and behavioral problems as discrete disorders. For example, this phase of the study included the prediction that a deviant score on Attention Problems of the CBCL ( T-score > 67) would also indicate the presence of an Attention Deficit Hyperactivity Disorder according to the DSM. In fact, the data showed this particular relationship to be true 100% of the time.Currently, the data collection phase of the Vermont Family Genetic Study is underway. The primary hypothesis predicts siblings of children who are deviant on CBCL syndromes / meet a DSM diagnosis are at increased risk for the same emotional / behavioral problems than siblings of a control group.Potential participants are selected from a pool of patients seen either at the Center for Children Youth and Families or the Pediatric Clinic at the University Health Center, University of Vermont. Eligibility is determined by the availability of at least one biological parent, at least one full sibling between the ages of 6-18, age of the patient must be between 6-18, and deviance on the Attention Problems, Aggressive Behavior, both, or neither of these syndromes on the CBCL. Parents are contacted by telephone and invited to participate. The study consists of two parts: completing several questionnaires, and a two hour visit during which parents are interviewed in person about their children's behavior, and children are administered a brief battery of cognitive tests in order to rule out learning disabilities or developmental delay. Parents are given the option of coming in to the Clinical Research Center at Fletcher Allen Hospital or be provided with a home visit. Saliva for genetic testing is obtained at the time of the visit.Data has been collected in this manner since 07/96. To date 75 families have participated. The goal is to schedule and interview 4 families a week for at least another year.The main implication if this study is to develop more accurate and efficient diagnostic methodology to ensure early intervention of common childhood emotional and behavioral problems."}
{"text": "Histologic and ultrastructal studies of heart, lung, liver and kidney were made of mice treated with interleukin-2 singly and in combination with interleukin-1 and or interferon-alpha. The vascular leak syndrome induced by interleukin-2 was ameliorated by concomitent treatment with interleukin-1. This reduction in toxicity may be useful in the treatment of neoplasms with interleukin-2."}
{"text": "Calpain 3 (C3) is the muscle specific member of the calcium dependent protease family, mutations in which result in a form of muscular dystrophy called limb girdle muscular dystrophy type 2A (LGMD2A). In the previous funding period for this grant, we made good progress in understanding the biological function of C3. We showed that C3 is important for proper sarcomere structure, in vitro and in vivo. Based on our findings, we hypothesize that during normal development and muscle adaptation, C3, in conjunction with the ubiquitin/proteasome-system, displaces and removes myofibrillar proteins, and allows for the subsequent replacement by new proteins. Once sarcomeres are fully assembled they form such a dense compact entity that their subunits are protected from proteolytic action. C3's placement in the sarcomere, due to its interaction with titin, allows it accessibility to myofibrillar proteins that are to be turned over following damage or remodeling. We further showed that pathogenic LGMD2A mutations that do not affect C3s proteolytic capabilities can affect its ability to bind titin, and that titin serves as an important anchor for C3. Finally, we have shown that there is a generalized loss of protein turnover in C3KO muscles, leading to protein aggregates and a cellular stress response. The above observations have elucidated the biological function of C3 but have left unanswered questions. How does loss of C3's normal activity result in muscle disease? How does loss of anchorage to titin affect C3's normal cellular role? In our preliminary data, we show that C3 has cellular substrates that fall into two major categories that include myofibrillar proteins and mitochondrial proteins. This finding suggests that in addition to its role in myofibrillar protein turnover, C3 might be important in mitochondrial protein turnover. This hypothesis is supported by our preliminary data showing abnormal mitochondrial morphology and oxidative stress in C3 knock out mice. In this funding period, we will investigate the following aims: Aim 1: To characterize the biochemical consequences of disrupted calpain 3-titin interactions on calpain 3 activity and substrate accessibility. Aim 2: To characterize the biological consequences of disrupted calpain 3-titin interactions on muscle morphology and function. Aim 3: To characterize the mitochondrial defect and features of oxidative stress in calpain 3 KO muscles. [unreadable] [unreadable] [unreadable]"}
{"text": "Core A takes care of the tissue processing needs for all projects. It is directly supervised by Dr. Pelayo Correa and his assistant Dr. M. Blanca Piazuelo. In Nashville our personnel receive all biopsies from Colombia. Biopsies for histopathology evaluation are fixed immediately in buffered zinc-formalin, embedded in paraffin, sectioned and stained with hematoxylin eosin and modified Warthin- Starry silver stain. Biopsies for frozen sections are received in OCT and frozen immediately. The laboratory performs all immunohistochemical stains, including Ki67, TUNEL assays, inducible nitric oxide synthase, nitrotyrosine and a variety of white blood cell markers. Fully trained and experienced personnel are available for all procedures."}
{"text": "Racial and ethnic health disparities abound, with decreased quality of health care in these underserved communities. The reasons for these health inequities are complex and multi-layered. As noted by the Institute of Medicine, there are historic and contemporary social inequities that have been embodied in these populations and in the health systems that care for them. In addition, racial biases of health care providers also may play a role in the unequal treatment of minority populations. Racial stereotyping and bias have been identified as factors in the delivery of cardiovascular disease care, pain management, and mental health care. Given the well-documented disparity in the receipt of total knee replacements between African-American and white patients with severe osteoarthritis, research is sorely needed to better understand what role racial stereotyping and bias plays in creating these inequities in the provision of this treatment. In the proposed research project, we will objectively measure physicians'racial stereotyping and bias and evaluate its association with those physicians'recommendations of patients for TKR. We also propose an innovative educational intervention aimed at reducing the effect of racial bias on clinical decision-making and will assess its efficacy. Findings from this exploratory study will be used to develop a larger, more long-term study of racial stereotyping and bias in clinical decision-making and methods for intervening to mitigate their effects. Specific Aim 1 will test whether physicians express explicit racial bias, using a Web-based survey instrument. Specific Aim 2 will test whether physicians show implicit racial bias, using Web-based Implicit Association Tests (IATs). Specific Aim 3 will evaluate whether the magnitude of implicit bias predicts physician recommendation of TKR for African-American and white patients with severe osteoarthritis. Specific Aim 4 will assess whether use of the Web-based IAT as an educational intervention decreases the effect of implicit racial bias on physician recommendation of TKR. Public Health Relevance: The proposed research project tests an intervention to help physicians manage and, perhaps, eliminate their racial bias. If this intervention proves effective, we will have a powerful tool to help reduce racial health disparities."}
{"text": "A new method was developed, based on measurements of the kinetics of cell activation, for determining whether or not the descending limb of biphasic dose response curve falls because of insufficient cross-linking."}
{"text": "The Department of Urology at the University of Oklahoma has a 7-year history of collaborative multicenter research on the correlation of biochemical and clinical features of interstitial cystitis and the testing of new drug therapies. Within the Department are strong programs in all areas of clinical urology, including oncology, andrology, female urology and urodynamics, the basic sciences, emphasizing immunology, molecular oncology and biology, and cellular disease markers. Strong links with epidemiology and pathology have allowed us to be a leader in several areas of research. We maintain an active specimen collection and storage system for urines and other specimens collected from interstitial cystitis patients using standardized, quality-controlled procedures and a patient database and have a followup mechanism for tracking patients. The Department has a comprehensive residency training program. An informal regional network of community and academic urologists has also been developed to help meet patient needs for improved diagnostic and clinical care and for support as well as to recruit patients for specialized studies and experimental treatment protocols. Routine care is provided by community urologists or other physicians while Dr. Roy provides certain specialized services and experimental treatment protocols. The Department has drawn its patient base predominately from Oklahoma and neighboring states, a population base of 32.2 million, containing an estimated 5-6,000 undiagnosed interstitial cystitis patients with an estimated yearly incidence of roughly 200 patients. The largest out-of-state component being from Texas. Patients have come from as far as Michigan and Montana. The University of Oklahoma Department of Urology clearly meets the criteria for a clinical center. In order to meet the overall programmatic objectives, our specific objectives for the overall collaborative program: (1). Expand and further formalize the current informal network of community and academic urologists in the region to ensure standardized collection of data and specimens as well as long-term followup and to aid collaborating urologists who are responsible for routine care of patients to provide the best care based upon the latest findings of the clinical centers. (2) Expand current database and sample collection and storage programs in conjunction with other centers. (3) Expand clinical services offered to interstitial cystitis patients to include bladder retraining and reference to a university pain clinic, both to provide better services to patients while facilitating patient participation in research studies. (4) Recruit 50 patients the first year and 75 per year thereafter. (5) Present an annual seminal for physicians and workshop for patients as a means of educating physicians and the public about interstitial cystitis and reaching patients."}
{"text": "Youth with a history of maltreatment and foster care placement are at risk for a host of mental health, behavioral, and social problems, resulting in adverse life-course outcomes of great public health significance. This R01 application seeks support for a 5-year efficacy trial of the Fostering Healthy Futures Program (FHF), a preventive intervention designed to promote prosocial development, and to reduce problem behaviors for youth in foster care. FHF is an innovative, culturally-sensitive and multi-component intervention for 9-11-year-old children who have been maltreated and placed in foster care. Through a 9- month intervention that includes screening assessments, one-on-one mentoring, and skills groups, FHF targets cognitive, social and behavioral domains in order to build competencies, improve mental health functioning and quality of life, and reduce problem behaviors and adverse life outcomes (e.g. arrests, school dropout, restrictive placements). Assessments with youth, caregivers and teachers will be conducted at baseline (pre-randomization), post-intervention, and 1-year follow-up. Data will also include child welfare, educational, and juvenile justice records. A randomized controlled pilot trial of FHF in one county has produced program manuals for the mentoring and skills group components, and evidence of program feasibility, with high recruitment and retention rates. The FHF pilot study has demonstrated positive preliminary effects on putative mediators, including youths'social functioning, attitudes, coping skills, behavioral regulation, and extracurricular involvement (cfe from .23-72). It has also demonstrated positive effects on distal outcomes, including problem behaviors, competencies, number of restrictive placements, and life satisfaction (cfe from .2S-.75). Finally, the pilot study has generated preliminary effect sizes that have enabled us to specify the sample size necessary for a full-scale efficacy trial. A multi-county randomized controlled trial of the FHF program with 256 youth will enable us to: 1) examine intervention effects on both proximal and distal outcomes, 2) examine potential moderators of the intervention, 3) conduct mediational analyses to identify the mechanisms by which the program may enhance outcomes, and 4) conduct within-group analyses. The goal is to design more efficacious interventions, thereby reducing disability, morbidity, and mortality, not only for youth in foster care, but for all high-risk youth."}
{"text": "The long term objective of this application is for the candidate to become an independent investigator studying promising new neuroprotective botanical treatments for multiple sclerosis (MS) in human subjects using advanced MRI imaging measures. Axonal loss is thought to be the cause of irreversible progression of disability in MS. None of the available therapies for MS directly protect axons. Epigallocatechin gallate (EGCG) is a potent antioxidant and is the main component contained in polyphenon E, an extract from the leaves of the green tea plant (Camellia sinensis). Animal studies done by our group indicate that EGCG protects axons in the spinal cord from injury in an animal model of MS, experimental autoimmune encephalitis. The overall objective of this application is to determine if polyphenon E is a safe neuroprotective therapy that warrants further study as a treatment to prevent the progression of disability in [unreadable] MS. To achieve this objective, a double-blind, controlled, two-arm parallel group clinical trial will be [unreadable] conducted; 46 subjects with MS will be treated for two years with either polyphenon E 400 mg twice a day or placebo. The first specific objective is to determine if there is evidence of a neuroprotective effect. To accomplish this objective the difference between the two groups on the rate of change in n-acetyl aspartate (NAA) will be determined. NAA was selected as the primary outcome because it is a selective marker for neurons and axons that reflects their number and metabolic activity and thus is optimal to show a neuroprotective effect. NAA levels will be measured using magnetic resonance spectroscopy at baseline, six months, a year and two years. Changes in magnetization transfer ratio and brain atrophy will be secondary outcomes. The second specific objective is to determine the safety of polyphenon E in subjects with MS. The subjects will be monitored for adverse events with laboratory exams and physical exams. The third specific objective is to determine if plasma levels of EGCG predict changes in the imaging markers or the occurrence of adverse events. Conducting this study will allow the candidate to obtain extensive training advanced magnetic resonance techniques and pharmacokinetics. Relevance to public health: MS is the second most common cause of neurological disability among young people. This study is relevant to public health because it is expected to result in a new treatment for MS that can prevent the progression of disability. [unreadable] [unreadable] [unreadable]"}
{"text": "Background: Estrogen antagonists are widely employed in the treatment of breast cancer. They also comprise the most promising drugs for breast cancer chemo-prevention. Despite these facts, the molecular mechanisms by which these compounds inhibiting cellular proliferation are not fully defined. One of such compound, tamoxifen, leads to highly-significant decreases in the rates of both disease recurrence and death, but tamoxifen is limited by a less that 50 percent response rate in breast cancer prevention and the inevitable development of cellular resistance in breast cancer therapy. Our preliminary studies have demonstrated that depletion of prohibitin protein or introducing dominant negative forms of co-repressors of prohibitin overcomes 4-hydroxytamoxifen-induced growth suppression in breast cancer cells. A potential role for prohibitin gene in breast cancer has also been suggested in earlier studies, in which prohibitin was found to be mutated in some sporadic breast cancers. Objective/Hypothesis: I hypothesize that prohibitin and its co-repressors Brg-1/Brm play a role in estrogen antagonists-induced growth suppression of breast cancer, and that estrogen antagonists target the prohibitin/E2F pathway to affect breast cancer cell proliferation. The studies proposed will test and prove a critical and necessary role for prohibitin/Brm-Brg/E2F axis in the response of breast cancer cells to estrogen antagonists. Specific Aims: I. Determine the mechanism of estrogen antagonist-induced growth repression of breast cancer cells II. Determine the molecular level at which estrogen antagonists regulate prohibitin activity. Study Design: Northern blot or RT-PCR and immunoblot analysis will be carried out to assess the regulation of endogenous E2F responsive promoters by estrogen antagonists. The requirement for prohibitin/Brg-1/Brm in estrogen antagonist-mediated transcriptional repression of natural E2F-responsive promoters will be tested, using anti-sense and dominant-negative strategies. Chromatin immuno-precipitation assays (CHIP) will be employed to study the effect of estrogen antagonists on the interactions of prohibitin, Brg-1, and Brm with the natural E2F-responsive promoters. Relevance: A major problem facing breast cancer chemo-prevention is the low response rate. Estrogen antagonists treatment is further limited by the inevitable development of resistance. The lack of information about the molecular mechanisms of estrogen antagonists-mediated growth suppression prevents the development of strategies to overcome the problems. Our demonstration of the involvement of prohibitin in 4-HT induced growth arrest suggests that the prohibitin/E2F pathway is the/a cellular target for estrogen antagonists, and thereby also implicates prohibitin as a potentially important target for breast cancer therapy."}
{"text": "This project will investigate the ability of two or more potential metabolites of chloramphenicol to induce aplastic anemia in mice. The colony-forming cells (CFC) assay will be used to detect potential toxicity of these metabolites to the bone marrow. The carcinogenicity of the two compounds will also be investigated, as this action may relate to aplastic anemia. The hydroxylamine and hydroxamic acid metabolites will be synthesized by proven methods, then subjected to biological testing. A study of the in vivo metabolism of the compounds will also be performed. All chemical investigations will be conducted at Midwest Research Institute; the biological investigations will be performed at the University of Kansas School of Medicine."}
{"text": "Changes in gene expression patterns are a hallmark of the aging process. Important insight into the mechanisms controlling such gene expression programs has come from the study of replicative senescence of cultured cells (eg, human diploid fibroblasts), which recapitulates many features of cells from aging individuals. This Project has traditionally studied changes in RBP expression and function during replicative senescence. It has also examined the influence of RBPs in replicative senescence by interventions to elevate or reduce RBP levels, followed by the analysis of changes in senescence-associated mRNA expression patterns. We have studied if a given RBP binds a senescence-associated mRNA using a variety of in vitro binding assays (biotin pulldown, RNA EMSA, etc) and assays to measure binding of endogenous molecules ribonucleoprotein immunoprecipitation (RIP) or crosslinking IP (CLIP). To investigate RBP function during senescence, we employ approaches such as RBP silencing, RBP overexpression, mutant RBP analysis, and RBP-associated mRNA identification (using microarrays, RNAseq, and RT-qPCR). To investigate whether RBPs affect the stability of target mRNAs during senescence, we measure the steady-state levels and half-lives of the mRNAs of interest as a function of RBP abundance. We investigate whether RBPs affect the translation of target mRNAs by studying the relative association of the mRNA with translating polysomes and by quantifying the nascent translation rates of the encoded proteins. We also employ reporter constructs to gain additional insight into the processes modulated by RBPs and use various senescence-associated markers to examine changes in the senescence phenotype. Over the past 12 months, this Project has examined the changes in gene expression that occur in human tissues as part of physiologic aging. Much of our effort in this Project has been directed at understanding how RBPs and noncoding RNAs affect the process of cellular senescence, which is increasingly recognized as underlying age-related changes in tissue physiology and pathology. The studies in this Project examine the RBPs and ncRNAs that modulate cellular senescence and the consequences of their influence on the senescent phenotype. Among the cell systems used for these studies, human diploid fibroblasts have been particularly informative. Senescence-associated RBPs. Following a long-established line of research in our group, we have continued the characterization of several RBPs implicated in aspects of cellular senescence, including the loss of proliferation, the impaired ability to respond to stress, and the implementation of a senescence-associated secretory phenotype (SASP). We discovered that the RBP NF90, which lowers the stability and translation of several mRNAs Tominaga-Yamanaka et al., Impact Aging 2012, associated with many mRNAs encoding SASP factors such as IL-6, IL-8, osteoprotegerin, GM-CSF, MCP-1, HCC4, Gro1, and Gro2. In young, proliferating fibroblasts, NF90 was abundant and inhibited the expression of mRNAs encoding SASP factors; by contrast, NF90 was low in senescent cells, allowing the levels of SASP factors MCP-1, GROa, IL-6, and IL-8 to rise. In collaborative studies, we have continued to investigate the roles of RBPs HuR and AUF1 in senescence, finding that the decreased nuclear export of HuR mRNA by HuR was linked to the loss of HuR in replicative senescence, that the interaction of HuR and AUF1 with the 3UTR (untranslated region) of p16 mRNA was inhibited when NSun2 methylated the p16 3UTR, leading to a rise in p16 levels Zhang et al., Nat Commun. 2012, and that the senescence-associated decline in CARM1, which methylates and hence activates HuR, was associated with the loss of HuR function in senescent cells Pang et al., BMC Mol Biol 2013. Senescence-associated ncRNAs. During the past twelve months, we have also continued to investigate the influence of ncRNAs in senescence. We have reported a number of specific microRNAs differentially expressed in senescent cells. During this evaluation period, we have also contributed several reviews to this emerging field, covering the topics of microRNAs that influence senescence and tumor suppression, the regulation of microRNA biogenesis in senescence, and the global influence of microRNAs on gene expression patterns in senescence Abdelmohsen et al., Ageing Res Reviews 2012. We have also initiated efforts to investigate the role of long ncRNAs (lncRNA) in senescence. A recent high-throughput search using RNA-Seq in collaboration with Dr. Beckers core facility, revealed numerous lncRNAs that are differentially expressed during cellular senescence. Among these senescence-associated lncRNAs, termed SAL-RNAs Abdelmohsen et al., Impact Aging 2013, we identified several transcripts that played a direct role in cellular senescence. SAL-RNAs are the subject of many follow-up studies in our laboratory that will be reported in the near future. In addition, we collaborated with the Prasanth laboratory in reporting that the lncRNA MALAT1 promoted cell proliferation and that silencing MALAT1 triggered cellular senescence Tripathi et al., PLoS Genetics, 2013. Tissue aging. Although our understanding of the post-transcriptional factors that influence senescence is advancing quickly, we still know relatively little about the RBPs and ncRNAs that affect the aging process itself. We recently reported that the levels of several RBPs that regulate translation and stability (HuR, AUF1, TIA-1, and TTP) in human tissues revealed that these RBPs are abundantly expressed in most tissues in all age groups. Expression was widespread in all normal tissues, including nondividing, terminally differentiated tissues, suggesting that these RBPs play life-long physiologic roles in tissue homeostasis."}
{"text": "Marked improvement on the growth of mouse megakaryocytes was observed in cultures maintained in a liquid medium containing 20% horse serum in medium NCTC 135. Degeneration of megakaryocytes appeared less than before. The complete sequential events of maturation of megakaryocytes through the stages of megakaryoblast, promegakaryocyte, granular megakaryocyte, and demarcation membrane formation was observed regularly in all the eight experiments performed up to the present time. This is quite different from previous results in that good granular and demarcation membrane forming megakaryocytes were observed only occasionaly in some experiments. The number of megakaryocytes increased 3- to 5-fold in a period of 3-4 days. A few megakaryoblasts were observed in cultures as late as 15 days after cultivation."}
{"text": "This application, which deals with the effects of alcohol on the liver, requests the conversion of an individual research grant, which has been funded for 25 years to a Program Project. The four individual projects are concerned with (1) the effects of ethanol on signal transduction in intact hepatocytes, (2) mechanistic studies of the effects of ethanol on signal transduction in membrane systems, namely turkey erythrocytes and isolated liver plasma membranes, (3) the effects of ethanol on the interaction of liver cell membranes and extrinsic membrane-associated proteins, involved in signal transduction, and (4) the molecular characteristics of ethanol-hepatocyte membrane interactions. In these projects, we will study the effects of ethanol in vitro and of chronic ethanol feeding on membrane-associated parameters in the hepatocyte, including (but not restricted to) ligand-receptor coupled CAMP and phosphoinositide signal transduction systems, homologous and heterologous desensitization produced by ethanol, alterations in the interactions of phospholipase A2 and protein kinase C with liver cell membranes, and differential binding of ethanol in hepatocyte membranes in naive and chronically intoxicated animals, to distinguish specific from bulk effects. Techniques include ESR, NMR, time-resolved fluorimetry, digital imaging fluorescence microscopy, laser confocal microscopy, and preparative and analytical HPLC. The overall goal of the integrated biochemical and biophysical studies in this program project is a deeper insight into the mechanisms of ethanol-induced liver injury."}
{"text": "Trypanosoma brucei is a eukaryotic parasite that evades mammalian host immune response by antigenic variation: changing its Variant Surface Glycoprotein (vsG) ?coat? by alternatively expressing one among hundreds of highly diverged VSG genes in its genome Most VSG switches occur by the little-studied process of gene conversion. Here, a new strategy is proposed, replacing VSG ORFs with selectable markers, to investigate parameters affecting gene conversion of VSG cassettes. Drug selection will detect recombination of promoter-less markers with the active VSG, allowing isolation and study of rare gene conversion events. The hypothesis that recombination rates differ for internal versus telomeric VSGS will be evaluated by measuring gene conversion from markers placed at each type of site. Creating lines with latent promoters upstream of silent telomeric VSGs will detect otherwise ?silent? switches, which might shuffle and increase the availability of VSGS at ?preferred? chromosornal locations. The reporter constructs can be altered to test the role of conserved sequence motifs of VSG gene conversion cassettes for effects on the rates and order of VSG switching. The influence of chromosomal context and local sequence elements on recombination will be teased apart. A thorough understanding of VSG gene conversion will allow development of more accurate models to explain and predict patterns of antigenic variation. This work is highly relevant to host-pathogen interactions, especially in trypanosomes and malaria parasites."}
{"text": "The goal is to understand the essential hydrophobic and subunit interactions with the mitochondrial electron transport system that enable it to couple electron transport to ATP synthesis. Four specific projects will probe functionally important interactions within cytochrome c oxidase, which is the terminal electron transport complexes in the inner mitochondiral membrane. The first objective will be to determine the phospholipid specificity and the functionally important high affinity lipid binding sites on cytochrome c oxidase. The approaches to be used include measurement of functional binding of synthetic derivatives of cardiolipin that contain defined abnormal fatty acid tails, and photoaffinity labeling the cardiolipin binding sites with cardiolipin analogs. The second objective will be to determine the involvement of the tightly bound cardiolipin in dimerization of cytochrome c oxidase. The techniques to be used will utilize high speed sedimentation velocity to measure the aggregation state of the protein as a function of bound cardiolipin. The third goal is to structurally locate the redox centers within the protein relative to the membrane interior and one of the protein subunits, subunit III. The approach will be measurement of intrinsic protein fluorescence. The last goal will be a direct investigation of the structure of one of the subunits of cytochrome c oxidase, subunit III. The approaches in this project utilize detergent binding methods to measure surface hydrophobicity and hydrodynamic measurements to determine overall size and shape of the purified subunit. Together, these four approaches will probe the importance of specific lipids and subunits in the structure and function of this inner mitochondiral membrane protein."}
{"text": "There are two long-term objectives of the proposal. First, to understand in neuronal detail how simpler nervous systems are able to generate behaviors and how these nervous systems are able to accomplish simple forms of learning. The second is to understand what happens in cortex during epileptic seizures. In order to better accomplish these scientific goals, we also have the methodological goal of improving optical techniques for monitoring neuron activity. We have been using optical methods to monitor neuron activity in the Aplysia abdominal ganglion during the gill-withdrawal reflex. Our preliminary results suggest that between 250 and 420 neurons in the ganglion are activated by a mechanical stimulus to the siphon. However this estimate comes from a recording that is perhaps 35% complete. We propose experiments to improve the completeness of the recording, experiments to allow preliminary identification of many of these neurons, and analyses to allow us to follow the activity of individual neurons during a series of trials involving habituation and sensitization. We also plan experiments to try to see if the same behavioral plasticity can be achieved with fewer neurons. If this is successful we propose to make a model of the neuron interactions that generate the behavior. We have been able to monitor activity in bicuculline induced inter-ictal epileptiform discharges in rat somatosensory cortex. Our results were surprising in two regards. First, they indicated that the optical measurements and the ball electrode measurements could give very disparate results. Second, they indicated that there were qualitative differences between discharges induced by sensory stimulation and spontaneous discharges. Our first aim is to confirm these result. Then we plan a local application of bicuculline to study the spread (or absence of spread) of the epileptic activity away from the treated region. Finally we plan a simultaneous measurement from both the bicuculline treated and the untreated contralateral somatosensory cortex. These experiments are meant to lead toward measurements in more sophisticated models (primates) of the human disease. These experiment may also lead to the use of optical monitoring techniques for determining the location of epileptic foci in surgical patients."}
{"text": "The long term goal of this proposal is to identify mechanisms responsible for the aging associated rise in coronary smooth muscle excitability, with emphasis on the role of K channels. The main hypothesis postulates that KCa channels, as key players of coronary arterial tone, are regulated by potent vasoactive substances and that their expression and/or modulation may decrease with aging. Thus, the capacity of KCa channels to maintain an optimal arterial tone is diminished in the elderly. This hypothesis will be tested by studying in coronary arteries of young-adult, mature and senescent subjects the abundance of KCa channels, their molecular constituents (alpha +/- beta subunits), response to vasoactive substances and mechanism(s) of regulation. KCa channels are abundant in vascular smooth muscle of various species. We and others have shown that KCa channels set the level of smooth muscle contractility in cerebral arteries and myometrium. Our preliminary data indicate that: 1) KCa channels are abundant in human and rat coronary arteries and are positively regulated by a beta subunit; 2) they are modulated by potent vasoactive metabolites like thromboxane A2 (TXA2) and nitric oxide (NO); 3) aging of coronary smooth muscle is associated with a downregulation of KCa channel expression and function; 4) they play a critical role in the control of coronary arterial tone. The questions that we want to answer are: 1) is regulation of coronary arterial tone by KCa channels affected by aging? 2) does the functional expression and/or characteristics of KCa channels change in the aging process? 3) are the alpha and beta subunits of KCa channels equally expressed or distributed at different ages? 4) is the modulation of KCa channels by potent vasoactive substances (e.g. TXA2 or NO) affected by aging? We will use a combined electrophysiological, biochemical, immunochemical, and mechanical approach. The specific aims are to compare in young, mature and senescent subjects (rats and humans): 1) the contractile responses to KCa channel blockers and agonists; 2) the channel functional expression, biophysical properties, and pharmacology; 3) the relative abundance of the pore-forming alpha and regulatory beta subunits; and 4) the channel response to vasoactive substances (e.g. TXA2, NO) and their mechanism(s) of modulation. This study will aid our understanding of age-related changes of coronary vasoreactivity, and possibly in the design of clinical treatments that reduce coronary spasm and heart dysfunction."}
{"text": "Project Summary/Abstract Age-onset diseases including cancer, neurodegenerative disease, diabetes, cardiovascular disease, stroke, and osteoporosis are generating a public health burden that is quickly becoming insurmountable. Exacerbating this problem, co-morbidities are common among the elderly. The ideal therapeutic strategy to confront this crisis is to target a unifying risk factor, but patient age is the only risk factor common to all these diseases. Fortunately, it is increasingly clear that the biological processes of aging are malleable, thus the rate and quality of aging may be improved. Genetic and nutritional interventions causing real or perceived energy- depletion are robust, conserved mechanisms to promote healthier aging. Unfortunately these interventions, e.g. activation of the molecular low-energy sensor AMPK, also carry clinically unacceptable side effects, such as suppressed immunity and fertility. Translating these findings into therapeutics thus requires identification of downstream mechanisms that are sufficient for healthier aging. Through a genetic model of longevity in C. elegans via activation of AMPK, we demonstrated that the negative side-effects of energy-depletion can be uncoupled from the positive effects on healthy aging. Using unbiased, systems-level approaches, we found that the longevity-specific mechanism involves downstream regulation of mitochondrial dynamics and metabolic functions, and we now demonstrate that regulation of mitochondrial dynamics is causal to AMPK longevity. New data indicate that perturbing the unfolded protein response (UPR), which mediates homeostasis of the endoplasmic reticulum (ER), interacts with the AMPK pathway to extend lifespan through a mechanism that also requires mitochondrial remodeling. Given recent studies showing that the ER physically interacts with mitochondria to regulate organelle morphology and metabolic signaling, these data suggest a new paradigm in aging: ER/mitochondrial regulation of longevity occurs through an integrated metabolic mechanism. Physical changes in mitochondrial networks are a hallmark of aging, but how organelle dynamics are mechanistically involved in longevity is unknown. By genetically inactivating mediators of mitochondrial remodeling (fission/fusion), Aim 1 will define how mitochondrial dynamics drive the changes in mitochondrial metabolism associated with low-energy longevity. Through novel transgenics and training in high-resolution microscopy in Aim 2, I will test the hypothesis that UPRER perturbations promote altered mitochondrial morphology and signaling between organelles. Finally in the R00 phase, Aim 3 will build on the tools and insights developed in Aims 1 and 2 to identify genetic mechanisms in C. elegans by which ER-mitochondrial inter-organelle communication can be directly targeted to extend healthy lifespan and protect metabolic homeostasis. Taken together, the goal of this proposal is to identify how evolutionarily conserved energy-sensing pathways coordinately modulate inter-organelle signaling and metabolic function to promote longevity."}
{"text": "Puerto Rican children in the United States experience the highest asthma prevalence and morbidity rates of all racial/ethnic groups and these rates are increasing. Factors such as biological differences in severity and sensitivity, environmental conditions, access to care, and socioeconomic status contribute to, but do not fully explain, these prevalence and morbidity rates. Inadequate self-management behaviors, which are potentially modifiable, also contribute to these asthma morbidity outcomes. This exploratory R21 application will develop and test an intervention to improve asthma self-management for this high-risk population. The intervention is unique in that the education and support provided to all participants from the standardized Core Curriculum can be modified using a Toolkit based on individual needs and strengths. The education incorporates specific skills training in environmental modification, problem solving, self-monitoring, and enlisting social support. Specific Aim 1 is to explore influences of asthma self-management behaviors (including asthma belief models, the use of folk remedies, social support, stress, access to care, and socio-economic status) for Puerto Rican parents of children with asthma. Specific Aim 2 will incorporate these influences into a community health worker asthma intervention and use a small behavioral randomized controlled trial to test the ability of this intervention to improve asthma self-management behaviors for parents of Puerto Rican children with asthma. The long-term goal is to apply these pilot data in the design and implementation of a larger behavioral randomized controlled trial that will test a similar asthma intervention aimed at improving asthma control. The investigators in this application have significant experience with community agencies serving the target population and have expanded these relationships to prepare for this application using the principles of community-based participatory research. A prominent community service agency and a local community college contributed to the design of this application and have committed their services in the study implementation and results dissemination. The data generated from this research will improve our understanding of the specific influences of asthma self-management for Puerto Rican parents of children with asthma and the study will provide important pilot data on an intervention that may ultimately improve asthma control for this high-risk population that suffers disproportionately from asthma. The participatory design serves to build community capacity for research and improve research quality and effectiveness. PUBLIC HEALTH RELEVANCE: The data generated from this research will improve our understanding of the specific influences of asthma self-management for Puerto Rican parents of children with asthma and the study will provide important pilot data on an intervention that may ultimately improve asthma control for this high-risk population that suffers disproportionately from asthma. The participatory design serves to build community capacity for research and improve research quality and effectiveness."}
{"text": "Senior citizens typically fail to follow their prescribed medication regimens. Separate studies report that adherence to long-term therapy for chronic illness is less than 50 percent. Failure to take medicines as prescribed significantly increases the likelihood of negative health outcomes. For example, studies suggest that 11 percent of elderly hospital admissions and 24 percent of nursing home admissions are due to medication non-adherence. Our application is to develop an image enabled intervention system whereby a remote informal caregiver (e.g., a family member) can collaborate with a senior citizen living at home to improve medication adherence. A significant innovation is changing the self-medication process model into a real time collaborative one. Today's solutions require physical presence for objective verification and do not encourage dialog related to the root causes of non adherence. Starting with the information on the labels of the patient's Rx bottles, the proposed approach allows a remote participant to literally see that the prescribed medications are properly organized and that they have been removed from the organizer according to the desired schedule. There are a plethora of pill organizers and reminder systems on the market today. The proposed Collaborative Medication Adherence System builds upon perhaps their simplest form (i.e. the $5.00, 7 day compartmented pill box) and adds the dimension of collaboration. A key component of the system are \"MediCam\" devices that apply existing low cost video conferencing technology in a novel and highly constrained manner to address privacy and ease- of-use concerns. The associated software captures an image history and presents reports that encourage the collaborators to discuss how to improve adherence. T I Works Inc. will collaborate with Oregon Health and Science University (OHSU)'s Oregon Roybal Center for Aging and Technology (ORACTECH) to accelerate the work. This will build upon their expertise, processes, Health Coaching Platform infrastructure, and patient population. To verify feasibility, experiments will be conducted with a small number of home- resident senior citizens that are already participants in ORCATECH's health coaching project. This new dimension of intervention is likely to have both adherence and socialization benefits. Further, the approach has the potential to delay the senior from being institutionalized, saving up to $70,000 annually;because there is high confidence in their medication adherence. These benefits would be compelling motivators for commercial products and services. PUBLIC HEALTH RELEVANCE: Poor adherence to medication regimens by senior citizens is wide spread, costly, and increases the likelihood of negative health outcomes ( e.g. worsening of condition, relapse etc.). The research proposed is relevant to public health because it has the potential to improve the wellness and delay institutionalization of senior citizens. The approach can be implemented at low cost because it uses existing technology and engages a previously untapped resource, namely remote relatives and friends. The vision is that \"aging in place\" elders can extend their ability to live independently and safely at home, because there is high confidence in their medication adherence."}
{"text": "We plan to study the environment of divalent metal ions responsible for activating enzymes. We will attempt to identify the nature of the metal's ligands, the liganding geometry and the effects of substrates; inhibitors and allosteric cofactors on the nature of the metal-protein interaction. We will then try to interpret these features in terms of the metal's catalytic role. A systemic study of enzymatically active metalloproteins substituted with a number of transition metal ions will be made. We will take advantage of the fact that contributions of metal's d electrons to absorption, circular dichroism, and magnetic resonance spectra are very sensitive to the nature of the metal-enzyme interaction. We will also make kinetic studies, to determine the catalytic properties of the substituted metalloenzymes we will be using."}
{"text": "Synthetic methods for indole derivatives are being investigated. Of particular interest are methods for obtaining indoles with medium sized (7-10 atoms) C-rings which also contain nitrogen and other functional groups. The eventual goal is to couple such compounds with the alkaloid vindoline to provide semi-synthetic analogs of the anti-neoplastic alkaloids vincristine and vinblastine."}
{"text": "Inhibition of the enzyme NQO1 in pancreatic tumor cells using the non-specific inhibitor dicoumarol resulted in increased intracellular superoxide, inhibition of cell growth and inhibition of the in-vitro malignant phenotype of cells. The hypothesis proposed was that inhibition of NQO1 led to increased superoxide levels which inhibited pancreatic cancer cell growth and the in-vitro malignant phenotype. We have recently shown that NQO1 can directly scavenge superoxide providing both a potential approach and a mechanism of action for the use of NQO1 inhibitors in the therapy of pancreatic cancer. In this proposal, we will test the hypothesis that specific mechanism-based inhibitors of NQO1 are effective compounds for the therapy of pancreatic tumors both in-vitro and in-vivo. One of these inhibitors, the indolequinone ES936, is a potent inhibitor of pancreatic cancer cell growth and the in-vitro malignant phenotype. We will therefore test the hypothesis that ES936 can be employed as an effective therapeutic agent in pancreatic cancer and extend in-vitro data to in-vivo xenograft and orthotopic pancreatic tumor models. We will define the mechanism of action of ES936 and will attempt to dissociate NQO1 inhibition from effects on pancreatic cancer cell growth and the in-vitro malignant phenotype using both chemical/pharmacological and genetic approaches. Whether the effect of ES936 is due to increased levels of superoxide as a result of NQO1 inhibition will be characterized and downstream effects of ES936 on modulation of the cell cycle and apoptosis will be determined both in-vitro and in-vivo. This approach represents a novel therapeutic strategy for the treatment of pancreatic cancer, a disease where therapeutic options are very limited and where chemotherapy has made minimal impact."}
{"text": "The interplay of circadian timing and metabolic physiology represents a new frontier in biomedical research. Research on weight gain in humans has focused primarily on macronutrient intake and energy expenditure, the latter predominantly through physical activity, to understand and treat overweight and obesity. Emerging evidence from animal models indicates that circadian physiology impacts weight gain, including the observation of obesity in clock gene mutants and most recently the finding that food intake restricted to the habitual sleep time of mice leads to weight gain as compared to the same amount of food intake during the normal wake episode. We propose to determine if findings from these mouse models translate to humans. To do so, we have designed an exploratory Clinical and Translational Research Center (CTRC) study that uses whole room calorimetry to determine how restricting the majority of food intake to the biological night impacts energy metabolism in humans. Eating at night is common in work schedules with long work hours and with work operations during the nighttime hours (e.g., health care, emergency response, security personnel) and in circadian sleep disorders including, but not limited to, shift work and jet lag disorder. Night eating disorders are also newly recognized conditions that may contribute to obesity. The project addresses several themes outlined in NIH PA NUMBER: 09-124 Exploratory/Developmental Clinical Research Grants in Obesity by testing the following specific hypothesis: i) circadian misalignment will lead to reduced fat oxidation-a physiological state that could contribute to weight gain. This research effort is responsive to PA-09-124 as it will assess how a behavioral phenotype of eating at night increases risk for obesity and will contribute to identification of this behavior as a modifiable determinant of overweight and obesity."}
{"text": "We propose to extend the recently developed Medical Imaging Informatics Bench to Bedside (mi2b2) Workbench software to make medical images collected during routine clinical care available to clinical translational investigators. Today, this gold mine of patient information is not easily available. Well characterized medical images from clinical repositories would be an extremely valuable resource for clinical translational investigators who have specific ideas that require testing in large sets of images from many patients with different diseases. Our hospitals are international leaders in the development and deployment of advanced biomedical imaging technologies (MRI, high speed CT, ultrasound, PET and others) for clinical practice. The Picture Archive and Communication Systems (PACS) within each of the Departments of Radiology in our participating hospitals (Massachusetts General Hospital, Brigham and Women's Hospital, and Children's Hospital Boston) contain a wealth of medical images that often equal the quality of available clinical research imaging data, and greatly exceed its volume in terms of the number of patients and disease types. Though immediately extensible to other research applications and diagnostic scenarios, we apply our approach first to the difficult task of interpreting pediatric brain magnetc resonance images (MRI). The proposed Pediatric MRI module and Harvard Catalyst Radiological Decision Support toolkit that host it are targeted to address the immediate clinical challenge of understanding normal because normal is a constantly moving target, changing rapidly with brain development. These free and open source software tools are natural extensions of currently funded government projects. This includes software from two National Centers for Biomedical Computing; Informatics for Integrating Biology & the Bedside (i2b2) which provides tools that extract and integrate data in a secure, HIPAA compliant manner from electronic medical records, laboratory data, billing information systems and the National Alliance for Medical Image Computing (NAMIC), which provides tools for medical image analysis including registration, visualization and computation of individual subject and large cohort data sets and will form the foundation for the Harvard Catalyst Radiological Decision Support Toolkit."}
{"text": "Specification of the most effective evaluations and exclusion of specific disorders of intermediary metabolism are being sought by testing analyses and performing metabolic challenges. (This is an ongoing study.)"}
{"text": "Description (taken from application): The purpose of this program is to facilitate the exchange of information between investigators who have research interests in the areas of diabetes and hormone action. The Enrichment Core has utilized six approaches to achieve this goal: 1) guest speakers program, 2) yearly symposia, 3) workshops, 4) individual research data clubs, 5) insulin-IGF research group meetings and 6) signal transduction research group meetings. This format has proven to be highly successful as it involves all members of the DERC at several levels of information interchange."}
{"text": "Rotaviruses have been studied extensively in many parts of the world predominantly by cross-sectional approaches. Such studies have yielded essentially \"numerator\" data which indicated that rotaviruses are a major cause of diarrheal illness in infants and young children. There has been a paucity of longitudinal viral gastroenteritis studies that yield not only important \"denominator\" data but also valuable insights into the natural history of a pathogen or illness, with special emphasis on epidemiologic, immunologic and laboratory parameters. We, therefore, initiated an intensive examination of anal swab and serum specimens obtained during a previous LID long-term longitudinal study (1955-1969) at Junior Village, a welfare institution for homeless, but otherwise normal children. Our aim is to investigate the natural history of rotavirus infections in such a longitudinal setting employing recently developed techniques such as serotyping of rotaviruses with VP7 specific monoclonal antibodies, determining the epitope-specific serologic responses in sequential sera to determine the scope of homotypic and heterotypic responses in order to gain insight into the parameters influencing susceptibility or resistance to rotavirus disease. In addition with the availability of rotavirus strains obtained over 20 years ago, it is planned to compare such strains with current isolates at the genetic level to determine whether significant differences had occurred and the effect of such differences, if found, on resistance to illness."}
{"text": "The rapidly growing database of completely and nearly completely sequenced genomes of bacteria, archaea, eukaryotes and viruses (several thousand genomes already available and many more in progress) creates both extensive new opportunities and major new challenges for genome research. During the last year, we performed a variety of studies that took advantage of the genomic information to establish fundamental principles of genome evolution. We combined mathematical modeling of genome evolution with comparative analysis of prokaryotic genomes to estimate the relative contributions of selection and intrinsic loss bias to the evolution of different functional classes of genes and mobile genetic elements (MGE). An exact solution for the dynamics of gene family size was obtained under a linear duplication-transfer-loss model with selection. With the exception of genes involved in information processing, particularly translation, which are maintained by strong selection, the average selection coefficient for most nonparasitic genes is low albeit positive, compatible with observed positive correlation between genome size and effective population size. Free-living microbes evolve under stronger selection for gene retention than parasites. Different classes of MGE show a broad range of fitness effects, from the nearly neutral transposons to prophages, which are actively eliminated by selection. Genes involved in antiparasite defense, on average, incur a fitness cost to the host that is at least as high as the cost of plasmids. This cost is probably due to the adverse effects of autoimmunity and curtailment of horizontal gene transfer caused by the defense systems and selfish behavior of some of these systems, such as toxin-antitoxin and restriction modification modules. Transposons follow a biphasic dynamics, with bursts of gene proliferation followed by decay in the copy number that is quantitatively captured by the model. The horizontal gene transfer to loss ratio, but not duplication to loss ratio, correlates with genome size, potentially explaining increased abundance of neutral and costly elements in larger genomes. The evolution of bacterial and archaeal genomes is highly dynamic and involves extensive horizontal gene transfer and gene loss. Furthermore, many microbial species appear to have open pangenomes, where each newly sequenced genome contains more than 10% ORFans, that is, genes without detectable homologues in other species5,6. Here, we report a quantitative analysis of microbial genome evolution by fitting the parameters of a simple, steady-state evolutionary model to the comparative genomic data on the gene content and gene order similarity between archaeal genomes. The results reveal two sharply distinct classes of microbial genes, one of which is characterized by effectively instantaneous gene replacement, and the other consists of genes with finite, distributed replacement rates. These findings imply a conservative estimate of the size of the prokaryotic genomic universe, which appears to consist of at least a billion distinct genes. Furthermore, the same distribution of constraints is shown to govern the evolution of gene complement and gene order, without the need to invoke long-range conservation or the selfish operon concept. Much of our work aimed at understanding evolution of viruses and mobile elements. Among other developments in this area, a survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f, cas7f, and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates that evolution of the CRISPR-Cas-containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We showed that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences and, in some cases, chromosomal sequences adjacent to the transposon. We hypothesized that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. These findings suggest the existence of RNA-guided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element. The rapidly growing metagenomic databases have become a rich information source for discovery of new groups of viruses and microbes. We have performed several projects in this direction. One of these involved the discovery of a previously unknown but apparently abundant and ecologically important group of viruses. Marine group II Euryarchaeota (MG-II) are among the most abundant microbes in oceanic surface waters. So far, however, representatives of MG-II have not been cultivated, and no viruses infecting these organisms have been described. Here, we present complete genomes for three distinct groups of viruses assembled from metagenomic sequence datasets highly enriched for MG-II. These novel viruses, which we denote magroviruses, possess double-stranded DNA genomes of 65 to 100 kilobases in size that encode a structural module characteristic of head-tailed viruses and, unusually for archaeal and bacterial viruses, a nearly complete replication apparatus of apparent archaeal origin. The newly identified magroviruses are widespread and abundant and therefore are likely to be major ecological agents. Taken together, these studies advance the existing understanding of the general principles and specific aspects of genome evolution in diverse life forms, in particular viruses and mobile elements, and provide new insights into general principles of genome evolution."}
{"text": "DESCRIPTION (Investigator's Abstract): The principal aim of the proposal in this Phase I application is to develop a device with immobilized chelator of clinical capacity, for removal of excess iron from patients with iron overload. Dr. Ambrus and her colleagues have developed such a device, using polysulfon hollow fiber cartridges with immobilized chelators, for aluminum chelation in dialysis patients. Preliminary studies using this chelating device indicate that iron removal by such methodology is feasible. Other preliminary studies done in dogs using artridges with immobilized desferrioxamine showed that iron could be removed rapidly by such techniques. In Phase I of the proposed study, Dr. Ambrus plans to develop several types of immobilized chelators. Using activated agarose as a macromolecular support, she will immobilize some new oral chelators (EHPG, PIH) and alpha-aminoacyl hydroxamate, a chelator ligand with high iron affinity. She will prepare a series of carboxymethyl-dextrans with increase content of acid gropus for the binding of large amounts of the desferrioxamine for iron chelators. She will then measure iron binding capacity of each of the above chelating agents in batch experiments. Iron depletion by each of the chelators will be measured using saline solution with albumin which contains various concentrations of iron, between 50 and 500 mcg/dL. The strength of iron binding will be estimated by the eluted amount of iron by water soluble chelators, such as EDTA. Chelators that look promising in the batch experiments will be immobilized onto hollow fiber cartridges. Cartridges for immobilization will be those marked for hemodialysis with high ultrafiltration performance. Each cartridge contains about 12,000, asymmetric poly-sulfon hollow fibers consisting of a thin capillary membrane surrounded by a relative thick, macroporous shell which imparts mechanical strength to the membrane and serves as a support to the chelating agent which is deposited in the macropores and at the outside wall of the fibers. The nominal pore size of the membrane is less than 30 kilodaltons, and allows iron and other metals to pass through. Hollow fiber cartridges with the immobilized chelators will then be tested. Using aqueous solutions of iron, the device will be tested for kinetics of iron removal and iron binding capacity. Using blood from hemodialysis patients having iron overload, the devices will be tested for kinetics of iron removal. Identification of the sources of iron removed will be explored by measuring iron in individual serum fractions separated by HPLC in recirculating blood. The above Phase I studies will test the feasibility of this approach to iron chelation and identify the final chelator for the prototype to be developed in Phase II."}
{"text": "Project Summary/Abstract: Early biomarkers of Alzheimer's disease are urgently required for at least three reasons: (i) to determine which individuals are at greatest Alzheimer's disease risk, thereby (ii) offering preventative intervention, pre-disease onset, and (iii) further allowing nascent treatment intervention as early as possible in the disease process. All three goals demand a sensitive, non-invasive, affordable, accessible biomarker of Alzheimer's disease pathology/progression. Addressing these needs, we propose to test the hypothesis that a unique NREM sleep EEG signature provides a candidate early biomarker of A? pathology, one that may accurately track and forecast Alzheimer's disease risk and Alzheimer's disease pathophysiological progression. If correct, the proposal would establish a novel, inexpensive, and early diagnostic tool for determining Alzheimer's disease risk and pathology progression before clinical symptoms emerge, and one that is suitable for community settings. Moreover, such data would motivate an increased medical awareness regarding the importance of treating sleep difficulties across the lifespan, and further motivate the development (clinical or commercial) of sleep-based interventions that improve adult sleep and thus reduce Alzheimer's disease prevalence and its societal burden. More generally, such findings would argue for improved public health policies advocating for sufficient quality sleep throughout adulthood?a memorandum that may lower dementia risk and maintain cognitive health across the populous."}
{"text": "Objective: To determine a consensus on the criteria for pre-treatment workup and treatment delivery in five disease sites for which radiation therapy has a major curative impact. Develop method to present data to the specialty."}
{"text": "Hot flashes, the most common symptom of menopause, cause extreme discomfort and disrupt sleep patterns. Depression and mood changes during menopause also compromise quality of life. Hormone therapy, the primary treatment for menopausal symptoms, is associated with increased risk of breast and endometrial cancer. The majority of menopausal women refuses or fails to comply with hormone therapy, suggesting the need for alternative treatments. Black cohosh, a plant which has proven effective in reducing menopausal hot flashes in several small clinical trials, does not contain estrogenic compounds, but does contain agonists for serotonin receptors. Our hypothesis is that black cohosh alleviates hot flashes and depression in animal models through serotonergic action. Aim 1 will test the hypothesis that black cohosh extracts alleviate hot flashes using an ovariectomized rat model implanted with thermosensors. Aim 2 is designed to deliver solid behavioral data to support the antidepressant activity of black cohosh through the Forced Swim Test animal model of depression. Aim 3 will test the possibility that effects of estrogen therapy on menopausal complaints occur through modulation of serotonin action in a neuronal cell culture model. [unreadable] [unreadable]"}
{"text": "Charges are produced in organic materials homogeneously by a multiphoton ionization mechanism. It is then possible to determine wavelength dependent ionization cross-sections at energies where the sample is opaque, but using light to which the sample is transparent. The transport of charges in organic materials is detected by a new interferometric technique. Finally the act of photoinduced charge-transfer is being probed by fluorescence lifetime measurements on the picosecond time scale."}
{"text": "The contractile properties of smooth muscle are broadly classified as phasic (fast) and tonic (slow). Phasic smooth muscle is characterized by relatively rapid rates of force activation, force relaxation and Vmax, whereas tonic smooth muscle is characterized by slower rates of force activation, force relaxation and Vmax However, the molecular mechanism that regulates the contractile properties of smooth muscle is unknown. Others and we have demonstrated that the mechanism for this diversity in contractile properties between phasic and tonic smooth muscle lies at the level of the contractile filaments, and differences in the kinetics of the actomyosin ATPase (AMATPase). The overall goal of this grant is to investigate the molecular mechanism for the differences in the kinetics of the AMATPase in tonic and phasic smooth muscle. Our hypothesis is that differences in the kinetics of the AMATPase are due to splice variant expression of the myosin heavy chain (MHC) and the 17-kDa myosin light chain (MLC17). The Specific Aims to test this hypothesis are to determine if splice variants of MHC and MLC17 are molecular determinants for the kinetics of the AMATPase of smooth muscle (Specific Aims 1-3). In addition, we will determine if non-muscle myosin is responsible for force maintenance in smooth muscle (Specific Aim 4). To test Specific Aims 1-3, we will force the expression of both splice variant isoforms of MHC and MLC17 in cultured embryonic aortic and gizzard smooth muscle cells. We will over-express both the endogenous and the alternative isoform of MHC and MLC17 in cultured aortic and gizzard smooth muscle cells and tissue strips. After forcing the expression of a single contractile protein, we will determine the effect of elevations of inorganic Pi and MgADP on steady state force and stiffness of single cultured smooth muscle cells, as well as in smooth muscle strips and compare the results to those obtained in the non-transfected controls. These experiments will elucidate the effects of the expression of a single contractile protein, in isolation, on the kinetics of the AMATPase of smooth muscle. In addition, to determine if non-muscle myosin participates in the molecular mechanism for force maintenance (Specific Aim 4), we will determine the properties of force maintenance in a transgenic mouse line, which lacks non-muscle myosin IIB. The results of these studies should elucidate the mechanism that determines the kinetics of the AMATPase of smooth muscle, and form a foundation for future investigation of how smooth muscle contractility is altered by disease states."}
{"text": "The long term objective of this proposal is to produce and accurate physical map and complete molecular clones of human chromosome segment 18q21.3 and to use this to characterize human diseases mapping to 18q21.3. Six probes known to map 18q21.3 provide the initial toehold in this segment. A long-range physical map of this segment using contour- clamped homogeneous electric field electrophoresis (CHEF) and rare cutting restriction endonucleases will order these loci. However, long-range restriction maps have technical limitations and emphasize the need to obtain large contiguous molecular clones. This approximately 2 megabase segment constitutes a reasonably sized test case to obtain overlapping clones from a yeast artificial chromosome (YAC) library of the human genome. YAC isolates obtained with the six 18q21.3 probes will be assessed for their authenticity and further mapped. Distal ends of YAC genomic inserts will be rescued and used to rescreen the library to link clones of 18q21.3. Molecular clones of this region provide the framework to localize new genes reveal associations between geography and the regulation of neural and immune related genes. Follicular lymphomas translocate a new proto-oncogene, Bc1-2 from 18q21.3, into the immunoglobulin locus (14q32) resulting in a marked overproduction of Bc1-2 mRNA. However, CHEF analysis reveals that the Bc1-2 gene possesses and enormous approximately 350 Kb Intron II and that the deregulatory effects of translocation are transmitted across this distance. YAC technology provides the capability to clone this intron as a single fragment. Other genes located within this intron would be identified, cloned, and their effects upon neoplasia or other cell types assessed. The 18q- deletion syndrome is a congenital developmental abnormality manifesting as craniofacial abnormalities, mental retardation, and humoral immunodeficiency. The deleted segment common to these patients is band 18q21.3 18q- syndrome patients with interstitial deletions or constitutional translocations at 18q21.3 will be used to identify distinct breakpoints. This should determine whether a single gene is always altered or whether multigenic loss is responsible. The physical map and YAC clones of 18q21.3 open new avenues for assessing normal and pathologic gene regulation and serve as a model for approaching human genetic disease."}
{"text": "Astrocytes are an important component of the neuropil and their dysfunction has been associated with a variety of idiopathic and genetic diseases including Alzheimer's and Parkinson's disease and in energy deprivation syndromes such as thiamine deficiency and antimetabolite poisoning. The prevailing model of healthy astrocyte-neuron interaction is one of continuous and intimate physical contact between adjacent membranes that promotes neuronal homeostasis. While the loss of astrocytes in the neuropil is generally viewed as a negative event, we propose here that in the acute phases of intoxication loss of astrocytes may protect neurons against further injury mediated by release of adenosine. Specifically, in this proposal we hypothesize that DNB-induced oxidative stress in astrocytes induces the release of adenosine which in turn activates A1 receptors in neurons via paracrine mechanisms and self-regulates A2 receptors on injured astrocytes. Since oxidative stress is the precipitating event, the corollary to this hypothesis is that in neurons, oxidative stress converges on PI3K/ERK to regulate the activity of BCL proteins that promote mitochondrial fusion and stabilization of the cell. Additional neuronal protection is achieved via A1-mediated activation of AKT with blockage of pro-death Bcl proteins and activation of survival Bcl-proteins. Conversely, in astrocytes, activation of the A2 receptor exacerbates loss of calcium control, swelling and cell death. This hypothesis for the role of astrocytes in the protection of neurons from oxidative stress-induced cell death will be tested by addressing the following specific questions. AIM 1: Can adenosine released by astrocytes silence neurons and protect them from the effects of exposure to 1,3-DNB? Aim 2: Does A1 receptor mediated signaling through PI3K, AKT and/or ERK block death- related members of the Bcl-family of proteins in neurons? Aim 3: Is the course of mitochondrial fusion or fission determined by or dependent upon binding of Mfn1/2, Bax/Bad/Bcl-XL and Drp 1? Aim 4: Does binding of proteins that alter mitochondrial morphology also alter membrane potential and function? Dinitrobenzene (DNB) provides and excellent model of energy deprivation syndromes with selective damage to astrocytes. This experimental approach will enable dissection of the role of BCL-proteins, mitofusins and Drp-1 in coordinating the loss of mitochondrial function and may provide new insights into neuronal/glial interactions that form the foundation for pathoclisis, or selective cellular susceptibility to environmental neurotoxicants. PUBLIC HEALTH RELEVANCE Astrocytes are supporting cells in the central nervous system. Data from our laboratories show that they are a primary target of many environmental chemicals that result in dysfunction of the central nervous system. As injury to the astrocyte progresses, ATP is converted to adenosine and is released into the extracellular space where it can interact with A1 receptors on neurons (protective) and A2 receptors on astrocytes (injurious). We propose here that in the acute phases of CNS injury, loss of neuronal function (silencing) is elicited by adenosine and may spare the neuron from the deleterious effects of oxidative stress and excitotoxicity."}
{"text": "VECTOR In order to understand the manner by which specific mutations lead to malignancy, the Vector Core provides Cancer Center members with several technological platforms that mediate overexpression or reduced expression of specific genes and the corresponding mutants for in vitro and in vivo studies. These platforms include both recombinant non-viral and viral products that facilitate the transfer of specific genes into either normal or cancer cells. The Vector Core also provides intellectual and technical advice to Cancer Center researchers regarding the optimal use and production of these systems. Despite the wide application of molecular biology to the study of cancer, many investigators do not possess a working knowledge of recombinant vectors. Many of the operational techniques required for optimal use of these gene transfer vectors are specialized and difficult to acquire without expert assistance. In addition, the manufacture of these reagents needs to be performed in laboratory space that has been specifically configured to comply with NIH biological containment guidelines. The requirements for compliance with these biosafety guidelines inhibit many investigators from pursuing the use of these valuable reagents. The Vector Core has been established to provide a cost-effective source of these valuable platforms while minimizing Cancer Center members'need to spend time and money on new laboratory space and hiring their own vector experts. The Specific Aims of the Vector Core are to: 1. Provide a Core laboratory for the development, construction, purification and characterization of recombinant vectors containing genes relevant to the study of cancer disease models for use as in vitro and in vivo gene transfer reagents. These systems include both non-viral (expression plasmid) and viral (recombinant retrovirus, recombinant adenovirus, and recombinant AAV) technologies. 2. Collaborate closely with Cancer Center researchers to ensure the Vector Core provides the platforms Cancer Center members require 3. Maintain qualified staff and monitor the competition to continually improve the Vector Core and provide high quality, cost effective products to Cancer Center members."}
{"text": "Ubiquitin-like modification is a conserved biochemical mechanism that is used by cells for various purposes. These include targeted degradation of proteins, endocytosis of receptors, regulation of protein localization, and signal transduction. Apart from a role of ubiquitin itself to modulate insulin receptors and IRS proteins, ubiquitin-like modifications have not been described to play a prominent role in insulin signaling. TUG was identified as a target of insulin signaling, and is implicated regulating GLUT4 glucose transporter trafficking and glucose uptake in adipocytes. According to the proposed model, TUG acts by \"tethering\" GLUT4 transporters intracellularly in the absence of insulin, excluding them from the plasma membrane and limiting glucose uptake. Insulin \"untethers\" the transporters to redistribute GLUT4 and to enhance glucose transport into cells. The mechanism by which insulin acts on TUG and GLUT4 is not well understood. The present proposal will test the hypothesis that insulin stimulates rapid and site-specific cleavage of TUG to liberate an amino terminal fragment, TUGUL, that is a new ubiquitin-like modifier. Using cultured 3T3-L1 adipocytes, several biochemical and cell biological approaches will be used in Aim 1 to test whether insulin stimulates TUG cleavage, and whether this is required for insulin to mobilize GLUT4. Aim 2 will test the hypothesis that TUGUL functions as a ubiquitin-like modifier, and will study the role of the putative TUG C terminal cleavage product. Aim 3 will study how the insulin signal may act on TUG to cause its processing. It is anticipated that, together, accomplishment of these aims will begin to define a novel pathway for insulin regulated ubiquitin-like modification. Furthermore, it is anticipated that the results will have importance for understanding how insulin stimulates glucose uptake. Type 2 diabetes is a major public health problem that results in part from a defect in the ability of insulin to stimulate glucose uptake. It is anticipated that the proposed studies of insulin action and glucose uptake will lead to a greater understanding of mechanisms that may contribute to the development of type 2 diabetes. [unreadable] [unreadable] [unreadable]"}
{"text": "Thalamocortical afferents are the sole source of sensory input to the mammalian neocortex. Their role during cortical development is equally important. Recent work has established that thalamocortical afferents are required during development for the differentiation of the architectural, connectional and functional features that distinguish and define the functionally specialized \"areas\" in the adult neocortex. The four specific aims of this proposal are designed to test hypotheses on mechanisms postulated to control the development of area-specific thalamocortical connections. This work will be done principally in rats, an excellent model for studies of cortical development and plasticity. It will combine a variety of anatomical and cellular methods, using in vivo and in vitro experimental approaches, as well as time-lapse imaging of cultured tissue. The major experimental goals are: (1) to assess the specificity in the targeting of thalamocortical axons and their invasion of the cortical plate. (2) to investigate a role for putative position-dependent molecular cues hypothesized to control specificity in thalamocortical axon targeting. (3) to determine whether maturation-dependent changes in membrane- associate molecules on the surfaces of cortical plate neurons and/or their secretion of a diffusible chemoattractant promotes the invasion of the cortical plate by waiting thalamocortical axons. (4) to analyze the degree of dispersion of neuroepithelial cells and their progeny during the development of the neocortical preplate as a means to evaluate hypothesized mechanisms of establishing position-dependent information in the subplate. The long term objectives of this work are to elucidate critical early events in the development of the mammalian cerebral cortex, with a focus on the neocortex. It is expected that the information gathered will provide insights not only into normal developmental mechanisms employed in the cortex, but that it will contribute to our understanding of the basis of the many congenital cortical abnormalities that afflict humans, as well as later striking neurodegenerative diseases."}
{"text": "During fiscal year 2010 we accomplished the following: 1) We used Bim-deficient B cells to further distinguish transcriptional and post-transcriptional regulation of p100 protein production in response to acute and tonic BCR stimulation. These studies substantiated the idea of PI3K-dependent translational control of p100 production. 2) We used NF&#954;B2-deficient mice to establish unequivocally that p100 is essential for BAFF-dependent survival of B cells in vitro. It is interesting to note that p100-deficient mice have reasonable numbers of mature B cells, whereas BAFF- or BAFF-R- deficient mice do not. Our results suggest that BAFF-dependent survival in vivo can be mediated by pathways other than p100. Since BCR is essential for survival of mature B cells in vivo, these other pathways must also be initiated at the BCR. We initiated studies of Mcl-1 regulation by BCR to determine if this anti-apoptotic protein is responsible in part for p100 independent B cell survival in vivo. 3) Downstream of PI3K activation we found that suppression of mTOR by rapamycin did not abolish p100 up-regulation in response to tonic or acute BCR signaling. However, rapamycin treatment of Pim2-deficient B cells abolished BCR-induced p100 up-regulation. We infer that Pim 2 and mTOR provide redundant pathways to p100 induction."}
{"text": "CORE D ? ENVIRONMENTAL ASSESSMENT CORE PROJECT SUMMARY The goals of the Dog Aging Project are to create a cohort of 10,000 companion dogs through which it will become possible to identify the genetic and environmental determinants of healthy aging, discover the underlying mechanisms through which genes and environment influence aging, and test a highly promising drug for its ability to increase lifespan and healthspan. Given that the majority of variation in aging phenotypes within mammalian species is likely to be explained by differences in environmental exposures ? either alone or via interactions with inherited genetic factors ? measurement of environmental variables for each dog in the cohort is essential. In order to meet the needs of the scientific projects for coordinated expertise in determining which aspects of the environment should and can be characterized, and through which approaches, an Environmental Assessment Core (EAC) will be established. The overall goal of the EAC is to support the scientific aims of the research projects by developing and implementing strategies for acquiring data that characterize various aspects of the companion dog environment. Specific aims of this Core are to: 1) Design and implement annual dog owner-administered questionnaires that elicit each dog's travel history, non- prescription medications, diet and feeding, household environment, physical activity, reproductive activity, and behavior, as well as key behavioral and health conditions for owners; 2) Collect, process, validate, and prepare primary and secondary data related to conditions of the dog's environment; and, 3) Collaborate with Project Investigators to develop analytic strategies to assess the role of environmental conditions, and their interactions with genetic factors, in processes of aging."}
{"text": "An estimated 1.5 percent to 7.5 percent of children suffer from encopresis (fecal soiling). One of the most effective ways of treating encopresis is with Enhanced Toilet Training (ETT). ETT is twice as effective as intensive medical management alone at 3, 6, and 12-month follow-up when delivered by skilled and knowledgeable clinicians. We have successfully transformed ETT into an interactive internet intervention, a web program accessible by anyone with a computer and internet access. This program has been shown to have significant additive value to standard clinical care, and is based on a theoretical model we developed for therapeutic behavior change achieved through internet interventions. To date, this Internet intervention for pediatric encopresis has only been offered to individuals who are currently in treatment with a health care provider. In order to significantly increase its potential for wide-spread dissemination, a new clinical trial is proposed in which families can self-refer to the intervention. In addition, as adherence is often a significant issue with internet interventions, an enhanced condition involving a stepped care approach with graduated levels of support (automated email, personalized email, phone support) for different levels of non-adherence will be evaluated as part of a large, national, randomized clinical trial. We propose a four year project in which we will experimentally test the short and long term benefits of the intervention as compared to use of the system with a stepped care approach to enhance adherence and ultimately symptom improvement. A static, educational website will be used with the control group. Cost-benefit analyses will be evaluated and documented. We hypothesize that the pediatric encopresis internet intervention will be more effective than a static website at 6 weeks and 12 months post treatment in terms of encopretic symptom reduction (including fewer accidents, increased BMs in toilet, and increased trips to the bathroom), behavior change (measured by acceptable cleanout, laxatives, straining, toileting, and routine), and reduced costs (doctor visits, medication usage, missed school/work days, diapers used). We also predict that the stepped care version of the program, as compared to the standard program, will produce greater website utilization and thus improved behavior change and symptom reduction. While overall cost will likely be higher with stepped care, we expect cost-benefit analyses to show that this increased cost is acceptable for a certain segment of the population studied. Additional testing of our model for internet interventions will also be conducted. Relevance: Internet interventions can provide sophisticated and personalized treatment at low cost. This application plans to take a program for children with encopresis and evaluate how effective it is when offered \"direct-to-consumers.\" It also attempts to increase success by integrating methods to improve adherence to the program. Findings from this study will have far reaching implications for the management of encopresis in particular and internet interventions in general. [unreadable] [unreadable] [unreadable]"}
{"text": "Malaria and diarrheal diseases are major barriers to improving maternal and child health in sub-Saharan Africa (SSA). Each year about 900,000 people in SSA die from malaria. Pregnant women and infants are particularly vulnerable. An estimated 10,000 women and 200,000 newborns, mostly in Africa, die each year as a result of malaria infection during pregnancy and severe malanal anemia (WHO, 2007a). Worldwide, diarrheal diseases kill almost two million children in poor countries each year (WHO, 2002, Kosek, et al., 2003), and 640,000 children under the age of five in SSA alone (Rao, et al., 2006). Overall, malaria and diarrhea account for 18 percent and 16 percent of under-five mortality, respectively (UNICEF, 2007). Many of these deaths could have been averted using available preventative measures. For instance, using an insecticide-treated net (ITN) during pregnancy reduces the incidence of severe maternal anemia by up to 47 percent (Marchant, et al., 2002; Ter Kuile, et al., 2003). Overall, ITNs can reduce the incidence of malaria by 50 percent and mortality by 20 percent at an estimated cost of $29 per disability-adjusted life year (DALY) averted. Point-of-use chlorination of drinking water reduces the incidence of endemic diarrhea by 37 percent at an estimated cost of $53 per DALY averted (Morel, et al., 2005; Clasen, 2007). However, ITN ownership and point-of-use water chlorination coverage are both under 10 percent in SSA (Miller, et al., 2007; Stockman et al., 2007). These low coverage rates suggest that these preventative products are not reaching the most vulnerable populations, raising two important questions: First, how can demand for these products be increased? Second, how can distribution systems be made more effective?"}
{"text": "DESCRIPTION: The cri-du-chat syndrome is a well-described partial aneusomy resulting from the deletion of the short arm of chromosome 5. Patients with this syndrome present with microcephaly, a round face, hypertelorism, micrognathia, prominent nasal bridge, epicanthal folds, hypotonia, and severe psychomotor and mental retardation. Through the characterization of over 50 patients with 5p deletions, a chromosomal region that is 2 Mbp in 5p15.2 and the chromosomal segment involved in the cat-like cry has been mapped to a 750 region of 5p15.3 YAC contigs of both regions have been completed and nonchimeric YACS that span each region have been identified. While the long term goal of this proposal is to identify genes that, when present in one copy, cause the clinical features associated with the cri-du-chat syndrome, the initial goal will be to develop a transcriptional map of the region in order to identify candidate genes based on their location with respect to the cri-du-chat critical regions. The strategy to identify these genes is first to continue with the characterization of patients with small 5p deletions or partial cri-du-chat phenotypes such that the two critical regions can be further narrowed. Second, the construction of a cosmid contig which has been initiated will be completed. Third, several approaches to identify the genes mapping within the critical regions will be performed and include EST mapping, cDNA selection, exon-trapping and cloning HTF islands. After performing screening strategies to identify clones that are derived from the same gene, the pattern of expression of unique genes will be investigated. This investigation will include investigating the level of expression in human adult and fetal tissues as well as the level of expression in different murine gestational stages. The initial characterization of these genes will identify candidate genes that may be involved in the clinical etiology of the cri-du-chat syndrome based on whether a gene is expressed during fetal development."}
{"text": "Development and application of centrifugal precipitation chromatography has been continued. The system internally generates a concentration gradient of ammonium sulfate (AS) along a long channel to fractionate proteins according to their solubility in AS solution. The separation column consists of a pair of disks with mutually mirror-imaged spiral channels that are separated by a semipermeable membrane. The disk assembly is mounted on a sealless continuous flow centrifuge. Concentrated As solution is introduced into the upper channel while a water solution is passed through the lower channel in the opposite direction in a rotating column. A mixture of proteins injected into the water channel moves along an AS gradient of increasing concentration that has been established in the water solution. Each protein species precipitates at a different AS concentration along the gradient. The eluate is continuously monitored and collected using a fraction collector. A series of basic experiments was performed to study the rates of AS transfer and osmosis through the membrane, and the operational parameters including elution time,revolution speed, inclination of the gradient and sample size were optimized using stable protein samples. A new set of columns with different channel configuration was made to improve the efficiency of separation.Preliminary applications were successful in purification of monoclonal antibodies from cell culture supernatant and ascitic fluid, and affinity separation of recombinant ketosteroid isomerase from a crude E. coli lysate. The method was also successfully applied to fractionation of other biopolymers such as hyaluronic acid, chondroitin sulfate, and polymerized catechins using a gradient between suitable organic solvents and water."}
{"text": "This application to join the NICHD Maternal Fetal Medicine Units Network is a combined proposal from the Oregon Health & Science University (OHSU) and Kaiser Permanente Oregon/Washington (KPNW) region. It is the result of a long-standing collaboration between OHSU and KPNW in maternal fetal medicine and brings together university and community research capabilities, which will increase diversity in enrollment. The combined deliveries (4091 in 2003 and 4043 in 2004) concentrate high risk patients in principally two sites: the Perinatal Center at Doernbecher Children's hospital at OHSU and the KPNW Perinatal Center at St. Vincent's Hospital. Both centers serve to selectively funnel consultations for genetics, preconception counseling, high resolution ultrasonography, high risk obstetrics and fetal echocardiography and are staffed by Maternal Fetal Medicine Specialists. Both OHSU and KPNW have a long history of conducting respected clinical research independently and together. This collaboration is based upon shared laboratory services and maternal fetal medicine coverage, an integrated residency in Obstetrics & Gynecology, and the combined strengths in research of the KPNW Center for Health Research and the Oregon Evidence-based Practice Center. The Center for Health Research (KPNW) and the Women's Health Research Unit (OHSU) have extensive experience in clinical trials and the research staff to facilitate the enrollment of patients in clinical studies. Focusing patient recruitment in two sites staffed by maternal fetal medicine specialists committed to this proposal will enhance enrollment and participation by research staff as well as maximize the quality of data obtained. [unreadable] [unreadable] [unreadable] [unreadable]"}
{"text": "In the United States over 6% of individuals greater than 12 years old have used marijuana in the past month and this substance accounts for approximately 55% of the illicit drug use in this country. The perception that cannabis is a \"safe\" illicit drug, without long-term adverse consequences, may contribute to the higher rates of use of marijuana when compared with more \"hard\" drugs. However, there has been relatively few brain imaging studies exploring the neurobiological effects of cannabis. Two main areas of recent interest with regards to cannabis use, both potentially related to the dopamine (DA) system, highlight the importance of further in vivo human brain imaging studies examining DA function in cannabis users. First, a relatively strong association (odds ratio > 2) between premorbid cannabis use and schizophrenia has been observed, particularly when cannabis use occurs in adolescence. In schizophrenia, abnormalities of the DA system have been observed in receptor imaging studies. Given the epidemiological link between cannabis use in adolescence and schizophrenia a neuroreceptor imaging study of this nature in individuals who use/abuse cannabis is of particular interest. Second, early use of cannabis has been considered a risk factor for the future use of hard drugs. This has been termed the \"gateway\" hypothesis and neurobiological factors such as sensitization may play a role. Sensitization refers to the enhanced DA response in the reward pathway of the nucleus accumbens (NAc) that occurs following repeated and chronic exposure to drugs of abuse. If exposure to cannabis can induce a cross- sensitization to other drugs of abuse (i.e. prior exposure to cannabis results in an increased DA release in the NAc on subsequent exposure to another class of drugs) this would be supportive of the gateway effect. This application seeks to examine the effects of repeated cannabis exposure, beginning in adolescent, on DA transmission in vivo within reward system of the ventral striatum (VST), an area which includes the NAc,. This will be accomplished with positron emission tomography (PET) by measuring amphetamine-induced displacement of [11C]raclopride in 8 adult individuals with a history of repeated cannabis use beginning in adolescence and 8 matched controls. If the hypothesis we seek to test is confirmed - that marijuana use beginning in adolescence alters dopamine signaling in the striatum later in life - it would provide a strong neurobiological basis for the epidemiological link between this drug and schizophrenia, have implications for the theory that marijuana serves as a gateway drug, and strengthen the argument that marijuana use cannot be considered a \"safe\" drug of abuse. In the United States over 6% of individuals greater than 12 years old have used marijuana in the past month and this substance accounts for approximately 55% of the illicit drug use in this country. However, there has been relatively few brain imaging studies exploring the neurobiological effects of cannabis. If the hypothesis we seek to test is confirmed - that marijuana use beginning in adolescence alters dopamine reward signaling in the brain later in life - it would provide a strong neurobiological basis for the epidemiological link between this drug and schizophrenia, have implications for the theory that marijuana serves as a gateway drug, and strengthen the argument that marijuana use cannot be considered a \"safe\" drug of abuse. [unreadable] [unreadable] [unreadable]"}
{"text": "The long-term objective of this application is to elucidate the molecular mechanism(s) underlying specification of intestinal cell identity. We have identified a previously unrecognized patterning step that takes place at embryonic day 16.5 in the mouse. This step involves the upregulation of >1,000 genes in the intestinal epithelium, a process that creates the distinct epithelial border that defines intestinal vs. stomach identity. We call this process intestinalization. Our evidence also implicates the transcription factor Tcfec as a potential regulator of intestinalization. Thus, the Specific Aims of this application are to: 1) determine if Tcfec is an activator in intestinal epithelium, 2) characterize the downstream pathways affected by Tcfec during intestinal epithelial cell specification, and 3) analyze the contribution of a conserved non-coding element (CNE) near the Tcfec gene to the temporal regulation of its expression in intestinal epithelium. For Aim 1, we will clone intestinal forms of Tcfec by RLM-RACE and determine their transcriptional activity using cell-based, luciferase reporter assays. For Aim 2, Tcfec expression will be manipulated in Caco-2 cells, an in vitro system that recapitulates intestinal epithelial cell differentiation. We will also establish several mouse models to test the requirement for Tcfec in the intestine, as well as the effect of Tcfec misexpression in the stomach. For Aim 3, we will generate transgenic reporter mice to ascertain the pattern of expression directed by the Tcfec CNE. The requirement of specific transcription factor binding sites will then be tested by mutagenesis. In addition to their contribution to the understanding of intestinalization per se, these studies may well be relevant to the pathologic state known as intestinal metaplasia of the stomach, in which cells within the stomach take on intestinal character. Since intestinal metaplasia is thought to represent an early step in the development of some forms of gastric cancer, our findings could eventually impact diagnosis or early treatment of this often fatal cancer."}
{"text": "A.Protein kinase C project: The generation of novel chemical libraries of DAG-lactones using our new solid-phase method of synthesis continues to yield important lead compounds in various areas of potential therapeutic importance: 1) The results on our first C1 domain-selective DAG-lactone with exclusive activity for RasGRP was published recently. This compound has allowed us to dissect an ERK phosphorylation pathway via RasGRP, which is totally independent of PKC alpha. This DAG-lactone, identified as 130C037, is capable of exclusively translocating RasGRP to intracellular compartments while other PKC isozymes remain in the cytoplasm; 2) Optimization and structure-activity studies of DAG-lactones capable of stimulating alpha-secretase activity continues; 3) Research using the Balb/C mouse JB6 model has helped identify some DAG-lactones that retain AP-1 activation but are not tumor promoting agents; 4) A different set of DAG-lactones is capable of stimulating interferon production in combination with IL-12 to levels superior to those achieved with the paradigm PKC activator, phorbol myristate acetate (PMA) without inducing tumor promotion. Large-scale syntheses of some of these compounds are in progress to perform animal studies. B.A new azido-functionalized flavone acetic acid analogue was conjugated to a FLAG peptide phosphine derivative via the Staudinger reaction. When the conjugate was mixed with cell extracts from mouse macrophage cells, the FAA-FLAG conjugated proteins could be immunoprecipiated using an anti-FLAG antibody. This work was recently selected to be highlighted in the CCR bulletin. C.Zebularine [2(1H)-pyrimidinone riboside] has been the subject of numerous studies in the last 3 years (31 papers in major journals). Unfortunately its path towards the clinic was brought to an end by an unforeseen toxicity in rhesus monkeys, which was lethal when plasma levels reached 25 micromolar. This is in total contrast to the complete lack of toxicity in rodents (rats and mice), even at high doses. Efforts to circumvent toxicity and improve the activity of zebularine at lower doses in monkeys have been centered on preventing the high levels of a metabolizing enzyme, aldehyde oxidase, which neutralizes the action of zebularine. Despite the discovery of raloxifene, a clinically approved agent to inhibit this enzyme, our efforts have been directed to the development of pro-drugs of 2'-deoxyzebularine which were initiated last year with the intent to improve the incorporation of 2'-deoxyzebularine-5'-triphosphate into DNA. After studying an extensive series of pro-drugs, which resulted in inactive compounds, we have now identified two analogues that are 10-fold more potent than zebularine in the activation of silenced tumor suppressor genes, particularly in CF-Pac-1 pancreatic tumor cells. Securing intellectual protection of these findings is in progress. The selective incorporation of zebularine-5'-triphosphate opposite to G in was demonstrated using DNA pyrosequencing; whe zebularine is part of the template base paring to G is less selective.D.A stable NAD analogue, beta-methylene-TAD, synthesized at the LMC in years past was used for the high-resolution crystal structure of a mono-ADP -ribosylating toxin (similar to diphteria toxin). The structure has implications for the mechanism of translocation on the 80S ribosome."}
{"text": "Multiple inputs and controls participate in the maintenance of energy homeostasis, and numerous neural and endocrine systems are involved in the integrated responses that maintain energy stores in the body at a level. Since most stored energy is in the form of adipose tissue (fat), and since the brain is the major controller of energy homeostasis, a fundamental requirement is that the brain receive accurate and timely information about the amount and distribution of body fat. This information is then integrated with other factors to determine whether an individual seeks and eats food (i.e., energy intake), as well as the efficiency with which the ingested food is utilized (energy expenditure) or stored. Information regarding the amount of fat stored in the body reaches the brain via the circulating signals insulin and leptin. Each is secreted into the blood in direct proportion to total body fat, and each is transported from the blood into the brain where it interacts with neurons situated to influence energy intake and expenditure. Hence, insulin and leptin are considered to be adiposity signals, and the long-term goal of this project is to elucidate how adiposity signals interact with other factors to influence energy homeostasis. We anticipate that the coming decade will see considerable progress in this arena, as well as the translation of the new-found information into potential novel therapeutic strategies to tackle clinical problems of dysregulation of energy homeostasis (i.e., obesity, eating disorders). With this as an ultimate end point, the present proposal has three specific aims that address critical and as yet unanswered questions regarding the actions of adiposity signals in the brain. The first assesses the hypothesis that insulin and leptin act in the hypothalamus by enhancing the signal provided by local levels of nutrients, including glucose and fatty acids. The second assesses several hypotheses following from our observation that there are important gender differences in the actions of insulin and leptin in the central control of energy homeostasis, and that males and females utilize different strategies to regulate energy homeostasis and defend their body fat stores. The final aim assesses hypotheses regarding the effect of reducing insulin signaling in specific brain regions on food intake and body weight, and upon related levels of metabolic hormones and neuropeptides, utilizing lentiviral technology. PUBLIC HEALTH RELEVANCE: Hormones such as insulin and leptin that are secreted in direct proportion to body fat interact in the brain with nutrients such as glucose, fatty acids and amino acids to determine food intake and energy expenditure. Proposed research will determine how these signals interact within neurons in the hypothalamus and intervene by manipulating the ability of the brain of laboratory rats to detect and respond to insulin or leptin. Experiments will assess behavior as well as molecular signals within cells. [unreadable] [unreadable] [unreadable]"}
{"text": "The purpose of this study is to demonstate the vagal nerve stimulation via a surgically implanted investigational device in the left vagas nerve will reduce the frequency of seizures in epileptic patients refactory to medical therapy."}
{"text": "The amiloride-sensitive epithelial sodium channel (ENaC) mediates the final event in the reabsorption of sodium by the kidney. The activity of these channels determines the amount of sodium retained by the kidneys hence, ENAaC has a fundamental role in the maintenance of extra- volume and blood pressure. Many stimuli modulate the activity and expression of ENaC in the kidney but among them, aldosterone is the single most important one. There are two aims in this research proposal. The first one is to study one of the mechanisms that mediate the aldosterone-induced activation of ENaC in the distal nephron by a novel serine/threonine kinase known as sgk. Specifically, we proposal to investigate whether sgk is i) necessary and sufficient to mediate the aldosterone response in epithelial cells, ii) to elucidate the mechanism(s) triggered by sgk that results in activation of ENaC, iii) to find proteins that associated with sgk that may constitute substrates of the kinase or regulatory proteins, and iv) to generate animal models (transgenic mice) that exhibit decreased or increased activity of sgk selectively in the principal cells of the distal nephron. The design of the research allows us to obtain and correlate data from cells in culture, in kidney, and in whole animals. In the second aim of the proposal we will investigate the molecular mechanisms of one human mutation in the gene of the gamma subunit of ENaC that produces pseudohypoaldosteronism type 1. These studies will contribute to further elucidate the structure-function of ENaC channels."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Staphylococcus aureus (Staph) is a germ that can cause skin infections and other serious and deadly infections. Community-Associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are types of Staph infections that are resistant to many antibiotics. CA-MRSA infections are suddenly very common, whereas 10 years ago they were unheard of. CA-MRSA are resistant to many antibiotics so doctors are not sure which antibiotics work best against skin infections, which are now caused mostly by CA-MRSA. To understand how to best treat skin infections, specifically ones called ?uncomplicated skin and soft tissue infections? (uSSTIs), we need to do a clinical trial to compare antibiotics that experts think are best. However, there is no proof about how good (efficacious) or safe these antibiotics are. We will perform a clinical trial of treatment of uSSTIs --specifically treatment of abscesses (boils) and/or cellulitis (bland skin infection)--in both children and adults in areas where CA-MRSA is common. The primary objective of this study is to compare the cure rates of two antibiotics, clindamycin and trimethoprim-sulfamethoxazole (TMP-SMX) for the treatment of uSSTIs. Treatment will be done in conjunction with the standard of care for treatment of abscesses, which is incision and drainage (i.e., lancing a boil). There is preliminary information that incision and drainage alone without antibiotics may be sufficient for patients with small abscesses. Therefore, subjects with small abscesses will be randomized to be treated with either clindamycin, TMP-SMX, or placebo."}
{"text": "The postnatal development of both the optical and neural visual systems is dependent on visual experience. Visual experience is defined by the information available in the retinal images in the two eyes. The goal of the proposed research is to extend our previous examination of retinal image quality in one eye during infancy to full binocular viewing conditions. We will examine human infants' visual experience in the context of image clarity and image alignment, which are primarily defined by accommodation and vergence responses and their interaction. These studies will document the emergence of the interaction between accommodation and vergence and their role in the development of refractive and accommodative strabismus. There are three specific aims: i) To understand the normal maturation of the relationship between accommodation and vergence with emmetropisation and growth of the distance between the eyes. ii) To determine the relative bias towards accommodation or vergence accuracy during the critical period of human development. iii) To understand the effects of accommodation and vergence behavior on visual experience of infants and children with high hyperopia or strabismus. PUBLIC HEALTH RELEVANCE: This project will determine how young infants and children manage apparently conflicting focusing and eye alignment demands during typical development. It will also investigate why some children develop refractive or accommodative strabismus while others, with apparently matching visual systems, do not. The goal is to develop intervention strategies to prevent this strabismus and associated amblyopia."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Total chemical synthesis was used to prepare a series of unique chemical analogues of HIV-1 protease, where we systematically substituted the residues Gly51, Gly51'at the tips of the mobile 'flaps'(residues 37-61 in each domain of the protein homodimer) with L-Ala, D-Ala or Aib (alpha-aminoisobutyric acid) in both symmetric and asymmetric fashion. Such substitutions, although in regions distant from the catalytic aspartates, led in most cases to reduction of catalytic activity. In contrast to this, a 'covalent dimer'with L-Ala51 in one flap and D-Ala51'in another flap has shown native-like enzyme activity. We used continuous-wave and pulse EPR to characterize dynamic properties of flaps in spin-labeled analogues and found that the thermodynamic balance of the closed, semi-open and open ensembles (with respect to flaps) in HIV-1 protease analogues is perturbed in comparison to that of wild-type enzyme.We interpreted our results on the basis of Northrop's 'kinetic isomechanism'for aspartic proteases which employs a low-barrier hydrogen bond (LBHB) as the central part of the concept. We have developed Northrop's concept further to take into account dynamic properties of the protein and the non-equivalence of the flaps in the ligand-bound enzyme. We proposed a mechanical coupling of motions of the flaps and the catalytic residues Asp25 and Asp25'. On a mechanistic level, opening of the flaps corresponds to shortening of the distance between catalytic residues Asp25 and Asp25'thus promoting formation of the LBHB, and vice versa.Our goal is to employ NMR methods to detect and unambiguously prove presence of LBHB. In addition we would like to determine ionization states of catalytic residues for chemical analogues which demonstrate distinctly different protein dynamics and thus attempt to correlate dynamic properties of the enzyme with its chemical mechanism."}
{"text": "To examine if oxandrolone can promote weight gain in HIV-infected men. To ex-amine the effects of oxandrolone on body composition by DEXA, muscle performance by IRM and other functional tests, oxandrolone pharmacokinetics. To examine if anabolic effects of oxandrolone occur at doses that do not result in suppression of LH and FSH; this will help determine whether anabolic and androgenic activi-ties of oxandrolone can be dissociated, as has been claimed in the literature. Thus although this is a multi-center study, several important aspects of the study will only be addressed in studies being done at this center."}
{"text": "The Short-term Mentored Career Enhancement Award in Basic Behavioral and Social Sciences: Cross-Training at the Intersection of Animal Models and Human Investigation (K18) is a unique opportunity to fund significant and innovative research while simultaneously building the skill set, and thereby future scientific discovery potential, of a scientist. The current application proposes to secure training in human investigation for Gretchen Neigh, Ph.D., a behavioral neuroendocrinologist currently adept at the use of animal models to address questions of the neurobiological basis of behavior. Dr. Neigh's research portfolio focuses on potential metabolic underpinnings of behavioral disorders such as Major Depressive Disorder (MDD) and Post-Traumatic Stress Disorder (PTSD). The work proposed serves the dual purpose of extending Dr. Neigh's innovative hypotheses regarding glucose transport and origin of neuropsychiatric disorders from her current work in rodent models to assessments using human investigation. This training experience will provide a foundation for translational reciprocity in Dr. Neigh's research program and address a question of fundamental importance: assessment of the potential role of metabolism in manifestation of MDD and/or PTSD. Over 23 million people in the United States suffer from MDD or PTSD and these disorders compromise quality of life and generate significant economic burden. Both human and animal studies have provided evidence of changes in cerebral metabolism in these conditions and related animal models. The alterations in metabolic activity are generally attributed to reduced glutamate release from neurons which thereby decreases regional glucose transport. Counter to this traditional dogma, it is possible that a primary change in facilitated glucose transport subsequently suppresses neuronal activity. Facilitated glucose transport is mediated by a family of transporters (GLUT) that are responsible for glucose transport across the endothelial cells of the blood brain barrier, and for uptake of glucose into astrocytes and neurons. Deficits in the expression and translocation of members of the GLUT family have been linked to neuropathological conditions including Alzheimer pathology, post-ischemic brain function, and post- traumatic brain injury deficits. Alterations in the expression o translocation of members of the GLUT family in either the endothelial cells of the blood brain barrier, astrocytes, or neurons, could alter neuronal energy supply and thereby neuronal function, subsequently altering behavior. A polymorphism in GLUT1 has been demonstrated in humans and linked to altered progression and prognosis in cancer and diabetic nephropathy. Alterations in the availability of GLUT1, due to genetic differences in expression, may alter the relative risk 0f development of aberrant behaviors following trauma, leading to the manifestation of MDD and/or PTSD. Given the crucial role of GLUT proteins in the transport of energy substrates into cerebral tissue, evidence of altered cerebral metabolism in neuropsychiatric disorders, and evidence that glucose transporters are altered after stress exposure in animal models, the proposed work tests the hypothesis that a polymorphism in GLUT 1 will decrease the incidence of MDD and/or PTSD following trauma exposure. This work challenges the standard paradigm because cerebral GLUT is a novel point of origin for consideration in the pathophysiology of MDD and PTSD as it is generally accepted that the changes in glucose transport that are documented in imaging of patients are the result of altered neuronal activity as opposed to the cause of altered neuronal activity. Specific Aim 1 will determine the extent to which a polymorphism in GLUT 1 decreases the manifestation of MDD following trauma exposure. DNA samples will be assessed for the rs710218 polymorphism in GLUT 1. Multivariate analyses will be conducted to determine if individuals that have been exposed to trauma and have the polymorphism are resistant to the manifestation of MDD. Specific Aim 2 will ascertain the degree to which a polymorphism in GLUT 1 decreases the incidence of PTSD following trauma exposure. Similar to work described in Aim 1, Aim 2 will again assess the influence of the GLUT 1 polymorphism on outcome from trauma, but in this case the focus will be on PTSD. Completion of the proposed work will provide novel and innovative insight into a potential metabolic susceptibility of genetic origin to trauma-induced mental health impairments. Appreciation for the role of metabolic factors in the manifestation of behavioral disorders will provide a new direction of consideration for novel therapeutic options. In addition, the proposed work will facilitate the cross- training of an established behavioral neuroscientist, Dr. Gretchen Neigh, currently specialized in investigation of the neural substrates of behavior using animal models, in human investigation. Enhancement of Dr. Neigh's training will lead to increased translational reciprocity in her research program and through her interactions with trainees and collaborators will improve interactions between clinical and basic researchers in the behavioral sciences."}
{"text": "Gram-positive bacterial pathogens inflict an enormous burden of human disease world-wide. This closely related group of microbes includes Bacillus anthracis, the most notorius bioterror agent, as well as Staphylococcus aureus, which, judged by human morbidity, is currently the single most important infectious agent in the United States. Broad dissemination of antibiotic (methicillin) resistant S. aureus (MRSA) strains in American communities implies the return of the pre-antibiotic era unless new therapies can reduce human mortality. B. anthracis has been weaponized and engineered to acquire antibiotic resistance traits that render currently available antibiotics ineffective and human populations defenseless, if they had been exposed to drug-resistant anthrax spores. The GLRCE Research Project 3 proposal addresses the need for new antibiotics by unraveling molecular mechanisms that lead to assembly of siderophores, proteins, capsules or teichoic acids in the cell wall envelope of B. anthracis and S. aureus. Biosynthesis of all four types of compounds is either essential for bacterial growth or absolutely required for the pathogenesis of infection. An interdisciplinary team of researchers at Argonne National Laboratory, the University of Michigan and the University of Chicago applies multiple different technological platforms to focus on these questions: bioinformatics, molecular genetics, biochemical purification and assay development, structure determination, organic chemistry and small molecule inhibition, as well as infectious disease modeling. Products of this research are the in depth molecular appreciation of envelope function and pathogenesis in B. anthracis and S. aureus and the identification of small molecule inhibitors that will be tested for their property of antiinfective or antibiotic therapies. The specific aims are: 1. Inhibition of capsular biosynthesis in Bacillus anthracis; 2. Inhibition of lipoteichoic acid biosynthesis in Bacillus anthracis and MRSA; 3. Inhibition of iron siderophore biosynthetic pathways in Bacillus anthracis and MRSA; 4. Inhibition of protein assembly pathways in the envelope of Bacillus anthracis and MRSA; 5. Inhibition of siderophore amide hydrolases in B. anthracis and MRSA."}
{"text": "In up to 26% surgical patients, subtle yet persistent deficits in learning and memory occur postoperatively, referred to as postoperative cognitive dysfunction (POCD). POCD has become a serious public health concern as it is associated with worse clinical outcomes including increased mortality. The pathogenesis underlying POCD remains unclear. Both modifiable and non-modifiable factors may contribute to POCD. To date, studies on POCD have primarily focused on direct influences of surgery and anesthesia on the central nervous system, which have identified age and genetics as major risk factors in POCD. Unfortunately, these are non-modifiable factors and difficult to be translated into clinical treatment. As such, there is an urgent need to identify modifiable factors underlying POCD. Among many modifiable factors, dietary influences and gut microbiota have been implicated in many neurological diseases with inflammatory features. Whether gut microbiota influences POCD has yet to be examined. In our preliminary studies, we observed a previously unrecognized role for gut microbiota in the development of POCD in mice post femoral artery exposure under isoflurane anesthesia. Specifically, we found: 1) mice with normal gut microbiota did not develop POCD while mice with gut dysbiosis developed POCD; 2) oral ampicillin treatment led to a status of gut dysbiosis, characterized by gut microbiota community structure changes and a dramatic decrease of indoles, particularly indoxyl-3-sulfate (IS) and indole-3-propionic acid (IPA); 3) oral administration of IPA, but not IS, deterred the POCD development; 4) mice with POCD displayed increased oxidation and impaired mitochondria function in the hippocampus, suggested by an enhanced production of reactive oxygen species (ROS), decreased production of NADH, and decreased protein levels of NDUFS4 (a critical mitochondria complex I component), when compared with mice without POCD; and 5) oral administration of IPA decreased ROS generation, increased NADH production and NDUFS4 protein levels in the hippocampus of ampicillin-treated mice. Based on these preliminary findings, we hypothesize that gut microbiota has a key influence on the development of POCD through IPA. In the research program proposed in this grant, we will examine the hypothesis by addressing three key questions: 1) Does the observed effect of gut dysbiosis on POCD represent an epiphenomenon or a ?permissive? effect? 2) What are the mechanisms underlying the IPA?s protective role in POCD? and 3) Can we develop a strategy based on gut microbiota and metabolites to prevent and treat POCD? This grant is built on our novel preliminary findings and our established research platform that combines cutting-edge metagenomics and metabolomics with immunological and neurobehavioral assays. Successful execution of this proposal will establish a novel conceptual framework linking modifiable factors such as diet and gut microbiota with POCD, and lead to new therapeutic strategies."}
{"text": "This study will compare 4 different study drug combinations for treatment of HIV infection. The purpose of this study is to see which of these study drug combinations does the best job of reducing HIV in the blood. Safety and tolerability of these drugs in children will be assessed."}
{"text": "To understand the mechanism of contractile process of the cleavage furrow, we propose to study (1) isolated cleavage furrow by biochemical methods, (2) immunohistochemical localization of contractile proteins in the furrow and (3) ultrastructural analysis of the contractile ring using cells which are frozen rapidly and freeze-substituted. Isolating cleavage furrow will be attempted by using micromanipulators on stabilized furrow. Immunohistochemical studies will be done at both light and electron microscopic levels, using antibodies against actin, myosin, alpha-actinin, actin binding protein and calmodulin. Rapid freezing is done using Heuser's apparatus and freeze-substitution will be done by cold acetone in the presence of OsO4."}
{"text": "The aims of this proposal are to develop flexible, semiparametric statistical models, methods, and inferences for longitudinal data, and to apply these models and methods to analyze AIDS data. The proposal consists of two aims. The first specific aim is to develop flexible models, methods, and inference for longitudinal data, which will involve (a) applying the penalized spline methods to longitudinal data analysis, comparing the methods with local kernel, regression spline methods to determine which is best in practice, developing statistical inference methods for penalized spline with longitudinal data; (b) developing flexible and efficient methods for time-varying coefficient mixed-effects models with longitudinal data, including investigation of local kernel regression and the penalized spline methods; and developing flexible methods for generalized varying-coefficient mixed-effects models with longitudinal data; (c) developing flexible methods for general two-stage semiparametric nonlinear mixed-effects models; (d) developing computer packages to implement the proposed methods. The second aim is to apply the proposed models and methods developed to study HIV dynamics by using data from AIDS clinical trials run by the AIDS Clinical Trial Group (ACTG) and data from AIDS clinical trials conducted at St. Jude Children's Research Hospital. We will focus on (a) characterizing long-term HIV/T-cell dynamics in HIV-l-infected patients treated with highly active antiretroviral therapy by using flexible, nonparametric or semiparametric methods for longitudinal data; and studying the relation between long-term HIV dynamics and T-lymphocyte kinetics during long-term antiretroviral drug exposure; (b) investigating which clinical and specific factors affect HIV dynamics; (c) investigating the relation between clinical endpoints and HIV dynamics; and (d) comparing the effects of different antiviral agents, and presenting statistical evidence to justify which of several treatments is the most effective. The proposed research is expected to benefit studies of the immune system in HIV-infected patients (one of the main focuses of future AIDS research)."}
{"text": "Post-translational modifications (PTMs) of histone tails are major marks for transcription activation and silencing. Accordingly, detecting and capturing post-translationally modified histones represent a critical step in epigenetic research. A major technological bottleneck in this area of research is a paucity of high-quality affinity reagents. Polyclonal and monoclonal antibodies are the only widely available affinity reagents for histone tails, but they have fundamental limitations in reproducibility, scalability, storage and production throughput and expenses. The long-term goals of this project are to develop an innovative and powerful technology platform for facile production of high-quality affinity reagents for histone tails containing PTMs and to make a standard set of such affinity reagents broadly available to the epigenetics research community. This project is built on an innovative protein-engineering concept that our group has recently established. The concept, termed Affinity Clamping, harnesses the inherent specificity present in the so-called interaction domains and dramatically enhances their affinity and specificity by attaching an \"enhancer domain\" and subsequently optimizing its interaction interface by directed evolution of combinatorial libraries. The resulting affinity reagents with clamshell architecture, collectively termed \"Affinity Clamps\" thus \"clamp\" the target, leading to orders-of-magnitude higher affinity and specificity. Protein libraries made in this manner are predisposed to binding to a specific class of peptide motifs (e.g. histone tails with a methylated lysine), and they virtually guarantee successful engineering of high-performance affinity reagents for a predefined peptide motif. Affinity Clamping represents a paradigm shift in affinity reagent generation. Because Affinity Clamps are fully recombinant reagents produced in E. coli, they can be easily produced in large quantities and distributed. Also they can be reformatted into a variety of fusion proteins suitable for in vitro and in vivo applications. Our proof-of-concept experiments have successfully demonstrated the general feasibility of the Affinity Clamping concept and suggest its enormous potential. Because there exist a number of interaction domains that weakly bind to post-translationally modified histone tails, we are confident that we can apply the Affinity Clamping strategy to produce high-quality affinity reagents to a variety of histone motifs. The proposed project will critically evaluate the feasibility and potential of applying the Affinity Clamping technology to epigenetic histone marks. The specific aims of the initial project period are (i) to produce \"Histone Clamps\" for well-characterized histone lysine methylation sites and benchmark them against commercially available monoclonal antibodies for their performance in commonly used assays;and (ii) to produce Histone Clamps for histone methylation sites for which no high-quality antibodies exist. Such Histone Clamps will be provided to the epigenetic community, which will have a major impact on the quality, scale and types of epigenetics research. PUBLIC HEALTH RELEVANCE: Accurately measuring the type and amounts of chemically modified forms of histones and capturing them for downstream analysis are major technological challenges in epigenetic research. This project will establish a totally new approach to facile generation of high-performance reagents for these purposes. This innovative and powerful technology will fill a major void in the currently epigenetic research, and products from this project, termed \"Histone Clamps\", will make it feasible to establish a standard set of epigenetic capture reagents that can be distributed broadly to the community and open new avenues of epigenetics research."}
{"text": "We have continued our studies of chromatin structure in the neighborhood of expressed genes. The globin gene family in chicken erythroid cells serves as a model system in which it is possible to study the mechanisms associated with regulation of the cluster and individual members of the family during erythroid development. We have focused attention on the 1.2 kb insulator DNA sequence at the 5' end of the chicken beta-globin locus, which is capable of blocking enhancer-promoter interaction when it lies between them. We had earlier narrowed down this activity to a small DNA fragment within the 1.2 kb sequence. We also purified the protein responsible for this activity and identified it as CTCF, which is a known factor ubiquitous in vertebrates, with both stimulatory and inhibitory effects on expression of a variety of genes, and we showed that CTCF sites are present in other vertebrate gene loci, including the 3' end of the chicken beta globin locus. We were also led to investigate a potential insulator activity in the neighborhood of the Igf2 (Insulin-like growth factor 2)/H19 locus in mouse and human. This is an imprinted locus: In the maternally transmitted allele Igf2 is silent and H19 is expressed. The reverse is true in the paternally transmitted allele. A region between the two genes is methylated on the paternal allele only, and it had been suggested that the insulator was within this region. We have now shown that this region contains multiple binding sites for CTCF (four in mouse, seven in human). They serve as very strong enhancer blocking elements, preventing a downstream enhancer from activating Igf2 in the maternal allele. Deletion of these sites abolishes insulation. We also have shown that methylation of the sites prevents CTCF binding. Thus the selective activation of Igf2 on the paternal allele is explained by inactivation of the insulator, which allows the downstream enhancer to turn on this gene. Our results also show that insulator activity can be modulated during development by site methylation. This greatly widens the potential range of action of insulator elements. In other studies we have examined the pattern of chromatin structural changes over the globin locus and its neighborhood during development. We had shown earlier that an erythroid specific folate receptor (FR) gene lies upstream of the chicken beta-globin locus, separated from it by 16 kilobases of condensed non-expressed chromatin. Making use of high resolution immunoprecipitation methods, we have shown that there are major changes in histone acetylation levels over these genes in cells corresponding to various developmental stages, but that the condensed chromatin domain and certain regulatory elements maintain their low and high acetylation levels (respectively) throughout development."}
{"text": "A number of recent studies have found a schizophrenia-associated loss of cells or volume in the human thalamus. Unfortunately, these previous studies did not have the resolution necessary to precisely localize the abnormalities. The specific aims of the proposed research are to use efficient stereological methods to: 1) Survey the entire thalamus for schizophrenia-related loss of volume or cellularity, 2) Localize any detected abnormalities to particular cytoarchitectonic divisions, and 3) Investigate the specificity of any detected abnormalities to schizophrenia. The investigator will use well-characterized postmortem material from the Mount Sinai Medical Center/Bronx Veteran's Affairs Hospital Schizophrenia Brain Bank to test the following primary hypotheses: 1) Previously reported schizophrenia-associated abnormalities in the mediodorsal thalamic nucleus will primarily involve its parvocellular and caudal divisions which, in man, have a prefrontal cortical dependence. The magnocellular division which does not degenerate following prefrontal ablations may be relatively spared in schizophrenia. 2) Schizophrenia-associated loss of cells and volume will not be restricted to the mediodorsal nucleus. Other nuclei where abnormalities are likely to occur include those with predominately temporolimbic connections and those with patterns of connections suggesting the potential for a role in arousal or in gating signals to and from cortex. The more specific sensory and motor relay nuclei are likely to be spared. 3) The schizophrenia-associated thalamic abnormalities are not common to all chronic mental illnesses and will not be seen in subjects with affective disorders. The studies of the present proposal will lay the groundwork for more definitive future explorations that will more fully assess the main and interactive effects of age, chronic institutionalization, neuroleptic exposure, and gender on the anatomy of thalamic nuclei where schizophrenia-associated abnormalities are found."}
{"text": "Recently, we have successfully produced simian-human immunodeficiency virus-like particles (SHIV VLPs) which contain SIV Gag and HIV Env by using a baculovirus expression system. Furthermore, we have incorporated the influenza virus surface glycoprotein, hemagglutinin (HA), into SHIV VLPs. Taking advantage of HA having a high affinity to bind to the mucosa of the upper respiratory track, our central hypothesis is that use of SHIV VLPs containing influenza HA as a mucosal vaccine will enhance both systemic and mucosal immune responses against HIV infection. The major focus of this project is to investigate the efficiency and mechanisms of the built-in adjuvanticity of HA in SHIV VLPs for intranasal immunization in a mouse model. Specifically, we propose: 1). To determine the role of incorporation with influenza HA in SHIV VLPs as a mucosal vaccine in enhancement of immune responses against HIV. Proposed experiments will investigate: a) whether the built-in adjuvanticity of influenza HA in SHIV VLPs as a mucosal vaccine is more potent than soluble influenza HA in enhancement of both systemic and mucosal immunity; and b). whether the receptor binding or membrane fusion activity of influenza HA affects its adjuvanticity for SHIV VLPs. 2). To determine the role of incorporation with influenza HA in SHIV VLPs in dendritic cell (DC) binding, activation, cytokine production, and antigen presentation. We propose to investigate: a). whether HA/SHIV VLPs have an increased ability to DC binding, internalization, and subcellular localization; b). whether HA/SHIV VLPs have an increased effect on DC activation, and cytokine production; c). whether HA/SHIV VLP-activated DCs increase naive T cell proliferation; and d). whether HA/SHIV VLPs increase the efficiency of antigen cross-presentation of DCs to CD8+ T cells and what are the associated intracellular pathways. 3). To determine the role of incorporation with influenza HA in SHIV VLPs in specific B cell binding, activation, and antibody production without CD4+ T cell help. Experiments are designed to investigate: a). whether HA/SHIV VLPs increase their ability to bind to naive B cells; b). whether HA/SHIV VLPs have an increased effect on naive B cell proliferation; c). whether HA/SHIV VLP-activated DCs have an increased ability to adhere to naive B cells; d). whether HA/SHIV VLP-activated DCs increase naive B cell proliferation; and e). whether B cell activation and differentiation and cytotoxic CD8+ formation occur in nasal-associated lymphoid tissue (NALT), an inductive site after intranasal immunization with HA/SHIV VLPs in CD4+ T-cell-deficient mice. This project represents a novel approach to develop an effective and safe HIV vaccine. Understanding the cellular and molecular mechanisms of HA/SHIV VLP-enhanced immune responses in mice is critical for the future design and testing of a successful HIV vaccine in non-human primate models and in human trials."}
{"text": "The experiments outlined in this application are designed to address the role of the synaptic protein PSD-95 in the synaptic transmission and plasticity of excitatory synapses in the nucleus accumbens shell (NAc) and if these mechanisms underlie some of the effects of chronic drug exposure. Genetic manipulation via recombinant lentivirus will be used to acutely knock down or enhance expression of PSD-95 proteins in medium spiny neurons of the NAc shell in living wild type mice. Characteristics of basal excitatory synaptic transmission will be examined in infected vs uninfected neurons via whole cell patch clamp recording and pharmacological manipulation of acute NAc slices. The effects of PSD-95 depletion or enhancement upon synaptic plasticity in the NAc shell will be addressed by studying NMDAR dependant forms of LTD and LTP as well as endocannabinoid LTD in infected neurons. If time permits, in vivo manipulation of PSD-95 levels will also be combined with a chronic cocaine regimen in order to examine the contributions of this protein to the depression of synaptic strength seen in the NAc shell in response to long term drug exposure."}
{"text": "ABSTRACT: A significant need exists to address complications associated with the growing pandemic of diabetes mellitus and metabolic syndrome now estimated to affect >20 million people in the United States (~7% of the population!). Impaired wound repair, a major complication of poor glycemic control, costs >$10 billion annually. Our laboratory first identified and cloned rabbit (Larrick et al., 1991) and then human Cationic Anti- microbial Protein (hCAP18) based on its LPS binding and anti-microbial activities. Over the past 10 years hCAP18 has been shown to be an important component of the innate immune system with broad anti-microbial activity conferred by its C-terminal fragment LL-37. hCAP18 is constitutively produced in leukocytes and is induced in barrier organs (e.g. lung, GI, skin) upon inflammation and infection. Recent work demonstrated a key role of hCAP18 in vascularization and re-epithelialization of skin wounds. High levels of hCAP18 are produced in skin in vivo upon wounding with a peak at 48 h post-injury, declining to pre-injury levels upon wound closure. hCAP18 is detected in the inflammatory infiltrate and in the epithelium migrating over the wound bed. For example, using a noninflammatory ex vivo wound healing model, composed of organ-cultured human skin, Heilbron et al. (2003) showed that treatment with anti-LL-37 antibodies inhibits re-epithelialization. In chronic non-healing wounds, hCAP18 levels are low and immunoreactivity for hCAP18/LL-37 is absent in ulcer edge epithelium. We hypothesize that LL-37 plays a critical role in wound closure and that its absence in chronic wounds impairs re-epithelialization, promotes bacterial colonization and contributes to failure of the wounds to heal. Hence, the overall goal of this proposal is to develop a protease resistance form of LL-37 as a novel therapy for chronic, non-healing wounds. To this end in phase I we will prepare prLL-37, a protease resistant form comprised of D-amino acids, demonstrate resistance to degradation by proteases, and evaluate activity of prLL-37 in a wound healing model using diabetic mice. In phase II we will carry out preclinical studies to support submission of an IND. This therapy will address a major unmet need among patients suffering from burn injuries and chronic non-healing, diabetic foot, decubitus and venous stasis ulcers. ABSTRACT: A significant need exists to address complications associated with the growing pandemic of diabetes mellitus and metabolic syndrome now estimated to affect >20 million people in the United States (~7% of the population!). Impaired wound repair, a major complication of poor glycemic control, costs >$10 billion annually. Our laboratory first identified and cloned rabbit (Larrick et al., 1991) and then human Cationic Anti- microbial Protein (hCAP18) based on its LPS binding and anti-microbial activities. Over the past 10 years hCAP18 has been shown to be an important component of the innate immune system with broad anti-microbial activity conferred by its C-terminal fragment LL-37. hCAP18 is constitutively produced in leukocytes and is induced in barrier organs (e.g. lung, GI, skin) upon inflammation and infection. Recent work demonstrated a key role of hCAP18 in vascularization and re-epithelialization of skin wounds. High levels of hCAP18 are produced in skin in vivo upon wounding with a peak at 48 h post-injury, declining to pre-injury levels upon wound closure. hCAP18 is detected in the inflammatory infiltrate and in the epithelium migrating over the wound bed. For example, using a noninflammatory ex vivo wound healing model, composed of organ-cultured human skin, Heilbron et al. (2003) showed that treatment with anti-LL-37 antibodies inhibits re-epithelialization. In chronic non-healing wounds, hCAP18 levels are low and immunoreactivity for hCAP18/LL-37 is absent in ulcer edge epithelium. We hypothesize that LL-37 plays a critical role in wound closure and that its absence in chronic wounds impairs re-epithelialization, promotes bacterial colonization and contributes to failure of the wounds to heal. Hence, the overall goal of this proposal is to develop a protease resistance form of LL-37 as a novel therapy for chronic, non-healing wounds. To this end in phase I we will prepare prLL-37, a protease resistant form comprised of D-amino acids, demonstrate resistance to degradation by proteases, and evaluate activity of prLL-37 in a wound healing model using diabetic mice. In phase II we will carry out preclinical studies to support submission of an IND. This therapy will address a major unmet need among patients suffering from burn injuries and chronic non-healing, diabetic foot, decubitus and venous stasis ulcers. [unreadable] [unreadable] [unreadable]"}
{"text": "This contract is to assess the new third-generation SERM, Bazedoxifene, alone and in combination with CE and/or an AI, to prevent mammary carcinogenesis induced in virgin female Sprague-Dawley rats by N-methyl-N-nitrosourea (MNU). The tasks will involve optimizing the dose and timing of BZA administration, and PK profile of BZA in combination with the AI."}
{"text": "Our microchemical facility at Caltech has exceeded all expectations in being able to sequence small quantities of protein and synthesize small or large fragments of genes. Several gas phase microsequenators are now functional and each has the capacity to sequence 20 or so residues a day. We have a DNA synthesizer with a three column system that has the capacity to synthesize simultaneously three oligonucleotides with a 14 min. cycle time. Thus, the microchemical facility has the considerable ability to sequence proteins and synthesize genes. These abilities have been turned to a variety of projects that are directly related to the cancer problem. Microsequencing done in this facility on platelet derived growth factor demonstrated for the first time the serum polypeptide hormone appears to be strikingly homologous to the primate oncogene. We are now in the process of analyzing the sequences of a series of other growth hormones and will have the opportunity to determine whether additional similar correlations can be made. We have also started to analyze the gene structure organization and rearrangements of genetic elements in coding the T cell receptor. Once again this receptor molecule plays a central role in facilitating the immune response against foreign invaders including tumors. The microchemical facility has been used to synthesize a wide range of DNA probes that have been used to clone the corresponding genes (partially documented in the Progress Report). Indeed, the DNA synthesizer has been used to synthesize an entire functional E. coli serine tRNA gene. The computer facility has added valuable backup and data analysis capacity to these and many other projects. The cell sorter is functional and has provided valuable data for a number of different laboratories. Within the next year we expect to have a fully functional automated peptide synthesizer and be well along in developing an automated DNA sequenator. Thus, this complement of instruments will allow us to both sequence and synthesize genes and DNA in a sensitive and rapid fashion. This year the microchemical facility has been used by more than 15 professors at Caltech and a much larger number of investigators outside the Institute."}
{"text": "This project is designed to investigate the roles of monocytes in limiting blood-borne metastasis of tumor cells, and to provide a valid basis for effective immunochemo prophylaxis (and therapy) of metastatic spread. Utilizing both metastasing and non-metastasing syngeneic tumor lines of DBA/2 mice, host immunity is semi-quantitatively assessed in normal and tumor-bearing animals by i.v. injection of radio-labeled tumor cells and by measuring the rate of tumor cell clearance in various target organs of these animals. Non-metastasing T1699 mammary adenocarcinoma is employed as the model in which immune suppression of the host is a prerequisite for successful development of metastatic growth. Immune suppression is induced by treatment with anti-thymocyte serum, antimacrophage serum, tilorone HCl, or by over dosage of chemotherapeutic agents. Restoration of therapeutic activity by in vivo (or in vitro) armed monocytes is attempted in this system. Spontaneously metastasing P1534 lymphatic leukemia or PP388D1 macrophage-like tumor line are used to study the effect of monocyte stimulants, e.g., pyran copolymers and beta-1,3-glucan on chemotherapeutic regimens and also on augmentative effect of in vitro armed monocytes on such chemotherapeutic effects. Results will be evaluated in relation to actual inhibition of metastatic growth of these tumors."}
{"text": "The proposed research seeks to understand how the spatial and temporal control of actin dynamics is regulated differentially across the developing vertebrate embryo. This will be investigated within the specific context of mesodermal cell behaviors during gastrulation and segmentation in the zebrafish embryo. The potential role of actin regulatory molecules (ARMs) that are expressed in specific areas of developing mesoderm will be investigated by loss of function analysis, using confocal imaging and a transgenic line of reporter fish expressing actin-GFP fusion protein that reveals cellular actin distribution. The signaling pathways controlling these ARMs will be characterized using mutagenesis screening and protein-protein interaction assays. The misregulation of actin and cell movements has been implicated in multiple human conditions such as Wiskott- Aldrich syndrome and Spina Bifida, in which compromised cell polarity causes a failure of neural tube closure in early development. An understanding of actin regulation during vertebrate development in the zebrafish may thus provide insights into the causes and treatment of certain human diseases."}
{"text": "ABSTRACT Alzheimer?s disease (AD) is the most common form of neurodegenerative disorder. Inflammatory changes in the brain are thought to represent key processes in the onset and progression of AD, but it remains unclear whether neuroinflammation confers neuroprotection, accelerated degeneration, or possibly both. Such an understanding in living humans is critical if we are to begin clinical trials using the array of FDA-approved immunomodulatory drugs in the future. We propose that complement- mediated neuroinflammation is protective in the early AD stages, while suppression of complement activities is accompanied by the development of greater cognitive deficits and faster cognitive decline. Our preliminary data from multiple cohorts support this hypothesis by showing 1) reduced levels of cerebrospinal fluid (CSF) complement-related markers occur in the dementia stage but not mild cognitive impairment (MCI) stage of AD; 2) reduced CSF complement-related markers and elevated CSF interleukin-10 (IL-10) levels are associated with faster decline in AD; and 3) CSF inflammatory protein alterations reveal networks of cellular and protein regulations. In the In the current application, we will build on the association between complement related proteins and rates of cognitive decline in AD to identify associated changes in soluble CSF cytokines and chemokines, differential inflammatory cell type regulation, and imaging correlates of neuroinflammation. This application takes advantage of our group?s strengths in performing CSF cytokine measurements, CSF immunophenotyping, molecular imaging of neuroinflammation through positron emission tomography (PET) and iron-enhanced MRI, and network analysis through a novel biochemical-bioinformatics pipeline. We will directly identify individual and networks of soluble CSF cytokines that accompany the transition from the MCI to the dementia stage of AD, correlate the complement and other altered pathways with microglial activation through two modern PET tracers (11C-PBR28 and 18F-FEPPA), and measure changes in individual T helper cell (type 1, 2, 17) and non-T cell populations. This application represents the first attempt to correlate, at the individual level and at the group level, CSF and imaging measures of neuroinflammation. If successful, this application will advance the understanding of neuroinflammation in AD through parallel approaches, form the basis of a new biomarker panel (and algorithm) to diagnose AD through a combination of degenerative and inflammatory markers, and accelerate the target identification of future therapeutics aimed at modulating the immune system in AD."}
{"text": "The purpose of this protocol is to develop novel methods of performing magnetic resonance imaging (MRI) evaluations so that these methods can be transferred to the clinical environment. Normal volunteers are recruited to optimize imaging techniques and the protocol has been very successful in recruiting normal volunteers. Among the accomplishments of this protocol over the last year include optimizing contrast administration rates during magnetic resonance angiograms, automatic table motion techniques for peripheral run-off magnetic resonance angiography (MRAs), phase contrast angiography, motion tracking for knee and patella movement, functional MRI of the brain, gated MRI to image the soft palate, and stroke protocols. We have made substantial gains in technical development in all of these areas and there have been no complications. We have developed protocols to be used in conjunction with Suburban Hospital in MRA, stroke, and cardiac imaging, and will continue this study."}
{"text": "The long-term goals of this project are to understand how cell-to-cell signaling systems interface with regionally expressed transcription factors during development to generate correctly proportioned and patterned body parts. The experimental system that will be used is the fruit fly, Drosophila melanogaster. The starting point for these experiments are two genes, homothorax and extradenticle, that are important for specifying the identity of the proximal portion of the appendages in flies. These genes appear to limit or modify the activities of signaling pathways known to operate during appendage development. Both extradenticle and homothorax encode homeodomain-containing transcription factors. However, Extradenticle protein is only nuclear in the presence of Homothorax. In several tissues, Homothorax and Extradenticle are co-expressed with a Zn-finger-containing transcription factor encoded by the teashirt gene and, in the eye, these three proteins are co-expressed with another homeodomain protein encoded by the eyeless gene.The specific aims for this funding period are to: (1) determine the mechanism by which Extradenticle's nuclear localization is controlled by Homothorax, (2) characterize the roles of homothorax and teashirt in wing and eye development, (3) characterize the genes involved in repressing hth expression during leg development, and (4) characterize the role of novel genes, identified by a forward genetic screen, in proximo-distal patterning in the fly appendages. homothorax and extradenticle both have vertebrate homologs that, when misexpressed, can contribute to leukemias, suggesting that they also play a role in regulating growth and development in vertebrates. In addition, the transcriptional and post-transcriptional regulation of these factors appears similar in vertebrate and fly limb development, suggesting that many of their functions may also be conserved. An understanding of their basic functions in fly development should therefore generate information that is relevant to leukemia, as well as to defects that can occur in vertebrate limb development."}
{"text": "Fungal infections are becoming an increasing public health concern, particularly among immune compromised patients or those undergoing chemo- and/or radiotherapy. Two fungi of particular concern are Aspergillus niger and Candida albicans. We have obtained crystals of the essential DNA repair enzyme dUTP pyrophosphatase (dUTPase) from C. albicans. Determination of the structure of the fungal dUTPase, and comparison with the structure of the human enzyme, recently determined in our laboratory, may allow design of inhibitors specific for the C. albicans enzyme for use as anti-fungal agents."}
{"text": "The object of the proposed research is to further understanding about the mechanisms that all vertebrates have evolved to survive the thermal stresses of the environment. Four major areas will be examined with this objective: (1) Thermal acclimation in teleost fish. The importance of oxygen transport and acid-base imbalance will be evaluated by allowing cold acclimated fish to return to the final thermal preferendum while dissolved oxygen and carbon dioxide concentrations are varied. Appropriate measurements of blood gases will also be made; (2) The mechanism of torpor in teleost fish. Fish will be acclimated to a large number of temperatures. Metabolic rate and spontaneous activity will be monitored at each temperature. ECG, brain electrical activity, oxygen uptake, and ventilatory frequency will be monitored as fish are slowly cooled. The importance of anterior brainstem temperature in the induction of torpor will be evaluated. (3) Thermal inputs to respiratory-cardiovascular regulation in teleost fish. Dorsal aorta temperature, blood flow, and PO2, and ventilation frequency or ventilatory minute volume will be monitored during rapid thermal shifts. Effects of changes in the anterior brainstem temperature on the above parameters will be evaluated. (4) Phylogenetic development of CNS integrated responses to thermal stresses. Certain of the above experiments will be performed on lampreys. It is felt that answers provided by this research will aid in unifying -- for all vertebrates --the common mechanisms for sensing and integrating important thermal variables as well as serving to better understand the development of specialized vertebrate adaptations such as endothermy and hibernation."}
{"text": "Early knowledge of HIV status is critical to prevent transmission to others, to link those who are HIV positive to medical care and services that can reduce morbidity and mortality and improve quality of life, and to reduce health care expenditure. Consistently, the National HIV/AIDS strategy has established a goal of increasing the awareness of HIV status in Americans from 79% to 90% in the next four years. The proposed research is in direct response to PA-11-119, HIV/AIDS Testing and Follow-up Among the Underserved in the United States, and proposes to investigate the acceptability, feasibility, and initial efficacy of a rapid HIV testing and counseling protocol in residents of domestic violence shelters. Intimate partner violence (IPV) is a pervasive public health problem, and a growing body of research highlights the association between IPV and HIV/STI risk (e.g., increased unprotected sexual occasions, sex with risky partners, trading sex, multiple partners). Further, IPV is associated with many barriers to receiving adequate health care (e.g., transport difficulties, child care needs, safety issues, lack of health insurance). Domestic shelters present an opportune setting for providing health care services for women, providing a safe and supportive environment for testing, while addressing many of the barriers to testing and linkage to care faced by shelter residents. RESPECT-2 is an evidence- based CDC Diffusion of Effective Behavioral Interventions (DEBI), utilizing a client-focused, interactive HIV risk reduction counseling model delivered in conjunction with rapid testing. Residents of domestic shelters, however, face distinct HIV risk factors, such as difficulty in negotiating condom use out of fear of retaliation from their abuser, and thus needs to be tailored to meet the specific needs of shelter residents. Thus, in the proposed exploratory/developmental research plan, we aim to expand RESPECT-2 for our target population (i.e., RESPECT-IPV), and to collect preliminary data on RESPECT-IPV + rapid testing's feasibility, acceptability, and initial efficacy in a sample of 100 high-risk shelter women. Predictors of acceptability (e.g., PTSD symptoms, substance use, IPV severity, prior testing history, HIV knowledge, HIV risk behavior, HIV anxiety, stage of change) will also be explored. Our primary hypotheses are that RESPECT-IPV will be associated with reduced unprotected vaginal or anal sex occasions at 1-week and 3-months post-shelter and fewer cases of vaginal trichomoniasis 3-months after leaving shelter."}
{"text": "A workshop convened in 2013 by National Institute of Aging and the National Institute of Neurological Diseases and Stroke prioritized research on cognitive impairment of multiple etiologies, contributions of vascular brain injury (VBI) to neurodegeneration and Alzheimer's Disease (AD), and disparities involving race, ethnicity, socioeconomics, and rural residence. VBI is a leading cause of accelerated brain aging and a major risk factor for stroke, cognitive decline, depression, and probable Alzheimer's disease (AD). However, few studies have evaluated VBI, cognitive impairment, and AD in American Indians (AIs), who bear a heavy burden of risk factors for these conditions. This exclusion is unfortunate, since the distinctive history, risk profiles, environmental stressors, social environments, and healthcare systems of AIs likely result in patterns of disease that differ substantially from other populations. Our research team has conducted the only cohort study to date of covert VBI in AIs. In 2010-2013, Cerebrovascular Disease and its Consequences in American Indians study completed standardized clinical examinations, neuropsychological testing, and cranial MRI on more than 1,000 AIs aged 64-95 years from 10 tribes in 3 states. In 2016, we began re-examination of surviving participants using the same protocols, augmented by assessment for probable AD. However, the particular grant mechanism from the National Institute on Aging that provides funding for this follow-up examination is limited to data collection, with no resources allocated for analysis. In this proposal, we will capitalize on the accumulated longitudinal data to apply sophisticated imaging and analysis methods to quantify and evaluate associations for incident VBI, neurodegeneration, and changes in cognitive status in this elderly minority population. Our Specific Aims are to: (1) establish normative and diagnostic standards for mild cognitive impairment and dementia in elderly AIs, and evaluate associations and determinants for incident VBI, cerebral atrophy, cognitive decline, and probable AD; (2) conduct cluster analyses to identify novel neuroimaging profiles that predict subgroups of elderly AIs at highest risk for cerebral atrophy, cognitive decline, and probable AD; and (3) complete a rigorous career development plan that will position the applicant as an independent investigator and leader in innovative neuroepidemiology research with minority populations. This proposal leverages a timely opportunity to generate the first population-based estimates of and address novel scientific questions on VBI and cognitive impairment in AIs, a minority population with pervasive health disparities."}
{"text": "Practice-based research networks (PBRNs) are organizations of clinicians working in practices that engage in research. PBRNs have emerged as a vital lynchpin in efforts to translate research into practice by translating practice into research. However, the development and maintenance and the continued engagement of busy community practices in cancer research requires specialized expertise and ongoing support. Therefore, the mission of the PBRN Core Facility is: [unreadable] To develop and sustain practice-based research networks that engage in cancer research; [unreadable] To advise and assist Case Comprehensive Cancer Center (Case CCC) investigators in developing community-based cancer research studies for implementation in PBRNs; [unreadable] To provide infrastructure that supports the implementation of Case CCC investigators' research studies in PBRNs, and that supports network practice-initiated research. The Practice-Based Research Network Core Facility supports the engagement of patients, clinicians, practices, and health systems in cancer research by: 1. Starting new PBRNs; 2. Developing the infrastructure of existing PBRNs; 3. Providing PBRN methods consultations to Cancer Center investigators and participating practices; 4. Advising and assisting investigators on research project implementation in PBRNs. The PBRN Core has started and sustained 8 successful PBRNs and is developing 5 new PBRNs. The established PBRNs represent major healthcare systems and include family medicine practices, federallyqualified health centers, community oncology practices, pediatrics practices, and general dentistry practices. The PBRN Core has facilitated the development and implementation of 23 funded research projects within the PBRNs that it has developed and sustained. Fourteen of these projects have been funded by NCI, and 6 supported by ACS. Grant funding for these projects totals over $15 million. The demand for the PBRN Core's services is strong and growing, and the potential for continued and expanded use of the facility is excellent. At present, 19 members of the Case CCC are pursuing 15 different lines of inquiry likely to result in funded studies to be implemented in Core-supported PBRNs."}
{"text": "Electrical activity in circadian clock neurons plays an important role in cellular oscillation. However, the particular voltage-gated ionic conductances necessary for circadian function and the mechanisms by which electrochemical signals interact with transcriptional events in the nucleus of clock neurons remain unknown. To identify the particular ionic conductances necessary for cellular oscillation, ion channel subtype-specific membrane-tethered toxins are transgenically expressed in clock neurons to interfere with voltage-gated conductances. Preliminary studies reveal substantial effects of in vivo expression of Ca2+ and K+ channel tethered toxin blockers on free-running behavioral rhythms. The proposed aims are (1) to test dose- dependence of these behavioral effects, (2) to examine effects of tethered toxins on cellular oscillation, and (3) to determine the molecular identities of the blocked ion channels. The long-term goal of this proposal is to improve understanding of circadian clock function, an issue of substantial health relatedness, given the great costs to society of genetic and environmental disruption of human sleep/wake cycles. [unreadable] [unreadable] [unreadable]"}
{"text": "Transcription from Recombinationally Activated Immunoglobulin Genes. Specialized nucleic acid elements are involved in the expression of immunoglobulin genes. These elements are 'enhancers' which act positively to promote transcription. The DNA rearrangements that occur during the construction of an antibody gene bring the enhancer near an otherwise weak promoter, thus activating the promoter. Particularly significant is the fact that unlike other enhancers, which are carried on viruses, the immunoglobulin enhancers are tissue specific--they only function in B lymphocytes. This suggests that B cells contain, as a result of their differentiation, proteins which are designed to function as specific transcriptional factors at this site. This application proposes two general lines of experimentation aimed at identifying and isolating the transcriptional factors involved in the expression of immunoglobulin genes. The first proposed approach is biochemical and involves trying to isolate the enhancer recognition protein directly, for example by assuming that it will specifically bind to enhancer DNA. In addition, an in vitro transcription system will be developed in which messenger RNA synthesis from DNA fragments--containing immunoglobulin genes with or without the enhancer--is tested for stimulation by protein fractions derived from B lymphocytes. The second general strategy is to seek mutants in the gene which codes for the enhancer recognition protein, and then use the mutants to isolate the gene itself. The fact that the immunoglobulin enhancer works only in B cells has allowed the design of three specific experimental strategies. Each is based on the idea of placing a drug resistance gene (neo) under immunoglobulin enhancer control and introducing the construction into various cells. For example, in B cells which are also caused to contain extra copies of the cloned enhancer DNA sequence, only a mutation to overproduction of the enhancer recognition protein will allow the neo gene to be expressed. This mutation would aid the biochemical studies described above, and potentially also allow the regulatory gene which produces the enhancer recognition protein to be cloned. With both the protein and nucleic acid components involved in immunoglobulin gene expression in hand, it should be possible to study this example of a regulated eukaryotic gene to the same depth that has been possible with, for example, the lactose and tryptophan operons in bacteria. Ultimately, purification of the enhancer recognition protein and analysis of mutations in the enhancer site that affect the proteins' ability to bind to the DNA will begin to allow a basic molecular description of immunoglobulin regulation and may provide key insights into the processes of development and differentiation in general. Malaria, DNA Rearrangements and Burkitt Lymphoma. Another experiment related to the recombinational activation of antibody genes attempts to probe the relationship between malaria infection and Burkitt lymphoma, which show a geographic coincidence. By co-cultivation of malaria-infected erythrocytes in vitro with tester mammalian cells, we will test the idea that infected erythrocytes release a clastagenic (chromosome-breaking) compound which could trigger the chromosome translocation associated with Burkitt lymphoma."}
{"text": "(Revised) DESCRIPTION (provided by applicant): Adolescents are at great risk for infectious diseases including the human immunodeficiency virus (HIV). Though the CDC reports that overall AIDS incidence is on the decline, there has been no comparable decline in the number of newly diagnosed HIV cases among young people aged 13-19, and young people of color are particularly at risk. Compared to the general adolescent population, adolescents involved with the criminal justice system are at higher risk for unintended pregnancy and infections. Alcohol use is commonly cited as a reason for risky behavior among high-risk adolescents such as those involved in the criminal justice system (e.g., Morris et al., 1998) and recent data from our research suggests that it is heavy alcohol use that is most strongly related to risky behavior (Bryan, Rocheleau, & Robbins, 2002a). The goal of this research is to design, implement, and test a successful HIV/Alcohol risk reduction intervention that is theory-based, empirically targeted to adolescents, and articulated to a criminal justice setting. The study compares a risk reduction intervention that incorporates an alcohol risk reduction component to a standard risk reduction intervention and a no treatment control condition. We hope to show that: 1) A combined risk reduction intervention will result in larger decreases in risky behavior, 2) The intervention will exert effects through reductions in alcohol use and changes in other mediators derived from a theoretically-based model of intentions and behaviors, and 3) A risk reduction intervention including an alcohol component will be especially effective for those adolescents with higher levels of existing alcohol problems. Finally, if the hypotheses are supported, the long-term objective will be to disseminate the intervention curricula and materials for use in adolescent detention facilities throughout the state."}
{"text": "The low rate of cure of certain cancers such as lung cancer, the most common causes of cancer death, is an important health problem. Early detection appears currently to be the only way of improving the high mortality rate, but is quite difficult because of the lack of symptoms in early disease. Moreover, lung cancer is mimicked in its in vivo image appearance by benign lesions and processes that lower the specificity of detection. Current imaging methods include chest x-ray, CT, and MRI. While these current methods are able to identify curable lung cancer they also result in many false positives. They are also limited in the size of the lung nodules they can detect. Using available criteria, sensitivity for lung cancer detection is high, but specificity and positive predictive value are only moderate. Thus there is a need for enhanced sensitivity and specificity for cancer cells. Our Anti-transferrin Receptor scFv-antibody fragment (TfRscFv) immunoliposome complex (scL) is a nanoconstruct (~100 nm) for delivery of gene therapy to tumors. It has been shown to target various types of human tumor cells in vivo when implanted as xenografts in mice and is now in Phase I clinical trials for delivery of wtp53. What we are proposing in this application is a quantum jump in diagnostic accuracy, an approach specific to cancer and best for small cancers such as lung cancer. The method we are developing is a nano- sized immunoliposome complex delivering superparamagnetic iron oxide particles (SPIO). Iron Oxide particles are both paramagnetic and super-paramagnetic, giving a biphasic response with both T1 and T2* features. This complex targets cancer cells with high selectivity. Thus the efficient delivery of SPIO directly into the tumor cells by the scL-SPIO complex of this application can increase the conspicuity of the lung tumor cells. Moreover, based on previous studies, the nanocomplex delivered contrast agent which is the focus of this application should accumulate within the cancer cells themselves remaining for an extended period (hours) allowing the contrast in non-cancer areas to wash out, further enhancing cancer conspicuity. In preliminary studies using an as yet unoptimized scL-SPIO complex, we demonstrated tumor cell specificity as compared to free SPIO, and enhanced image intensity. More importantly, in earlier studies with scL complexed with another imaging agent, gadopentate dimeglumine (gad-d), in a lung tumor model, the scL-gad-d (and not free gad-d) was able to enhance and identify lung tumors as small as 1-4 pixels (0.1-0.4mm), a size smaller than possible with current technology. No toxicity was found with this complex. In this application we will optimize the scL- SPIO complex and fully characterize its capabilities for use in early detection of lung cancer in a mouse model of human lung cancer and extend our studies to a mouse model of primary lung cancer. In collaboration with investigators at NIST and NCI we will also asses the magnetic properties of the complex and determine it sub cellular localization and trafficking through the cell. Our goal is to perform the majority of the studies necessary for filing an IND as we aim to move rapidly towards clinical trials. If cancer is detected early (e.g. Stage I), it can in many instances be cured (lower mortality). The challenge is to be able to find and positively identify the cancer at this early stage, particularly lung cancer. While the current methods of detection are good, they can only detect tumors of a certain size. Moreover, lung cancer is often mimicked during imaging by non-cancerous lesions, resulting in uncertainty and many false positives, which are often resolved only by following growth of the tumor. Thus there is a pressing need for imaging agents that increase sensitivity and specificity. Our tumor-specific nano complex delivery of an MR imaging agent, e.g. gad-d and iron oxide, has shown great promise in our preliminary studies in this regard, demonstrating that its high affinity for cancer cells in the lung can result in improved sensitivity in detecting tumors and in overall specificity. The development of an imaging agent that can lead to earlier detection is a high priority in the war on cancer and could lead to increased survival."}
{"text": "In order to protect against the four epidemiologically important rotavirus serotypes, we developed a rotavirus vaccine that contains four distinct strains: rhesus rotavirus (RRV) representing serotype 3 (the Jennerian approach), and three human RV-RRV reassortants, each possessing ten RRV genes and a single human RV gene that encodes VP7 (a major outer shell protein) that is responsible for serotype 1, 2, or 4 specificity (the modified Jennerian approach). Efficacy trials of this orally delivered vaccine have been completed in over 10,000 infants and young children with single or multiple vaccine components with successful results in most but not all studies. In five of these trials which included over 7000 infants and young children, the quadrivalent formulation, given orally in three doses, has been shown to be effective: protective efficacy against severe rotavirus diarrhea ranged from 69% to 91% in various locations. The vaccine was 75% to 100% effective in preventing dehydrating diarrheal illnesses. Because of the demonstrated safety (transient fever) and efficacy of the quadrivalent vaccine, on January 31, 1997 Wyeth Laboratories, the licensee, submitted a Product License Application to the Food and Drug Administration (FDA) and on December 12, 1997, the FDA Advisory Committee concluded that the clinical data presented supported the safety and efficacy of the quadrivalent rotavirus vaccine. On February 11, 1998, and again on June 25, 1998, the Advisory Committee on Immunization Practices recommended its use for infants at ages 2, 4, and 6 months of age. Subsequently, on August 31, 1998 the FDA granted a Biologics License to Wyeth Laboratories. and on November 4, 1998, the American Academy of Pediatrics recommended the rotavirus (Rv) vaccine. In addition, on May 7, 1999, the European Commission issued marketing authorization for the Rotavirus Vaccine with this approval covering all 15 member countries of the European Community.In related continuing rotavirus vaccine studies, we are evaluating collaboratively the feasibility of administering the licensed vaccine to neonates to determine its reactogenicity and immunogenicity with different administration schedules. This study will not only provide information on the immunogenicity of the vaccine when the initial dose is given during the neonatal period, but is also designed to compare the immunogenicity of a two-dose vs a three-dose schedule administered at 2 and 4 months and at 2, 4, and 6 months, respectively, the latter the currently approved schedule. We are also evaluating collaboratively several second generation vaccines. One of these, is a quadrivalent reassortant rotavirus vaccine that possesses a single VP7 gene with serotype 1, 2, 3, or 4 specificity and the remaining ten genes from bovine RV (UK). - Human Subjects"}
{"text": "The proposed study is a third follow-up of 581 male narcotic addicts admitted to the California Civil Addict program (CAP) during 1962-1964. The subjects were interviewed once in 1974/75 (DA01146), and again ten years later in 1985/86 (DA03425). The combined prospective and retrospective follow-up interviews provide a unique natural history database that can address issues specifically related to long-term narcotics addiction, patterns and consequence of use. Similar interview instruments will be used to collect self-reported measures about narcotics use, legal supervision status, criminal involvement, drug trafficking, employment, marital status, and treatment episodes over the entire addiction career. Corroborative data will be obtained from official arrest records for each interviewed subject, from a voluntarily provided urine specimen to be taken at interview, and from official death certificates for identified decedents. Specific aims of the study are: (1) to provide an additional ten years of data to allow a detailed natural history description over an almost 40-year addiction career of a sample of narcotics addicts; (2) to assess addiction patterns over time identifying factors which influence the divergence of outcomes, in particular, relapse, cessation of use, the process of \"maturing-out \", and mortality; (3) to analyze and describe morbidity and death among this aging addict sample; (4) to evaluate the extensiveness of criminal activity and to identify specific criminal career patterns in relation to narcotics use; (5) to conduct a treatment intervention history analysis, examining successive and cumulative treatment effects and the extent to which these individual and cumulative treatment episodes have reduced narcotics use; and (6) to assess long-term costs and consequences of prolonged careers of addiction. Augmentation of the existing addict career database provides a unique opportunity for investigation into the current status of addicts now between 47 and 73 years old."}
{"text": "The object of these investigations is to elucidate the biochemical mechanisms responsible for the reduced cardiac function and mortality that may follow open heart surgery. The availability of additional sufficiently refined methods of studying the cellular chemistry and ultrastructure of relatively intact human myocardium obtained by drill biopsy at the time of cardiac surgery has created a need for this supplementary application to support this work. This will be in addition to the histochemical and biophysical studies already being carried out on biopsy specimens at different stages of an operation. It is hoped that once the abnormalities are fully identified, it may be possible to begin to discover what is required to restore the function of such dysfunctional myocardium (or possibly what modification of the surgical procedure will be necessary to avoid further damage to a diseased heart)."}
{"text": "Immune dysregulation may contribute to the pathophysiologic findings seen in SCLS. A monoclonal gammopathy of unknown significance (MGUS, a premalignant precursor to multiple myeloma (MM), in which a clonal plasma cell population secretes monoclonal immunoglobulin (Ig, also referred to as a paraprotein) detectable in patient sera, is present in a majority of SCLS cases. Several patients with SCLS in whom MGUS evolved into myeloma or plasma cell leukemia experienced fewer capillary leak episodes after chemotherapy for their hematopoietic disorder. These findings suggest that the monoclonal paraprotein from the dysregulated plasma cell population may be the direct or indirect source of the pathophysiologic findings observed. We are characterizing the transcriptome of blood cell RNA and the proteome of SCLS serum/plasma, both pre- and post-attack, to determine whether specific biomarkers of acute symptoms and/or etiological factors can be identified. We have now evaluated 58 patients with a confirmed diagnosis of SCLS under this protocol in the last 7 years. We are the primary referral center in the U.S. for SCLS. The transient episodes of hypotensive shock and anasarca in SCLS are thought to arise from reversible microvascular barrier dysfunction. Application of episodic but not convalescent SCLS sera to human microvascular endothelial cells (ECs) caused vascular endothelial cadherin internalization, disruption of interendothelial junctions, actin stress fiber formation, and increased permeability in complementary functional assays. EC contraction and temporary attenuation of adherens junctions may thus permit leakage of solutes and proteins into the extravascular space during acute episodes. Circulating permeability factors, vascular endothelial growth factor (VEGF), angiopoietin 2 (Angpt-2), CXCL10, CCL2, and IL-6, were elevated in episodic SCLS sera compared to remission sera. Thus, angiogenic proteins and proinflammatory cytokines that induce EC hyper-permeability may contribute to transient EC barrier dysfunction around SCLS flares. To test the possible contribution of the Angpt2 pathway and/or other permeability inducers to SCLS, we have expanded blood-outgrowth ECs (BOECs) from circulating precursors obtained by venipuncture. These cells possess structural and molecular characteristics of mature ECs. Preliminary results suggest abnormal gene expression patterns in SCLS BOECs compared to those from healthy controls. Current studies are aimed at examined responses of ECs derived from subjects with SCLS and controls to various mediators of permeability to test the hypothesis that the SCLS endothelium is prone to exaggerated responses to otherwise mundane inflammatory stressors. The role of specific gene defects in SCLS, if any, is also unknown; e.g. whether the endothelium is genetically programmed for hyper-responsiveness to routine stimuli. There are no consistent familial aggregations in SCLS. Using Affymetrix Single Nucleotide Polymorphism (SNP) microarrays, we performed the first genome-wide SNP analysis of SCLS in a cohort of 12 disease subjects and 18 controls. From unbiased high-density mapping of single-nucleotide polymorphisms (SNPs), a small genetic interval, 3p25.3, was identified as the highest-ranking candidate susceptibility locus (p 10-6) with an odds ratio of 41. Odds ratios (7-41) and p values (10-4 and 10-6) for the top SCLS-associated variants were outsized for such a small sample size. These results imply high penetrance for a rare disease allele that remains to be identified. An inbred mouse strain was identified (SJL/J), whose phenotype bears similarity to SCLS, including spontaneous vascular hypersensitivity to a variety of permeability factors including histamine (HA), serotonin, and bradykinin, important mediators of endothelial hyperpermeability in humans, and B cell neoplasms characterized by the presence of serum paraproteins. A recessive locus in SJL mice controlling systemic vascular hypersensitivity to HA (Shs) was mapped to an interval on mouse chromosome 6 syntenic with human 3p25.3, which harbors the primary genetic association with SCLS in humans. This interval contains several gene candidates, which will be analyzed in the SCLS cohort and various mouse strains."}
{"text": "PROJECT SUMMARY Organophosphates (OPs) pose a constant threat to human health due to their widespread use as pesticides and their potential employment in terrorist attacks. The acute toxicity of OPs has been extensively studied; however, the consequences of prolonged or repeated exposure to levels of OPs that produce no overt signs of acute toxicity are poorly understood. Further, there is clinical evidence that such low-level exposures to OPs leads to prolonged deficits in cognition, although the mechanism for this effect is unknown. One long- term goal of our laboratories is to elucidate the mechanisms responsible for the prolonged neurobehavioral deficits associated with chronic low-level OP exposures such that more effective therapeutic strategies can be developed. The results of our experiments conducted during the initial funding period established that low- level exposures to the commercial pesticide, chlorpyrifos, resulted in protracted deficits in prepulse inhibition (a model of pre-attentive processing) and spatial learning without significantly affecting locomotor function. Further, chlorpyrifos was associated with decreases in neurotrophin receptors and cholinergic proteins in brain regions that are important to cognitive function. These deficits were accompanied by decreases in axonal transport measured in sciatic nerves ex vivo. However, the molecular mechanisms for the deficits in axonal transport and the extent to which such effects on axonal transport occur in the brain are unclear. The objective of this application is to identify the mechanisms responsible for alterations in axonal transport as well as to further define the long-term effects of low-level OP exposure on cognitive function. Our central hypothesis is that OPs covalently modify key proteins that are involved in axonal transport and that such modifications compromise the function of neuronal pathways that support cognitive function. To achieve our objective, we propose three specific aims: 1) Determine the consequences of chronic low-level exposure to representative OPs on attention and cognitive flexibility, 2) Determine the consequences of chronic low-level exposure to representative OPs on axonal transport in the brain, and 3) Identify the molecular mechanisms responsible for OP-induced deficits in axonal transport. To address these aims, we will use a five choice serial reaction time task to assess sustained attention, a water maze task to measure extinction (a form of cognitive flexibility) and stereotaxic injections of traceable dextrans, immunohistochemistry, and mass spectrometry to determine OP effects on axonal transport in the brain and the consequences of its impairment. The significance of this project and its relevance to public health is that by mechanistically defining OPs based on their long-term effects on essential components of information processing in animals, we will have addressed a fundamental gap in our knowledge of how OPs likely affect humans over time. The experiments will contribute to a better understanding of the toxicity associated with a class of chemicals that continues to pose a significant environmental risk to millions of people worldwide."}
{"text": "Hepatitis C may in time result in progression damage to the liver and lead to cirrhosis or liver cancer. Interferon alone or in combination with Ribavarin are the only FDA-approved therapies for Hepatits C infection. Treatment in the early stages may prevent progression to cirrhosis. Interferon has several undesireable side effects, therefore, investigators are looking for new drugs that may be useful in the treatment of Hepatitis C. Amantadine is a drug that was developed 35 years ago for the treatment of the flu. Previous research has shown that amantadine may be useful for patients with Hepatitis C and results in a 30% response rate when given at a dosage of 200mg daily. The purpose of this study is to evaluate the efficacy of amantadine in hepatitis C with higher doses of the drug."}
{"text": "Sickle cell disease (SCD) is a systemic ilness effecting over 50,000 patients in the U.S. Transfusions are an important component in the the treatment program for many of the serious complications of SCD. Although a life saving therapy, transfusions also present complications and the persistence of donor white blood cells following transfusion may underlie the immunomodulatory phenomena. This study will examine, prospectively, the natural history of transfused white blood cells and correlate the observations with clinical and laboratory features considered etiologically related."}
{"text": "Project Summary. Estrogen receptor positive (ER+) breast cancers comprise the majority (~70-80%) of breast cancers, the majority of late recurrences emanating from indolent or dormant breast cancer, and the majority of breast cancer deaths resulting from metastatic disease. Anti-endocrine therapy with tamoxifen remains the cornerstone of adjuvant therapy for ER+ breast cancers, particularly in premenopausal women, but also following treatment with aromatase inhibitors in the post-menopausal setting. Nevertheless, many women do not respond to tamoxifen in initial therapy, and of those that do, one third will relapse with resistant and metastatic disease within 15 years. Endocrine resistant ER+ breast cancers therefore remain one of the major causes of breast cancer metastasis and mortality. In fact, initial or acquired resistance to tamoxifen is involved in more than half of all ER+ breast cancer deaths. Reversing resistance to tamoxifen therapy is a crucial overarching breast cancer challenge. We provide preclinical research demonstrating the discovery of a crucial axis that provides tamoxifen resistance to ER+ breast cancer cells mediated by the epidermal growth factor receptor (EGFR)/estrogen receptor ? (ER?) pathways, and their impact on selective mRNA translation through the kinase mTOR. Moreover, we demonstrate that there are several experimental drugs developed for other indications that can be repurposed to target this axis for the treatment of tamoxifen resistant ER+ breast cancers. Tamoxifen is an estrogen receptor antagonizing small molecule used for treatment for ER+ breast cancer worldwide. Resistance is well established to commonly involve overexpression of EGFRs on breast cancer cells, and hyper-activation or increased signaling of the MAPK-ERK and PI3K-Akt-mTOR pathways. We have now identified two novel hyperactivated mediators of resistance to tamoxifen therapy that lie at the intersection of these key pathways, and we show their importance in resistance. The two effectors of tamoxifen resistance are the inhibitor of translation initiation factor eIF4E, known as 4E-BP1, and the phosphorylation of eIF4E by hyper- activation of its ERK associated kinase, MNK1. Both are therapeutic targets for existing experimental drugs developed for other purposes with good toxicity profiles. eIF4E comprises the basic translation component for loading ribosomes onto mRNAs. Many studies by my group and others have shown that increased phosphorylation of eIF4E is controlled by the ERK-MNK1 pathway, and its increased abundance is controlled by the mTOR/4E-BP1 pathway, which selectively upregulates translation of specific mRNAs required for survival, proliferation and metastasis of breast cancer cells."}
{"text": "Eye pathologies, such as cataracts and macular degeneration, are the leading causes of impaired vision and blindness in the world. Although these pathologies form as a natural process of aging, there are several environmental and lifestyle factors that increase the risk of these diseases, such as cigarette smoking, exposure to light and ultraviolet radiation, oxidative stress, steroid treatment and stress-induced DNA damage. We propose to investigate the role of the metal-responsive transcription factor 1 (MTF-1) in the transcriptional regulation of a suite of genes involved in eye development that may also contribute to environmental stress- related eye pathologies. The main objectives of this proposal are to determine what role MTF-1 plays in normal eye development and whether or not inappropriate activation of MTF-1 signaling by cellular stress in the eye can lead to misexpression of genes that promote eye pathologies. Zebrafish embryos and adults will be used as our model organism and comparative experiments will be performed in a human primary lens epithelial cell line. Targeted gene expression assessments by in situ hybridization, real-time PCR and global transcriptomic profiling via RNA-seq will be used to determine the effects of MTF-1 activation or inhibition on the regulation of eye-specific genes. We have chosen the toxic metal cadmium as our model environmental risk factor because it is one of the best known activators of MTF-1 signaling and because it is believed to be a direct causative factor in the increased risk of cigarette smokers to cataractogenesis and macular degeneration. ChIP-Seq analysis complementary to the microarray expression profiling will be used to determine DNA binding by MTF- 1 as confirmation of a direct role in the regulation of genes responsible for eye development and stress-related pathologies. Additional promoter characterization via transient transfection assays and chromatin immunoprecipitation will be used for final confirmation of MTF-1 regulation of the eye-specific genes in zebrafish and humans. The results obtained from these experiments will yield public health significance by providing novel insight into mechanisms of action by which environmental stressors contribute to eye pathologies. In addition to the discovery of a potential new regulatory pathway involved in eye development with new insight into cataractogenesis and macular degeneration, successful completion of the proposed research will further expand the current use of zebrafish as a model organism for eye disease-related research."}
{"text": "The long-term objective of the research program is to induce immune tolerance to allogeneic organ transplants such that immunosuppressive drugs are not required to maintain permanent graft acceptance. Previous studies in the program have shown that tolerance to heart allografts can be achieved using combined heart and bone marrow transplantation in wild-type MHC-mismatched murine hosts that have been conditioned with total lymphoid irradiation (TLI)and anti-thymocyte serum (ATS). The hosts become stable mixed chimeras without the development of graft versus host disease (GVHD). Tolerance induction and prevention of GVHD is dependent on host regulatory natural killer (NK)T cells that become the predominant residual T cell subset after TLI and ATS conditioning. Our recent studies show that tolerance and GVHD prevention is also dependent on the development of donor Treg cells and host Treg cells that are not NKT cells. A hypothesis that explains the results is that host NK T cells interact with host and donor APC's, and then augment/activate the non-NK Treg cells that provide alloantigen specificity for tolerance induction. Whereas the regulatory activity of the NK T cells is IL-4 dependent, that of the non-NK Treg cells is IL-4and IL-10 dependent. The hypothesis will be tested by adding back purified NK T cells and non-NK Treg cells from wild-type, Treg deficient, and cytokine deficient (i.e. IL-4\"'\", IL-10\"'\") host and donor type mice to appropriate TLI/ATS conditioned hosts. The conditioned hosts will receive combined MHC-mismatched organ and bone marrow transplants, and graft acceptance and GVHD will be monitored. The phenotype and cytokine dependence of the non-NK Treg cells will be determined as well as the dependence of NK T cell activation on interaction with APC's. Our recent studies show that the NK T cells in wild type mice are far more resistant to apoptosis induced by TLI/ATS conditioning than conventional T cells. However, the differential resistance is lost in p53\"'\" mice and in Bcl-2 transgenic mice. We will determine whether the ability to induce tolerance and prevent GVHD is also lost in the latter genetically altered mice."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The child obesity epidemic threatens a worldwide health care crisis. Prenatal, early infancy, and age at adiposity rebound are critical periods for development of persistent obesity and its co-morbidities. Early childhood growth patterns are associated with risk of obesity at later age. Interventions targeting the prevention of child obesity could be more effective if directed at the individual children who are likely to become overweight or obese later in life. Our aims are to characterize the trajectories of temporal change in age and sex adjusted BMI-z score, to explore the relationship of the pattern of trajectories with known correlates and consequences of child obesity. These aims will be achieved in two preliminary studies. In the first study, we will include children who were born between January 2003 and December 2005 and had the first well child visit at a Nemours clinic within the first month of birth and had at least one well child visit each year for the next 5 years. We will collect data related to demographics, socio-economic and co-morbidities from Nemours Electronic Medical Record (EMR). We will calculate age and sex adjusted BMI-z score for each subject and classify subjects in groups based on pattern of the correlation structure of change in BMI-z score of each individual. In study 2, we will select a sample of 600 patients from the stratified groups in study 1 and collect retrospective data on known correlates of childhood excess weight gain that are not available in the EMR. The proposed study will enable us to classify children in terms of their risk of excessive weight gain to ensure a stratified randomization in a future randomized control trial to evaluate three family based interventions targeting the prevention of excess weight gain in early childhood."}
{"text": "Congenital cataract is both an ophthalmologic and social problem. The role of hereditary has shown it forms 8.25% of all congenital cataracts. A genetic analysis in humans is not possible due to generation times and small numbers of progeny. Mouse congenital cataract mutants Cat-Fraser (Cat-Fr) and Lop provide an excellent system to study inheritance of cataract. They also provide a method to undertake in vivo and in vitro research on the growth, replication and gene expression in lenses of the cataractous mice. Breeding experiments are presently in progress to assign a specific chromosome for the dominant inherited Cat-Fr mouse. Chromosome 10 has already been mapped for the Lop cataract. Experimental chimeras formed from the aggregation of preimplantation embryos of cataractous and non-cataractous mice will attempt to \"rescue\" the abnormal lens defects. Blastocyst injection of cataractous cells will analyze the contribution of these cells to normal tissue. In vitro studies on clonal populations of mouse lens epithelial cells derived from Cat-Fr, Lop and normal lenses, will characterize morphological changes with replication and growth patterns, e.g. nuclear and cell size, distribution of microtubules, intermediate filaments and microfilaments, synthesis of specific lens proteins. Those in vitro parameters which are found to be associated with certan phases of the normal lens cells replication and/or growth pattern, will serve as a base line for similar studies on the lens epithelial cells derived from the cataractous mice. Somatic cell hybridization and micromanipulation techniques will be employed to study the possibility of manipulating the in vitro parameters."}
{"text": "This pilot study aims to evaluate the safety & toxicity & tolerance of combination cyclophosphamide, doxorubicin, vincristine, and prednisone (modified-chop) in combination w/triple antiretroviral therapy consisting of the protease inhibitor indinavir and 2 anti-retroviral transcriptase agents, 3tc and 4d4t."}
{"text": "The interaction of HDL apolipoproteins, particularly apoA-I, with the cell membrane transporter ABCA1 removes excess cellular cholesterol and protects against atherogenesis. This process is mediated by lipid- poor apolipoproteins generated by either de novo synthesis or dissociation from HDL particles. Thus, factors that impair the generation or lipid efflux activity of apoA-I could have profound atherogenic effects. Oxidative damage is implicated in the pathogenesis of atherosclerosis, a chronic inflammatory disease. 3- Chlorotyrosine and 3-nitrotyrosine, stable products of protein oxidation generated by phagocyte-derived hypochlorous acid (HOCI) and reactive nitrogen species (RNS), have been detected in human atherosclerotic lesions. However, the underlying factors that control tyrosine oxidation in proteins remain poorly understood. We found that oxidation of apoA-I by HOCI severely impairs its ability to remove cellular cholesterol by the ABCA1 pathway, consistent with the possibility that oxidation of HDL apolipoproteins in vivo would be atherogenic. We propose to test the hypothesis that site-specific oxidation of tyrosine residues by phagocyte-derived HOCI and RNS alters the biological and atheroprotective function of HDL. We will characterize the effects of site-specific oxidation of tyrosines in apoA-I on its ability to remove cellular cholesterol, identify protein motifs that direct site-specific tyrosine oxidation by HOCI and RNS, and determine if HDL is a physiological target for oxidative modification in the artery wall. This project will use HPLC and tandem mass spectrometry to locate and quantify the sites of tyrosine oxidation in apoA-I and model peptides, cell biology procedures to characterize the effects of apoA-I oxidation on lipid transport activity and interactions with ABCA1, tissue analysis to identify and characterize oxidized apoA-I in human atherosclerotic lesions, and mouse model approaches to test for the effects of macrophage-generated HOCI on apoA-I oxidation and atherogenesis in vivo. The proposed studies will provide insights into the structural features that direct oxidative modification of proteins, with important implications for the physiological significance of oxidative reactions in atherosclerosis and other inflammatory diseases."}
{"text": "The tick borne flaviviruses (TBFV) includes Tick borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus, Kyasanur forest disease virus, Powassan virus and Langat virus (LGTV). The TBFV are listed among the NIAID category B and C lists of priority for research on pathogenesis, to identify novel targets for therapeutic and vaccine development. As their name suggests, these viruses are transmitted by ticks, and following infection of humans, cause encephalitis, meningitis or hemorrhagic fevers resulting in approximately 10,000 to 13,000 hospitalizations annually with mortality rates as high as 40%. The TBFV belong to the Family Flaviviridae, genus Flavivirus, which comprise some of the most medically significant emerging and re-emerging pathogens. Other members include the mosquito-borne West Nile virus (WNV), Japanese encephalitis virus (JEV), dengue virus (DEN) and yellow fever virus (YFV). Hence, research into the pathogenesis of TBFV will reveal insight into the biology of this globally important group of viruses. The research in our laboratory aims to identify and understand interactions between the TBFV and their hosts (both the tick and the mammal) critical to virus replication and pathogenesis. We have been studying LGTV which is a naturally attenuated member of the TBFV that shares approximately 80% identity with TBEV at the amino acid level. This makes LGTV an excellent model to gain insight into the TBFV. The main studies ongoing in the laboratory are outlined below. 1. Study of virus interactions with the invertebrate host. Ticks represent the natural reservoir of TBFV and therefore are critical for virus persistence in nature and are the major source of infection for humans. We have investigated global transcriptional changes in Ixodes scapularis nymphs infected with LGTV using custom Agilent microarrays. The microarray was based on sequences from an mRNA library derived from salivary glands of Ixodes scapularis ticks. The aim of this work is to identify tick host proteins important for the replication or transmission of TBFV. These proteins can be used for the development of novel anti-tick vaccines that prevent virus transmission to the immunized mammalian host. We first examined transcriptional changes in uninfected ticks during feeding on mice. This revealed clusters of genes that were differentially regulated, particularly on day 0, 1 or 3 post tick attachment. Preliminary results from experiments with LGTV-infected ticks have identified a subset of these genes that are affected by infection. We are currently working to confirm these results and will then determine the immunogenic potential of those gene products. 2. Interactions between TBFV and host innate responses to infection. Following the bite from an infected tick, dendritic cells (DCs) resident in the skin of the mammalian host are among the first cell types infected by TBFV. These cells have important roles in innate immunity through the production of interferon (IFN), cytokines and chemokines, as well as in orchestrating adaptive immunity. Thus, early interactions between these cells and TBFV are likely to have a major influence on the outcome of infection. We have initiated a project in the lab to investigate the effects of TBFV infection on primary DCs and the ability of TBFV to modulate the signal transduction pathways involved in detecting virus infection and producing IFN. Following infection with LGTV, flow cytometry was used to distinguish between infected and uninfected bystander DCs. Maturation profiles revealed that infected DC, but not bystander cells, increased cell surface expression of MHC class II. However, infected DC did not upregulate CD80 and CD86, costimulatory molecules involved in activating T cell responses. Moreover, infected DC did not respond appropriately to stimulation with toll-like receptor (TLR) 3 or TLR4 ligands. The failure of LGTV-infected DC to express key molecules involved in anti-viral immunity in vivo may contribute to viral evasion of immune responses. Hence, examining these interactions will lead to an understanding of the mechanisms that contribute to TBFV pathophysiology. Type I and type II IFNs are crucial elements of the innate immune response to flavivirus infection, restricting virus replication, dissemination and lethality in mouse models. Type I IFN is a potential therapeutic candidate for flavivirus infection. However, such treatment often fails. We have previously shown that LGTV utilizes its nonstructural protein NS5 to interfere with IFN signaling by directly inhibiting Janus kinase-signal transducer and activator of transcription (JAK-STAT) signal transduction. Over the past year, we have shown that NS5 from other flaviviruses including TBEV also suppress IFN responses. We also found that the virulent New York (NY99) strain of WNV suppressed IFN-dependent JAK-STAT signaling. In contrast, NS5 from Kunjin virus (KUN), a naturally attenuated subtype of WNV, was a poor suppressor of IFN responses. Importantly, mutation of a single residue in KUN NS5 to the analogous residue in WNV-NY99 NS5 (S653F) rendered this protein an efficient inhibitor signaling. Thus, a naturally occurring mutation is associated with reduced ability of NS5 to function as an IFN antagonist and hence may influence virulence of WNV field isolates. This work identifies NS5 as a potential virulence factor of WNV and has implications for live-attenuated vaccine design."}
{"text": "In order for sister chromosomes to be faithfully segregated to opposing daughter cells during cell division, each chromosome must possess a single centromere. The lack of a centromere or the creation of dicentric chromosomes (those possessing two centromeres) leads to chromosomal breakage and missegregation of chromosomes during mitosis. In turn, aberrant chromosome segregation may give rise to developmental abnormalities and neoplastic disease. Furthermore, a better understanding of the components of the human chromosome that govern transmission of genetic information may be important in the development of novel gene therapies. Specific Aim 1: By developing a transgenic mouse containing a conditional disruption of the endogenous mouse CENP-A gene, these studies seek to determine whether CENP-A protein is required for the establishment and maintenance of the mammalian centromere. Specific Aim 2: Furthermore, using a yeast two-hybrid screen, this study endeavors to identify proteins that interact with CENP-A that may be involved in centromere specification and kinetochore assembly. Specific Aim3: Finally, the studies proposed here are designed to determine whether CENP-A itself is sufficient to recruit centromere components and ultimately specify the centromere."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Glycoxidation or advanced glycation endproducts (AGE) accelerate oxidative mechanisms, resulting in activation of transcriptional pathways, excessive proliferative/growth-related phenomena and sustained inflammation leading to clinical aging. These are further accelerated by diabetes or by conditions associated with increased production or decreased AGE clearance, eg. renal insufficiency. A major source of AGE precursors and oxidant-stress is identified to be the western diet. The turnover of AGE involves specific AGE receptors and depends on renal function. Advancing age is known to be associated with increased OS, increased prevalence of cardiovascular disease, impaired glucose tolerance, diabetes mellitus, renal decline, as well as with accumulation of AGE. While efforts have linked AGE-mediated OS and chronic complications in aging animals, human studies are strikingly lacking. We plan to test the hypotheses that: 1) older men and women (ages 60+) who consume standard (high AGE) diets will have higher serum AGE in conjunction with higher OS, and markers of vascular dysfunction or inflammation, compared to age-matched subjects consuming low AGE diet, and to younger (less than 35 years) subjects and 2) AGE-restriction, by dietary modification, will reduce the total AGE burden, oxidative and inflammatory mediators and attenuate the differences between older and younger groups. The Specific Aims are to determine: 1) the correlation of circulating levels of defined AGE and inflammatory markers (eg CRP, TNFa, and IL-6) in older and younger groups with the dietary intake of AGE. 2) The effect of dietary AGE restriction on AGE and AGE-dependent parameters of OS and inflammation. 3) The effect of environmental (dietary) glycotoxic burden on AGE receptor-mediated removal mechanisms in PBMN from older and younger persons. It is hoped that the data will lay the groundwork for future studies evaluating optimal methods of nutrient preparation as ways to ameliorate a major environmental promotor of pro-oxidant events, and thus aging."}
{"text": "PROJECT SUMMARY/ABSTRACT ? PROJECT 1 The CD226/TIGIT pathway is a novel co-stimulatory/co-inhibitory pathway that closely parallels the CD28/CTLA-4 pathway. Similar to CD28 and CTLA-4, CD226 and TIGIT share ligands (CD112 and CD155) and ligand engagement of CD226 co-stimulates T cell activation while engagement of TIGIT inhibits T cell responses. Over the last few years, TIGIT has emerged as an important co-inhibitory receptor. While initial experiments indicated that TIGIT inhibits T cell responses indirectly by promoting tolerogenic phenotype in dendritic cells, we, and others, have shown that TIGIT has T cell-intrinsic inhibitory functions. We have now found that TIGIT is co-expressed with other co-inhibitory receptors (PD-1,Tim-3,Lag-3) on CD8+ T cells that exhibit dysfunctional/exhausted phenotype in chronic diseases such as cancer and chronic viral infection. Importantly, we find that expression of TIGIT marks the most dysfunctional subset of CD8+ T cells in these conditions, thus raising the important issue of how TIGIT co-operates with other inhibitory receptors to drive severe dysfunctional phenotype in effector T cells. In addition to its role in effector T cells, we have recently identified that TIGIT marks a distinct subset of regulatory T cells (Treg) that exhibit heightened expression of known Treg effector molecules and increased suppressive capacity. Importantly, we find that TIGIT+ Treg are highly enriched in inflamed tissues and that they exhibit specialized function; selectively suppressing pro- inflammatory Th1/Th17 responses while sparing Th2 responses. However, how TIGIT may function in CD8+ T cells to dampen their responses while in Treg function to direct specialized Treg function is not known. Given the data supporting important roles for TIGIT in regulating effector T cells and Treg, it is surprising that nothing is known regarding the molecular signals that induce TIGIT expression. Our preliminary data indicate that the immunomodulatory cytokine IL-27 induces TIGIT expression both in vitro and in vivo. We have now leveraged this information to identify candidate transcription factors that are downstream of IL-27 and may drive TIGIT expression in effector T cells. Based on our data, we hypothesize that TIGIT has a dual role in regulating T cell responses: 1) TIGIT co- operates with other co-inhibitory molecules to regulate effector T cell phenotype and 2) TIGIT drives highly active and specialized Treg phenotype in tissue. We have generated a number of tools including agonist and antagonist antibodies and conditional knock-out mice to study the function of this emerging pathway in the regulation of T cell responses. Our specific aims are: 1) Dissect the T cell intrinsic and extrinsic roles of TIGIT in regulating T cell activation and dysfunction 2) Determine the role of TIGIT in directing Treg function in inflammatory conditions"}
{"text": "This project involves a study of the regulation of histidine and threonine catabolism by concentrating on the control properties of the key enzymes histidase, urocanase and threonine dehydratase. The majority of the investigation deals with these enzymes in, or isolated from Pseudomonas putida. Threonine dehydratase of this organism is a dual function enzyme, being involved in both the biosynthesis of isoleucine and in the catabolism of threonine. Moreover, extensive mutant analyses indicate that the enzyme is required for efficient expression of the genes controlling the formation of the histidine utilizing enzymes. In the project we are attempting to confirm the existence of a covalently modified form of the enzyme by enzymological studies of the purified enzyme and by mutant construction and analysis. We propose that the covalent modification, probably a nucleotidylation, is regulated by the availability of an efficient nitrogen source and is employed by the cell as a means of directing threonine dehydratase activity towards isoleucine biosynthesis. Also planned are related studies on other control phenomena which may affect the biosynthesis of histidase, urocanase and other histidine-degrading enzymes. Of primary concern are the roles of cyclic AMP and glutamine synthetase in favoring transcription of the genes for these enzymes. Mutants altered in cyclic AMP production or glutamine synthetase activity are being isolated to facilitate these studies. The nature of allosteric regulation of histidase activity by pyrophosphate will also be examined with purified histidase. Further studies will be made on the urocanase of P. putida which has been recently found to be NAD positive dependent. Mechanistic analyses, primarily isotope exchange and NMR studies, will be conducted along with NAD positive binding measurements. Purification of beef liver urocanase will be attempted to permit generalization of the role of NAD positive as a coenzyme for urocanase."}
{"text": "DESCRIPTION: (from applicant's abstract) An important new hypothesis for understanding and treating drug addiction is that it may be an inappropriate form of learning. This hypothesis is based on studies demonstrating that glutamate, a transmitter critical for learning and memory, is required for the development of neuroadaptations believed to underlie addiction and relapse. We have shown that the repeated administration of psychostimulants (amphetamine or cocaine) causes profound alterations in glutamate systems. These include alterations in AMPA receptor expression and responsiveness in the nucleus accumbens (Nac), a brain region critical for the rewarding and addictive properties of psychostimulants. Medium spiny GABA-containing neurons, which represent 95% of all neurons in the Nac, receive convergent inputs from midbrain dopamine (DA) neurons and glutamate neurons originating in cortex and limbic regions. Our long-term goal is to understand how psychostimulants, which initially target the DA transporter, ultimately produce adaptations in glutamate transmission in the Nac. This is an important question, because glutamate systems are likely to mediate the neuronal plasticity underlying the transition from drug experimentation to drug dependency. Unfortunately, we know little about chronic interactions between DA and glutamate receptors in the Nac. A logical starting point is to determine how acute DA receptor stimulation can influence glutamate transmission. This application will use postnatal Nac cultures to test the hypothesis that DA receptors modulate the phosphorylation of the AMPA receptor subunit GluR1. We are focusing on AMPA receptors because they are the primary mediators of excitatory transmission in medium spiny neurons and on phosphorylation as a regulatory mechanism because GluR1 phosphorylation leads to marked enhancement of AMPA receptor-mediated currents. Phosphorylation of GluR1 will be detected using phosphorylation site-specific antibodies to GluR1 developed by Dr. Richard Huganir. One selectively recognizes GluR1 phosphorylated on ser-845 [protein kinase A (PKA) site]. The other recognizes GluR1 phosphorylated on ser-831 [protein kinase C (PKC)/Ca2+ -calmodulin dependent protein kinase type II (CaMKII) site]. The first Aim is to characterize conditions regulating basal phosphorylation of GluR1 and the kinases involved in its phosphorylation. First, control cultures will be compared with cultures incubated in the absence of Ca2+, the absence of synaptic activity (TTX, bicuculline, MK-801, CNQX), the absence of inhibitory activity (bicuculline), and the absence of excitatory activity (MK-801, CNQX). Second, we will use activators and inhibitors of PKA, PKC and CaMKII to determine which kinases phosphorylate GluR1 in our cultures. The second Aim is to determine if DA receptor stimulation modulates GluR1 phosphorylation. Experiments will be designed to detect both stimulatory and inhibitory effects of D1 and D2 DA receptor activation. The identity of the residue phosphorylated, combined with experiments involving selective protein kinase inhibitors, will help identify the kinases involved in DA receptor-mediated effects."}
{"text": "Serum-free conditioned media from several human tumor cell lines (e.g., epidermoid carcinomas, melanomas, bronchogenic carcinomas, and rhabdomyosarcomas) and from NIH/3T3 cells transfected with DNA from a human lung carcinoma were observed to produce a class of factors which inhibits growth of human melanoma and carcinoma cells in soft agar and in monolayer cultures. These inhibitors of tumor cell growth have been designated tumor inhibiting factors (TIFs). Normal human fibroblasts and epithelial cells, however, are stimulated to proliferate by these same factors. This project is directed towards the purification and characterization of this new class of factors that inhibits tumor cell growth. We have determined some of the biochemical and biophysical properties of TIF. We have also identified a rich source of TIF in certain normal human tissues. This should facilitate its purification and characterization. This source should yield sufficient TIF for sequencing, antibody production, and in vivo experiments."}
{"text": "Project Summary Studies on the health effects of sex tourism report that sex workers are one of the most vulnerable groups for acquiring HIV due to their high rates of sexual risk behaviors1 and their disadvantaged positions to negotiate safe sex because of social, economic, and cultural factors.2 In low prevalence countries with concentrated epidemics, HIV prevalence rates among sex workers are often 3 to 4 times that of the general population.3 This risk is often perpetuated by the availability and accessibility of illegal drugs in tourism zones. While studies have examined the association between HIV risk-related behaviors including drug use and sexual risk-taking and the social and physical environment that characterize neighborhoods;4, 8-11 there is a fundamental gap in the current scientific evidence-base about which extra-individual contexts, ?activity spaces? (spaces in which daily activities occur) and social interactions or networks interact, and how that impacts HIV risk behavior. The goal of the proposed research is to utilize innovative spatial-temporal and social network methodology to describe the relationship between activity spaces and social network characteristics of male migrant sex workers in tourism zones in the Dominican Republic (DR), and to assess how such daily activity patterns and networks jointly affect HIV risk behavior, testing and infection. Considering that the DR has one of the largest sex tourism industries in the Caribbean and is currently experiencing a dramatic increase in the use and transport of illegal drugs, spurred by a shift in smuggling routes to the United States,27, 28 the country provides a unique environment for examining how environmental and social structures shape HIV risk and related behaviors among sex workers. A deeper understanding of the multiple factors that influence day-to-day experiences of male migrant sex workers is paramount for reducing the HIV burden in the DR, and for preventing international transmission through tourism and migrant routes. This study has three specific aims: (1) To utilize novel spatial-temporal methodology to characterize the activity spaces of male migrant sex workers in the Dominican Republic by linking activity space GPS data to already collected, novel ethnographic mapping data from a current NIDA-funded R01, and to examine the impact of risky activity spaces on HIV risk and related behaviors, specifically drug use, risky sex, HIV testing, and self-reported HIV; (2) To describe the social networks (e.g. size, composition) of male migrant sex workers, and assess the role social networks have on HIV risk and related behaviors in this population; (3) To examine the interaction between daily activity space and social network characteristics on HIV risk and related behaviors. If activity spaces can be identified and play a significant role in drug use and other HIV-related behaviors?including their combined role with network characteristics?it will provide critical information for creating effective structural and interpersonal HIV prevention interventions. To address the current barriers to HIV prevention and treatment, it is paramount that we focus not only on multiple social determinants, but also on the social experiences and lives of at risk populations."}
{"text": "DESCRIPTION: This application is a request for continued support of an ongoing project. The overall goal of the project is to better understand the cognitive and brain bases of covert motor processes, particularly motor imagery. The project focuses on the activity of three cortical motor areas (primary, premotor, and supplementary) during overt movement and covert motor processes. The principal measures employed are based on event-related brain potentials. These measures have the temporal resolution to detect changes in brain activity occurring on the order of milliseconds and sufficient spatial resolution to monitor separately each of the above cortical areas. In the proposed new research, these measures will be combined with magnetic resonance images, in order to correct for distortions resulting from conduction of the electric signal through the skull and to locate sources of activation on an accurate map of the cortical surface. \"Functional coupling\" between the cortical areas will be examined through measures of correlation between their moment-by-moment activities. The experiments conducted in the project to date have monitored the three motor areas under a wide range of conditions in which covert motor processes are known to take place. The new experiments will monitor these and other cortical areas during cognitive events in which covert motor processes have been hypothesized to play a role. These include observation of a skilled performance prior to its imitation, use of an \"articulatory loop \"to maintain information in working memory, and illusions evoked by viewing a moving phantom image of one's own limb . Also examined will be the covert motor processes responsible for the timing and serial order of hierarchical movement sequences, as well as the continuity between motor imagery and gentle overt movements. Findings from the project may help to 1) improve the efficacy of mental practice, observational learning, or treatment to aid motor recovery, 2) diagnose disorders of the motor system, and 3) determine the direct consequences of activity in the studied brain areas for learning, remembering, or tuning movement. At a broader level, the work will enhance our understanding of the motor system at both a functional and physiological level. Further information concerning its less known covert side may lead to unexpected insights about the motor system in general, including its possible roles in cognition."}
{"text": "A number of 4-X-bioimidazoles are accessible by direct electrophilic substitution (nitro, halo, etc.); 2-X-bioimidazoles are far less accessible and can be obtained ,only by indirect and, often, very tortuous routes. Prior to our major efforts in this area, the majority of 2-X-bioimidazoles were unknown. By far the 2-substituted imidazoles most easily obtainable are 2-iodo and 2-(trifluoromethyl). The 2-iodo derivatives of histidine and histamine are of interest, not only because of their potent antimalarial activities, but because they can serve as valuable intermediates for the synthesis of other 2-X-bioimidazoles, as can the trifluoromethyl analogues. 'We are currently exploring the utility of such conversions to prepare photosensitive bioimidazoles and affinity labels for in vivo use. Over the past 15 years, we have found consistently that 2-X-bioimidazoles (especially fluoro and iodo) have a broad range of strong biological activities but ,the corresponding 4-X-bioimidazoles are essentially inactive. Various explanations for this remarkable selectivity in biorecognition have been invalidated on experimental grounds. Our analyses of (13)C NMR spectra have now revealed that the differentiation may be based solely on tautomer preference. While 4-X-imidazoles exist preferentially as the unnatural pi-tautomers, 2-X-bioimidazoles exist preferentially natural tau-tautomers. This discovery generates broad implications in many areas of drug design, e.g., selective antihistamines, imidazole-based antimalarials and antimicrobials, CVS and CNS-selective TRH analogues, and analogues based on pyrrole and triazole regioisomerism. Thus. novel synthetic methods are being explored to make available even more 2-X-bioimidazoles."}
{"text": "The long-term goals of this project are to understand the molecular mechanisms that underlie the degeneration and death of neurons in Parkinson's Disease (PD) and to use this information in turn to devise treatments to suppress the progression of this disorder in patients. The proposed studies will test the over-arching hypothesis that neuron degeneration and death in PD are due to inactivation of the key enzyme Akt which is required for nerve cell function and survival, and that this is mediated by induction of the stress-responsive protein Trib3, an inhibitor of Akt activation. The specific aims will assess four hypotheses: (1) that Trib3 expression is elevated in PD models and in PD; (2) that Trib3 mediates neuron degeneration and death in models of PD; (3) that Trib3 promotes neuron death by interfering with phosphorylation/activation of Akt; and (4) that induction of Trib3 in PD models and PD is mediated by the transcription factor ATF4. Multiple experimental approaches will be employed. These will include assessing Trib3 expression in toxin- and alpha- synuclein based cell culture models of PD, in animal models of the disease and in post-mortem brain tissues from PD patients and controls; determining whether loss of Trib3 expression, either via shRNAs or gene deletion, protects neurons from death in cell culture and animal models of PD; ascertaining (by over- expression or loss-of function studies) whether Trib3 is responsible for mediating the loss of Akt activity that occurs in cellular models of PD; and establishing whether manipulating expression of ATF4 in culture and animal models influences Trib3 induction in culture and animal models of PD. If successful, these studies will establish Trib3 as a required element in the mechanism that leads to neuron death and degeneration in PD and will provide insight as to how it does this, and how it is induced in this disorder. Such information will be exploited in the long term to formulate new approaches to suppress progression of PD in patients. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is characterized by progressive degeneration and death of specific types of nerve cells. Current treatments are aimed at ameliorating the symptoms of this disease rather than its progression. The proposed studies seek to uncover the molecular mechanisms underlying nerve cell degeneration and death in PD so as to lead to generation of new strategies to suppress disease progression."}
{"text": "All patients developing non-A, non-B hepatitis following open heart surgery will be followed with serial studies of ALT to determine the incidence of chronic liver disease and to compare this incidence with those having type B hepatitis. Liver biopsy will be obtained when the ALT is elevated for more than six months and then again at one and three years. Biopsy material will be obtained for flourescent and EM studies, as well as light microscopy with multiple stains."}
{"text": "Ubiquitination is a versatile post-translational modification that regulates a large number of different cellular processes including signal transduction, protein trafficking, DNA repair, gene transcription and regulated protein degradation. Typically, ubiquitin is appended to a target protein as a signal that is then received and interpreted by proteins carrying ubiquitin-binding domains (UBDs). UBDs are numerous and diverse, and, in most cases, little is known about how they function in the physiological context of a full-length protein. Several protein interaction SH3 domains were recently discovered to act as UBDs, whereas SH3 domains were previously thought to act almost exclusively by recognizing proline-rich peptides. The known ubiquitin-binding SH3 domains are components of the endocytic machinery. The first-discovered and perhaps best-characterized role of ubiquitin outside proteasome degradation is as a regulator of receptor endocytosis. Numerous components of the endocytic machinery that act to internalize cargo are ubiquitinated and/or carry UBDs. Furthermore, many endocytic cargo proteins are themselves modified by ubiquitin at the plasma membrane, where ubiquitin acts as a regulated internalization signal to direct these proteins into endocytic vesicles. Specific cargo proteins that are ubiquitinated and regulated by ubiquitin-dependent endocytosis include leptin receptors, growth factor receptors, glucose transporters, ion channels, the aquaporin-2 water channel and the anthrax toxin receptor. Impairment of ubiquitin-dependent endocytosis results in, or is linked to, diseases including hypertension, cancers, immune disorders and viral infections, as well as inflammation and bacterial toxin entry into cells. Three components of the endocytic machinery that bind to ubiquitin through SH3 domains have been identified: amphiphysin, CIN85 and the yeast homologue of CIN85, SlA1. Biochemical and genetic experiments proposed in this grant will define the function of ubiquitin in regulating these proteins, and will provide a general paradigm for how ubiquitin regulates the formation of the protein-protein interactions mediated by SH3 domains."}
{"text": "This investigation is a family study on the effect of heterozygosity for an inactivating mutation of the estrogen receptor gene on bone density and other parameters of estrogen action. Excluding one nuclear family, the lumbar spine bone densities suggest that carrier status is associated with decreased bone mineral density compared to wild type; the mean spine BMD z scores were -0.9 for heterozygotes vs. -0.1 in wild type individuals (p = 0.02). In this past year, there were no outpatient visits, but the Core Laboratory of the CRC performed determinations on the collected samples. The analysis of the data will be forthcoming."}
{"text": "A central issue in neuroscience is to understand how experience shapes brain function. Recent studies in visual cortex suggest that the maturation of intracortical inhibitory circuits is necessary to initiate and drive ocular dominance plasticity during the critical period. However, the maturation and regulation of the specific type of inhibitory circuit involved is not understood. Critical period plasticity is also modulated by visual deprivation and BDNF over-expression, although the cellular mechanisms remain unknown. Here we hypothesize that the functional maturation of a specific subtype of GABAergic neurons, parvalbumin (Pv) basket interneurons, are a component of the inhibitory mechanism underlying critical period plasticity, and a cellular target of experience deprivation and BDNF regulation. Using bacterial artificial chromosome transgenic (BAC) mice expressing GFP in Pv-interneurons, we will characterize the morphological and physiological development of Pv-interneurons using two photon laser scanning microscopy and electrophysiology in brain slices. We will then examine the effects of dark rearing on the maturation of Pv-interneurons. Finally, we will test the role of BDNF in experience-dependent maturation of Pv-interneurons by blocking trkB signaling using BAC transgenic mice expressing a dominant negative form of trkB receptor specifically in Pv-intemeurons. These results will reveal a mechanism by which experience shapes function of an identified inhibitory network and provide strong evidence that maturation of the Pv-interneuron circuit facilitates the coding of visual information necessary to drive ocular dominance plasticity. The knowledge gained will aid in the design of drug treatments in diseased states such as epilepsy and schizophrenia."}
{"text": "Complex cellular networks underlie the functional foundation of the mammalian central nervous system (CMS). Understanding the physiological dynamics of these networks, in other words understanding how signaling between interacting groups of cells produce and modulate meaningful physiological information, will directly contribute to our understanding of how the CMS functions in health and how it fails in disease. At present, our mechanistic understanding of the dynamics of neuronal and glial networks is very limited, even though we understand the molecular functional unit that underlies it (i.e. the synapse). One approach is to apply network theory to characterize neuronal and glial networks. Network theory is a branch of statistical mechanics that classifiescomplex networks independent of the physical details of the network and provides an understanding of its dynamical behavior. Applying network theory to neuronal and glial networks requires knowing their structure or topology. However, high throughput computationally intensive measurementsof molecular signaling between neurons and glia, and the extraction of quantitative information about their underlying network structure is not possible given current techniques. What is needed therefore, are algorithms and softwarethat will allow the high throughput characterization and analysis of physiological neuronal and glial networks. Here, we propose to develop computational tools that will allow us to map the spatial and temporal topology of functional neuronal and glial signaling networks, and classify and analyze them within the context of network theory. We present a detailed discussion on the algorithms and programming required to do so, and illustrate the operation and validation of a beta version of such a program. We propose that using this approach, neuronal and glial networks can be classified within known mathematical networktypes and behave as dictated by the quantitative properties of the network types they are classified into. We also present preliminary experimental data showing for the first time that calcium signaling in astrocyte networks, mapped using our software tools, have a previously unidentified toplogy. We propose that the networktopologies of healthy neurons and glia remodel following injury and underlie the induction and maintenance of neuropathological disease states, making the clinical significance of these findings and the development of the computational tools required to investigate them very important."}
{"text": "The crowded environments inside cellular compartments are very different from the typical dilute conditions of in vitro and in silico biophysical studies of biomolecular systems. The long-term objective of this project is to bridge the in vitro-in vivo gap, by quantitatively reconstructing the influences of cellular environments on the thermodynamic and kinetic properties of biomolecules. Exploiting tremendous opportunities opened by our postprocessing approach for modeling effects of crowded cell-like environments and other recent advances, in this project we will (1) advance FFT-based postprocessing to achieve high accuracy in modeling crowding; (2) quantitatively delineate temperature dependence of crowding effects; and (3) characterize conformational ensembles and binding of intrinsically disordered proteins under crowding. Through capitalizing on FFT-based postprocessing and carrying out our own wet-lab studies, we will closely integrate computation and experiment to overcome challenges toward gaining insights into in vivo biochemical processes. The ability afforded by this research to use dilute-solution experiments and simulations for predicting the conformational ensembles of intrinsically disordered proteins under cell-like conditions will move us forward in elucidating their cellular functions. The conceptual advance that macromolecular crowding in cellular environments may serve as an important factor for protein stability in thermophiles could have broad implications for protein evolution and design."}
{"text": "Need for new lead compounds against Leishmania and Trypanosoma intensifies the desirability of improved screening adapted to large-scale work. We wish to exploit a hardy Leptomonas of proven high sensitivity to several standard antitrypanosomatid agents; Crithidia fasciculata; and T. mega of amphibia -- a trypanosome easily cultivable on autoclaved media, and whose position hints it may share chemotherapeutic responses with stercorarians, e.g., T. cruzi and salivarians. Minimal media for the Leptomonas and T. mega would be applied to broaden the tolerances --at present apparently narrow -- in the published medium for T. brucei. Detection of novel agents acting as antimetabolites, and mode-of-action studies of likely targets of antimetabolites, make development of physiologically lean, highly reproducible media, desirable. By playing one flagellate against the other we hope: a) to detect lead compounds, and how best to deploy the few available effective drugs; b) use of these drugs to uncover new targets for antimetabolite therapy, especially polyamines, for in related work; the Leptomonas has already served to chart cross-resistance and collateral sensitivities to several established antitrypanosomatid agents. The organisms would be grown in physiologically minimal defined media at blood pH. They would be used to explore interrelationships among polyamines, their precursors, and Mg2 ion; the former compounds are likely targets, along with nucleic acids, of the strongly cationic trypanocides. These organisms would be used also to chart the effects of other agents on trypanosomatid growth, applied singly and in combination with cationic trypanocides. Such agents might significantly inhibit: a) biosynthesis of polyamines; 2) methylation of tRNA's; 3) heme metabolism. Work would focus on ethidium, crystal violet, Antrycide, pararosaniline, methylglyoxal-bis(guanylhydrazone) (a potent inhibitor of polyamine biosynthesis), and transmethylase inhibitors such as 6-dimethylaminonicotinamide. Effective drugs and combinations would be tested against our trypanocide-resistant Leptomonas strains. Inhibition analysis would be directed at identifying metabolites (e.g., polyamines) counteracting these drugs, then assembly of these metabolites into \"rescue\" cocktails to be tested on all 3 organisms in the presence of drugs."}
{"text": "Photodynamic therapy (PDT) utilizing hematoporphyrin derivative (HPD) and a cw argon-pumped dye laser tuned to 630 nm has been shown to be a promising treatment for a number of forms of cancer. However, because tissue penetration is maximized for wavelengths greater than 700 nm, substantial effort has been directed towards finding new dyes which absorb in the near-ir (670-900 nm). The tuning range of the recently developed Ti:sapphire laser overlaps this regions, thus providing an all-solid-state alternative to the dye laser. The Phase I effort will involve cell-level experiments using two new photosensitizers: sulfonated chloraluminum phthalocyanine (CASPc) and benzoporphryin derivative monoacid A (BPD) which absorb at 680 nm and 690 nm respectively. Although a Ti:sapphire system can be operated either pulsed or cw in this wavelength region, the initial experiments will be performed with a pulsed laser, since the cw Ti:sapphire would produce results that were very similar to the cw dye. Although the primary cause of cell necrosis under cw irradiation is the creation of singlet oxygen, the high intensity levels generated by the pulsed laser may photosensitize by non-oxygen-dependent mechanisms. The long term goal would be to develop a clinically qualified solid-state laser for PDT."}
{"text": "Influenza viruses are classic examples of antigenically variable pathogens, and have a seemingly endless capacity to evade the immune response. For example, since the influenza A(H3N2) subtype entered the human population circa 1968 the vaccine against it has had to be updated 24 times to track the evolution of the viral quasispecies and to remain effective. Most of the bioinformatics methods for analyzing viral evolution are based on genetic analyses; however, it is the phenotypic (antigenic) properties of the virus that determine its success at escaping prior immunity and causing infection. Indeed, the antigenic data are the primary criteria for selecting the virus strain used in the influenza vaccine, and which are important for much basic and applied research on influenza. However, there is no reliable method to determine quantitatively antigenic differences. Antigenic differences are typically determined using some form of binding assay (for influenza virus, the hemagglutination inhibition assay, for other pathogens a neutralization assay, ELISA, etc.). In such assays, typically a panel of antisera is titrated against a series of antigens and the data are organized in tabular form and analyzed by eye. This has been the procedure for over 50 years. These data are difficult to interpret quantitatively, and sometimes even for experts give an inconsistent picture. The primary reason for this difficulty is that the data contain irregularities, or paradoxes. On such irregularity is that one antiserum might detect a difference between two antigens, while another will not. Another irregularity is that heterologous titers are sometimes higher than homologous titers. Furthermore, it is often difficult to compare data from different laboratories. These, along with other irregularities result in binding assay data only being considered reliable enough to judge large antigenic differences?in the case of influenza virus differences of sufficient magnitude that they necessitate an update of the vaccine strain. By only being able to judge gross differences among the thousands of influenza strains characterized each year, one misses opportunities to optimize the vaccine strain choice, to detect signals in the evolution of the viral quasispecies that could give advance warning of the necessity to update the vaccine, to understand the epidemiology of influenza, to judge how vaccination affects the viral evolution, and to devise novel intervention strategies which have the potential to fundamentally change our options to control epidemic and pandemic influenza. Influenza virus is one example of an antigencially variable pathogen;others include human immunodifficiency virus and hepatitis C virus. The degree of antigenic diversity will increase as interventions increase selection pressure to generate escape mutants, and the characterization of these phenotype differences will thus only increase in importance."}
{"text": "Vasopressin (AVP), the antidiuretic hormone is a peptide hormone actively involved in the regulation of body fluid osmolality, blood volume, blood pressure, and cell proliferation via the stimulation of specific membrane-bound receptors classified into V1a-vascular, V2- renal, and V3-pituitary subtypes having distinct pharmacological profiles and intracellular second messengers. The secretion of AVP is an absolute requirement for the maintenance of fluid homeostasis as shown in human and experimental models of diabetes insipidus. The usefulness of natriuretic agents has been demonstrated in conditions associated with water and salt retention. However, there are diseases (hyponatremia of congestive heart failure, liver cirrhosis, nephrotic syndrome, and the syndrome of inappropriate secretion of antidiuretic hormone, SIADH) due to the inappropriate release of vasopressin and characterized by an excess retention of water. An agent that increases urine output by producing a free water diuresis would be of significant advantage in these conditions. This study is designed to evaluate the efficacy, safety, and tolerance of a single oral dose of VPA-985 in patients with hyponatremia due to SIADH. In addition, preliminary pharmacokinetic and pharmacodynamic parameters will be assessed. This study has just begun patient recruitment. Four to six patients with diagnosed SIADH between the ages of 18 and 75 years will be enrolled at this site."}
{"text": "This application addresses the broad Challenge Area (01) Behavior, Behavioral Change, and Prevention and specific Challenge Topic, 01-AA-102*: Functional Roles of Neuroimmune Factors in Mediating Behavior. The title of this proposal is \"Alcohol-chemokine interactions and neurotransmission\". Emerging research implicates a role for the innate immune system of the brain in the effects of alcohol on behavior. Astrocytes and microglia, the primary cell types that comprise innate immune system of the brain, are normal components of the brain and serve essential roles in normal brain development and function. Microglia and astrocytes also play critical defensive and repair roles during adverse conditions. Basic to their roles during both normal and adverse conditions is the production of neuroimmune factors such as cytokines and chemokines, which are signaling molecules that initiate or coordinate cellular actions appropriate to the need, be it cellular development, normal cell function or a defense/repair response to adverse conditions. Recent studies provide strong support for a role of neuroimmune factors, and in particular the chemokine CCL2, in alcohol use and abuse. Importantly, acute alcohol has been shown to increase levels of CCL2 in the hippocampus, a brain structure that is essential for cognitive functions such as short-term memory. CCR2, the receptor for CCL2, has been shown to be expressed in abundance in the hippocampus. Moreover, research has identified the hippocampus as one of several brain regions that play a central role in the cognitive deficits produce by alcohol abuse. An important target of alcohol action in the hippocampus is synaptic transmission and plasticity at the Schaffer collateral to CA1 synapse. Our studies show that CCL2 also alters the functional properties of this synaptic pathway. Thus, hippocampal synaptic function is a target of both CCL2 and alcohol action and a likely site for interactions between alcohol and CCL2 that could be a critical factor in the behavioral effect of alcohol. Alcohol actions in the hippocampus have been well documented but Information on the effects of CCL2 on hippocampus function is limited and interactions between alcohol and CCL2 at the level of neuronal function and synaptic transmission have not been studied. This information is critical to an understanding of the role of CCL2 in alcohol use and abuse and is the topic of the proposed studies. The studies will test the hypothesis that activation of the CCL2 signaling pathway in the hippocampus alters the actions of alcohol on hippocampal synaptic function and that these interactions between CCL2 and alcohol are also manifested at the behavioral level. To test this hypothesis, we will use variety of experimental approaches including electrophysiological recording of synaptic function, biochemical assessment of signal transduction pathway activation and behavioral testing. The studies will be carried out in transgenic mice that express elevated levels of CCL2 in the brain and their non-transgenic littermates as controls. Taken together, these studies will provide important new information that will significantly advance our understanding of the role of neuroimmune factors in alcoholism."}
{"text": "During the past year we have done extensive analyses of the data from both the child and adult components of the NHANES surveys. Two papers have been published or are in press and four others have been submitted. Below we describe the substantive issues addressed in this work. [unreadable] 1) We have completed the analyses of the prevalence estimates of GAD, panic, eating disorder, major depressive disorder/dysthymic disorder, ADHD and conduct disorders for the overall sample, by informant (child or parent), gender, race/ethnicity, and age group. In order to assess the severity of these conditions, we have also calculated prevalence by four alternative impairment algorithms. These data will provide the first population prevalence data on these seven major mental disorders in children ages 8 through 19 in a national probability sample of the US. [unreadable] 2) A separate project set out to describe the prevalence, demographic correlates, comorbidities, and service patterns for nocturnal enuresis. There are no published nationally representative prevalence estimates of nocturnal enuresis among children in the United States using standardized diagnostic criteria. The overall 12-month prevalence of nocturnal enuresis was 4.45%. The prevalence in boys was significantly greater than that in girls. Nocturnal enuresis was more common at younger ages and among black youth. ADHD was strongly associated with nocturnal enuresis. Only 36% of the enuretic children had received health service for nocturnal enuresis. Nocturnal enuresis is a common condition among children in the US. Few families seek treatment for nocturnal enuresis despite the potential for adverse effects on emotional health. Child healthcare professionals should routinely screen for nocturnal enuresis and its effects on the emotional health of the child and the family. Assessment of ADHD should routinely include evaluation for nocturnal enuresis and vice versa. Research on the explanations for the association between nocturnal enuresis and ADHD is indicated.[unreadable] 3) We investigated the prevalence of mental and physical comorbidity and health service utilization among 3,042 children aged 8 to 15 from the 2001 2004 NHANES. There was a strong association between mental and physical disorders. After adjusting for social and demographic characteristics, comorbidity was associated with a 2.5 times increase in healthcare utilization compared to those without comorbidity. Our findings confirm prior local population studies of comorbidity among youth and suggest that physical and mental comorbidity has a strong impact on healthcare utilization. Integrated treatment of mental and physical disorders is clearly indicated. Future studies should examine the explanations for systematic patterns of comorbidity in youth.[unreadable] 4) To determine the prevalence, sociodemographic correlates and comorbid medical conditions of recurrent headache in U.S. children, we investigated 10198 children aged 4 to 18 years old who participated in the 1999 to 2004 National Health and Nutrition Examination Surveys. We found that frequent or severe headaches including migraine in the past 12 months were reported in 17.1% of children, with greater rates among girls and older adolescents. Children with headache had more school absences and health care utilization. Asthma, hay fever, and frequent ear infections were more common in children with headache, with at least one of these occurring in 41.6% of children with headache vs. 25.0% of children free of headache (p<.0001). Children with headache were 13.6 times more likely to have all three of these conditions. We conclude that recurrent headache in childhood is extremely common and is associated with a variety of other medical conditions, particularly those with immunologic and inflammatory bases. The high frequency of recurrent headaches and their association with school absences suggest that headaches in U.S. youth have major public health significance. These results are a significant first step towards research to understand biologic mechanisms, identify more homogeneous subgroups in clinical and genetic studies, and develop better clinical management.[unreadable] 5) Our analytic team investigated patterns of mental and physical comorbidity among adults with severe headaches and migraine using merged data from the 1999-2004 survey years. The analyses include a comparison of the sociodemographic characteristics of individuals with versus without headaches as well as a description of the rates of comorbidity of both mental and physical disorders within both headache groups. In order to assess the relative impact of these comorbid conditions, we compared the responses to a series of questions on health care utilization and health perception across five groups (no headache, headache only, headache plus any physical condition, headache plus any mental condition, headache plus a physical and mental condition). [unreadable] 6) To understand the association between severe/recurrent headache and a range of cardiovascular and immunologic measures in the adult U.S. population, we used NHANES 1999-2004 surveys data including 14,503 adults and found that the major laboratory correlates of cardiovascular disease assessed in the NHANES including C-reactive protein, folate, homocysteine and HDL cholesterol differed among those with severe headaches/migraine compared to their non-headache counterparts. Moreover, people with severe headaches or migraine were more likely to have high BMI and diastolic blood pressure. These findings suggest that inflammatory mechanisms may play an important role in the pathophysiology of migraine as a risk factor for stroke and cardiovascular disease.[unreadable] 7) A parallel study to investigate the association of childhood headache with biomarkers for cardiovascular and cerebrovascular disease was also conducted among 11,770 children aged 4 19 years old in NHANES 1999 2004 data. We found mean values for homocysteine, C reactive protein, and body mass index were higher in children with than without headaches, and more children with headaches were in the highest quintile for these factors. Serum and red cell folate were lower in children with headache. More children with headache (32% in children < 12 years) thanMore children with headache (32% in children < 12 years) than without headache (18%) were in the highest quintile of risk for 3 or more of these factors. The findings indicate that these important risk factors for long-term vascular morbidity cluster with severe or recurrent headache in childhood or adolescence. Recognition of the subgroup among children with headache in whom these risk factors concentrate may permit improved preventive management. [unreadable] [unreadable] 8) These data provide new information on the significant role of comorbid disorders, specifically comorbid mood and anxiety disorders, on the impact of severe headaches or migraine, on health care utilization and health perception. Future analyses will then examine the risk factors and biological correlates of these conditions in the general population. We will prioritize our analyses to address the key study questions that are the focus of our own research."}
{"text": "The long range goal of this project is to develop methods of using supercritical carbon dioxide (scCO2) to achieve terminal sterilization that meets or exceeds the USP standard of 10-6 microbial survival probability and that produces a dry drug product in a single process. The objective of this application is to identify at least one set of conditions in which the act of drying drugs by the scCO2 process known as lyophobic precipitation (LP) can also sterilize the drug product in its dispensing container as well as the chamber in which the process is conducted. The rationale behind this project is that recent observations that scCO2 can be used to effect terminal sterilization of food and medical products without damaging the product will be adaptable to the LP drying process. Many drugs, including many anticancer agents, must be administered by non-oral routes (intravenous or intraperitoneal) and therefore must be sterile for injection. For stability reasons such drugs often must be dry as well as sterile. Current technology requires a two-step process of filter-sterilization followed by lyophilization under aseptic conditions to achieve safe, stable dosage forms. To accomplish the objectives of this application two specific aims will be pursued: (1) determine the level of sterilization of drug substance and container under normal LP conditions and using tested and untested added sterilizing agents, and (2) determine the level of sterilization of the chamber under normal LP conditions and using tested and untested added sterilizing agents. The strategy to accomplish the specific aims is to measure the level of killing of standard sterility-testing biological indicators added to containers of model drug under conditions that mimic the conditions that will eventually be used commercially. Similarly, challenge doses of biological indicators will be placed in the LP chamber to determine effectiveness of sterilization of the process with and without added sterilizing agents. The expectation is that at the conclusion of this Phase I SBIR period of support, precise conditions necessary to achieve a sterilization activity level of 10-6 for drug substance contained in a dispensing container and the chamber in which the lyophobic precipitation occurs will be identified. This information will guide a Phase II application to determine the effect of treatment conditions determined in this study on the chemical stability of existing oncology drugs that possess various reactive groups (e.g., alcohols, amines, thiols, carboxylic acids, etc.) that may prove susceptible to damage. PUBLIC HEALTH RELEVANCE: This project proposes a new technique to safely dry and terminally sterilize drugs in their dispensing containers. If successful, this approach will lead to simpler processing of injectable drugs that would increase safety and lower production costs."}
{"text": "This is a resubmission of the previously submitted proposal investigating the regulation of luminal breast cancer stem cells by HER2. We have addressed the previous critiques by adding an investigator with expertise in bone biology, Evan Keller, and a biostatistician, Kelley Kadwell. In addition, we have added new preliminary data and refined the research plan. The development of HER2 targeting agents represents one of the greatest advances in the therapy of breast cancer. Although the use of HER2 targeting agents such as trastuzumab has been limited to breast cancers with HER2 gene amplification, recent retrospective analyses of adjuvant trastuzumab studies suggests that a much wider group of breast cancer patients may benefit from this therapy. We propose that the biological basis for these surprising clinical findings relates to the cancer stem cell (CSC) model. We hypothesize that HER2 is selectively expressed in CSC populations in luminal breast cancers where it plays a major role in the regulation of CSC self-renewal. Furthermore, we hypothesize that the bone microenvironment induces HER2 expression expanding the CSC population in luminal breast cancer bone metastasis. This is mediated by RANKL-RANK-NF?B signaling in a process independent of HER2 gene amplification. We propose that the clinical efficacy of adjuvant trastuzumab in women whose breast cancers do not display HER2 gene amplification (HER2-negative) is due to the effective targeting of CSCs in micrometastasis in these patients. We propose to utilize established breast cancer cell lines, primary human xenografts and primary and metastatic breast cancer tissues from patients to directly test these hypothesies. These studies directly challenge the current clinical paradigm that only the 20% of women with HER2 amplified breast cancers benefit from adjuvant HER2 blockade which, if confirmed would have significant clinical implicaitons. These studies provide a strong biological rationale for the current intergroup trial NSABP-B47, a phase III study designed to determine whether adjuvant trastuzumab benefits women with HER2-negative tumors. Furthermore, the proposed studies have broader implications for the development of adjuvant cancer therapies. If CSCs mediate tumor recurrence, then effective adjuvant therapies will need to successfully target this cell population."}
{"text": "The primary objective of this project is to support the advanced development of candidate products for use in post-event settings following the intentional release of or in response to naturally occurring outbreaks of infectious diseases caused by NIAID Category A, B, and C Priority Pathogens. This contract may support formulation and manufacture of the individual vaccine components, as well as stability testing, nonclinical immunogenicity and efficacy testing in nonhuman primates, IND enabling GLP repeated dose toxicology, submission of an IND and eventual clinical evaluation."}
{"text": "Ca2+ entry through voltage-dependent Ca2+ channels is important in cardiac and vascular muscle excitation-contraction coupling. The Ca2+ regulated multifunctional protein kinase, Ca2+/calmodulin-dependent protein kinase II (CaMKII), has a very important role in signal transduction in nervous system, such as long-term potentiation (LTP) and memory. However, little i known as to whether this protein kinase also modulates the function of cardiac cells. Our studies demonstrate both spatially resolved and temporally distinct novel effects of CaMKII on L-type Ca2+ channel current (ICa) in cardiac cells. Either depolarization alone or calcium influx can increase the amplitude and slow the inactivation of ICa. The distinct volt e- and Ca2+-dependent effects persist with time constants of about 1.7 s and 9 s, respectively. Both effects are completely abolished by a specific peptide inhibitor of CaMKII. This CaMKII inhibitor also suppresses the prolongation of ICa induced by depolarizing holding potentials. An antibod specific for the autophosphorylated (activated) CaMKII, PY-66, is localized close to sarcolemmal membranes and the profile of CaMKII activation is qualitatively correlated with the changes in ICa under various conditions. Thus, the action of CaMKII on ICa is dually regulated by membrane depolarization and by calcium influx: the latter directly activates CaMKII while the former likely promotes the interaction between constitutive CaMKI and the membrane channel proteins. In contrast to the active CaMKII distribution, the intracellular distribution of the total CaMKII enzyme (visualized by using an antibody which specifically reacts with CaMKII k isoform) but does not sense its activation state is uniform with a higher nuclear distribution. This suggests that CaMKII is translocated to the cel sarcolemma following activation in cardiac myocytes. These findings provid new insights toward understanding the physiological function of the ubiquitous protein kinase, CaMKII in cardiac muscle cells as possibly in other type of cells as well."}
{"text": "The objective of the proposed project is the characterization of the properties of the different types of serotonin (5-hydroxytryptamine, 5-HT) receptors in the mammalian central nervous system (CNS). One part of this involves the use of in vitro binding assays utilizing ligands such as [H-3]5-HT, [H-3]PAT, and [H-3] ketsanserin, which are thought to label specific populations of serotonin receptors. A major emphasis of this work will be the pharmacological characterization of these receptors to determine the structural requirements of compounds for discrimination between different types of 5-HT receptors. In conjunction with the ligand-binding assays a program for the synthesis of new compounds will be carried out. Thus, information form the binding assays will be used in the design of new compounds for testing at 5-HT receptors. Another part of the project will be the characterization of functional 5-HT receptors, both in vitro and in vivo. This will include examination of 5-HT receptors that mediate contraction of the cerebral vasculature and 5-HT autoreceptors that regulate the release of 5-HT, as well as examination of central 5-HT receptors involved in the control of respiration, blood pressure, body temperature, and release of certain pituitary hormones such as prolactin and thyrotropin. The study of functional receptors will provide information as to whether the compounds are agonists or antagonists of 5-HT and whether the properties of the functional receptors correlate with those of the putative receptors measured by ligand-binding. As with the ligand-binding studies, a major emphasis of this portion of the project will be the attempt to determine the structural requirements of compounds for discrimination between different types of 5-HT receptors. Once these groups of receptors are classified and characterized, it is hoped that more specific drugs can be designed that will facilitate the study of the roles and actions of 5-HT in the CNS. It is hoped that information from such studies can be used for the design of new, more effective therapeutic agents for the treatment of behavioral or mental disorders that are thought to be linked to abnormal serotonergic function in the central nervous system."}
{"text": "In response to increase in ambient temperature or other noxious stimuli, all organisms generally respond by a rapid synthesis of heat shock RNA's and proteins. The regulation of heat shock gene transcription occurs by heat shock factor (HSF) which binds to heat shock elements (HSEs) in the gene. Even though HSEs are highly conserved among the species, there appears to be marked difference in HSFs. Two human forms of HSFs have been recently cloned from human B cell lymphoma and HeLa cells. In this study we have used both reverse - transcriptase polymerase chain reaction (RT - PCR) and standard cloning techniques to clone an HSF gene from human retinal cDNA library. Northern blot analysis of poly A+ RNA isolated from human retina indicated the presence of RNA of 2.4 kb size hybridizing with human [32P] HSF probe. The same probe was used to identify a number of cDNA clones of HSF in the human retinal library. These clones are presently being subcloned for sequence analysis. RT - PCR technique has also indicated that a form of human HSF is expressed in human retina. The existence and expression of a form of HSF gene in human retina underscores the possibility of induction of heat shock factor(s) by change in ambient temperature or by noxious stimuli as a means of protection of the retina from injury such as high temperature or oxidative damage."}
{"text": "NO ABSTRACT ON FILE"}
{"text": "PROJECT SUMMARY (Description) Microglia are intimately involved in brain homeostasis and synaptic maturation. Recent studies suggest that developmental brain disorder may induce pathological microglia to impair the neural network. In this project, we will test whether these perinatal injury-induced pathological microglia have a unique lineage origin. The current prevailing view holds that microglia are derived exclusively from early CNS settlers of a yolk sac, and do not receive contributions by peripheral monocytes and derivtatives. Yet, our pilot studies suggested a far more complex situation, which bears great implications for perinatal brain injury/infection-related cognitive derangements. This project will test the hypothesis monocytes readily contribute to the brain microglial pool prenatally, but cease doing so shortly after birth. However, perinatal brain injury or infection widens the window of monocyte-to-microglia conversion to produce chronic pro-inflammatory microglia in adult brains. Aim 1: Generate CCR2-CreER mice using BAC recombineering to identify monocyte derivatives. We will produce tamoxifen-inducible CCR2-CreER mice using bacterial artificial chromosome recombineering transgenesis to detect monocyte-derivatives in the R26R-GFP reporter line. We will donate CCR2-CreER mice to the Jackson Laboratory to make this powerful transgenic tool readily available the research community. Aim 2: Compare monocyte-derived microglia in normal development or maternal immune activation (MIA) with and without mild hypoxia-ischemia (HI) in the neonatal period. We hypothesize that Monocytes transform to pro-inflammatory microglia to damage nearby synaptic structures after maternal immune activation (MIA) or endotoxin-sensitized hypoxia/ischemia (LPS/HI) in neonates. Aim 2a compares the number and morphology of monocyte-derived microglia in normal development and after MIA with and without mild HI injury. Aim 2b uses fluorescence-activated cell sorting (FACS) followed by RT- PCR analysis to compare gene expression by monocyte-derived microglia. Aim 2c employs confocal imaging analysis to compare the relationship of monocyte-derived microglia with adjacent synapses."}
{"text": "The transport of all classical neurotransmitters into synaptic vesicles depends on a H+ electrochemical driving force (??H+) generated by the vacuolar-type H+-ATPase. However, different transmitters depend to varying extents on the two components of ??H+, the chemical component (?pH) and the electrical component (??). Most of the vesicular transporters depend on ?pH, and mediate exchange of cytosolic transmitter for lumenal H+. However, the vesicular glutamate transporters (VGLUTs) depend primarily on ??, with an unclear role for H+. They also exhibit allosteric activation by Cl- and recent work has conflicted about their expression of a Cl- conductance. Adapting the use of electrophysiology to record currents associated with the VGLUTs at the plasma membrane, we have found that the VGLUTs exhibit a Cl- conductance allosterically activated from the lumenal side by H+ as well as Cl-. The Cl- condutance exhibits many of the properties previously described for vesicular glutamate transport and the two anions compete, suggesting they use a related permeation pathway. The long-term objective of this program is to determine how these mechanisms influence synaptic transmission, and the strategy is to characterize the mechanisms in heterologous expression systems, then test the role of these mechanisms in excitatory neurotransmission. We propose the following aims: 1) Identify residues responsible for allosteric activation of the VGLUTs by Cl- and H+. We will determine the effect of point mutations on the Cl- conductance associated with VGLUT expression in Xenopus oocytes, then extend the analysis to glutamate transport by functional reconstitution of purified protein. 2) Determine the role of allosteric regulation by Cl- and H+ in excitatory neurotransmission. Using VGLUT1/2 double knockout mice, we will rescue excitatory transmission with mutant VGLUTs defect in allosteric activation by Cl- and H+. We will also determine the effect of these and other properties on synaptic vesicle acidification. 3) Characterize the properties of currents associated with vesicular glutamate transport by whole endosome recording. We have recently detected currents associated with the expression of wild type VGLUTs on endosomal membranes. Importantly, these currents reflect the flux of glutamate as well as Cl-, and their characterization will provide basic new information about glutamate transport as well as the associated Clconductance. 4) Identify residues responsible for the differences in ionic coupling by different VGLUT family members\\ (continuation of prior Aim 3). Bacterial members of the SLC17 family mediate the cotransport of organic anions with H+, raising questions about their relationship to the ??-drlven activity of the VGLUTs. In this aim, we will test the relationship between these closely related protein by introducing mutations that convert their coupling to that of the other."}
{"text": "We recently documented a case of naturally-acquired leprosy, near the LL end of the spectrum, in a sooty mangabey monkey (Cerocebus torquatus atys). The etiologic agent, identified as Mycobacterium leprae, and M. leprae isolated from a human leprosy case were each used to inoculate two normal manabeys. All four inoculated animals developed clinical and histopathologic signs of disseminated leprosy. This indicates that the mangabey may represent the first known nonhuman primated species that is susceptible to the experimental transmission of leprosy. The primary goal of the proposed studies is to establish the mangabey as a model for the study of experimental leprosy. Since the mangabey is rare in capativity and unvailable from importers, and additional goal is to establish a breeding colony to provide mangabeys for the proposed studies and to allow for future leprosyt research. Susceptibility will be studied by varying the dose and route of M. leprae inoculation. Natural transmission modes will be investigated by exposure of mangabeys to aerosols and to mangabeys with untreated, advanced leprosy. Each animal will be completely characterized prior to and at intervals after inoculation or exposure to M. leprae. The characterizations will include: serum chemistry values and hemograms; physical, clinical and neurological characteristics; and bacteriologic and histopathologic examinations of blood cells and biopsy materials. The following immunologic parameters will be studied: in vitro mitogen and antigen (e.g., lepromin) responses of lymphocytes (L); in vitro L immunoglobulin (Ig) production; B- and T-L numbers and subpopulations; suppressor/helper T-L activity; natural killer 9nk0 and antibodyt-dependent cellular cytotoxicity (ADCC); blood mononuclear cell cyclic-AMP levels; skin test responses; cross-reacting antigens between mangabey M. leprae and other mycobacteria; and serum levels of Ig's, C3 and C4, immune complexes, prostaglandins, sialic acid, rhematoid factor, and anti-M leprae antibody. The proposed studies will relate disease progress to changes in clinical, bacteriologic, histologic, and immunologic parameters. These relationships should define the extent to which the mangabey can serave as a useful model for the experimental study of human leprosy."}
{"text": "Cardiovascular disease is the leading cause of death worldwide. Current therapies for cardiovascular disease are associated with partial success, restricted access, delays, possible neurotoxicity and other important limitations. Our goal is to develop a novel therapeutic agent that is safer and more effective at dissolving the blood clots (thrombi) that cause heart attacks and strokes. Studies of humans and mice with lifelong deficiency of a2-antiplasmin (a2AP) have shown that it is the major regulator of blood clot dissolution. We have produced high affinity monoclonal antibodies that induce functional a2AP deficiency. We have shown that these monoclonal antibodies cause venous thrombi and pulmonary emboli to dissolve in vivo. They also accelerate the dissolution of cerebral arterial thrombi-thereby reducing stroke size without increasing bleeding. In this Phase I application, we will modify these promising antibodies by molecular engineering techniques to convert them into potential therapeutics suitable for human trials. In Aim 1 we will engineer and express a chimerized antibody and antibody fragment (Fab). In Aim 2 we will evaluate the relative abilities of the antibody and antibody fragment to bind and inhibit a2AP and enhance blood clot dissolution. With successful completion of these aims we will pursue a Phase II application to optimize the production of these molecules in order to examine their safety and efficacy in suitable pre-clinical models. PUBLIC HEALTH RELEVANCE: Cardiovascular disease is the leading cause of death worldwide. Each year ~ 1.6 million Americans suffer a heart attack or stroke. The resulting death and disability costs the U.S. a staggering $316 billion a year. Current therapies are associated with partial success, restricted access, delays, possible neurotoxicity and other important limitations. This project seeks to develop a novel therapy for heart attacks and strokes that could markedly reduce death, disability and costs."}
{"text": "The University of Louisville proposes to establish a Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases (CPMBEID). Our long-term objective is to support the development of gene-based countermeasures and diagnostic approaches based on system changes that occur during host-pathogen interactions. Focusing on host-based bio-threat and emerging infectious disease research should allow the development of broad-spectrum interventions to infectious agents rather than the current paradigm of \"one bug/one vaccine\" approach so prevalent today. Our specific aims are to 1) meet the expanding needs of current researchers who work with Category A, B, or C priority pathogens at the University of Louisville; 2) expand facilities so that we can add an additional six faculty investigators who work with these agents; 3) assemble a unique mix of technologies in a free standing BSL3 facility so that the instrumentation and expertise is available on site to manipulate experimental materials and avoid the need for shipping material off site; and 4) to serve as a resource for investigators from other institutions in our region who can benefit from the unique mix of technologies we will assemble in the facility as well as be prepared to bring those technologies to bear in the event of a bioterrorism emergency. The University created a superb team of investigators and administrators to shepherd this project from application to operation. We are committed to working with NIAID and believe our missions can be mutually reinforced by building a RBL in Louisville."}
{"text": "The goal of the proposed research is to identify the functional groups at the active site of the mitochondrial F1-ATPase that participate in substrate binding and in the bond-making and bond-breaking steps that occur during catalysis. Analogues of ATP that are potential affinity labels for the enzyme will be synthesized for these studies. These analogues will also be used to determine if a nucleotide binding site exists in the enzyme in addition to the one that we have identified in the beta subunit with p-F-sulfonyl(14C)benzoyl-5'-adenosine. In addition studies on the topography of the ATP synthetase complex will be carried out. These studies will involve the modification of soluble coupling factors with soluble mercaptoalkylimidoesters followed by modification with Ellman's reagent to produce zwitterionic mixed disulfides that reduce the positive charge on the lysine residues to which they are attached by one. The zwitterionic derivatives will be fractionated by ion exchange chromatography and the resolved species will be converted to mixed disulfides which contain mitroaryl azides by reaction with 4-azido-2-nitrobenzenemercaptan. The modified coupling factors will be photolyzed in the presence of the F1-ATPase and depleted membrane preparations. Specific cross-links will be identified by two dimensional polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate in which the cross-links are cleaved after the first dimension. Since it contains the catalytic site for ATP synthesis and hydrolysis, the complete amino acid sequence of the beta subunit is being determined."}
{"text": "This competing renewal application is to provide for the continued maintenance of and activities associated with the SWAN Repositories of serum, plasma, urine, DNA and transformed cells generated from a 10-year study of a population of 3302 women from 5 ethnic groups who have been evaluated annually prior to, during and following the menopausal transition. These Repositories, an arm of the Study of Women's Health Across the Nation (SWAN), is meant to support, facilitate and extend the Core SWAN; additionally, the Repositories provide a mechanism for opening the resources of SWAN to the greater scientific community. Implementing activities associated with three proposed specific aims of this competing renewal will 1) provide for the continued management of the current 1.7 million Repository specimens and the additional specimens that will accrue as a result of fielding SWAN IV in 2009 to 2014; 2) expand the DNA Repository, the most frequently requested specimen type that is uniquely renewable because of our investment in cell immortalization; 3) promote effective information interchange about the SWAN Study, its data and the Repository resources through development of a 2-level web-based data warehouse; 4) provide for continued administration of the application review process for specimen utilization and administrative management of specimen distribution including Material Transfer Agreements; 5) engage in strategies to promote utilization of specimens; and, 6) expand the scope of the genetics studies associated with the SWAN study and its Repository."}
{"text": "Sequencing of the human genome and those of other organisms has revealed that a large percentage of pre-mRNAs are spliced in multiple patterns to produce mRNAs encoding distinct proteins. Although alternate splicing is widespread and must regulate the expression of thousands of gene products, the fundamental mechanisms that control it are not yet well understood. The RNA binding protein Tra2 has been shown to affect the regulated alternative splicing of several pre-mRNAs. In humans these include splice site choices in disease-associated genes such as SMN, tau and CD44. In Drosophila, Tra2 is required for the alternate processing of multiple mRNAs that are necessary for the regulation and realization of sexual differentiation. These include those of the doublesex, fruitless and exuperantia genes as well as tra2 itself. While the splicing activation function of Tra2 in doublesex RNA processing has been well studied and has served as an important paradigm for developmental control of exonic splicing enhancers, little is known about how Tra2 affects splicing of other pre-mRNAs. In this project the mechanism by which Tra2 represses splicing of a specific intron in its own pre-mRNA will be investigated and compared with regulatory elements used in the activation of doublesex splicing. Using an in vitro splicing assay in which recombinant Tra2 specifically blocks splicing of the M1 intron by Drosophila nuclear extracts, the regulatory elements through which Tra2 represses splicing will be identified and the hypothesis that Tra2 blocks the activity of a required exonic splicing enhancer upstream of the intron will be tested. Regulatory complexes that form on the Tra2 pre-mRNA will be compared to those that it associates with in the dsx splicing enhancer to define key elements that determine activation or repression of splicing. The veracity of mechanistic models developed from in vitro splicing studies will be tested by generating transgenic fly strains in which pre-mRNA and regulators with altered sequences are expressed in the tissue and stage of development where Tra2-dependent repression normally occurs. In further studies genetic approaches will be used to define the requirements for SR proteins and other splicing factors that cooperate with Tra2 in the control splice site recognition. These studies will elucidate the fundamental mechanisms by which individual splicing regulators can alter splicing in different ways for different pre-mRNAs. [unreadable] [unreadable]"}
{"text": "A significant part of our investigation is concerned with the structure elucidation of fifteen new substances that we have isolated from cancer urine. Eleven of the fifteen unknowns (E,F,G,H,J,K,L,M,N,O, and P) are from the urines of colon carcinoma patients, three (unknowns D,I and Y) are from the urine of chronic myelogenous leukemia, and one (unknown X) is isolated from the breast carcinoma urine. In addition to these fifteen, three (unknowns alpha, beta, and gamma) are isolated from the normal urines. Preliminary information through uv, nmr and chromatography methods indicates that these compounds can be classified into the purines, pyrimidines, imidazoles and nucleosides. We suggest that these substances are derived most probably from the anabolic and catabolic pathways of tumor nucleic acids in man. Another part of our investigation deals with the study on the source of origin and on biochemical and clinical significance of the four new substances that have recently been identified in the Principal Investigator's laboratory. The new urinary compounds are: N6-succinyladenosine (1), N'-beta-D-ribofuranosylpyridin-4-one-3-carbox-amide (2), 2'-O-methyluridine (3), and 6-amino-3-methyl-5-(N-formylmethylaino)uracil (4). The source of origin of the compounds 2 and 4 will be determined by administration of selected radioactive precursors to normal and tumor bearing rats, followed by urinary analysis. The new anabolic modified nucleosides (1 and 2, Chart II) will be estimated by radioimmunoassay in the urines of the patients with colon carcinoma, chronic myelogenous leukemia, Hodgkin and non-Hodgkin lymphoma and in normal subjects. For the estimation of the compounds 3 and 4, a gas-chromatography-mass-spectrometry method using single and multiple ion monitoring techniques will be developed. On the whole this study will provide structures of several new urinary purines, pyrimidines, nucleosides and related compounds. Some of them in conjunction with other substances may well serve as quantitative tumor markers or indicators of tumor activity in assessing the treatment of certain cancers."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Detailed understanding of the molecular mechanisms of muscle contraction and the many other motile mechanisms involving actin-myosin type interactions is now facilitated by the availability of high intensity synchrotron X-ray sources. Limiting factor in this research, however, is current X-ray detection systems which fail to provide required time and spatial resolution simultaneously. The most promising design for detectors use scintillating phosphors coupled to charge coupled device (CCD). As the current phosphor screens exhibit long persistence effects and lower light conversion efficiencies, the suitability of these systems for proposed demanding time-resolved studies is contingent on having a phosphor with the fast decay characteristics, high spatial resolution, and high conversion efficiency. To address these specific needs, we propose to develop a large area imaging detector based on a novel structured scintillator. The scintillating screen will be fabricated using a sputter deposition process. For detecting X-ray energies typically used in macromolecular crystallography, this sensor will provide high resolution, high detection efficiency, and a fast time response, allowing the full potential of the modern synchrotron sources to be realized. Testing at BioCAT involves pairing these new scintilators with commercial EMCCD based cameras which have the promise to be useful, very cost-effective, X-ray detectors for specialized time-resolved applications"}
{"text": "This is an open label Phase I study. At least 10 HIV-infected pregnant women between 14 and 28 weeks gestation will be enrolled. The women will receive triple combination therapy with indinavir, 3TC, and ZDV during the antepartum period, discontinue indinavir at the start of the intrapartum period while continuing 3TC, and ZDV, and restart indinavir, 3TC and ZDV during the post partum period thru the first 12 postpartum weeks. The mother and her primary care physicians will decide if the mother will continue the same regimen after 12 weeks post partum. The infant will receive 3TC and ZDV at birth and for six weeks following birth. Pharmacokinetic evaluations on cervical secretions and plasma will be performed at the antepartum, intrapartum, and postpartum visits in the women and postnatally in the infants. Baseline and study safety evaluations will include the monitoring of adverse experiences, clinical laboratory tests, physical examinations and vital signs. Temporarialy closed to enrollment awaiting FDA approval for modification."}
{"text": "7. PROJECT SUMMARY/ABSTRACT High-reliability organizations (HROs) such as aircraft carrier flight decks and nuclear power plants are organizations that operate hazardous technologies in a nearly error-free manner under trying conditions rife with complexity, interdependence, and time pressure. Case studies of HROs as well as in a few healthcare organizations that follow the principles of HROs suggest that a robust safety culture enables more reliable work processes, and thus safer performance. More tangibly, safety culture can be seen ?coming to life? in HROs through specific behavioral processes observed in front-line employees collectively termed collective mindfulness (CM). These five interrelated behavioral processes (also called safety organizing behaviors) are: (1) preoccupation with failure; (2) reluctance to simplify interpretations; (3) sensitivity to operations; (4) commitment to resilience, and (5) deference to expertise. Healthcare is increasingly examining and adopting the principles of CM as a means to improve quality and safety of care delivery. The critical need for achieving HRO status in healthcare is no more apparent than in neonatal perioperative care, which is among the highest risk services hospitals may provide. Neonates are highly vulnerable to iatrogenic events due to their small size, fragility, and exceptional sensitivity to environmental stressors. The objective of this pilot study is to measure the prevalence of CM in neonatal intensive care unit (NICU) and operating room (OR) teams and its impact on non-routine events (NREs) ? defined as any event that is perceived by care providers or skilled observers as a deviation from optimal care based on the clinical situation - during neonatal perioperative care. The project will leverage an active large prospective observational study (IRB-approved NICHD R01) that is defining the epidemiology of risk for neonates in the perioperative environment through an innovative analysis of NREs and contributory factors. We propose a two-year pilot study to characterize CM behaviors in NICU and OR teams and to measure their impact on patient safety as measured by the incidence and severity of NREs during NICU-to-OR handovers and subsequent care. Our Specific Aims are to: 1) Conduct a prospective observational pilot study of NICU and OR teams to: a) estimate the prevalence of perceived CM (i.e., self-reported using the SOS) during the perioperative period and b) delineate the relationship(s) between team attributes, case attributes, and perceived CM scores; 2) Determine the impact of perceived CM on the incidence and severity of NREs occurring during and across phases of neonatal perioperative care; and 3) Conduct a preliminary validation of a provisional behavioral marker system, by assessing the concordance of observed (expert ratings of A-V recordings) and perceived CM (self-reported SOS scores) in the same perioperative teams. We anticipate that knowledge gained from this study will lay the groundwork for a multi-center study on the impact of team-based HRO interventions on neonatal safety in the perioperative environment."}
{"text": "A method is outlined for obtaining information about the effectiveness of drug therapies in reducing the coagulability of blood in contact with biomaterials. This information will be gathered such that the relative effectiveness of current clinical drug therapies can be compared quantitatively in their ability to reduce the coagulatility of the blood with specific biomaterials as encountered in surgical procedures. The biomaterial will be exposed to the circulating blood of a canine via an ex-vivo (extracorporeal) test chamber and arterio-venous shunt. The ex-vivo technique has several advantages for evaluating the effect of pharmacologic agents on thrombus formation and coagulation dynamics: a) Ease in controlling blood flow, b) Continual exposure of the test surface to \"fresh\" blood so that cofactors are not depleted, c) Multiple samples can be run simultaneously, d) The material surface can be easily removed and the resulting thrombus quantitatively analyzed for its structure and composition, including platelet, fibrin, and erythrocyte content."}
{"text": "Identification of persons truly allergic to penicillin and cephalosporin antibiotics is still a major unresolved and expensive public health problem. Existing licensed skin testing material detects less than half of persons susceptible to anaphylactic reactions to penicillin, is of no value in diagnosing hypersensitivity in non-urticarial drug associated rashes, and when positive in urticarial rashes has the effect of over-estimating the extent of cross-hypersensitivity with other penicillins and cephalosporins. A major improvement in clinical skin testing materials for diagnosis of non-penicilloyl reaginic hypersensitivity will be sought by characterizing antigenically active fractions obtained by chromatography and isoelectric focusing from minor determinant solutions. Polylysines bearing sidechain-linked penicillin with nucleus modified to mimic minor determinants will be evaluated for their relevance in detecting allergy to minor determinants and compared with isolated purified MDM antigens. Evidence for a cell mediated immunopathogenesis will be sought in patients experiencing non-urticarial drug rash responses or allergic interstitial nephritis reactions to beta-lactam antibiotics. A guinea pig model of cell mediated immunity (CMI) to beta-lactam antibiotics will be used to validate in vitro assays for CMI (antigen-dependent DNA synthesis; agarose leukocyte migration inhibition) and to study the mechanisms and treatment of allergic interstitial nephritis. The CMI response of normal and hypersensitive humans will be compared using in vitro assays, and evidence will be sought for tolerogenic and suppressor mechanisms. Diagnostic testing systems that are practical and clinically useful as well as new information on mechanisms of reactions and potential modes of desensitization will result from these studies on penicillin allergy."}
{"text": "Melanona represents an important cause of cancer-related mortality in the Western world, and its incidence has increased steadily over the past 3 decades. Advanced melanoma remains largely refractory to most therapeutic approaches;in part, this may reflect the considerable genetic complexity underlying melanoma and most other solid tumors. To this end, a main goal of cancer genomics is to characterize genetic alterations at a large scale within human tumors, in hopes of deriving molecular classification schema that might aid targeted therapeutic interdiction. High-density SNP microarrays, which simultaneously interrogate more than 100,000 SNP alleles, have recently been adapted for high-resolution cancer genome analysis. Prior work by the Sellers/Meyerson group and others showed that SNP arrays enabled detailed characterization of chromosomal gains, losses, and loss of heterozygosity (LOH) regions, even in the absence of matched normal samples. In addition, preliminary studies on the NCI60 panel of cancer cell lines demonstrated that combined analysis of SNP array and gene expression data, followed by functional validation, represented a powerful means of tumor classification and cancer gene identification. We are therefore applying an integrated, functional genomic approach to the study of metastatic melanoma. Specifically, high-density SNP array and comprehensive gene expression data will be collected on a large set of patient-derived, short-term melanoma cultures. Computational algorithms will then be employed to define and order the meaningful deletions, amplifications, and LOH regions across the sample set. Next, gene expression data will be combined with chromosomal information to identify candidate cancer genes within relevant genomic lesions. Finally, selected candidate oncogenes will be validated functionally by a lentiviral shRNA approach. These approaches should provide robust grounds for melanoma classification and gene discovery, thereby offering new options for future therapeutic interdiction."}
{"text": "Dental caries is the single most prevalent chronic disease of childhood. Early childhood caries (ECC), a particularly severe form of caries, affects infants, toddlers and preschool children younger than 6 years. Among the general U.S. population, the prevalence of ECC is 25% among children 2-5 years (NHANES III). More recently, NHANES data from 1999-2002 found that the prevalence of caries in young children had increased from the 1988-94 time period, from 24% to 28% in 2- to 5-year-old children (Beltran-Aguilar, et al, 2005). As in the previous surveys, the 1999-2002 NHANES data revealed demonstrated profound disparities in Dental caries prevalence and severity based on race/ethnicity and poverty status. Among American Indian and Alaska Native (AI/AN) children, however, the disease burden is much higher, with 79% of 2-5 year old AI/AN children having ECC (IHS, 2001). The reason for this disparity is not understood, partially because a comprehensive study on the etiology of ECC in this high-risk population has not been undertaken. Until the etiology of the disease is fully understood, there is little hope of developing effective preventive strategies. The overall goal of this study is to identify risk factors for ECC among AI/AN infants and toddlers and to determine if Streptococcus mutans (SM) alone, or in combination with environmental and behavioral factors, increases risk of caries in Native American children. The Specific Aims are: 1) To determine the temporality and fidelity of transmission of Streptococcus mutans in Native American children, 2) To characterize expression of virulence determinants of specific clones of Streptococcus mutans, 3) To determine the composition of total cultivable flora, total Streptococcus mutans and total acid flora in plaque samples of Native American subjects over time, 4) To determine the incidence of ECC in Native American children through age 36 months, and 5) To identify dietary and nutritional risk factors for acquisition of virulent SM clones and ECC in Native American infants and toddlers. Working closely with the Health Board of the Aberdeen Area Tribal Chairman, we will work with the Oglala Sioux tribal community at Pine Ridge, South Dakota from which we will recruit 200 Native mother/child dyads within 1 month of delivery. Information on potential risk factors and caries status will be collected every 4 months until the child is 36 months. As part of our effort to train AI/AN researchers, we will hire a local program coordinator and Dental hygienists. We believe that this assessment of vertical transmission of specific SM clones using DNA fingerprinting approaches will provide valuable information on the acquisition of these cariogenic bacteria in these Native American children. [unreadable] [unreadable] [unreadable]"}
{"text": "The major focus of research in my laboratory is transcriptional control of the pro-opiomelanocortin (POMC) gene. In particular we are interested in transcriptional factors that mediate the effects of stress and neuroimmune modulation on the POMC gene. We have previously described a transcriptional activator of POMC, designated PO-B, that interacts with the POMC promoter. PO-B represents the first defined transcriptional activator of POMC whose function has been verified by mutational analysis. Mutation of the PO-B binding site decreases basal transcription by 70% indicating a major role for this protein in POMC regulatory mechanisms. However, we do not know the physiological or pharmacological agents that regulate signal transduction pathways involving PO-B. Towards this goal we have recently purified PO-B to more than 90% homogeneity from HeLa cells. Interestingly deophosphorylation of the purified protein leads to a more 30-fold increase in DNA binding affinity. In this proposal we wish to rapidly extend these early observations by obtaining a full length cDNA clone of the PO-B gene. This is a defined project of limited duration that will enhance our knowledge of POMC transcriptional regulation and be a useful resource for other work ongoing in the laboratory. Since this laboratory was the first to identify and purify PO-B we would also like to obtain the full length clone in a timely fashion. The specific aims are: 1) To obtain reliable N-terminal and internal amino acid sequence data from purified PO-B. \"Best-guess' oligonucleotide probes will be designed from these sequences. Specific PO-B cDNA sequences will be amplified with PCR from a human cDNA library using pairs of these oligonucleotides representing N-terminal and internal sequence. Amplified fragments will be recovered and sequenced to determine if they code for PO-B amino acid sequence. If this approach is unsuccessful, or only N-terminal sequence is obtained, we will use conventional screening of a HeLa lambda gt10 cDNA library identifying positive PO-B cDNA clones by hybridization with the 'best- guess' oligonucleotide probes. 2) PCR and conventional screening methods are not always successful approaches to identifying correct cDNA clones. Therefore, in parallel with the above experiments, we will also use expression screening. This method does not rely on obtaining any amino acid sequence from PO-B. For these studies we will screen a human fibroblast plasmid cDNA library that can be transiently expressed in mammalian cells. The identity of the full length clone will be confirmed by specific binding of the in vitro translated product to the PO-B cognate DNA binding element. The in vivo activity of the full length clone will be confirmed by the ability of transfected PO-B expression vectors to induce transcription from promoters harboring the PO-B binding site. If progress on PO-B cloning is rapid, we will examine its precise role in POMC transcription. We will perform mutational analysis of the protein to determine which domains are required for DNA binding, phosphorylation status and transcriptional activation. It is expected that these experiments will yield valuable information on the signal transduction pathways in which PO-B is involved."}
{"text": "Organisms capable of dramatic regeneration maintain adult cells that have the flexibility, or plasticity, to change their fate. This feature enables thm build replacement parts, like organs or limbs, to recover from damage or disease. Previous work on the project and other studies have revealed a broad propensity for specialized cells to display some properties of stem cells; they give rise to new cell types in response to injury. The dynamic changes that cells undergo during the transition from one fate to another could reveal the mechanisms that cells employ to enable them to switch their fates. Such insights will reveal basic features of cellular plasticity that could improve regeneration therapy in human cells. However, due to limitations in analyzing the global status of regenerating cells in vivo, little is known about how cells make the traversal from one state to another. The experimental plan proposes to provide a comprehensive view of cells as they transition to new fates during regeneration. The study utilizes plants because the model system provides a rare opportunity to model cellular plasticity. In addition, the mechanisms that regulate plasticity are well conserved across plants and animals and the plant's adept ability to regenerate will illustrate the full potential of cells to change their identity. The proposal takes advantage of a powerful model system that permits continual imaging of regenerating tissue and the analysis of the transcriptional contents of single cells as they traverse fates. This system includes an inventory of active genes for each cell fate, permitting quantitative analyses to track the complex identity of cells as they regenerate. Thus, the complete analysis will generate a model of the trajectory of regenerating cells in order to address basic questions about regeneration: What is the origin of the highly plastic cells that participate in regeneration? Do regenerating cells show proliferative behavior that resembles stem cells? Do fate transitions require losing all identity and reaching a ground state or younger developmental stage (dedifferentiation)? Or, do cells traverse directly to new fates? The results will point to processes that can be targeted to make regeneration more efficient. The innovation of profiling the complete transcriptional dynamics of cells and projecting them onto images of regenerating tissue is widely applicable to many developmental model systems. Thus, overall, the proposed work has broad impact on the field of regeneration."}
{"text": "To construct lotteries with abstract individual choice structures identical to critical organizational collective-outcome experiments involving voluntary participation. Two essential characteristics to be built into the lotteries are these: 1) the collective-outcomes (momentary rewards in these experiments) will be available to the subject whether he is present at a drawing or not, but the probabilities of the various outcomes will differ according to whether or not he is present; and 2) attendence at a drawing entails opportunity costs (e.g. the effort and time required to attend a drawing.) In the organizational research (by McMahon and Camilleri), the ratio of the utility $0.75 to $2.00 varied from nearly zero to close to unity in organizations differing by authority and consensus structures. The primary objective of the present research is to determine whether variations in the utility of monetary pay-offs observed in the organizational experiments can be attributed strictly to the number of possible outcomes in choice structure, or whether these variations are due to aspects inherent in the social aspects of the organizational experiments. S's will be offered 10 opportunities over five weeks to participate in a pure lottery. Three experimental conditions will be developed and executed, differing according to the number of outcomes that can occur, each outcome entailing a specific monetary reward ($2.00, $0.75, $0.00.) The lotteries will be performed by a remote terminal connected to the central CDC 6500 computer. The probabilities of participation (attendence) and the utility ratios at issue will be estimated by use of a choice model developed by Camilleri and Berger."}
{"text": "The objective of this proposal is the continued synthesis and development of anti-convulsant drugs related to 1) those dopamine agonists which can be considered as congeners of apomorphine, 9,8-dihydro-6H-dibenzo[c,g]azonines, and amonotetralin, and 2) aminoisoquinolines, and their evaluation as anticonvulsant drugs in the Anticonvulsant Screening Project (ASP) carried out by the NINCDS. In this research program we shall also investigate 1) the relationship of dopamine agonist response on the basis of the activity on DA sensitive adenylate cyclase, the competition of binding to various 3H-ligands compared with their anticonvulsant activity both from the ASP screen and from audio- or photic-induced seizures in rodents and baboons; 2) the continued study of the chemistry of aminoisoquinoline to make structural changes in this heterocyclic system and to assess the SAR in a systematic approach to structural modification; 3) the study of the acid catalyzed rearrangement of opium alkaloids which would lead to novel aporphines and related structures of interest for biological evaluation; 4) the continued development of sensitive methods for the quantification of N-n-prophylnorapomorphine (NPA), 2,10,11-trihydroxyaporphine (TNPA) and certain pro-drug derivatives of these agents in biological fluids using electron capture gas chromatography in combination with mass spectrometry. In order to facilitate absorption and pharmacokinetic properties, pro-drug derivatives of the most active anticonvulsant compounds will be investigated."}
{"text": "Bacterial identification using DNA hybridization probes has become increasingly sophisticated and accurate as probe technology and methods have improved. Up to now, however, instrumentation for detecting fluorescently- labeled probes has not been able to fully exploit the capabilities of such probes to identify high complex mixtures of genotypes in situ. Positive identification of single variant organisms in large populations of similar cells is also problematic. Fluorescent labeling methods are ultimately limited by the number of different spectral 'fingerprints' that can be distinguished by the imaging system that is used to measure and sort them. We propose to use a set of compatible fluorophores and recently developed KAIROS instrumentation and spectral deconvolution and sorting algorithms to increase the number of fluorescent tags that can be simultaneously distinguished, thus enabling accurate 'fingerprinting' of bacteria. In Phase 1 we will develop a set of these probes and demonstrate their efficacy on microscopic test targets. In Phase 2 we will apply the library of probes and the spectral deconvolution software to correctly identify highly complex mixtures of cells in situ by spectral sorting. This new technology for multispectral bacterial identification (Mbid) will benefit clinical and environmental microbiology as well as biotechnology. PROPOSED COMMERCIAL APPLICATION: Rapid and accurate fingerprinting of bacteria by multispectral fluorescent probes and fluorescence imaging microspectrophotometry is an enabling technology for both clinical and environmental aspects of microbial ID."}
{"text": "The only solution to the HIV epidemic in the developing world is a vaccine that either prevents infection or reduces transmission. Recent data from our laboratory suggest that CD4+ T cell responses along with CD8+ T cell responses against subdominant epitopes can lead to control of replication of SIVmac239. The goal of this proposal, therefore, is twofold. First, we would like to investigate the importance of CD4 help in an HIV vaccine. Second, we want to use innovative new technologies to vaccinate for specific peptides rather than whole proteins. This would eliminate the use of large, commonly seen vectors, thereby circumventing immunodominant responses against vector-derived epitopes. Our new vaccination regimen should induce both CD8+ and CD4+ T cells that efficiently control viral replication. Using newly developed, novel vaccine regimens, we hypothesize that vaccinating macaques with CD4 and subdominant CD8 epitopes will generate immune responses that should control SIV replication. In Specific Aim I of the R21, we will induce CTL specific for subdominant Mamu A*01-restricted SIV epitopes. In Specific Aim II we will engender multiple CD4+ T cell responses against epitopes that are commonly seen in SIV-infected elite controller rhesus macaques. In the R33, we will use the most effective vaccine regimen(s) from the R21 phase to engender CD4+ T cell responses and subdominant CD8+ T cell responses. Animals will be vaccinated with subdominant CD8+ T cell epitopes, CD4+ T cell epitopes, or a mixture of the subdominant CD8+ T cell and CD4+ T cell epitopes. These experiments will allow us to elucidate the role of CD8+ T cell responses against subdominant CD8+ T cell epitopes and CD4 help in an effective HIV vaccine. Project Narrative: Recent vaccine trials have failed to protect individuals from HIV infection or reduce transmission of the virus. Using newly developed vaccine modalities, we now have the tools to circumvent problems characteristically seen in common vaccine vectors as well as address the contribution of individual immune responses against HIV."}
{"text": "The Center for the Neural Basis of Cognition offers an interdisciplinary Ph. D. level educational program intended to create a cohort of excellent researchers who bring the skills, insights, and perspectives from a wide range of existing disciplines into the emerging new discipline of Cognitive Neuroscience. This program has now been in operation for almost 10 years. The program takes students from seven different departments spread over Carnegie Mellon and the University of Pittsburgh. Students are required to pursue a course of study including four core courses (Basic Neuroscience, Systems Neuroscience, Cognitive Neuroscience, and Computational Neuroscience) that often overlap very little with the core requirements of their home departments. Students also participate in a series of student and faculty research presentations, a student-run colloquium series and an annual retreat in which current issues are considered through a combination of research presentations and discussion groups focusing on special issues within the broad range of scientific activities encompassed by the Center for the Neural basis of Cognition, and students are encouraged to attend national and international meetings to gain exposure to contemporary research. There are currently 51 participants in the program, and the program's 17 graduates are all pursuing research careers, many of them either as assistant professors or as post-doctoral fellows in outstanding laboratories. We are adding a new research ethics series to the program and are joining in a broad recruitment effort to increase diversity among the participants. The program has been supported since its inception by an NSF research training grant that has now run its course and is not renewable. The current proposal seeks funding to allow the continuation of this program."}
{"text": "Studies on the regulation of cell proliferation and growth are important to our understanding of developmental biology and tumorigenesis. The long-term goal of the proposed research is to investigate the molecular mechanisms that regulate cell proliferation and growth. In order to address this question, the investigators will analyze the Tsc1 and Tsc2 tumor suppressor genes as well as genes interacting with them in the developing imaginal tissues of Drosophila melanogaster. Mutations in either TSC1 or TSC2 result in a hereditary disease - tuberous sclerosis. The disease occurs in 1/6000 births and is characterized by benign neoplasms known as hamartomas, in multiple organs. Tsc1 and Tsc2 are functionally conserved from flies to humans. The investigators have previously shown that Tsc1 and Tsc2 function together in the Insulin/PI3K/Akt signaling pathway, downstream of Akt and upstream of S6K. Tsc1, Tsc2, and other upstream components of the pathway affect both cell proliferation and growth, however S6K only affects cell growth. The mechanism by which Tsc1 and Tsc2 regulate cell proliferation is not yet known. The investigators propose to investigate the mechanism by which cell proliferation is regulated by Tsc1 and Tsc2. Specifically, the investigators propose to identify Tsc1/Tsc2 interacting genes by performing genetic modifier screens and by using microarray to identify genes regulated by Tsc1/Tsc2. The investigators will then select several Tsc1/Tsc2 interacting genes and further characterize the interaction between these identified candidate genes and Tsc1/Tsc2 as well as their role in the regulation of cell proliferation. This application for the mentored K08 award is submitted so as to enable the candidate to gain necessary didactic and scientific training in developmental and molecular genetics. The eventual goal is to independently investigate molecular mechanisms underlying human disease and develop targeted therapeutic strategy utilizing model organisms."}
{"text": "The focus of this U19 project, \"Immunobiology of Acute Environmental Asthma\", is to conduct mechanistic studies of the role of innate immune processes in mediation of acute environmental asthma. Epidemiological studies have clearly shown that asthma exacerbation linked to air pollutants is a major cause of asthma exacerbation. Endotoxin is a commonly encountered environmental pollutant found in ambient particulate matter and in occupational and domestic settings as well. Endotoxin can induce neutrophilic inflammation at high levels, and at low levels enhance response of asthmatics to airway allergen challenge. We have shown that endotoxin causes changes in airway monocytes and macrophages (increased CD14, CD80, Fc?RI and HLA-DR) which are associated with enhanced response to either innate or acquired immune stimuli. Our team has recently identified novel regulatory molecules of inflammation in the CATERPILLER family, specifically cryopyrin (which upregulates neutrophilic inflammation and monocytic function) and monarch-1, (which downregulates response to innate activation). Cryopyrin is of particular interest as it acts through formation of a Caspase-1 based inflammasome to cleave pro-IL-l? to active IL-1? (with downstream production of IL-6 and other mediators) and IL-18. Furthermore, cryopyrin is activated after ligation of either pathogen associated molecular pattern receptors (including CD14 facilitated binding of endotoxin by TLR4) or the P2X7 receptor by ATP (which is increasingly recognized as an endogenous danger signal released by host cells following non-specific cell injury). These dual activation pathways likely account for the similar actions of a wide variety of inhaled environmental contaminants, and represent novel targets for treatment of acute asthma. We will conduct mechanistic studies of the role of the CATERPILLER family members cryopyrin and monarch-1 (Project 1-J. Ting PI), and the role of NALP-1 and the purinergic receptor P2X7 (Project 2-B. Koller, PI) in rodent models of environmental asthma, in conjunction with a translational project (Project 3-D. Peden, PI) designed to determine the effect of interaction of endotoxin- and allergen-induced inflammation on the airway biology of allergic asthmatics. Human studies will be focused on the biology of airway monocytic cells and the expression of innate immune and the CATERPILLAR family of immune regulators and P2X7 receptors in the airway. In addition to assessment of the role of innate immune processes in regulating airway inflammation, we will examine the relationship of this inflammation on airway physiology, specifically mucociliary clearance. Decreased mucociliary clearance is an understudied process which mediates asthma exacerbation and is a feature of increased asthma severity. [unreadable] [unreadable] [unreadable] PROJECT 1: Novel and innate immune genes in asthma (TING, J) [unreadable] [unreadable] PROJECT 1 DESCRIPTION (provided by applicant): We recently discovered the CATERPILLER family which share structural similarities with the NB-LRR (nucleotide-binding, leucine-rich repeat) super-family of disease resistance (R) proteins that constitutes the plant immune system. In the animal kingdom, this family is also known as NOD or NLR. The clinical importance of this family is underscored by the genetic linkage of family members to a number of immunologic disorders. Among the human gene family members, several of these appear to mediate negative regulatory function in controlling an overzealous inflammatory response. Most notable is the Monarch-1 protein which blocks the function of NF-?B inducing kinase (NIK). Inhibition of NIK reduces the expression of an array of chemokines with relevance in asthma. Gene profiling of induced sputum from mildly asthmatic individuals suggests that the Monarch-1 gene is reduced in these individuals relative to controls, supporting the inhibitory role of this gene during inflammation. Another group of family members regulates IL-1 production. Most notable among these is cryopyrin which mediates formation of the inflammasome complex upon stimulation with a number of inducers. The inflammasome complex is required for procaspase 1 processing to mature caspase 1. In turn, caspase-1 is required for the processing of pro-IL-1 and pro-IL-18 to their mature forms. IL-1 and IL-18 are respectively important in inflammation and TH2 skewing. Cryopyrin is also important in mediating macrophage necrosis which exacerbates inflammation. Thus there are compelling reasons to believe that Monarch-1 and cryopyrin have crucial roles in asthma, however there is no in vivo data to indicate that this is the case. Furthermore we have shown that both of these proteins are ATP-binding proteins, and they exhibit ATPase activity, thus providing ways to modulate their function, which might be important leads to drug discovery. The goals of this proposal are: (1) To study the relevance of Monarch-1 in three animal models of asthma (OVA-induced, endotoxin, and house dust mite and delineate the biochemical effects of Monarch-1 in vivo and ex vivo. (2) To study the relevance of cryopyrin and a cryopyrin-adaptor (ASC) in asthma. (3) To study and identify factors which modulate the nucleotide-binding properties and ATPase function of Monarch-1 and cryopyrin. [unreadable] [unreadable] [unreadable]"}
{"text": "The purpose of the proposed project is to provide minority (Black) undergraduate students in the Department of Biology, a carefully designed biomedical research that focuses on the use of video microscopy and digitized image analysis to monitor morphological changes in normal and malignantly transformed cells and to assess the impact of morphological modulations on tumorigenesis. The initial investigation will attempt to initiate morphologically variant cell types from transformed hepatocytes and a tumor cell line derived from mouse adenocarcinoma. Clonal derivatives from these cells will be examined, through the use of video counting and microdensitometry, for differences in their uptake and retention of fluorescent dyes (cell density), total cell area, shape factor (fraction for estimating the amount by which a cell varies from a circle), parameter, and nuclear/cytoplasmic ratio of normal and malignantly transformed cells. The clonal isolates will be used to determine the histological types of tumors arising from implantation of specific phenotypically variant cell lines. Karyomorphology of phenotypically variant cell lines will be determined to monitor possible chromosomal changes that may attend in vitro cytodifferentiation. It is felt that morphological and karyomorphological analysis of cells from a single tumor but variant phenotypes will provide valuable information on the series of cytological changes associated with cellular differentiation."}
{"text": "Continuation of the animal resource diagnostic and investigator laboratory in the Unit for Laboratory Animal Medicine is requested. The program consists of clinical, laboratory, and research elements which are guided by the academic standards of excellence prevailing at the University. The objectives are enhancement of experimental medicine and advancement of laboratory animal medicine. The specific aims are to diagnose and control naturally occuring diseases in animal colonies at the University, to call attention to problems which could disrupt research; to conduct research in laboratory animal medicine, including research on spontaneous or induced animal diseases, particularly diseases which could serve as models of human disorders; and to promote research training and education in laboratory animal medicine. Methods to be used will continue to include those of clinical medicine, clinical and anatomic pathology, immunology, and other relevant disciplines. Current research includes characterization of pathologic changes associated with aging in rats, immunobiology of Pasteurella multocida in rabbits, neutrophil function in rabbit pasteurellosis, animal model of thyroid dysfunction, latency and transformation of herpes viruses, and others."}
{"text": "Ultimate long term success of an auditory prosthesis requires an electrode design which is flexible enough for easy insertion, can bond sufficiently with metals to withstand bending and eliminate wicking of physiological fluids initiated at electrode contact points. Three fundamental objectives related to a flexible scala tympani electrode will be addressed: (1) Adhesion of metallic conductors to polymer (insulator) substrate, (2) etching and processing of metallic conductor to obtain optimum geometry and (3) evaluate physical properties of metal/polymer composite. A porous polysulfone will be evaluated for metal adhesion under hydrated conditions and with appropriate current stimulation. Delamination is a severe problem with present electrode configurations. Preliminary work has demonstrated that porous polymer surfaces enhance metal adhesion by mechanical interlocking. Preliminary studies with metal coating on porous polysulfone have demonstrated excellent adhesion even under prolonged hydrated conditions. Control of polymer surface energetics and surface porosity appears to enhance metallic coating adhesion sufficiently to warrant further investigation. A porous polysulfone will be evaluated for long term (greater than 2 months) adhesion, using Titanium undercoatings followed with platinum and rhodium coatings. Adhesion will be evaluated under hydrated conditions with appropriate levels of current stimulation. Flexural properties of electrode composites will be evaluated and compared to existing discrete 6-wire electrode systems currently in use for cochlear stimulation. Physical properties of the polymer-metal composite will be evaluated for processing via photolithography techniques. Both chemical and reactive ion etching (RIE) processing will be studied for generation of suitable electrode geometries compatible with current microcircuit fabrication technology."}
{"text": "We propose to develop Gene Therapy vectors that are targeted to particular cell membrane receptors, cell types and/or tissues. Development of these precise delivery systems will help overcome potential adverse reactions and high dosing requirements imposed by the current, non-targeted, traditional Gene Therapy vectors. The goal in Phase I is to construct a combinatorial library of chimeric Adeno-associated virus (AAV) vectors to screen for tissue-specific targeting during Phase II. Because of technical limitations, AAV-based combinatorial libraries were not previously available. We plan to use a unique new technology for building and subsequent selection of AAV vector libraries. Specifically, we will exploit molecular breeding of viruses by DNA shuffling to direct evolution in vitro of AAV capsid genes through recombination of multiple homologous parental sequences of known AAV serotypes. To construct the library, we will use a new AAV production technology based on insect cells and genome pseudotyping. Researchers at AGTC and UF Powell Gene Therapy Center, with proven track records in the AAV field, will join efforts to complete the project. During the feasibility study, we will demonstrate whether it is possible to build an AAV-based library of viable viral clones of at least 106 complexity using recombination of capsid genes of AAV1 and AAV2 serotypes. The successful completion of this step will allow us to proceed to the next stage of selection of vectors with a particular cell/tissue tropism completed during Phase II of the project. We anticipate to market AAV combinatorial libraries to users within the gene therapy community, as a product to select for delivery vectors targeted to a particular tissue or tailored to a particular application, e.g. targeted delivery of pro- apoptotic or suicidal genes to tumors. This project will represent a significant advance in the development of therapeutic gene delivery systems with precise targeting to improve patient outcomes."}
{"text": "This project has concerned the development of animal models of neurological disease produced by exposure to synthetic and naturally occurring neurotoxins in the environment. The interaction of various toxins and neurotransmitters in the environment. The interaction of various toxins and neurotransmitters and hormones in the CNS have provided the focus for combined behavioral and neurochemical studies emphasizing basic mechanisms of action of proposed neurotoxins. Major topics studied this past year were: (1) effects of heavy metals on neurotransmitter and neuroendocrine function; (2) neurotixic mechanisms of chemical and hereditary porphyrinopathies; (3) effects of artificial food colors on neuronal membranes and neurotransmission; (4) role of vestibular function in posture and motor function."}
{"text": "Production of candidate chikungunya vaccines for human clinical trials will be done in compliance with current Good Manufacturing Practices (cGMP) and released for use in human clinical trials. This will necessitate developing production methods based upon novel cell substrates and If these trials demonstrate safety and immunogenicity of this vaccine in human trials, further evaluation may take place in larger (Phase 2) trials which will necessitate production of the clinical trial materials (CTM) at large scale."}
{"text": "Accumulated clinical experience contains information relevant to clinical management and the clinical decision process. Utilization of prior clinical experience has been impeded by the frailties of the memory, the statistical biases of retrospectively recorded data, and most importantly, by difficult access to recorded clinical information. These problems are particularly acute in the major rheumatic diseases, where long patient courses, frequent fluctuations, interactions of problems in many organs, and uncertain natural history combine to create complicated clinical data. This project establishes a pilot Rheumatic Disease computer data bank network (ARAMIS), building upon previous national projects in standardization of clinical description. The network enables pooling of clinical data between institutions, inter-institutional studies, rapid access to large quantities of clinical data, and provides computer consultation and clinical decision procedures based upon this data. It demonstrates the feasibility of a chronic disease network, including successful function, fruitful collaboration, and documentation of appropriate levels of utilization. Evaluation of the contribution of the system to patient care, teaching, and research is being carried out by an evaluation team of biostatisticians, health economists, and physicians. The data bank is an essential tool to relate medical process to patient outcome. It can pinpoint areas of non-productive effort, leading to more efficient practice. By increasng the efficiency of clinical management studies it provides new information to the clinician. And by recognizing the unique characteristics of each patient it can assist in the development of individualized approaches to patient management. ARAMIS provides access to detailed, uniform clinical data for larger numbers of patients than previously amassed. Proceeding in concert with the National Arthritis plan, it seeks to provide the physician and the investigator with better information with which to manage the patient."}
{"text": "?DESCRIPTION (provided by applicant): Colorectal cancer (CRC) is the third most common cause of cancer death in the USA. Germline mutations in the apc tumor suppressor gene, one of the key players in the development of CRC and an important component in the Wnt/beta-catenin signaling pathway, are responsible for familial adenomatous polyposis (FAP). Mutations that result in constitutive activation of the Wnt/-catenin signaling pathway can lead to colon cancer. Wnt(s) have diverse roles in regulating cell fate, proliferation, migration, and death. beta-Catenin is highly expressed in many cancer cell types and promotes growth and tumor formation. Elevated beta-catenin activity in carcinogenesis model systems and neoplastic tissues suggests that this enzyme is a valid target for chemoprevention. By using computational biology with BlueGene/L and GPU supercomputers, we have identified/synthesized several small molecule inhibitors of beta-catenin that are highly effective or have good potential to suppress colon carcinogenesis. In this application, we propose to use state of the art technologies to continue to identify, characterize, test and validate novel, nontoxic small molecule inhibitors of beta-catenin. Our approaches include determination of binding, binding affinities, identification of the specific binding sites, and examining the resulting structural changes by computational simulation using our supercomputer systems. We will validate the effectiveness of these inhibitors by performing protein binding assays, cell transformation assays and in vivo animal experiments, including xenograft models and the AOM-induced colon cancer and APCMin mouse models. Through these studies, we will develop more effective agents targeting beta-catenin with fewer side effects for the chemoprevention of colon cancer."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This project studies avian heart development. This portion of the project concerns the processing of 4D (time-lapse + multiple focus plane) image sets to deconvolve image features between focus planes, to improve particle (cell tracking) accuracy and apply PSC-VB and other tools to view and analyze image sets."}
{"text": "The long-term goal of this proposed work is to characterize protein-protein interactions involving the enamel extracellular matrix proteins, as well as proteins integral to the plasma membrane of ameloblast cells, and characterize how these interactions regulate enamel formation. To achieve this the molecular biochemistry of biglycan, Cd63, Anxa2 and Lampl, and their relationship to the major enamel matrix proteins, will be studied. The hypothesis of this grant application is \"protein-protein interactions involving secreted enamel matrix proteins and the secretory surface of ameloblast cells (Tomes' processes) play a key role in the formation of the enamelprismatic structure.\" To test this hypothesis we propose three specific aims. These are to: 1) define, using in situ hybridization and mouse-specific antibodies, the spatiotemporal expression patterns for Cd63, Anxa2 and Lampl within the murine developing incisor tooth, and relate these expression profiles to those of their respective ligand (enamel matrix protein partner), and the stages of enamel biomineralization; 2) demonstrate that the protein-protein interactions previously identified for the enamel matrix proteins and biglycan, Cd63, Anxa2 and Lampl occur in vivo and have physiological significance, and to define the minimal binding domains of Cd63 and Lampl that enable their interaction with amelogenin; 3) define and disrupt the receptor-mediated molecular mechanism that permits the enamel matrix expression levels of biglycan to influence or control amelogenin expression levels, and the subsequent events of enamel biomineralization. Knowledge gained from this and related studies may lead to better dental and non-dental materials, or biomimetics. This data will be critical to others in their pursuits to regenerate an entire tooth. For tooth regeneration to become a reality, the protein- protein interactions involving the key dental proteins must be known and understood."}
{"text": "This is the second competing renewal of our Program Project Grant, \"Drugs of Abuse: Role of Protein phosphorylation\" (DA 10044), which was initially funded in 1996 and again in 2001. The grant has been highly successful based on research productivity and initiation of new research projects. The overall objective of the Program Project Grant remains the same as the original, namely, to elucidate the molecular basis of the actions of drugs of abuse, particularly psychostimulants, in the dorsal striatum and nucleus accumbens. The addictive properties of drugs of abuse such as psychostimulants depend on their ability to augment dopamine neurotransmission in the basal ganglia. The Program Project Grant is organized in four Projects, a Scientific Core and an Administrative Core. The Administrative Core staff will support the integration of the researchers and institutions involved in the Program Project Grant. The main goal of the Scientific Core is to provide a research and technical support facility for all of the Program projects. Responsibilities of the Scientific Core will include the creation, characterization, and breeding of genetically altered animals;the design and execution of yeast two-hybrid studies;the production and maintenance of key reagents stocks and new reagents;and the performance of certain routine tasks required to accomplish the studies described in Projects I-IV. Project I, \"Psychostimulants and dendritic spines,\" will focus on the role five regulators of actin dynamics play as targets of psychostimulants in the dendritic spines. Project II, \"The Role of DARPP-32, CK1, and CK2 in Mediating the Molecular and Behavioral Effects of Drugs of Abuse,\" will extend previous studies of four key phosphorylation sites of DARPP-32 upon in vivo administration of drugs of abuse and chronic exposure to drugs of abuse. Project III, \"Striatal Phosphoproteins and the Actions of Psychostimulants,\" which will be a subcontract carried out at Yale University School of Medicine, study the role(s) of RCS and ARPP-16 in mediating or modulating the actions of psychostimulants as well as study the roles of the three isoforms of PP1 (PPla, P and y) in the actions of psychostimulants. Project IV, \"The Role of GPCR-interacting Proteins in the Actions of Pscyhostimulants,\" will utilize yeast two-hybrid screening to further investigate the multiple intracellular signaling pathways of GPCR and its protein-interacting subunits in mediating actions of cocaine, D-amphetamine, ecstasy and caffeine."}
{"text": "The focus of the proposal is in the area of breast cancer imaging and tumor characterization using oxygen sensitive phosphorescent labels and hybrid imaging modality. We propose to use simultaneous diffuse optical tomography (DOT) and magnetic resonance imaging (MRI) to obtain high spatial-resolution and quantitative functional images of breast tumors. Imaging in pre-clinical models with injected contrast agents and imaging the oxygen concentration within tissue are important for a number of clinical and scientific reasons. Low oxygen content in tumors is a marker of radiation-resistant tumor regions, and is also correlated with a poor treatment outcome. For basic research applications, it is also very important to understand how hypoxia develops in tumors and how it is correlated with exogenous and endogenous markers of low oxygen. During Phase I, in conjunction with standard diffuse optical tomography (DOT), we obtained high contrast images of hypoxic regions in breast tissue simulating phantoms using long-lived oxygen sensitive contrast agents and a simple lifetime imaging system with cooled avalanche photodiodes. In Phase II, a prototype of the CW and time domain lifetime imaging module will be developed which will have parallel acquisition across a cooled 16-element avalanche photodiode (APD) array for high resolution and high throughput imaging. The low-cost, compact lifetime imager module will be designed to be compatible with high magnetic and RF fields associated with MR units in hybrid imaging systems. Using this APD module in a dual-modality imaging setup at our collaborator's facility at Dartmouth College, NH (Prof. Brian Pogue, Thayer School of Engineering), phantom and animal model imaging will be performed to obtain functional optical images with high resolution and high contrast. The optical image reconstruction will be aided by the spatial and anatomical information obtained from simultaneously obtained high-resolution MR images in order to improve the accuracy of these images. The compact APD based detector prototype will have a small footprint and will be mounted inside the magnet of the MRI unit, dramatically decreasing the complexity of the current PMT based detection systems. PUBLIC HEALTH RELEVANCE: Hybrid imaging with simultaneous diffuse luminescence imaging and magnetic resonance imaging can be used to obtain high resolution functional maps of tumors based on their oxygen concentration. Hypoxic breast cancer detection is the primary target application, with the goal of improving diagnostic accuracy and treatment prognosis using exogenous contrast agents. Other applications could include brain imaging to monitor cerebral function or cerebral hemorrhage, and monitoring tissue and muscle oxygenation, histotoxicity, etc. [unreadable] [unreadable] [unreadable]"}
{"text": "The Metabolic Physiology Shared Resource (MPSR) is developed as an institutional core that will allow DRTC investigators to study metabolism using a range of animal models and human subjects. MPSR components include laboratories for study of mice, rats, canines, and human subjects. This resource is shared with two other centers. They are the Mouse Metabolic Phenotyping Center (MMPC) and General Clinical Research Center (GCRC). The MMPC provides comprehensive services for study of mice. The GCRC provides support for human studies in such areas as nutritional services, nursing, physical resources, biostatistics, and bioinformatics. The MMPC and GCRC both have NIH support independent of the DRTC. The DRTC will support laboratories for rat and canine experimentation and for the study of energy balance and nutrition in human subjects. The Rat Laboratory is an expansion of the DRTC efforts to centralize and develop techniques for studying in vivo metabolism (e.g. energy balance, insulin sensitivity, insulin secretion) in the conscious, unrestrained rat. While distinct from the MMPC, this component greatly benefits from and synergizes with the MMPC. The Canine Laboratory is a continuation of DRTC support of our novel approach to study in vivo metabolism (e.g. liver substrate balance, hormone action) using the conscious dog. The Human Energy Balance Laboratory is an essential complement of existing services available to DRTC investigators by way of the GCRC. It will support services for assessment of energy expenditure, body composition, and physical activity. The MPSR is directed by faculty (D. Wasserman, S. Davis) experienced in the range of experimental models supported by the resource and provides specialized technical assistance to investigators using the resource. The MPSR is structured to facilitate translational studies from genetically modified mice to humans and to allow investigators working in human subjects to explore more basic mechanisms in animal models. The MPSR represents the most efficient and scientifically sound approach to utilizing institutional resources that have common goals (i.e. study of metabolism in the whole organism). The MPSR effectively pools expertise and in some cases physical resources, allowing DRTC investigators the ability to study metabolism in scope that is unprecedented."}
{"text": "The Postmenopausal Estrogen/Progestins Intervention (PEPI-1) trial of 875 participants followed at 7 centers for three years is the largest and longest randomized double-blind placebo-controlled clinical trial of hormone treatment in postmenopausal women -- and the only long-term trial of estrogen-progestin regimens. It is the only long-term trial designed to compare the effects of Estrogen Replacement Therapy (ERT) and combined (estrogen-progestin) hormone replacement therapy (HRT) on heart disease risk factors. It is also unique for the number of women who began ERT or HRT more than 5 years after the menopause. Details of the study design, hormone replacement regimens and study population are given in Project I, as are the major analyses of PEPI-1 data proposed for completion during the extended follow-up period. Project II, Studies of Repository Samples, was planned to utilize plasma, urine and other samples already collected but not analyzed by the close of PEPI-1. Both Project I and Project II, submitted by the Coordinating Center to reflect the centralized activity necessary for a multicenter project, will be conducted with the Clinical Investigators as a cooperative project. Project III, submitted in parallel from each of the 7 PEPI Clinical Centers, describes a three year prospective observational follow-up study designed to: ensure PEPI women's safety and learn more about possible delayed adverse hormone effects; study treatment choices, compliance and reasons related to these decisions; and monitor long-term effects on heart disease risk factors and bone density. All participants will be invited to an annual clinic visit, (conducted according to the established PEPI-1 protocol) which includes a medical history and limited examination, mammogram, endometrial biopsy, and standardized questions about quality of life, sexuality, symptomatology, and medication use. We will measure primary PEPI endpoints (HDL-cholesterol, blood pressure, insulin and fibrinogen) and their covariates, and bone density at the hip and spine."}
{"text": "The proposed research is designed as an analytical and manipulative investigation of mechanisms critical to cell migration in the patterning of the developing nervous system. The studies will focus on two developing cranial nerve nuclei in the chick embryo: The oculomotor ventromedial subnucleus and the trigeminal motor nucleus. In studying the pattern of migration and possible mechanisms important for the proper execution of these patterns, a combination of approaches will be used, including interspecific embryonic transplantation, sequential electron microscopic analysis of intercellular interactions during disparate migration phases, analysis of cellular-substrate interactions, and dual embryonic microsurgical-autoradiographic study. Within the oculomotor ventromedial subnucleus, the experiments will determine the pattern of migration whereby the primordial cells attain their definitive position, the nature and sequence of intercellular contacts formed during migration, (both within and between populations), and will examine pinocytosis as a possible mechanism for mediation of guided cell movement. Within the trigeminal motor nucleus, the proposed studies will examine the nature and sequence of intercellular contacts formed prior to, during and after migration and will experimentally analyze the importance of extracellular influences (i.e., ingrowing ganglionic afferents) to migration of the nucleus."}
{"text": "Drug-resistant tuberculosis is a serious public health threat. Diagnosis of drug-resistant strains presents a particularly urgent challenge, as such strains are responsible for an increasing burden of morbidity and mortality worldwide. Genome-wide scans that have revealed multiple genetic variants that are associated with drug resistance. The goal of this project is to identify and validate which ofthe resistance-associated mutations are causally connected to either high-level drug resistance or an increased risk of developing resistance. In addition, for selected mutations and agents, we plan to further explore the spectrum of potential mutations that either have not yet been found in clinical isolates or whose association with drug resistance is unclear. Specifically, we will construct isogenic strains containing resistance-associated mutations, producing strains with single point mutations in an otherwise wild type genetic background. We will determine the drug resistance phenotype of the resistance-associated mutations, and we will also predict mutations associated with resistance not yet seen in clinical isolates. Taken together these studies will lead to validated SNPs that will serve as the basis for much needed sequence-based diagnostic tests for tuberculosis."}
{"text": "BIOLIBRARY & PATHOLOGY SHARED RESOURCE Directors: Devon Kelly, M.S. and George Thomas, M.D ABSTRACT/PROJECT SUMMARY The Knight Cancer Institute (Knight) sponsored BioLibrary & Pathology Shared Resource brings together tissue collection, processing, clinical annotation, and histopathology services into a single core. The unit provides members with: 1) a repository of well-annotated human tissue, blood and saliva, 2) a database of annotated clinical variables including patient demographics, treatments and clinical outcomes; research and clinical trial participation; genetic profiles, research analyses and specimen imaging, 3) tailored data in the form of client-specified case reports, 4) pathology services for studies on human and animal tissues, including cryostat sectioning, paraffin-embedding, microtome-sectioning, histochemical staining, immunohistochemistry, immunofluorescence and fluorescent in situ hybridization, 5) support in producing high quality RNA and DNA samples from both frozen and fixed specimens, and 6) pre-award consulting for preparation of grant and other funding applications. The BioLibrary & Pathology Shared Resource is accredited through the College of American Pathologists (CAP) and is Clinical Laboratory Improvement Amendments (CLIA) certified. It is staffed with members who have decades of combined experience in handling samples from small animals and human to ensure that tissues are properly prepared to meet the various needs of the research community, both for immediate analyses as well as long-term storage and use."}
{"text": "Description: The International Union of Operating Engineers (IUOE) has developed a training plan to provide hazardous materials health and safety training to its membership, in particular the Hoisting and Portable (H&P) engineers and Stationary Engineers. They plan to expand and administer their on-going training effort to ensure that their membership who could work in hazardous waste cleanup have the skills to recognize and control hazards. The IUOE is a current grantee in the NIEHS program. They claim to be the largest provider of hazardous waste worker training in the United States since the inception of their training program and grant funding in 1988. Under their current NIEHS cooperative agreement they trained 7,439 workers in the 40-hour worker course and 22,803 workers in the 8 hour refresher. This training program is one part of the IUOE s long tradition of providing worker training programs, including a formal apprenticeship program and Job Corps Program."}
{"text": "Cell signaling mediated by the Hedgehog (Hh) family of secreted proteins plays crucial roles in animal development and human diseases. The Hh pathway is operating in a similar way among organisms ranging from insects to human. Drosophila has been a powerful model organism to study Hh signaling mechanisms, as sophisticated genetic, molecular, and biochemical tools are available to dissect this important pathway in whole organisms as well as in cultured cells. The long-term goal of my laboratory is to delineate the complex regulatory network that governs Hh signal transduction in order to understand how graded Hh signal is transduced to generate multiple developmental outputs. Hh exerts its biological influence through a conserved signaling cascade that culminates in controlling the balance between the activator and repressor forms of the transcription factor Ci/Gli (CiA/GliA and CiR/GliR). The goal of this research is to investigate the multifaceted regulatory mechanisms that control Ci activity. Our recent study has uncovered a dual role of the Ser/Thr kinase Fused (Fu) in the regulation of both the production of CiR and the activity of CIA, and revealed that Fu is activated through dimerization-mediated phosphorylation of its activation loop. These findings provide a critical inroad into a mechanistic dissection of Ci activation. We will explore the mechanism by which Fu promotes the maturation of Ci into CiA and investigate how the Hh gradient is translated into a Ci activity gradient (Aim 1). In a genetic modifier screen, we have discovered that the SUMO pathway can modulate Hh signaling activity and identified Ci as a SUMO substrate. We will further characterize this new post-translational modification of Ci to explore its role and mechanism of action in Hh signaling (Aim 2). The molecular mechanism by which Sufu inhibits Ci is still poorly understood. We have uncovered a previously unidentified nuclear localization signal (NLS) that overlaps with the Sufu binding domain in Ci. We will further study the function of this NLS and its regulation (Aim 3). Finally, how Ci functions in the nucleus to control Hh target gene expression has not been fully explored. We have identified multiple domains required for CiR-mediated repression. Identifying cofactors that interact with these domains and investigating their roles in Hh signaling should shed important lights into how Ci regulates its target gene expression. Therefore, we will carry out protein- protein interaction screen and in vivo RNAi screen to identify Ci co-repressors (Aim 4). The proposed study should provide a much deeper understanding of the Hh signal transduction mechanism and shed new light into how graded Hh signals are translated into different developmental outcomes. PUBLIC HEALTH RELEVANCE: The Hh pathway is a major signaling pathway that controls embryonic development and adult tissue homeostasis. Deregulation of Hh signaling has been attributed to many human disorders including birth defects and cancers. Investigation of the multifaceted and conserved mechanisms that regulate Hh signaling activity will not only provide insights into fundamental developmental problems such as how cells interpret different levels of spatial signals but may also provide new avenues for developing diagnostic tools and therapeutic treatments for cancers caused by deregulation of Hh signaling activity."}
{"text": "This application requests the competing renewal of UNC's MSTP award. Our primary goal is to train an outstanding group of young men and women who are committed to become physician-scientists, fully capable of bridging the gap between basic science and clinical medicine. These individuals will go on to become the next generation of leaders in biomedical science, thereby making significant contributions to and improvements in human health. At the same time, they will become teachers and scholars not only at many of the best medical schools, but also at major research institutes and leading organizations in the biomedical and pharmaceutical industry. We hope to achieve our goal by identifying and then recruiting to UNC candidates from diverse backgrounds who bring with them a great variety of academic and research interests. Once here, they find a strong education in clinical medicine that is well integrated with superb research opportunities. Since the time of our last competing renewal, we have expanded our Leadership Team to include: Kim Rathmell, MD-PhD, the Director for Translational Science and Mohanish Deshmukh, PhD, the Director for Basic Science. In addition, based on the feedback that we received at the time of the last competing renewal of this MSTP grant, we have implemented several new initiatives designed to link the graduate school-based research activities of each student with relevant clinical experiences. Examples include: a) a Longitudinal Clinical Clerkship: this provides each student with clinical experiences that are related to and complement his/her research project; b) a monthly Clinical Case Conference: this is organized and directed by our senior MD-PhD students working in conjunction with Chief Residents from Medicine and Pediatrics; and c) a focus on the Clinical Relevance of Doctoral Dissertation: we are asking that each thesis committee include one clinician- scholar (often a physician-scientist), whose role is to ensure that the clinical relevance of the research is always at the forefront of the student's thinking. W believe that these additions have addressed the concerns of the last review panel and at the same time served to integrate the two phases of our training program. Over the past five years, our program has grown from 64 to 76 students, drawn to UNC from many of the best colleges and universities all across the USA. Their academic credentials are superior: this year's incoming class, for example, had a mean GPA of 3.75 and a mean MCAT of 37.6. Once here, our students are performing at a high level in both the classroom and the laboratory. They are pursuing their graduate training in 20 individual departments and curricula representing the Schools of Pharmacy, Public Health, Medicine, and the College of Arts and Sciences. They are receiving a high percentage of honors, publishing on average four manuscripts, successfully competing for a variety of awards and independent funding (e.g., 26 of our students hold F30 awards from the NIH), and completing the dual degree program in eight years. After completing their post-doctoral training, many of our graduates are becoming faculty members at many excellent institutions."}
{"text": "The photometabolism of bilirubin is to be studied by broad band fluorescent radiation of jaundiced Gunn rats fitted with a bile duct cannula. The photoproducts of bilirubin are to be isolated from the bile by thin layer and high pressure liquid chromatography and identified as either photo-isomers, photo-oxygenations and/or photo-adducts of bilirubin. Similar studies are to be performed using as the light source monochromatic light from an argon tuneable lasar. The latter will permit the determination of the in-vivo action spectrum of bilirubin. The diarrhea that is associated with the clinical use of phototherapy for neonatal hyperbilirubinemia is being studied in experimental rodents by small bowel perfusions with bile pigments. The mechanism of the enterocyte secretion of sodium and water that is induced by non-conjugated bilirubin is being compared with other classic secretogogues. Reversal of the bilirubin-induced secretions is also being investigated by blocking the enterohepatic circulation of bilirubin to prevent enterocyte exposure to non-conjugated bile pigment. The microsomal enzymatic abnormalities for the conjugation of bilirubin in the Gunn strain of rats is to be further defined by comparison with the other microsomal deficiencies in this strain of animal. Ohter microsomal activities and lipid composition are to be examined. Novel amino acid conjugates of bilirubin have been qualitatively found in the meconium of some infants, in bile of patients with hemolytic disorders and in the Crigler-Najjar syndrome Type I. Attempts to induce synthesis of these conjugates in experimental animals will be made."}
{"text": "Disruptive behavior disorders (DBDs; conduct disorder and oppositional-defiant disorder) with severe aggression constitute a public health problem for which evidence-based treatment options are limited. Increasing numbers of youth with aggression are being treated with atypical antipsychotics without good evidence of safety or incremental advantage over safer stimulants. This double-blind, placebo-controlled, parallel groups study will compare the effectiveness of (a) parent training in behavior management (PMT) + placebo (PBO), (b) PMT + d-methylphenidate (d-MPH), and (c) PMT + d-MPH + risperidone (RIS) in children with severe aggression, primary DBDs, and comorbid ADHD. Participants must exhibit a clear history and current pattern of serious physical aggression (i.e., moderate or higher scores on the Modified Overt Aggression Scale (OAS-M), a Clinical Global Impressions (CGI) Scale Severity score of 4 or higher for aggression), and high scores on the on the Disruptive-Total of the Nisonger Child Behavior Rating Form (NCBRF) . The primary aims are to determine (a) if PMT + d-MPH are superior to PMT+ PBO and (b) if PMT + d-MPH + RIS are superior to PMT + d-MPH and to PMT + PBO. Secondary aims include determining whether type of aggression (reactive vs. proactive) moderates treatment response. Design: Two hundred sixteen children across 4 sites (Case Western Reserve, Ohio State, Pittsburgh, & Stony Brook) will be randomized to 9 weeks double-blind of PMT + PBO (n=72), PMT + MPH (n=72), or PMT + MPH + RIS (n=72). All groups will receive a 12-session course of carefully monitored, empirically-based PMT. Responders will be followed on their assigned treatments in a 12-week Extension, and all participants will be assessed at one year after baseline. Clinical change will be measured by (a) parent ratings on the NCBRF & ADHD Symptom Checklist (CL) ; (b) teacher ratings on the ADHD Symptom CL; (c) clinician interview of the child with OAS-M; (d) clinician CGI-Improvement score (CGI-I); and direct observations of child-parent behavior. The primary outcome measure is the NCBRF Disruptive Total score; secondary outcomes are CGI-I, response rate (NCBRF reduction of at least 25%, plus CGI-I score of 1 or 2), other NCBRF and ADHD Symptom Checklist subscales, and cognitive tests. Baseline score on the Antisocial Behavior Scale will assess type of aggression (reactive or proactive) as a potential moderator. AEs and tolerability will also be assessed. This study will assess the use of placebo, d-methylphenidate (Focalin), and d-methylphenidate plus risperidone (an atypical antipsychotic drug; brand name Risperdal) against the back-drop of behavior therapy which will be taught to the parents of participants. The participants will be children ages 6 to 12 years, inclusive, who have been diagnosed with a disruptive behavior disorder plus ADHD and who display significant aggressive behavior. Given the increasing rates and severity of violence in our society, this investigation will help to determine if drug therapy, combined with parent-provided behavior therapy, can reduce child aggression. [unreadable] [unreadable] [unreadable]"}
{"text": "The Emergency Responder Health Monitoring and Surveillance (ERHMS) Technical Assistance Document (TAD) aimed at National Response Teams highlights the need for a technology platform for collecting data, before, during and after disasters, to assess the effect training has on response-related illness and injury for emergency responders. This SBIR grant application is aimed at developing a software platform that will not only provide the means to efficiently collect data for the assessment of training impact; but also will provide the means to efficiently deliver pre-deployment and site specific training (SST), against which the assessment of training impact on responders' illness and injury can be evaluated. If successfully implemented, the software platform will be a useful tool both for delivering just-in-time training and for supporting environmental health research relating to responders' exposure to potentially harmful environmental contaminants during and after disaster events. This research project proposes an innovative system for generation and distribution of incident-related knowledge (including manuals, general and site-specific trainings) with integrated monitoring and reporting functionality to address the needs described above. The system will allow the incident command official to generate training materials and data collection tools by simple and straightforward import from existing materials, as well as to customize previously generated tools to better-fit the current situation. All the materials will be delivered to emergency responders in the form of instantly generated smartphone (iOS, Android) and web applications, which will significantly reduce costs of logistics and latency of materials delivery. Materials encapsulation into mobile apps will address the prevalence of mobile devices (smartphones, tablets) at the scene and will ensure that emergency responders can access the content of any situation and circumstances anywhere in the field in the most convenient form, independently of what kind of device they have. Phase I will concentrate on assessing, validating and prioritizing the most demanded tools together with a representative set of key stakeholders; developing a system prototype for digitization and modularization of training and data collection tools, and instant distribution via rapidly created mobile application; prototyping specific interfaces for emergency responders and incident command personnel; testing and finally, evaluating the first prototype. Phase II will concentrate on capabilities of electronic collection and distribution of health status and surveillance data, on integration with an external IT system for two-way communication of hazard data outside incident command, and on generation and delivery of additional materials in response to changed environmental conditions."}
{"text": "Project Summary Oxidation reactions are among the most important reactions in organic chemistry and play a crucial role in the synthesis of pharmaceuticals, natural products, and other bioactive compounds. Advances in catalytic oxidation reactions have potential for major impact in the discovery and production of pharmaceuticals. The majority of existing catalytic oxidation methods face challenges in their efficiency and selectivity, including chemo-, regio- and stereoselectivity, limiting their use in small- and large-scale applications. The proposed research will develop new oxidation and oxidative coupling methods that form carbon-carbon and carbon-heteroatom bonds, including C(sp3)?H functionalization reactions. Some of the resulting methods will streamline the discovery of new bioactive molecules with diverse three-dimensional architectures, addressing key challenges in medicinal chemistry and drug discovery, while others will provide the basis for streamlined process-scale synthesis of pharmaceuticals. Three complementary project directions are outlined in this proposal. The first focuses on the development of oxidase-type aerobic oxidation catalysts that feature a transition metal and a redox active organic co-catalyst. New bioinspired catalyst systems will be explored that exhibit second-order biomimicry, whereby simple organic precursors undergo oxidative self-processing to create the essential co-catalysts. This process resembles the post-translational modification of amino acid side chains to generate reactive cofactors in Nature. The second project will pursue new electrochemical oxidation methods for the synthesis of organic molecules that are difficult to access via classical synthetic methods. These efforts target the identification of versatile mediators and electrocatalysts that permit the reactions to proceed at low electrode potentials, thereby tolerating diverse functional groups and enabling broad scope and utility. Finally, we will develop radical relay methods for benzylic C?H oxidation and oxidative coupling to afford new C(sp3) C?O, C?N, C?X, and C?C bonds. These efforts will be applied to pharmaceutical building-block diversification, core-modification, and late-stage functionalization. In each of these project areas, empirical reaction discovery efforts will be complemented by mechanistic studies of the catalytic reactions. Close interactions and collaborations with pharmaceutical companies in all phases of this project will play an important role in ensuring the broadest possible impact of our efforts."}
{"text": "The specific goals of this study are: a) to investigate whether the cellular tropic and cytopathic characteristics of distinct HIV-1 isolates allows for infection and death of hematopoietic stem/progenitor cells; and b) to investigate whether HIV-1 infection reduces the \"stem cell capacity\" of the putative CD34++CD38+/- stem cells engrafted NOD/SCID mice transplanted with a human thymus."}
{"text": "Annual Report 2001 This project investigates signal transduction pathways that regulate survival, growth, and differentiation in the lens and corneal epithelium. Recent studies have focused on the signaling pathway linking EGF receptor activation with c-fos transcription in the lens and on the functions of Cdk5, a member of the cyclin dependent kinase family, in both lens and corneal epithelium. Our previous studies of lens epithelial cells have shown that 12(S)HETE, a lipoxygenase metabolite of arachidonic acid, is required for EGF-dependent activation of PKCalpha and PKCbeta.. Further studies of this pathway have now demonstrated that exogenous 12(S)HETE alone leads to activation of these two PKC isoforms and of PKCgamma, an isoform that is not activated in the presence of EGF. We are now investigating the possibility that EGF-dependent signals may prevent 12(S)HETE-dependent activation of PKCgamma. Since we have also shown that 12(S)HETE-dependent activation of PKCalpha and PKCbeta is necessary but not sufficient for c-fos transcription in lens epithelial cells, we are investigating sequences in the c-fos gene that mediate the response to PKC. Our studies of Cdk5 suggest that this enzyme may regulate cell adhesion, migration, and survival in a variety of non-neuronal cells, including lens and corneal epithelial cells. To investigate the possible mechanism of these effects in the lens, cDNA library has been constructed from E18 embryonic rat lenses for yeast two-hybrid screening using Cdk5, p35, and p39 as 'baits'. Interacting clones are being characterized. To extend our previous studies of Cdk5 and p35 in corneal epithelial cells, we have stably transfected a mouse corneal epithelial cell line, A(6)1, with Cdk5 or Cdk5T33 and have used these lines for in vitro studies of cell adhesion and migration. Results indicate that Cdk5 promotes cell adhesion to a fibronectin or collagen matrix and inhibits cell migration, as measured by an in vitro scratch assay. Similarly, we have found that overexpression of Cdk5 in the corneal epithelium of transgenic mice retards corneal wound healing. However, this inhibitory effect on cell migration seems to be relatively specific for corneal epithelial cells. In several other cell types that we have examined, overexpression of Cdk5 increases, rather than decreases, the rate of cell migration. Possible explanations for this cell-type specific difference are being explored. Finally, we have found that Cdk5 overexpression induces apoptosis in COS1 cells and in certain other cell types. This effect is also produced by the Cdk5 activator, p39, but not by the alternative activator, p35, or by the kinase deficient form of Cdk5, Cdk5T33. Cdk5-transfected COS1 cells were found to release a substance to the medium that causes apoptosis even in non-transfected COS1 cells. The nature of this substance is being examined."}
{"text": "Project Summary: Coronary artery disease can lead to myocardial infarction (MI), which is a major cause of morbidity and mortality in the US. Blockage of the coronary arteries prevents blood from reaching downstream target tissue, causing cardiomyocyte necrosis. This results in infarct expansion, dilation of the heart, scar formation, and depletion of cardiac pump function. Current therapies to restore blood flow to the affected tissue causes ischemia reperfusion (IR) injury which increases cardiomyocyte death and can lead to heart failure and mortality. Our lab has recently discovered a novel stem cell in the bone stroma, which we have called cortical bone stem cells (CBSCs). In both murine and porcine MI models, CBSCs were demonstrated to reduce scar size, promote cardioprotection of cardiomyocytes, induce new myocyte formation, and improve cardiac pump function post MI. Additionally, cardiomyocyte mass was shown to increase following CBSC treatment in pig MI/IR models at 3 months post MI. However, it is unclear if this increase in viable mass mainly results from protection of cardiomyocytes from death or the induction of myocyte growth from preexisting cardiomyocytes, rather than CBSCs. We will determine if CBSC secreted exosomes (CBSC-exo) are responsible for improving cardiac structure and function post MI/IR as seen with CBSC therapy. We will also define how CBSCs and/or their exosomes (CBSC-exo) directly protect adult cardiomyocytes from cell death, induce cardiomyocytes to reenter the cell cycle, and promote angiogenesis. For experimental studies, mice will undergo MI/IR surgery followed by injection of GFP+CBSCs, CBSC-exo, or saline. Cardiac repair will be assessed by echocardiography, invasive hemodynamics, histology and molecular studies to evaluate cardiac function, structural remodeling, cardiomyocyte density, and new blood vessel formation. To study the direct effects of CBSCs and their paracrine factors on myocytes and endothelial cells, adult feline cardiomyocytes or human endothelial cells will be subject to hypoxic damage and then treated with CBSCs, CBSC-exo, or CBSC cultured media. This proposed research should provide insight on how stem cells mediate cardiac repair through paracrine signaling. Results from these studies will determine whether exosomes can be used as a novel cell-free therapy to better repair the heart after MI/IR."}
{"text": "Activities in this area are focused on the development of agents with novel mechanisms of action, and the development of novel combinations. A range of compounds are under study. Currently, phase II studies being conducted in ovarian cancer include: 9-AC, a topoisomerase I inhibitor; MGI-114, a novel DNA damaging agent; and, CAI, an anti-angiogenesis agent. A phase I study is beginning, using SU5416 with carboplatin in recurrent ovarian cancer. SU5416 is an anti-VEGF agent with molecular properties that inhibit angiogenesis, and may impair platinum-DNA adduct repair. Preclinically, gene therapy approaches in ovarian cancer are being explored, using dominant negative constructs to AP1 that have been developed by Dr. Charles Vinson of DBS. Also, proteasome inhibition is being explored on the preclinical level in human ovarian cancer cells. In prostate cancer, a range of agents have also been studied. Such agents include gallium nitrate (a heavy metal with a nubmer of activities), high dose tamoxifen (PKC inhibition), thalidomide (anti-angiogenesis) and thalidomide combinations, CAI (anti-angiogenesis), and ketoconazole (anti-androgenic agent) and ketoconazole combinations. - angiogenesis, ovarian cancer, pharmacology, platinum compounds, prostate cancer, - Human Subjects & Human Tissues, Fluids, Cells, etc."}
{"text": "Genomes can be altered in a variety of ways including single base pair changes, deletions and duplications of sequences, and chromosome rearrangements (translocations and inversions). The rates at which these events occur affect 2 quite different processes. Genetic changes are required for evolution and the rate of these changes, therefore, can in principle affect the rate of evolution. Second, many cancers are associated with elevated rates of chromosome rearrangements, and this type of instability appears to be strongly associated with metastatic tumors. When mammalian cells are treated with aphidicolin (a DNA polymerase inhibitor), the chromosomes are broken at specific sites (fragile sites) that are related to translocation breakpoints observed in tumor cells. High levels of chromosome breaks and other aberrations are also observed in mammalian cells with mutations in the ATM and ATR genes. Our general goal is to mimic this type of genetic instability in the yeast Saccharomyces cerevisae, and to characterize the mechanisms responsible for this instability. Our specific aims are: 1) to determine the DNA sequence properties that characterize yeast fragile sites and to characterize the chromosome rearrangements that are associated with DNA breaks at fragile sites, 2) to characterize chromosome aberrations in yeast strains with mutations in genes affecting DNA damage checkpoints, telomere length regulation, recombination, and/or chromatin assembly; for example, strains with mec1 and/or tel1 genes (yeast homologues of the mammalian ATR and ATM genes, respectively) will be examined, and 3) to understand the genetic instability induced by ionizing radiation. These studies will emphasize techniques that allow examination of the entire yeast genome, including DNA microarray analysis (to detect changes in gene dosage and to map translocation breakpoints), and gel technologies that allow separation of intact chromosomal DNA molecules. [unreadable] [unreadable] [unreadable]"}
{"text": "The olfactory system is able to recognize thousands of odorants thus maintaining food consumption in humans. The ability to identify odors by humans is dependent on odorant receptors within olfactory neurons that line the nasal epithelium. This epithelium is under continuous cell division by a basal stem cell population that produces immature olfactory neurons, which then become mature olfactory neurons. Odorant receptors (ORs) are clonally expressed in immature olfactory neurons. The mammalian genome consists of ~1500 OR genes; these genes belong to the seven-transmembrane receptor superfamily. However, each allele of each gene is treated as a separate entity leading to ~3000 OR alleles to be expressed in a clonal (singular) manner by olfactory neurons. The ability for this singular OR allele expression is a multi-step manner. A critical step is the production of a functional OR protein. Olfactory neurons are found in domains within the epithelium such that a given olfactory neuron chooses (in general) between ~100 OR genes (~200 alleles). The selection process occurs in two-steps: one of ~200 alleles is chosen for expression (1st choice) within an immature neuron, followed by OR protein production, which is tested for functionality. Failure to produce a seven-transmembrane protein allows for the singular choice mechanism to choose another OR allele in that same immature olfactory neuron (2nd choice). If the chosen seven-transmembrane OR is unable to couple with the signal transduction machinery, then that olfactory neuron will fail to mature and die. The critical step in the expression of an OR gene is the initial choice of one OR allele within an immature neuron (1st choice). To date, it is completely unknown how this singular choice process occurs at a molecular level. Analysis of very small OR promoter regions (~300bp) have lead to the identification of two transcription factors that may play a role: an O/E-1 type binding protein and a homeodomain type biding protein. The identification of more candidate DNA binding proteins and their interactions is necessary to understand the singular choice process. This project fully exploits the small regulatory regions used to produce OR singular choice in immature olfactory neurons. In Aim 1, the most minimal OR control region will be defined in order to identify other candidate transcription factors. There are at least 10-15 different olfactory neuronal cell types each fated to express one of ~200 OR alleles. In Aim 2, two minimal OR promoters normally expressed in different olfactory cell types will be used to identify additional transcription factors. This will allow the understanding of how specific patterns within the olfactory epithelium are obtained. In Aim 3, a candidate homeodomain protein will be deleted within immature and/or mature neurons in order to determine its role on OR gene expression. In Aim 4, I will make every attempt to promote this work into publications and greater funding."}
{"text": "The proposed research can be divided into two broad sections: 1) A. -Special histological studies will be continued in cases demonstrating alterations in fluid pathophysiology of the inner ear. This would include study of the endolymphatic sac and cochlear aqueduct systems together with their respective vascular channels, as well as the vestibular organs and cochlea. B. -Detailed study of cochlear otosclerosis, with and without sensorineural deafness, will be performed, relating possible etiological factors to certain specific tissue changes in some cases of cochlear otosclerosis. 2) The laboratory has well over 500 temporal bones, received either as UCLA autopsy specimens or from the National Temporal Bone Banks Program. Histological examination of these temporal bones creates interest in detailed studies of particular middle and inner ear diseases. Many specimens demonstrate unexpected or unusual changes, and one cannot precisely predict in which direction such findings may lead."}
{"text": "To identify alcoholism risk genes, we are collecting and testing for linkage and pattern of genetic transmission families from three different American Indian populations which are relatively homogeneous and in which alcoholism is highly prevalent. This study also addresses the genetic epidemiology and psychiatry comorbidity of alcoholism in American Indians using structured psychiatric diagnostic interviews of subjects in large families. Data sets of Cheyenne Indian and Pima Indian subjects from large families have been collected. A large panel of random polymorphic probes is being typed in Pima Indian and Cheyenne Indian alcoholics and controls and genetic linkage and transmission analyses are in progress for these groups. An analysis of psychiatric comorbidity in Pima Indian alcoholics and nonalcoholics has been completed."}
{"text": "An estimated 23 million Americans, including adolescents and the elderly, suffer from circadian rhythm sleep disorders, such as delayed sleep phase disorder, advanced sleep phase disorder and winter depression. These conditions are characterized by persistent insomnia and/or excessive daytime sleepiness, impaired performance, reduced well being and lower quality of life. The negative symptoms result from a misalignment between the timing of sleep and the internal circadian clock. Clinical research has demonstrated that circadian rhythm sleep disorders are most effectively diagnosed (differentiated from other causes of insomnia) and treated if each individual patient's circadian phase is known. The timing of the master internal circadian clock is most reliably measured from the onset of the endogenous circadian rhythm of melatonin, a neuroendocrine hormone, as measured in dim light (dim light melatonin onset, or \"DLMO\"). However to date the reliable and valid assessment of the DLMO is limited to the research laboratory setting. This application answers PAR-08-212 \"Methodology and Measurement in the Behavioral and Social Sciences\" which calls for research to \"improve and develop methodology and measurement in the behavioral sciences through innovations in data collection techniques and measurement...approaches that integrate behavioral science research with biological science research are particularly encouraged...developing and validating research instruments that fill a gap in research needs...improve measurement in the real-world setting.\" Our goal is to develop a streamlined procedure for the accurate assessment of circadian phase (DLMO) outside of the laboratory that will provide clinicians and researchers with a novel diagnostic and research tool. In this way the underlying neurobiological cause of a patient's insomnia and/or circadian rhythm disorder can more readily be diagnosed and treated. Specific Aim 1 is to validate procedures for at-home circadian phase assessment in a large sample of healthy people (n=120). Validation will occur by (1) objectively measuring compliance to the at-home procedures and (2) comparing DLMOs collected at home to DLMOs collected in the laboratory, in a within-subjects counterbalanced design. We have pilot data to support the validity of our at home procedures. Specific Aim 2 is to validate the same at home procedures in patients with delayed sleep phase disorder (n=30). Specific Aim 3 is to conduct rigorous analyses to inform future users which subject characteristics and light levels predict (1) compliance to the at home procedures and (2) valid at-home DLMOs. The results of this 3 year study will have substantial implications for the translation of basic and clinical research to the community: (1) the diagnosis and treatment of insomnia and circadian rhythm sleep disorders will be significantly enhanced, thus improving public health and safety, mood and quality of life, (2) community participation in research will be improved, particularly in vulnerable and under represented populations, thus increasing scientific knowledge and (3) research and clinical costs will be substantially reduced. PUBLIC HEALTH RELEVANCE: This research will test a new method to measure the timing of the body clock at home, instead of in the research laboratory. The results will facilitate the diagnosis and treatment of circadian rhythm sleep disorders such as advanced sleep phase disorder, delayed sleep phase disorder and also winter depression, thereby improving public health and safety, well-being, mood, mental function, and quality of life."}
{"text": "The major aim of this project is to examine which subjects are likely to succeed in a behavioral obesity treatment. The first objective is to empirically derive predictors of successful response to a behavior modification approach. Predictors which will be examined are weight loss history (prior dieting), severity of overweight, extent of binging, and attitudes about dieting (perfectionistic goal setting). The second objective is to cross-validate these predictors on another sample of subjects. Moderately overweight females, from 150-200 lbs, at the time of their application and ranging in age from 25-45 will be studied. Treatment will be conducted in groups led by experienced behavior therapists."}
{"text": "The first aim of this research is to investigate the properties of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) whose constituent fatty acyl chains are highly asymmetric in length. There are many important membrane components, such as the ceramide, sulfatide, ganglioside, and sphingomyelin molecules of nervous tissue, which contain two hydrocarbon chains of inequivalent length. The results of the research proposed in this project will help lead to an understanding of the effect of chain length asymmetry on the properties of lipids in natural membranes. It has recently been demonstrated that the asymmetric-chain phospholipid, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine, possesses important physiological functions as a platelet activating factor (PAF) and as an antihypertensive agent. A long term research project is being initiated in my laboratory with the objective of studing the physical and physiological properties of this important phospholipid. Thus, a second aim of this proposal is to begin this project by studying the physical properties of synthetic PAF and to attempt to isolate the receptor for PAF that has recently been shown to exist in human platelet membranes. Specifically, we propose to (1) investigate the properties of saturated mixed-chain PEs, C(18):C(18)PE through C(18):C(2)PE. This work will complement the studies that we have already carried out on the properties of saturated mixed-chain PCs. (2) to investigate the properties of binary mixtures of mixed-chain PCs and PEs with symmetric-chain phospholipids with particular emphasis on the mixing properties of the two lipid types as well as the possible existence of nonlamellar phases in the binary combinations; (3) to study the properties of synthetic PAF in comparison to those of C(18):C(2) PC. As C(18):C(2)PC is physiologically inactive, a comparison of the properties of these two lipids may serve to elucidate some of the important structural properties of PAF necessary for its physiological function; (4) to attempt to isolate the membrane receptor for PAF that has been shown to exist in human platelets by employing a combination of detergent solubilization and affinity chromatography. Principal techniques to be used are differential scanning calorimetry and densitometry, 31P-NMR, electron microscopy, quasi-elastic light scattering, Raman spectroscopy, detergent solubilization, and affinity chromatography."}
{"text": "Intravascular administration of cell-free hemoglobin appears to be strongly correlated with a rise in pulmonary and systemic blood pressure. This hypertensive response has been attributed to the reaction between hemoglobin and the endothelium-derived relaxing factor (EDRF), which is now believed to be nitric oxide (NO). We explored the interactions between NO and the oxidized forms of a number of chemically modified hemoglobins. These hemoglobins are blood substitute candidates due to their lower oxygen affinity and greater stability of their tetrameric structures. Using fast reaction techniques, the stopped flow apparatus, we documented that there are differences in the rate of the initial NO binding to the ferric forms of these proteins and in the subsequent NO-driven reduction step. Since NO oxidizes oxyhemoglobin to ferrihemoglobin (metHb) extremely rapidly in vivo, we hypothesized that a redox cycle between NO and metHb could be partially responsible for the depletion of NO as a biological transducer, leading to altered vascular tone with variable effects observed for different hemoglobin preparations. This and other related work was presented at a recent international meeting on blood substitutes and recently appeared in full in Arch. Biochem. Biophys. Work is in progress to explore the mechanism(s) of interactions of NO with other hemoproteins to fully understand the degree to which heme pocket chemistry and other structural features determine the binding of NO to hemoproteins. This will ultimately contribute to the design of hemoglobin that, along with desirable oxygen binding characteristics, exhibits a suppressed ability to react with NO."}
{"text": "Summary: With the potential threat of smallpox as a bioterrorism agent, a major effort should be invested in two main areas: a) development of new effective preventive vaccines, and b) production of high titer vaccinia IGG (VIG) that can be used as a first line of defense in exposed and/or high risk populations. The current stocks of VIG are limited and outdated. Scientists at CBER and elsewhere (MPH) were encouraged to produce new lots of VIG and subject them to careful titration in validated neutralization assays. In addition, experiments are underway to compare the neutralization capacity of different VIG subclasses, in order to optimize viral neutralization in future production of VIG.The current neutralization assay for vaccinia (plaque-reduction), is labor intensive, slow (up to 7 days), and depends on scoring plaques by eye (i.e., subjective). This assay is difficult to validate and to transfer to sponsor-based laboratories. As an alternative approach, our laboratory initiated late last year the development of an alternative vaccinia neutralization assay, which is fast, quantitative, and easy to perform and transfer to other laboratories.This assay is based on the use of recombinant vaccinia virus expressing a bacterial gene coding for the enzyme b-Gal under the control of the vaccinia late promoter. Several recombinant viruses were tested and two of them, vSC8 and vSC56 are currently under investigation in our laboratory. We also tested several cell substrates for the assay: BSC1, Vero, and HeLa. Results: - We have established and standardized a novel assay to measure vaccinia neutralization. It is based on the expression of a reporter gene coding for the bacterial b-galactosidase enzyme (b-Gal) under the control of a synthetic E/L promoter. We demonstrate that the new assay is rapid (24 hr), of equal sensitivity to PRNT assays, reproducible, objective, and easy to transfer. In addition, we describe preliminary results with a second reporter gene assay based on a vaccinia-EGFP recombinant virus. The expression of the GFP is induced by IPTG. The two assays provide high throughput capabilities and may be established in clinical laboratories for the evaluation of multiple samples from clinical trials. - Thus far, it was found that the MPH VIG is somewhat better than the Baxter (4oC) VIG with ID(50) values of 10-15 ug/ml and 25 ug/ml, respectively. - We also tested Ig subclasses derived from the MPH VIG and found that the neutralization efficiency of the IgG1 subclass is superior to all other subclasses and to the total (unseparated) VIG preparation. - We tested multiple lots of IVIG from 5 different manufacturers. Several lots were found to have significant anti-vaccinia neutralization titers."}
{"text": "This revision of U24 l\\/lH068457-08, \"NIMH Center for Collaborative Studies on Mental Disorders,\" (NIMHC) requests funding for a \"Repository Supporting Stem Cell Research Relevant to Mental Disorders\" in response to NOT-MH-10-024. Since late 1998, the NIMHC, whose biorepository is at Rutgers University and whose data repository is at Washington University School of Medicine, has established a robust infrastructure for the receipt of blood samples and externally produced cell cultures. DNA and RNA has been extracted from blood and over 75,000 lymphoblastoid cell lines have been produced in-house for the NIMHC, with 99.8% success on the first attempt. In the past 5 years, the NIMHC has distributed -450,000 DNA, ~15,000 RNA and ~8,000 cell lines to researchers throughout the world. Based on serving the needs of NIMH Genetics Initiative grantees, the NIMHC maintains secure clinical (phenotypic) and genomic databases and a password protected, secure website for NIMH-approved researchers to access. The NIMHC has also developed and made available web-based bioinformatics resources for the design, analysis and interpretation of studies in psychiatric genetics. We propose to utilize the facilities, expertise and experience of the NIMHC to serve researchers wishing to use human patient and control primary cells, adult stem cells and induced pluripotent stem cells (iPSCs) for investigation of the cellular bases of mental disorders. The NIMHC will receive primary tissue samples for the establishment and banking of primary cell cultures or adult stem cell lines. The NIMHC will also receive cultures of primary cells, adult stem cells and iPSCs for characterization and banking. These cell lines, along with subject clinical and genomic data and molecular and phenotypic data describing the cell lines, will be distributed to NIMH-approved researchers. The NIMHC will also provide consultation regarding the nature and use of these stem cell lines. To a lesser extent, the NIMHC will also produce, characterize and bank iPSCs made from primary cells. Freely sharing primary and stem cells within the research community is likely to greatly accelerate progress in understanding the cellular bases of mental disorders. Public Health Relevance: Mental disorders are a common, widespread and heterogeneous group of diseases whose biological bases are poorly understood. Elucidation of the underlying genetic and physiological bases for these disorders is necessary for the creation of effective therapeutics. Nerve cells or other cells derived from adult stem cells offer the possibility of modeling these disorders in cell culture and for preliminary screening of pharmaceuticals."}
{"text": "This study will enroll 10 people currently participating in the extension phase of ACTG 306 plus an additional 60 people not participating in ACTG 306. The study will last six months. The purpose of the study is 1) to compare the effects of two ZDV-containing drug combinations to see how many people ahve an undetectable viral load at both weeks 20 and 24; 2) to compare the safety of each of the three drug combinations."}
{"text": "Project Summary Problems frequently encountered social and behavioral science researchers concern heterogeneity of the: 1) individuals needing treatment, 2) interventions responding to individuals'specific needs, and 3) array of potential outcomes resulting from treatment (Goldenberg, 1978;Gordon, Powell, Rockwood, 1999;Rockwood, Joyce, &Stolee, 1997). A common response to these problems is to identify various outcome measures that encompass the variability in patients, treatments, and outcomes (Kazdin, 2005). In these studies, the patients treated are often assessed on all outcomes. Analyses often involve multivariate approaches or latent class models. A problem with this design and analysis strategy is that when patients are assessed on all potential measures, many are assessed on outcomes that their specific treatment plan was never designed to address. The project is designed to expand the research base on analytic methods to assess the statistical significance associated with interventions targeted at heterogeneous populations. Specifically, the project will continue the study of the maximum individualized change (MIC) procedure developed by Boothroyd, Banks, Evans, Greenbaum, and Brown (2004). The method was initially developed as an analytic alternative to the more traditional multivariate and latent model approaches often used in studies examining individually-tailored interventions. Our initial developmental work on this approach suggests that it offers a number of significant advantages over traditional statistical approaches in studies where a number of measures are used to assess potential treatment outcomes. These advantages include increased statistical power to detect smaller program effects and few problems associated with missing data. Despite the MIC's early promise, more basic work is necessary to determine whether this approach should become a standard analytic tool in future studies. The study will consist of two phases. First, an expanded simulation study of the MIC procedure will be conducted (Boothroyd et al. 2004) broadening the initial set of assumptions. Specifically, the impact of seven variables will be systematically examined. The variables include varying the: 1) level of correlation among outcome measures, 2) number of outcome measures/domains being assessed, 3) distribution of response effects, 4) number of outcome measures on which a person improves, 5) sample size, 6) weighting the outcome measures based on qualitative assessment regarding the likelihood of change, and 7) amount of missing data. In phase two, the MIC procedure will be used in the re-analysis of data collected as part of SAMHSA-funded multi-site national study examining the impact of managed care. In a recently published article summarizing the study findings, the authors concluded, \"Managed care and fee-for-service Medicaid programs did not differ on most measures;however, a lack of sufficient power was evident for many measures\" (Leff, et al., 2005)."}
{"text": "This is the first submission of an application to investigate the mechanism of Zn2+ toxicity in the CNS. Recent findings indicate that this cation is elevated during stroke, traumatic brain injury, and oxidative stress, and that elevated intracellular Zn2+ is associated with the death of cultured neurons. Elevated Zn2+ is also associated with mitochondrial dysfunction, and with the death of cells by necrosis and apoptosis, although the mechanism of these effects is unknown. The investigator and his associates have recent findings that seem to bear upon the mechanism of Zn2+ toxicity at the cellular level. They have found that submicromolar concentrations of Zn2+ inhibit the mitochondrial alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase complexes, and that Zn2+ stimulates the mitochondrial permeability transition. They also note that oxidative stress can release Zn2+ from metallothioneins and that the metallothionein expressed in brain is associated with the mitochondrial outer membrane. Putting all of this together, the investigator hypothesizes that cell injury caused by oxidative stress arises, at least in part, because Zn2+ is released from metallothionein, inhibits the TCA cycle at the level of one or both of the dehydrogenases, and thereby, or through other mechanisms, provokes the permeability transition and subsequent cell death. To begin evaluating this hypothesis, Dr. Brown will investigate the mechanism by which Zn2+ inhibits the dehydrogenases using enzyme kinetics and the study of partial reactions. Mechanisms transporting Zn2+ in mitochondria will be determined, and respiration studies will be conducted to ascertain the relative importance of Zn2+ action on the dehydrogenases, compared to the electron transport chain, in producing mitochondrial dysfunction. Finally, the mechanism by which Zn2+ influences the permeability transition will be determined. In several cases data obtained from brain, heart, and liver mitochondria will be compared, to ascertain the generality of the findings, and to allow a cross comparisons of literature, which is developed to different extents for mitochondria from these various organs."}
{"text": "Mechanisms and Importance of Opioid and ORL-1 Receptor Coupling to Ca2+ Channel Subtypes 1. What are the molecular identities of opioid and orphanin FQ-sensitive Ca2+ channel subtypes? a. Do ORL- 1 receptors couple like opioid receptors to L-type Ca2+ channels when expressed in GH3 cells? b. Which different L-type Ca2+ channel a1 subunit transcripts are expressed by GH3 cells? c. Do antisense oligonucleotides to the a1 subunit abolish the opioid- and orphanin-sensitive Ca2+ current component? d. Do opioid and ORL-1 receptors expressed in HEK293 cells couple to Ca2+ channels formed by a10 subunits? 2. What G protein mechanisms couple opioid and ORL-1 receptors to CA2+ channels? a. Is there a consensus sequence at which bg binds to all opioid and orphanin FQ regulated Ca2+ channels? b. Does a peptide mimicking this sequence prevent receptor coupling to L- type Ca2+ channels in GH3 cells? c. Can a similar approach uncouple opioid receptors from a1a, a1b and a1d Ca2+ channels in HEK293 cells? 3. What is the contribution of L-type Ca2+ channels, relative to other effectors, in the modulation of vesicular release by opioids and orphanin FQ? a. Other than Ca2+ channels, what effectors are regulated by opioids and orphanin FQ in GH3 cells? b. Do opioids inhibit pro[actin release from GH3 cells through G protein bg subunits? c. What is the contribution of L-type Ca2+ channel inhibition in the opioid regulation of prolactin release from GH3 cells? 4. Do specific Ca2+ channel subtypes have a role in the central actions of opioids and orphanin FQ? a. Are a10 subunits (or other opioid and orphanin FQ sensitive subunits) coexpressed with opioid and/or ORL-1 receptors in central neurons? b. Do specific Ca2+ channel subtypes participate in the opioid and/or orphanin FQ regulation of dopamine release in the reward pathway?"}
{"text": "The mission of the University of Toronto MHSc in Bioethics International Stream is to strengthen individual and institutional leadership capacity in bioethics, with particular emphasis on research ethics, in low- and middle-income countries. We work in partnership with existing and emerging programs in strong institutions in the developing world to identify leaders who will make those programs successful and sustainable. Building upon the success of the program over the past 8 years, (32 graduates as of June 2008, program experience rated highly by graduates), the reach of the program will refocus on West Africa (especially Ghana and Nigeria), and continue to focus on South Asia (extending beyond India and Pakistan to Bangladesh). Key emerging issues of global ethics (such as genomics/biotechnology and public health ethics) will be incorporated into the curriculum. We will build on continuous professional development program for graduates. The University of Toronto Component consists of 10 months of course work covering breadth and depth areas in bioethics, and resulting in the MHSc degree. The Home Country Component consists of 14 months of mentored research, educational, and leadership activity in bioethics in the trainee's home country culminating in a workshop in Toronto to facilitate intertrainee exchange of experiences, ideas, successes, and challenges. The program draws upon an internationally-distinguished faculty from a variety of disciplines. Over 4 years, the program will train at least 12 highly-qualified individuals nominated by home institutions and carefully selected for potential impact in their home country. We believe our targeted and strategic approach, intellectually rich environment for bioethics scholarship, legitimizing effect and discipline of the graduate degree, emphasis on adult learners and career development, continuous evaluation and improvement of our programs, and long-standing and trusting partnerships with institutions in developing countries, are the key success factors of our program. PUBLIC HEALTH RELEVANCE: As clinical research expands globally it is imperative to build capacity in the developing world for the ethical assessment and regulation of research in the contexts where the research will be conducted. The University of Toronto, Masters of Health Sciences in Bioethics (MHSc), International Stream is devoted to contributing to capacity building efforts in international research ethics. Over the past 8 years, working with established organizations in the developing world, the program has graduated 32 fellows with MHSc degrees who are leaders in scholarship and practice in ethics and regulation of human research. In this renewal we intend to continue our efforts in capacity building with a specific focus on Nigeria, India, Pakistan and Bangladesh, four of the most populous nations in the world, with pressing health needs. [unreadable] [unreadable] [unreadable]"}
{"text": "PROJECT ABSTRACT Most neurodegenerative disorders exhibit highly heterogeneous genetic underpinnings. For example, with Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and Parkinson's disease (PD), mutations in different genes are causal in distinct subsets of families. In addition to this genetic heterogeneity among inherited cases, the majority of individuals exhibit sporadic forms of these disorders in which there are no known causal mutations. In part because of this sporadic onset, it is widely accepted that (largely unknown) environmental forces are at play in the initiation and/or progression of each of the above disorders. This proposal will use a systems approach in Drosophila to identify forces that drive initiation, as well as common cellular responses that may modulate progression. This will be accomplished by three scientific aims. First, we will test a series of cellular stressors, behavioral stressors, and models of injury/inflammation. The effects of these manipulations will be assayed by following 7 different neurodegenerative phenotypes and biomarkers, including a novel assay of endogenous retrovirus replication. Second, we will use a relatively new approach to purify the population of cells that are most impacted, and profile active transcription within the nuclei. This experiment will identify common downstream cellular responses. Finally, we take advantage of high throughput genetic approaches in Drosophila to systematically test for functional impact of identified gene targets."}
{"text": "The overall goal of our grant proposal is to evaluate the possibility that natural HIV viral env sequences that emerge during the course of HIV infection, can be used as immunogens to elicit broadly-reactive anti-HIV neutralizing antibodies. These env sequences will be derived from HIV infected subjects which during a very short period of time (2-3 years) developed broad and potent anti-HIV cross-neutralizing antibody responses. In Project 1, we will monitor HIV infected subjects to identify those that develop broad cross-neutralizing antibody responses shortly following infection and we will characterize in detail these responses. We will amplify viral env from longitudinal samples from these patients and in conjunction with Project 2 we will examine what role the CD4+ T cell helper responses have in the development and maintenance of broad anti-HIV neutralizing antibody responses. The Envs identified in this Project 1 will be used as immunogens in Project 3 to test the hypothesis that they can elicit broad cross-neutralizing antibody responses in animals. RELEVANCE (See instructions): Our proposed studies will explore a novel concept in the development of an immunization protocol that will lead to the elicitation of broad cross-neutralizing antibodies against HIV. As such, the proposed studies are highly relevant to the development of an effective anti-HIV vaccine."}
{"text": "Please reference the abstract RELEVANCE (See instructions):"}
{"text": "The investigators objective is to determine the physical and molecular basis for the cooperativity found between the troponin-tropomyosin complexes which make up the regulatory strand of the thin filament. To this end, methods have been developed or are under development to manipulate the regulatory proteins of chemically skinned mammalian muscle fibers. Troponin C, I, T and tropomyosin will be extracted or replaced either singly or in various combinations with structurally different isomers or with chemically modified ones purified from various muscles. When extraction without replacement is possible, the effect of extraction on cooperativity during thin filament activation by Ca2+ (slope of the pCa/tension relationship) and by rigor crossbridges (slope of the pS/tension relationship) will be examined. Through replacement techniques, fluorescent probes or other structural modifications will be introduced into the regulatory strand of skinned fast and slow muscle fibers. Fluorescence changes in single fibers will be used to measure Ca2+ binding, crossbirdge binding and the state of the regulatory strand simultaneously with tension. The apparatus developed for these studies automates solution mixing, protocol execution, and the collection of fluorescence and tension data; similar apparatus without the fluorescence measurement capability is used to develop better methods of exchanging the regulatory proteins of the thin filament. The cooperative regulation of thin filaments will be modeled with the classical formalism for allosteric proteins and with an induced shift formalism under development. By comparing pCa/tension and pS/tension data from control, extracted, and reconstituted fibers with the model expectations, experimental tests of proposed cooperative mechanisms are developed and new insights into the relationship between molecular form and thin filament physiology are realized. The cooperativity found between the regulatory complexes of the thin filament is more extensive than any molecular interactions hitherto described; it integrates the actions of at least 75 regulatory protein molecules and unifies control over hundreds of actin molecules. To explain it in physical terms may require the development of new ways to envisage molecular interactions in biological structures. Through this contribution to basic knowledge, the proposed work will contribute to the capacity of biomedical researchers to develop treatments for disease."}
{"text": "Guanosine triphosphate-binding proteins regulate numerous processes in all cells. The types of process include synthesis, signal transduction, growth regulation and intracellular transport, and several others. These proteins vary in size and structure, but they share regions of conserved protein sequence, in their GTP-binding domains. Moreover the GTP-binding domains of two of these proteins, ras p21 and elongation factor Tu, possess nearly identical tertiary structures. The similarity of these structures suggests that GTP and GDP similarly alter the structures of these proteins and indeed those of all GTP regulatory proteins. This conclusion warrants the use of reasoning by analogy to define the functions of these proteins. The peptide chain elongation factors Tu and Ts comprise a GTP-regulatory system whose interaction with a variety of cellular components make it an informative subject both for the study of GTP-promoted processes and for the identification of protein-protein and protein-nucleic acid interactions. EF-Tu presents a useful model for these processes because it is an abundant, soluble protein of tractable size whose tertiary structure has been largely solved. EF-Tu engages in three separate but interdependent processes-GTP-mediated aatRNA-binding, ribosome-mediated GTP hydrolysis, and EF-Ts-mediated GDP exchange. Other GTP-regulatory proteins engage in analogous processes. To understand how these processes occur, we are determining how single amino acid replacements alter each of the functions. The structural consequences of these changes are determined by reference to the EF-Tu structure, and the effect of the analogous change on other GTP-regulatory proteins is predicted. The reliability of this approach can be assessed by comparing the effects of analogous changes on the functions of EF-Tu and ras p21, whose structure is also known."}
{"text": "This Phase II SBIR will develop Continuous Real Time (CRT) ShuntCheck, the first portable, non- invasive device for real time, continuous monitoring of changes in flow in CSF shunts. This device will result in improved clinical management of hydrocephalus by providing a non-invasive method for monitoring and researching shunt function. Hydrocephalus, a common condition in which CSF accumulates in the brain ventricles, is corrected by placing a VP shunt that drains excess CSF to the abdomen. Shunts frequently malfunction, usually by obstruction, but the symptoms of shunt failure are unspecific - headache, nausea. Diagnosis of shunt malfunction is expensive and presents risks (exposure to radiation from CT Scans, risk of infection from radionuclide testing). Additionally, ongoing clinical management of shunted patients is complex (due to a lack of tools for investigating CSF over drainage, for assessing the performance of specific shunt valves and siphon control devices and for streamlining the adjustment of programmable shunt valves). NeuroDx's existing device, ShuntCheck-Micro-Pumper, is a shunt obstruction detector and addresses the need for a non-invasive test for shunt malfunction. While this makes it a valuable tool for the Emergency Dept, the short duration of the test limits its utility for shunt valve adjustment, investigating suspected shunt over drainage, etc. A non-invasive, non-radiologic device which can track changes in CSF flow rate would address many ongoing clinical management needs and become a valuable tool for the neurosurgery clinic. In our Phase I studies, we developed a laboratory prototype CRT based upon a breakthrough innovation in our thermal dilution technology and validated its safety and accuracy in bench and animal studies. CRT can reliably differentiate between no, low and robust shunt flow and can track changes in shunt flow rates over extended time periods. The goal of this Phase II project is to refine CRT ShuntCheck from a laboratory prototype to a production-ready device, validate its safety and accuracy in bench and animal testing, and complete a 510k submission for FDA clearance. Post-Phase II clinical studies will demonstrate the clinical utility and cost effectiveness of CRT ShuntCheck for streamlining valve adjustment in pediatric and in adult NPH patients. NeuroDx's business model for this product involves the generation of revenue primarily from the ongoing sale of single-use, disposable sensors for these tests. Shunt management testing constitutes approximately 105,000 shunt flow tests annually in the United States alone. The need for new diagnostic tools for managing hydrocephalus patients is highlighted by the NIH announcement Advanced Tools and Technologies for Cerebrospinal Fluid Shunts (PA-09-206), to which this application is responding. Our application directly responds to the request for Diagnostic tools for use in a hospital or outpatient setting that work in real-time to quantitatively determine shun function."}
{"text": "Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine phosphatase that is a critical component of signaling pathways regulating cell function. The heterodimeric core of PP2A associates with a variety of regulatory subunits that contribute to the specificity and subcellular localization of the enzyme. PP2A also interacts with a number of proteins that are not considered to be conventional regulatory subunits of the enzyme. The investigators anticipate that identification of additional interacting partner for PP2A will provide new insights into the cellular organization of the enzyme and that such organization dictates specificity of enzyme action. One sub-population of PP2A is associated with microtubules in neurons. Tau and MAP2 are neuronal microtubule-associated proteins (MAPs) that are highly phosphorylated in vivo and are targets for numerous protein kinases in vitro. The phosphorylation state of tau and MAP2 determines their abilities to bind to and thus stabilize microtubules. Results from investigators' laboratory indicate that PP2A dephosphorylates tau and MAP2 in vivo, suggesting the enzyme regulates the neuronal cytoskeleton. An important aspect of this work is that one of the pathological hallmarks of Alzheimer's Disease includes hyperphosphorylation of tau and disruption of axonal microtubules. The proposal has three specific aims. 1) The first aim will test the hypothesis that an anchoring protein that promotes targeting of PP2A to microtubules is required for efficient dephosphorylation of tau and MAP2. The molecular basis of PP2A interaction with the microtubule cytoskeleton will be explored. Putative anchoring protein(s) will be purified, characterized, and cDNAs cloned. The role of anchoring in regulating PP2A will be examined with the assumption that direct protein-protein interactions define the specificity of PP2A toward neuronal MAPs. 2) The underlying hypothesis of aim 2 is that suppression of PP2A in neurons of intact animals will lead to increased phosphorylation of tau and neuronal degeneration. SV40 small-t antigen, a specific inhibitor of PP2A, will be expressed in transgenic mice using neuron-specific promoters. The effects of reduced PP2A activity will be assessed by measuring tau phosphorylation and neuronal morphology. 3) The hypothesis to be tested in this aim is that two novel PP2A- interacting protein influence the activity and function of PP2A. Characterization of p31 and p17, two PP2A-interacting proteins identified in a two-hybrid screen, will be pursued with high priority. The effects of these proteins on activity, subcellular localization, and in vivo function of PP2A will be determined. Additional candidate interacting proteins, identified in the two-hybrid screen will also be characterized."}
{"text": "Autologous skin grafts are the preferred method for covering large burn surfaces. The healing of autologous skin grafts, and the re-harvesting of skin donor sites for autologous skin grafts, is often slowed by the altered metabolic state of burn victims. Accelerating the healing of the skin graft donor site would increase the number of sites that could be used and decrease the re-harvest time between grafts. One of the single most important factors in healing a skin graft donor site is the neovascularization and reestablishment of oxygen tension and nutrient levels in the wound. Ultimately the oxygen, and some of the nutrients, are used to produce adenosine triphosphate (ATP). ATP is used for nearly every aspect of the wound healing process, including protein synthesis, growth factor production, and mitosis. We have developed a technique for the rapid and controlled delivery of ATP using fusogenic lipid vesicles (VitaSol). VitaSol, when applied topically, provides wounds with ATP during hypoxic periods and accelerates the healing process of acutely wounded skin. The long-term objective of this proposal is to develop a safe and effective technique to accelerate the healing of partial-thickness wounds in swine using VitaSol, and prepare the compound for a Phase I clinical trial for autologous skin grafts. The specific aims of phase I project are: 1) Determine the mechanism by which VitaSol accelerates partial-thickness wound healing in swine. We hypothesize that the beneficial effects of increasing wound ATP levels are related to increased angiogenesis, increased angiogenic growth factor synthesis, and accelerated mitosis of keratinocytes and fibroblasts; 2) Optimize the formulation of VitaSol for maximal acceleration of partialthickness wound healing in swine. Once the mechanism of how VitaSol accelerates wound healing is known, the formulation will be mixed with an excipient to create a hydrogel which can be easily applied to wounds, and helps maintain ATP levels during a 24 hour period; and 3) Stabilize the optimized VitaSol formulation and examine efficacy in swine. The optimized VitaSol formulation will be prepared as freezedried powder and examined for stability and efficacy in partial-thickness wound healing. The success of this project is likely to have a major impact on medicine. It will enhance our understanding of how ATP delivery to wounds affects the wound closure rate and time to complete wound closure. Our paradigm shift in wound healing will provide a totally new therapeutic approach that could significantly increase number and frequency with which skin grafts can be harvested and used clinically. [unreadable] [unreadable]"}
{"text": "The lateral geniculate nucleus (LGN) is a major relay station in the visual system. Relay cells in the LGN summate direct excitatory inputs from retinal ganglion cells. Also, these ganglion cell inputs provide inhibitory inputs to the relay cells, via LGN interneurons. This project will attempt to characterize the way in which all of the retinal ganglion cells that overlap the general region of an LGN neuron's receptive field contribute to, or influence that LGN unit's firing. This will be done in the cat by recording simultaneously from individual ganglion cells and from individual LGN neurons. At both the level of the retina and the LGN, attention will be paid to the W/X/Y classification scheme, as well as to the familiar ON and OFF-center types of receptive fields. It is known that in terms of direct excitation, the anatomically based W/X/Y pathways are kept relatively separated all the way from the retina to the visual cortex. At the LGN, interneurons are used to provide inhibitory influences that modify the direct excitatory signals that pass along these channels. By recording from ganglion cells and LGN neurons simultaneously we will ask what, if any, is the specific organization of this inhibition? That is, to what extent are various pathways segregated in inhibitory terms as they are known to be in excitatory terms? Inhibitory influences from both eyes onto any particular relay cell will be studied. In a related series of experiments, we will record independently from two neighboring ganglion cells and an LGN relay cell to ask the basic question, to what extent is simultaneous firing of these ganglion cells important as an influence on the relay cell's firing? Various levels of background illumination, including complete darkness, will be used."}
{"text": "This research examines the effects of variations in serum testosterone levels on GH secretion and basal metabolic rate in normal men. Subjects will be studied 3 times, in random order, under conditions of low, normal, and elevated testosterone. Both stimulated and non-stimulated GH secretion, BMR, and lipids will be measured during each admission. CORE LAB ONLY."}
{"text": "DESCRIPTION: The main goal of the project is to study the mammalian metabolism of the anticancer agent betulinic acid using microorganisms as in vitro models of mammalian metabolism. Microorganisms, particularly fungi, have been utilized successfully in studying the mammalian biotransformations of drugs and other xenobiotics. The use of microorganisms as in vitro models to mimic and predict mammalian biotransformations for initial metabolism studies has many advantages including the ability to produce large quantities of metabolites for full structural characterization and biological evaluation, substantially reduced cost over in vivo studies, and lessened animal demand. Befulinic acid, a pentacyclic lupane-type triterpene, is a relatively common natural product that was recently reported as a melanoma-specific cytotoxic agent following both in vitro and in vivo studies. Because of its high antitumor activity and lack of toxicity, the drug is currently undergoing preclinical development for the treatment or prevention of malignant melanoma. An essential part of preclinical development of a drug is to elucidate its mammalian biotransformations and evaluate the biological activity and/or toxicity of its mammalian metabolites. Since there have been no reports on the mammalian metabolism of betulinic acid, it is the main objective of this study to utilize microbial cultures as model systems to predict and prepare potential mammalian metabolites of betulinic acid. In addition, the generated metabolites of betulinic acid will be evaluated for antitumor activity and/or toxicity."}
{"text": "Our ability to analyze hazardous material in toxic waste sites has improved dramatically in recent years. However, we are very limited in our ability to trace the movement of hazardous materials from Superfund sites through various media or to prioritize and mitigate the hazards involved. Our ability to predict exposure, much less susceptibility or effect, of these materials on humans and their environment is still more limited. This Program consists of eight integrated projects, three research cores, a training core and an administrative and outreach core to address these problems. We will determine the fate and transport of hazardous materials in ground water, surface water, and air as they move from toxic waste sites. Concurrently we will develop sensitive systems for evaluating the exposure and effect of populations to these materials. These biological markers will be based upon immunochemical and other detection systems and based on a fundamental understanding of the toxicological processes involved. The project will emphasize pulmonary, dermal, and reproductive systems in mammals as well as microbial and fish systems in the environment. We also will explore new technologies for thermal and bioremediation of toxic waste and address possible health risks associated with these technologies. Rapid immunochemical analysis will supplement classical technologies for the evaluation of sites, validating models of transport from these sites, as well as determining human susceptibility, exposure and effect. The biomarkers developed in this project will serve as biological dosimeters in epidemiological and ecological studies in this and sister projects. The technologies developed in the project will be tested at field sites and transferred to end users."}
{"text": "This project will target the enzyme, fatty acid synthase (FAS), for the treatment of lung cancer. Our preliminary studies have found that the vast majority of non-small cell lung cancers express high levels of this enzyme compared to normal tissues. This increased expression of FAS is significant because inhibition of this enzyme in cancer cells leads to a metabolic imbalance and cellular apoptosis. In a series of in vivo experiments, we found that treatment of orthotopic xenografts of human mesothelioma cells with an agent that inhibits FAS essentially abolished the growth of established tumors. Furthermore, in preliminary experiments, we found a promising anti-tumoral response of lung cancer orthotopic xenografts (in nude rats) treated with an FAS inhibitory compound. Importantly, these treatments did not result in any recognizable damage to normal tissue but did lead to dose-limiting anorexia. The proposed studies will further develop the use of FAS inhibitory therapy for lung cancer treatment. In the first phase of our preclinical studies, we will compare several novel FAS inhibitory agents, using in vivo and in vivo experimental systems, to identify a lead compound with high level of activity against lung cancer cells and tolerable levels of toxicity/anorexia. In the second phase of the preclinical studies, we will optimize dosing protocols for the treatment of lung cancer orthotopic xenografts using this compound. This optimization of treatment protocols could be useful for designing treatment protocols to be applied in the clinical setting. The third pre-clinical aim of this project is to examine the effects of the FAS inhibitory compound when used in combination with other agents, such as those currently used to treat lung cancer or being evaluated for treatment of lung cancer. Because the FAS target represents a pathway distinct from those targeted by other compounds, there is a significant potential that such combinations could have synergistic antineoplastic activity, thus allowing reduction of doses of the respective agents. The final aim of this project is to initiate a phase I clinical trial for a compound identified by the preclinical studies to have the best potential for lung cancer treatment. Successful completion of this phase I trial and the preclinical modeling studies will provide a framework for further evaluation of an FAS inhibitory compound in the treatment of lung cancer."}
{"text": "People with HIV/AIDS survive to older ages, but we still do not know if or how HIV-associated neurocognitive disorders (HAND) is exacerbated in the aging brain. We will evaluate the hypotheses that brain inflammation drives an interaction between HAND and brain aging by modifying the ubiquitin proteasome system (UPS). The UPS is the main route of neuronal protein turnover;it is required for normal minute-to-minute physiological changes in synaptic potentiation, learning and memory formation. The UPS plays a key role in preventing the accumulation of misfolded neuronal proteins in pathological neurodegeneration during brain aging. We established that the UPS is abnormal in brain cortex of people with HAND: Immunoproteasomes (IPS) are induced in response to interferons. IPS induction was linked solidly with HIV-associated neurocognitive disorders (HAND). It was relevant pathologically because it was prevalent in subjects with HIV encephalitis (HIVE) and/or replicating HIV concentration in the brain. It was anatomically pervasive in critical neuronal compartments including synapses, axons and neuronal nuclei, perikarya, and isolated synaptosomes. The proposal will test the hypothesis that IPS induction perturbs the synaptic protein economy by diverting the UPS substrate repertoire pathologically, which alters the substrate repertoire, synaptic protein turnover, and synaptic plasticity in HAND and brain aging. The first aim will use human brain specimens to determine how IPS induction affects macromolecular assembly and enzymatic functions of the UPS, including the dynamic interaction with synaptic proteins. The second aim will use interferon-stimulated mixed brain cell cultures to determine how IPS induction influences synaptic protein turnover and holoenzyme kinetics, and will determine whether inhibiting the IPS reverses these changes in the synaptic protein economy. A final aim will use autopsy specimens to determine if the age-of-onset or rate-of-accumulation of misfolded proteins during pathological brain aging is perturbed in association with IPS induction. The program overall addresses a highly novel mechanism for synaptic dysfunction in HAND and has important implications for the mechanism of interaction between pathological brain aging and HIV/AIDS. PUBLIC HEALTH RELEVANCE: \"The project is important because it will clearly define a novel basic mechanism regarding how HIV infection in the brain disturbs learning and memory, and how HIV worsens brain degeneration during aging. The studies will determine if changes in brain proteins that occur can be blocked, in order to stop dementia and brain aging in HIV infected people\""}
{"text": "This project is the synthetic organic chemistry component of this program project. Our tasks is the synthesis of research quantities of lead compounds. These compounds are supplied from other projects for biological testing. Amounts ranging from 100 mg to 1 g are sufficient for confirmation of initial in vitro activity and testing in secondary systems, such as mouse mammary organ culture (MMOC). If the results are promising, short-term animal testing can also be performed with these quantities of test materials, as can mechanistic evaluations. In this context, this project is also responsible for derivatization and synthetic modification of lead compounds for structure-activity relationships (SARs), which will be used for lead optimization. In addition, project 5 will perform scale-up syntheses of lead compounds for use in full-term animal studies for the assessment of chemopreventive activity. Scale-up synthesis will involve multi-step processes and possibly stereoselective transformations for quantities ranging from 500 g to 2 kg. The goal of project 5 is the efficient and timely synthesis of compounds from the discovery part of the program to the stage of complete biological evaluation. To accomplish this goal, a flexible, competent, and responsive synthetic group has been assembled. In ongoing studies, this project will focus on large scale syntheses of sulforamate, oxomate, sulforaphane, brassinin, cyclobrassinin, 4'-bromoflavone, E-resveratrol and 4'-bromorot- 2'-enonic acid for inclusion in animal carcinogenesis models (project 3). We will also continue analog syntheses of resveratrol, rotenoids and isoflavones. Thus, in a highly coordinated manner, project 5 will contribute to the further development of lead compounds that have or will emerge from this program project."}
{"text": "To advance the work already performed in our laboratory with rats, fetal substantia nigra or adrenal medulla was grafted to the denervated caudate of the rhesus monkey in our continuing research on brain tissue transplantation. Although only moderately successful thus far, the experiments have demonstrated for the first time that peripheral tissue autografts can survive transplantation into nonhuman primate central nervous system."}
{"text": "Variola (smallpox) remains a bioterrorism threat in the 21st century. The Dryvax vaccine is too toxic for many persons. Little is known about the specific T cell response induced by intradermal Dryvax. We propose detailed studies of the epitopes and antigens recognized by vaccinia-specific CD8 and CD4 T-cells in humans. The planned antigen discovery method, expression cloning, interrogates the viral proteome with libraries of cloned vaccinia genomic DNA. We have successfully used this method with HSV-2, which has a complex genome similar to that of vaccinia. The responses of vaccinia-specific human T-cells lines and clones will be validated in quantitative measurements of immune responses using direct ex vivo assays. In Aim 1, we will derive CDS CTL lines and clones that are specific for vaccinia, and determine which vaccinia open reading frames and peptide epitopes they recognize. Epitopes will be validated in both CTL and IFN- gamma assays and dose-response curves done to document high-avidity recognition. We will then measure the diversity of the CDS response and the evolution of the response over a one year period in a subset of primary vaccinees. We predict that the immunodominant CDS antigens will be non-structural early proteins, and that immunodominance will also be controlled by HLA type and will be stable over time. CD4 T-cells provide important help for antibody and CDS responses. Therefore, in Aim 2, we will detemine the fine specificity of vaccinia-specific CD4 T-cell lines and clones using our proven molecular library method. Selected vaccinia proteins that stimulate CD4 T-cell responses will be expressed as full-length proteins in baculovirus to study CD4 T-cell immunodominance in a population of vaccinated subjects. We predict that structural proteins will be dominant and that the CD4 response will include reactivity with vaccinia proteins that are the targets of neutralizing antibodies. Several such proteins are already available as baculovirus constructs. The data will be medically useful in evaluating the immunogenicity of candidate smallpox vaccines that are being evaluated for widespread use in the case of possible variola exposure. Poxviruses expressing heterologous microbial or cancer proteins are being modified for increased immunogenicity, with the goal of prevention and therapy of other infections and malignancies. Our data will assist in evaluating the immunogenicity of these constructs."}
{"text": "Reactivation of BK virus (BKV) following renal transplantation (KTx) is common and can lead to BK virus- associated nephropathy (BKVN), a serious and increasing problem. Current therapeutic options are limited to tapering of immunosuppression and experimental use of cidofovir. Available evidence suggests that BKV- specific T-cell responses are important in controlling infection, and we hypothesize that viral reactivation in KTx drives the expansion of functional BK-specific cells which control BKV in the majority of patients, but in a minority, this immune response is absent or dysfunctional, which may predispose to progression to BKVN. If true, functional BKV-specific T-cells could represent correlates of protection against BKVN. We will test this hypothesis by using state-of-the-art flow cytometry methodologies to track and characterize BKV-specific T-cell responses in two cohorts of KTx patients at UCLA Medical Center. The first cohort will comprise 40 Ktx recipients who will be prospectively followed beginning 1 month and until 1 year post- transplant. A second cohort will consist of KTx patients with clinically confirmed BKVN and enrolled at the time of diagnosis. The patients will be monitored by plasma PCR for BKV viral load. Global analyses of immunological responses to BKV will consist of flow-based intracellular cytokine assays employing as antigens overlapping peptide libraries spanning the majority of the viral proteome. These will be supplemented by more sensitive immunological probes to a panel of immunodominant BKV T-cell epitopes we have previously identified. The functionality of these BKV-specific CD4 and CDS T-cells in terms of cytolytic potential and production of different cytokines will be characterized and correlated with viral load and clinical status to see if these cells represent markers for BKV reactivation and/or protection against BKVN. Immunological probes to detect these cells may form the basis for tests to guide clinical management. Relevance: With the increasing problem of Type II diabetes, the number of kidney transplants being performed within the US is rising. Although BK virus is ubiquitous in human populations and establishes a life-long infection, it is only life-threatening in the context of a minority of immunocompromised KTx recipients. Information from the proposed study may allow identification of patients at risk of progression from BKV reactivation to clinical BKV disease and guide therapeutic interventions. [unreadable] [unreadable] [unreadable]"}
{"text": "Numerous human disorders arise from microdeletions and microduplications of relatively large genomic regions. These rearrangements can result in copy number alterations (CNAs) of one or more genes. Conditions arising from microdeletions and microduplications can manifest as multiple congenital anomalies (MCA) in patients. The most common phenotypic features observed in these patients include global developmental delay, mental retardation, cardiac defects and cranio-facial differences. Thus, diseases mediated by CNAs are referred to as 'genomic disorders' as large regions of the genome are altered leading to disorders in multiple organ systems. Many of the recurrent genomic disorders are mediated by aberrant homologous recombination between highly identical blocks of DNA sequences referred to as low copy repeats or segmental duplications (SDs) which may comprise up to 5% of the human genome. Despite this known correlation between SDs and genomic disorders, a significant proportion of SDs have not yet been associated with disease- causing genomic rearrangements. This observation suggests that other SD-mediated genomic disorders may exist but are currently undetectable mainly due to the low resolution of diagnostic techniques standardly used in molecular cytogenetics. We hypothesize that a significant proportion of children with MCA have a submicroscopic CNA that is not evident on standard genetic testing. We believe that a substantial number of these CNAs may be mediated by SDs or other unstable architecture within the human genome. We will test this hypothesis by analyzing patients with MCA using high resolution microarrays in order to detect disease-causing CNAs. Further, analysis of the genomic sequences at the rearrangement breakpoints will help determine what proportion of CNAs are mediated by predisposing genome architecture like SDs. The goal of this proposal is to identify previously undetectable, disease-causing CNAs in patients with MCA. The identification of genomic regions altered in MCA patients will allow a better understanding of the mechanisms underlying this group of disorders. We can then begin to assess the role of gene(s) in these regions that may be critical to early human developmental pathways. Relevance: Many children have genetic diseases that are undiagnosed due to limitations in current methods used for testing. These children may have very small deletions and duplications which can only be detected by techniques that allow a genomewide analysis. The identification of genomic regions that are deleted or duplicated in patients with birth defects will allow the identification of genes that are important in normal development."}
{"text": "The Lab of Immunoregulation is using molecular, biochemical and cell biological approaches to explore the viral envelope glycoprotein of HIV as a target of immunity. We know that envelope is a target of potent neutralizing antibodies made by people after infection. A mixture of just three human monoclonal antibodies to envelope is capable of protecting monkeys against challenge. Our goal is to develop vaccines that can elicit an immune response as strong as the response to infection. We have two approaches, comparable to the Salk and Sabin vaccines for polio. Our Salk-type vaccine is a particle with a lipid core and an array of HIV envelope glycoprotein on its surface, closely resembling a noninfectious version of HIV virus. These particles have the potential to enhance vaccine potency by up to 1,000-fold, as they have for other successful partulate vaccines. Our Sabin-type vaccine is a live rubella virus containing additional genes coding for the HIV envelope. Each cell infected by this attenuated virus looks to the immune system like a cell infected with HIV. However, the virulence of HIV is gone, only its antigens remain in the vaccine. If these vaccines can elicit a response as strong as to infection, they may be able to prevent or control a subsequent HIV infection. HIV-1 Neutralizing Antibodies from Live Attenuated Vaccines. Many of our most effective viral vaccines are live-attenuated viruses. However, for HIV vaccines, it seems that almost no degree of attenuation would be safe enough for human use. Instead, a number of live recombinant vaccines have been proposed. These have generally been defective viruses, capable of expressing HIV antigens in a single round of infection, but unable to replicate further. In this project, we have begun to make a live viral vector safe enough to express HIV antigens over multiple rounds of viral replication. Like HIV, rubella is an enveloped plus strand RNA virus which infects at mucosal surfaces. A single dose protects for life against mucosal and systemic infection with rubella virus. Live attenuated rubella vaccine is safe for human use. It has been given to millions of children around the world, including asymptomatic HIV+ children. It has no DNA intermediate, so there is no risk of integration and no reservoir of chronic infection. It is minimally pathogenic for adults and children, and its genetic stability is shown by the fact that it has not changed serotype in over 40 years. Ideally, a rubella/HIV hybrid could combine the growth and safety of rubella with the antigenicity of HIV gp120, but without the pathogenicity of HIV. With this vaccine, we may be able to induce a level and duration of immunity to HIV comparable to what has been achieved for rubella. We have used a full length, infectious cDNA clone of rubella, provided by Dr. Teryl Frey. We have identified an acceptor site in rubella where foreign sequences can be inserted without disrupting essential viral functions. For example, we inserted green fluorescent protein as a reporter gene at this site. When Vero cells were transformed with the DNA, they produced GFP in large amount. Significantly, the culture supernatants were infectious with virus that expressed GFP in the next round of infection. These viruses expressed all structural proteins, as measured by western blot, indicating that second strand RNA synthesis and the subgenomic promoter were both active. However, GFP expression was lost over two more passages of virus, although the rubella virus continued to grow without GFP expression. We are working on strategies to stabilize expression of the insert by preventing recombinants from outgrowing the GFP expressing strains. We will then express gp120 in the same manner. The resulting rubella/gp120 hybrid will be tested for growth and gp120 expression in vitro. It will then be tested for propagation in rhesus macaques and for the ability to elicit anti-gp120 antibodies (serum IgG and mucosal IgA), as well as safety and viral shedding and persistence. Macaques are the ideal host for demonstrating protection, since they can be infected with rubella and then challenged with an SHIV or SIV challenge strain expressing envelope glycoproteins of the same type or different from the vaccine strain. Novel Reagents for Making gp120 Conjugate and Particulate Vaccines. HIV envelope glycoprotein gp120 contains epitopes which are targeted by broadly cross-reactive neutralizing antibodies in humans and which depend on the native protein conformation. Monoclonal antibodies to these sites can neutralize a broad range of HIV-1 isolates and have protected monkeys against viral challenge by iv and oral routes. We have found a site on gp120, where foreign protein sequences can be inserted without disrupting the native folding of the neutralizing sites. By linking gp120 to a carrier protein capable of self-assembly, we could enhance the intrinsic vaccine potency of gp120 by assembling virus-like particles, while retaining important conformational sites needed to elicit these antibodies. When hepatitis B surface antigen (HBsAg)was inserted at this site, the resulting HBsAg-gp120 hybrids assembled particles efficiently. The particles sedimented at large size and banded at light density (1.22-1.25), consistent with a lipoprotein composition. On electron microscopy, they were 20 to 30 nm in diameter, similar to the spherical particles formed by native HBsAg. By analogy with native HBsAg particles, these contain about 100 to 200 copies of HBsAg-gp120, arrayed at a lipid/water interface. This recreates the natural milieu of gp120 on the surface of virions. Gp120 in the hybrids showed normal glycosylation, high affinity binding of the natural receptor CD4, and bound a panel of broadly reactive human neutralizing monoclonals, indicating that it was folded correctly in the native conformation. This is the first multimeric gp120 vaccine, and we anticipate that it may elicit a strong immune response in mice, rabbits and monkeys. We have now tested the immunogenicity of gp120 particles in mice and rabbits. Both species showed comparable antibody ELISA titers for particles as for monomeric gp120. However, the virus neutralizing activity of the particle-immune sera was much greater after two doses than for the monomer-immune sera. In some rabbits, the neutralizing titer was nearly as great as the ELISA titer, indicating efficient induction of neutralizing antibodies. The ratio of neutralization over ELISA titer for the rabbits was the same as for high-titered HIV-Ig from infected humans and was 30-fold greater than for animals immunized with gp120 monomers. If monkeys also produce high titered neutralizing anti-bodies, they will be challenged with titered stocks of SHIV virus of the same or different envelope type. We have expressed three envelope types: IIIB, 89.6, and SIV453 as particles. The first two correspond to SHIV challenge strains. The last one could be used to protect monkeys from a potent SIV challenge, which may more closely represent a natural HIV infection with a highly adapted virus. Through potent carrier and multimer effects, particulate forms of gp120 and other viral antigens may have a profound effect on HIV immunology. This project is funded by an intramural NIH grant. Transmembrane glycoprotein gp41 is another important target for HIV neutralizing antibodies. Human monoclonals such as 2F5 bind a site on gp41 close to the lipid bilayer. They can neutralize a broad range of HIV isolates, including fresh isolates. So far, vaccines have been unable to elicit similar antibodies. We hypothesize that, because they lack lipid, they cannot fold or display the 2F5 site correctly. We have expressed HBsAg-gp41 hybrids, which form gp41-rich particles. In these particles, gp41 is displayed at a lipid-water interface, and its transmembrane domain may span the lipid layer, just as it does on virus. We have produced sufficient quantities for immunological studies. These particles may be the first immunogen to recreate gp41 as it exists on the virus. Since the neutralizing sites on gp41 are conserved among diverse HIV isolates, antibodies elicited by these antigens have the potential to be broadly neutralizing. Antibodies to functional sites on viruses and prions. We have recently found that a variety of viral antigens, when expressed in tandem with HBsAg, will spontaneously assemble into 20-30 nm virus-like particles. The particles contain about 20% lipid, based on their density. The proteins are displayed on the surface of a lipid micelle. This milieu resembles the surface of an enveloped virus, as well as the local environment of a bacterial toxin bound to the surface of a cell. The HBsAg carrier is quite flexible in accepting other viral envelope proteins, while still forming particles. For example, the E2 and E1 envelope glycoproteins of VEE virus, as well as both E2E1 together, were expressed as HBsAg hybrids. All three forms assembled into particles. When expressed together, the E2E1 precursor was processed correctly into E2 and E1, and both proteins remained attached to the particles. This may be an ideal immunogen, since envelope proteins on the surface of a lipid particle resemble the same proteins on the viral surface. Immunogenicity will be tested in rabbits, and the resulting antibodies will be tested for the ability to protect cells in vitro from infection with VEE. Similarly, we have created hybrid particles containing protective antigen PA of anthrax. Antibodies to this protein are known to protect against anthrax. We anticipate that the particulate form of PA may greatly increase immunogenicity, allowing us to immunize with fewer doses and to maintain immunity with less frequent boosting than the current anthrax vaccine. In addition, PA was expressed in its active, cleaved form, which may elicit antibodies to sites involved in critical functions such as toxin subunit binding and translocation into the cell. PA particles will be tested for immunogenicity, and the resulting antibodies will be tested for the ability to block toxin-mediated killing of cell lines in culture. If successful, we will immunize mice and challenge them with anthrax spores, in collaboration with NIH scientists. This project incorporates FY2002 projects 1Z01BK009002-11, 1Z01BK009003-11, and 1Z01BK009008-04."}
{"text": "The general goal of this project is to study the cellular and molecular determinants of leukocyte interactions with components of the rheumatoid synovial microenvironment. In the past four years, we have shown that the transmembrane hyaluronate receptor (CD44) is a central, multifunctional proinflammatory molecule in RA, and is the same as the Hermes class of lymphocyte homing receptors that mediates mononuclear cell adhesion to synovial endothelial venules. Recent data have shown that CD44 represents a family of molecules with isoforms. In this project, we propose to define to define the proinflammatory functions of each of the isoforms of the CD44 with the goal of devising novel strategies of inhibition of CD44 proinflammatory function as potential new therapies for RA. Individual specific aims in this project are: 10 We will develop oligonucleotide probes that are specific for known isoforms of CD44. We will develop mabs that specifically react with the human CD44R1 (CD44E) form of CD44, and profile the expression of CD44 isoforms in normal and RA PB T cells, PMNs and monocytes, and in normal, OA and RA synovial T cells, macrophages and fibroblasts. 2) We will characterize the size of CD44 isoforms on purified purified synovial macrophages, T cells and fibroblasts, and compare with T cells, B cells, monocytes and PMNs and various cell lines. 3) We will We will study the origin of soluble CD44 in serum and synovial fluid. 4) recombinant (r) CD44-immunoglobulin (Ig) fusion molecules (rCD44Rg-2), 2), rCD44, rCD44R1 (CD44E), affinity purified transmembrane CD44, and purified soluble CD44S from synovial fluid and serum, we will determine the determine the immunoregulatory effects of various CD44 isoforms on in vitro monocyte function. 5) Using purified proteins, we will determine the the ability of regions of CD44 to bind to cytokines (IL3, GM-CSF, TGF-beta, FGF) and to bind to components of the extracellular matrix. Using cytoskeletal components, we will determine the ability of various forms of purified CD44 to associate with the cytoskeleton, and, using deletion mutants of CD44 cytoplasmic domain, define functional sequelae of CD44- cytoskeletal interactions. 6) To map functional domains of CD44, we will prepare a series of deletion mutants of CD44 extracellular or cytoplasmic regions. 7) Using a novel system of synovial tissue grown in vivo in severe combined immune deficient (SCID) mice, we will study the ability of autologous and allogeneic T cells to migrate to human synovial xenografts. Taken together, these studies should provide critical new knowledge necessary for the design of new therapies to interrupt multiple stages of inflammation in RA."}
{"text": "This loss of membrane-bound beta-adrenergic receptor recognition sites in frog erythrocytes during subsensitivity induced by exposure of cells to isoproterenol is due at least in part to the internalization of beta-receptor recognition sites. When the beta-adrenergic receptors are resensitized, the amounts of internalized receptor recognition sites are also returning to the normal. The internalized recognition sites of beta-adrenergic receptors have properties identical to those of membrane-bound receptors. Glycoprotein seems to play an important role in triggering this event. A series of tricyclic antidepressant drugs was found to inhibit the receptor internalization; these effects can be attributed to an inhibition of these drugs of the agonist binding to beta-receptor recognition sites in erythrocyte membranes. Inhibitors of transglutaminase diminish the extent of recognition site internalization with a concomitant prevention of the loss of membrane-bound receptors elicited by isoproterenol. There is an excellent correlation between the inhibition of recognition site internalization and the potency of these compounds to block the transglutaminase activity in vitro."}
{"text": "The Adult Clinical Core at the Beth Israel Hospital will serve to provide clinical specimen to the Dana-Farber Cancer Institute CFAR investigators, facilitate trials of therapies evolving from the basic research of CFAR members and encourage scientific exchange and collaborations between clinical and basic science investigators. The Beth Israel Hospital provides primary care to approximately 480 HIV-infected adults and has extensive NIH funded clinical research programs at the hospital, including a component of the Harvard/Boston City Hospital Aids Clinical Trials Unit (ACTU). The Adult Clinical Core will provide partial support for both a research nurse and a laboratory technician. The research nurse will provide CFAR investigators with clinical specimens from patients and carefully document the clinical histories of these patients for the investigators. The laboratory technician will establish and characterize HIV isolates from patients and maintain a bank of clinical specimens for the use of CFAR investigators. Both the research nurse and laboratory technician will also participate in the trials of therapies initiated through the Dana-Farber Cancer Institute-Beth Israel Hospital interactions. CFAR Cores will be made available to AIDS clinical investigators at the Beth Israel Hospital to strengthen the scientific base of their clinical studies. Finally, joint institutional conferences will be held to foster scientific collaborations and encourage the initiation of clinical trials of novel AIDS therapeutics."}
{"text": "A third of the world's population is infected with Mycobacterium tuberculosis (Mtb). Tubercle bacilli can remain inactive in lung lesions only to emerge decades later to seed new outbreaks of tuberculosis. In addition, tuberculosis is one of the most difficult bacterial infections to treat and continues to cause more deaths than any other bacterial infection. Bacilli exist in replicating and non-replicating states in a range of microenvironments that vary in oxygen concentration and nutrient availability. The bacilli that survive during latent infection likely exist in a non-replicating state and antimicrobials, effectie against actively growing bacteria, are often not effective against non-replicating bacteria. Population heterogeneity, the presence of more than one phenotypic variant in a clonal population, provides bacteria diverse mechanisms to endure environmental challenges. We have demonstrated that Mtb exists in two semi-stable states that appear to be epigenetically controlled. When mycobacteria were grown as a dense population, either by pellicle or settled growth, the vast majority of the population took on characteristics of a form termed pellicle. If bacilli were passaged starting with a solitary bacillus or just a few cells, however, the ensuing population shifted to a form termed solitary. One of the most striking contrasts between the solitary- and pellicle-prepared Mtb was the solitary form adapted to hypoxic and anaerobic conditions by maintaining high transcriptional activity, while the pellicle form failed to adjust t hypoxia and became truly dormant under anaerobic conditions. We hypothesize that populations of Mtb contain at least two phenotypic variants that allow for divergent responses to imbalances in proton homeostasis, particularly resulting from hypoxia. To test this hypothesis we will determine the differential abilities of the solitary and pellicle variants in adjusting to conditions that exert pressure on internal pH homeostasis. We will also identify regulatory elements that control the shift between the variants. Pellicle growth has historically been used to increase or maintain Mtb virulence during in vitro growth. Therefore, we will investigate if the pellicle-passaged variant is more virulent than the solitary form by using a mouse model of TB that reproduces hypoxic necrotic granulomas."}
{"text": "One in nine women suffer from pelvic floor dysfunction, including urinary incontinence and vaginal wall or uterine prolapse (VUP). Stress urinary incontinence (SUI) affects 38% of women over the age of 65 years and over 13 million women in the United States. Pelvic muscle strength is commonly assessed in these patients. However, current measurement techniques are either subjective or produce artifact, due to their non-isometric nature or contamination by intraabdominal pressure. During Phase I, we developed a second generation system that measures the isometric strength and contractile properties of female pelvic floor muscles. The system centerpiece is a novel intravaginal transducer that differentiates between intraabdominal pressure and levator ani force. During Phase II, system mechanics, electronics and software will be refined to improve system sensitivity, accuracy, and ease-of-use. Laptop- and Personal Data Assistant-based systems will be developed and validated. Clinical device performance will be confirmed by testing the null hypothesis in 120 women (40 healthy continent, 40 with VUP, 40 with SUI) that localized pelvic floor muscle defects visible on MR scans will correlate with pelvic muscle weakness. The system allows assessment of pelvic floor function and exercise intervention efficacy, and can provide biofeedback and adherence data during training."}
{"text": "DESCRIPTION: To date, no vaccine strategy has successfully induced potent broadly neutralizing antibodies (BnAb) against HIV-1. In vitro analysis, passive antibody transfer studies and analysis of antibody responses in the RV144 vaccine efficacy trial suggest non- neutralizing antibodies might contribute to protection against HIV-1 transmission. Three linear regions in the HIV-1 Env MPER, V2 and V3 have been implicated as potential targets of protective non-neutralizing antibody. We have constructed a novel recombinant Lactobacillus acidophilus vaccine platform that is orally delivered and induces antigen-specific mucosal IgA and systemic IgG against MPER peptides inserted into the bacterial surface layer protein. We have developed two different adjuvants for use with recombinant L. acidophilus, IL1 and flagellin (FliC). Specifi Aim 1 will determine the optimal adjuvant for mucosal immunization and whether responses are T-cell dependent or independent. Specific Aim 2 will test whether recombinant Lactobacillus acidophilus expressing candidate MPER, V2, and/or V3 epitopes can induce mucosal and systemic antibody responses that are protective against vaginal HIV-1 challenge in the HLA-DR transgenic (DRAG), humanized mouse model."}
{"text": "This project is designed to develop a means of replacing radioiodine, the radionuclide presently employed in the labeling of antibodies, with a radionuclide that reduces the absorbed radiation dose to the patient and has physical properties that can be more efficiently utilized by modern radiation detecting systems, specifically the gamma camera. During the past year, we have developed a procedure for labeling antibodies with indium 111. This procedure involves the conjugation of the IgG fraction of the antisera with iron-free transferrin using glutaraldehyde as the coupling compound. Indium 111 ions are then added to the solution containing the transferrin-IgG conjugate and are instantaneously chelated by transferrin. The projected goals for this year include the refinement of the conjugating procedures and the evaluation of the effectiveness of intravenously injected 111 In-transferrin-anti-CEA conjugate obtained from antisera raised in goats in detecting colonic tumors transplanted in animals. The results will be compared with those obtained with anti- CEA preparations labeled with I131 in the standard manner."}
{"text": "This research will focus on the management of diabetes and cardiac risk factors in elderly patients. Cardiovascular disease (CVD) and diabetes are two major public health problems, particularly in the elderly. By the year 2040, the elderly population in the United States will make up 20 to 24 percent of the population. It is projected that 19 percent of the elderly will be aged 85 or above and this population will grow to at least 12.8 million. An extensive literature review revealed that there has been research focusing on short and long-term outcomes in patients with either acute myocardial infarction (MI) or diabetes. Despite this, little is known about these combined conditions and their relationship to outcomes. In addition, management of diabetes and cardiac risk factors before and after MI have not been studied in the context of outcomes in these patients. The purpose of this preliminary clinical trial is to determine the relationship between management of diabetes and cardiac risk factors, and selected outcomes in elderly patients with diabetes following MI."}
{"text": "The incidence of infertility has increased 4% since the 1980s, with up to 20% cases having no known cause. One of the prevailing hypotheses is incompatibility between cognate egg and sperm proteins; however, very few pairs of interacting reproductive proteins have been identified in any organism. The best model for studying fertilization remains the marine gastropod abalone, where one of the first steps in fertilization involves the interaction between the sperm protein lysin and its egg coat receptor VERL. As a major component of the abalone egg coat, VERL is a giant, fibrous glycoprotein composed of ~22 ZP-N repeats that form intermolecular hydrogen bonds to create the highly stabilized and protective egg coat. Lysin creates a hole in the egg coat by competing for the hydrogen bonds, allowing sperm to pass and fuse with the oocyte. The VERL repeats are homologous to human egg coat proteins, and likely share a similar protein topology. However, the precise structural mechanisms that drive egg coat dissolution remains to be determined. To address this fundamental question in fertilization, two specific aims are proposed using state-of-the-art structural and proteomic approaches. In aim 1, multidimensional NMR will be used to characterize the structural basis of lysin-VERL interactions for three species of abalone. In aim 2, deep mutational scanning will be utilized to explore the specific adaptations acquired by lysin and VERL that permit species specific interactions, and simultaneously test theoretical models of sexual selection. The proposed research is innovative for its combined use of proteomic and structural techniques to characterize the evolutionary history of rapidly evolving reproductive proteins. The results are expected to shed insight into the core mechanisms that mediate egg-sperm interactions in abalone, and provide foundational information towards understanding the more complex mammalian system."}
{"text": "The formation of flagella and the synthesis of proacrosin will be investigated in primary cultures of mouse round spermatids. Initial experiments are designed to determine whether spermatids that do not make flagella in culture lack the centrioles necessary for axonemal nucleation. The requirements of protein, RNA, and tubulin biosynthesis for flagella formation will also be examined. The length of time for cell culture will be increased to ascertain the extent of tail maturation that can be obtained in vitro. Antibodies to the major fibrous sheath protein will be used to determine whether this component appears in the newly-formed flagella. The synthesis of proacrosin will also be examined to determine whether proacrosin contains N-linked oligosaccharides and whether these carbohydrate side chains are processed to the \"complex\" type. The carbohydrate side chains will be analyzed for mannose-6-phosphate groups. If these residues are found, it suggests that proacrosin is directed to the acrosome by the same phosphomannosyl receptor system that transports lysosomal hydrolases to the lysosome. The experiments of this proposal will provide the bases for long-term biochemical studies of mammalian spermiogenesis."}
{"text": "We plan to isolate and analyze zebra fish mutants with altered visual systems. Of particular interest initially will be mutants with altered organizations of retinal cell types, and specific enrichment procedures will be utilized to aid in the isolation of such mutants. The mutants will be analyzed by behavioral, anatomical, physiological and genetic methods. In order to increase the frequency of mutants in the populations subjected selection, we will expose fish to mutagens at appropriate stages in their life cycle. Since most mutations are recessive, we plan to generate homozygous fish from mutagenized eggs in order to enable us to recognize mutant phenotypes. Procedures for generating homozygous fish have already been developed in our laboratory."}
{"text": "Leukotriene B4 (LTB4) is a potent lipid chemo attractant that classically has been associated with myeloid cell chemotaxis. We isolated a novel murine seven transmembrane spanning G protein-coupled receptor, which we identified as BLTR1, an LTB4 receptor, and generated BLTR1-deficient (BLTR1-/-) mice by targeted gene disruption. Characterization of this mouse revealed that BLTR1 is responsible for LTB4- mediated leukocyte calcium flux, chemotaxis, and firm adhesion to endothelium. Further, despite functional redundancy with other chemo attractant-receptor pairs in vitro, loss of BLTR1 function significantly reduced eosinophil recruitment into the peritoneum in a model of peritonitis. Follow-up studies of BLTR1-/- mice have allowed us to make several novel observations about the roles of LTB4 and BLTR1 in leukocyte recruitment that will be the focus of this grant proposal. We have found that loss of BLTR1 function significantly and specifically impairs neutrophil transendothelial migration (TEM) in vitro and in vivo. Secondly, we have found that BLTR1 is highly expressed, functional on certain subsets of activated T cells, and important for their trafficking in vivo. Additionally, we have found that recruitment of BLTR1-/- T cells into the airways early in allergic pulmonary inflammation is profoundly impaired, despite preserved trafficking of these cells into the lung parenchyma. Based on these novel preliminary findings, the central hypothesis of this proposal is that LTB4 and BLTR1 play a critical role in facilitating the ability of leukocytes to traverse endothelial, interstitial and epithelial barriers. We hypothesize that interactions between leukocytes and barriers to migration they encounter, e.g. endothelial cells, tissue interstitium, and epithelial cells, induces the production of LTB4. We hypothesize that the LTB4 produced by these interactions in turn activates leukocyte BLTR1, which facilitates penetration of these barriers by the migrating leukocytes. We believe that the LTB4 biosynthetic pathway uniquely positions this chemo attractant-receptor pair for this role. LTB4 is produced by serial enzymatically catalyzed reactions, and hence can be produced much more rapidly than peptide chemokines, which require transcription and translation. The rapid kinetics of LTB4 biosynthesis enables leukocytes, endothelial cells, and epithelial cells to produce LTB4 contemporaneously with leukocyte migration across cellular and extracellular barriers. In this grant, we propose to investigate this hypothesis as it relates to neutrophil and lymphocyte recruitment. Specifically we propose to: (1) determine to cellular source of the LTB4 that facilitates neutrophil transendothelial migration; (2) define the roles of BLTR1 and CXCR2 in neutrophil recruitment into the airways; (3) determine the role of LTB4 and BLTR1 in antigen specific T cell trafficking; (4) determine the role of LTB4 and BLTR1 in T cell recruitment into the airways of the lung. [unreadable] [unreadable]"}
{"text": "All forms of diabetes are due to a relative deficit of functional beta cells, yet adult human beta cells are resistant to attempts at replication, and serve as a poor experimental model for replicating human beta cells. Because of this paucity of models for adult human beta cell replication, we have developed a large Biorepository of rare human insulinomas, benign pancreatic adenomas that grow and over-secrete insulin, believing that they may provide information that will inform attempts at clues and drug targets and pathways for therapeutic human beta cell regeneration. Recently, we have provided the results of intensive genomic, transcriptomic and bioinformatic analysis of human insulinomas, comparing them to FACS-sorted pure human beta cells. Remarkably, we find that insulinomas display three cardinal features: 1) they almost universally contain recurring mutations, copy number alterations and gene expression abnormalities in members of the Trithorax Group of chromatin modifying enzymes, notably including KDM6A, MLL3 and/or MEN1; 2) they also almost universally display alterations in the Polycomb Repressive Complex of chromatin modifying enzymes and their targets, particularly YY1, EZH2 and H3F3A; 3) they almost universally display abnormalities in the chromosome 11, such as allelic loss of all or part of chromosome 11, and/or allele-specific expression and/or DNA methylation/imprinting abnormalities of the imprinted 11p15 region of chromosome 11, reminiscent of the other beta cell proliferative disorders, such as the Focal Variant of Hyperinsulinism and Beckwith-Wiedemann Syndrome. On the other hand, the mechanisms through with these events lead to beta cell proliferation and while maintaining the beta cell phenotype are unknown. More specifically, exactly how MEN1, KDM6A, MLL3, YY1, EZH2 and H3F3A modulate beta cell function and proliferation are largely unknown. The three Specific Aims of this application address this important knowledge gap. Aim 1. To Elucidate the Abnormal Pathobiology of Three Key Trithorax Members In Insulinoma vs. Beta Cells. Aim 2. To Define Abnormal Biology of Three Key Polycomb Repressive Complex Members in Insulinomas vs. Beta Cells. Aim 3. To Define 3-D Chromosome 11p15 Architecture in Insulinomas and Beta Cells."}
{"text": "This proposal is to study the local molecular interactions in cytochrome c oxidase from beef heart that control reduction of dioxygen to water and the conservation of chemical energy for sythesis of adenosine triphosphate. These studies will include freeze-trapping of intermediate species at temperatures between 10K and 280K as required. The chemical nature of stable or freeze-trapped cytochrome oxidase derivatives will be characterized by U.V. visible, and near infrared spectra, electron paramagnetic resonance spectra, and by vibrational spectra of ligand complexes with a3Fe and CuB. Fourier transform infrared spectroscopy has provided new information about the reduced oxidase in studies of the carbon monoxide complexes, a3FeCO in the dark, and CuBCO that results from photolysis at low temperature. This has opened new ways to measure structural interactions that control electron flow and oxygen reduction with minimum release of intermediates such as superoxide or peroxide. Pathological states that interfere with this control may cause altered forms that we observe by FTIR spectroscopy of the CO complexes of a3Fe or CuB. Conditions that affect the relative amount of these states will be explored. These studies apply areas of physics, spectroscopy, transition metal chemistry, and computer analysis of band shapes and reaction kinetics to problems of heart physiology and heart disease. The technology that is being developed in the conduct of these experiments will open yet new windows in our study of the heart."}
{"text": "PRODUCTION OF MUTANT CONGENIC MICE"}
{"text": "Quantitative Imaging: Biostatistics and Medical Physics (SR-C). This Specialized Resource provides support for a wide range of imaging (MRI, nuclear and optical imaging) procedures and statistical functions for the clinical trials in ICMIC-3 as well as all the pre-clinical animal research for all Research Projects and Developmental Projects. The tasks and services to be performed by SR-C fall into six broad categories; 1) to make additional computational and centralized Image storage facilities available to lCMIC-3 investigators thereby facilitating data sharing between projects; 2) to make specialized image processing and display software available, allowing the sharing of the software licenses and centralizing support; 3) to provide software development capabilities for the performance of specialized processing and analyses including the automation of bulk processing tasks, format conversions and the development of new algorithms; 4) to consult on issues regarding imaging protocols, optimal acquisition settings, quantitation and on the modeling of tracer kinetics; 5) to provide biostatistical expertise in the design and analysis of research studies and to conduct statistical analyses and modeling of data generated by those studies; and 6) to conduct research leading to the development of new capabilities of common benefit to the iCMlC-3 projects."}
{"text": "Melanoma is a devastating form of skin cancer with limited therapeutic options. This project focuses on the signaling pathways in NRAS mutant melanomas, which accounts for approximately 20% of melanomas. Therapies that target RAS and MEK, a RAS effector, have not been clinical effective in mutant NRAS melanoma patients. In cell-based studies, we show that the response of NRAS mutant melanomas to the MEK inhibitor AZD6244 is heterogeneous, with a sensitive and an insensitive group. For the AZD6244 insensitive cells, there is a need to identify specific effectors that mediate mutant NRAS actions and additional pathways that are synergistic for NRAS signaling to form the basis of new therapeutic strategies. One protein that may prove to be important in this group is TANK-binding kinase 1 (TBK1), an atypical IkB kinase family member. In KRAS transformed cancer cells, TBK1 mediates oncogenesis through its phosphorylation and activation of the pro-survival kinase AKT. We show that NRAS expression increases TBK1 activation in melanoma cells. In mutant NRAS melanoma cells, we will determine if TBK1 regulates AKT activation, as measured by Western blot and kinase assays, through TBK1 depletion. To assess its potential as a therapeutic option, we targeted TBK1, via RNA interference or the pharmacological inhibitor BX795, in conjunction with MEK. We found that the combination leads to increased cell death in AZD6244 insensitive NRAS mutant melanoma cells. We have obtained a more specific inhibitor of TBK1, Compound II, and we will use it alone and in combination with AZD6244 in in vitro 3D collagen survival assays (which mimic the dermal environment) as well as in in vivo intradermal xenografts. In these experiments, we will be utilizing melanoma cell lines as well as cells dissociated from surgically resected NRAS mutant tumors. Additionally, we will analyze expression and activation of TBK1 in tumor microarrays of metastatic melanomas, associating our findings with NRAS genotype. In addition to TBK1, we predict that NRAS mutant melanoma cells also rely on genes that are not directly downstream of NRAS for survival. To identify signaling alterations in AZD6244 insensitive cell, we will use a short hairpin RNA (shRNA) library in a negative selection screen to find shRNA that are selectively toxic to AZD6244-treated AZD6244 insensitive cells. To validate the targets, we will knockdown the target using RNA interference to see if the lethal phenotype can be recapitulated. The validated targets will be examined in AZD6244 sensitive and insensitive cells and overexpressed in AZD6244 sensitive cells to see if they confer resistance to MEK inhibition. If targets or their involved pathways have inhibitors, then we will examine the effects, either alone or in combination with AZD6244, on cell death in 3D collagen survival assays. The data generated from this proposal may lead to the identification of TBK1, along with other proteins, as novel targets for NRAS mutant melanoma to be used in the clinical setting as part of a combination therapy regimen. Our long-term objective is to improve the treatment options for NRAS mutant melanomas."}
{"text": "Common use of condoms by young adults across all racial/ethic groups remains a formidable public health challenge, especially for African American males (Kennedy et al., 2007a,b,c; Kennedy et al., under review). As a call to action, a collaborative team of researchers proposes to conduct a four-year behavioral-driven research study to modify, implement and evaluate the efficacy of a promising brief condom promotion program in a two-arm randomized trial among African American males 18-24 years from 12 community centers on Chicago's South Side. Accordingly, we propose the following specific aims and related set of research questions: 1. Modify, implement, and assess the efficacy of a condom promotion program for African American males. 1.1. Will participants randomly assigned to the intervention report a statistically significant increase in the frequency of condom use and decrease in unprotected sexual intercourse (primary outcomes) relative to those assigned to the comparison group? 1.2. Will the intervention produce statistically significant positive effects on secondary outcomes (e.g., condom use self-efficacy, condom use decisional balance, etc.) relative to the comparison group? 1.3. Will the secondary outcomes mediate the relationship between intervention status and condom use (the primary outcome)? 2. Assess characteristics of program implementation and its relationship to changes in behavioral outcomes. 2.1. What was the level of implementation fidelity? 2.2. How is variation in implementation fidelity related to change in primary outcomes? For a nine-month formative phase, we will conduct a series of qualitative interviews with key informants and the target population to modify our existing program. Over the next 16 months, we will recruit 520 study participants from our recruitment sites to implement the intervention and comparison programs and assess the efficacy of the intervention program on a series of primary and secondary outcomes. Assuming 30% attrition rate (n=156), we expect to collect complete outcome data via Audio Computer Assisted Self Interview (ACASI) from about 364 participants over 12-months. In addition, we will conduct biological assessment of STDs to validate participants' self-reports on sexual behaviors at all assessment points. This proposed study is an extension of a recently completed NIH-funded R21 (PI: Kennedy) to develop, implement, and pilot a brief condom promotion program for African American males (Kennedy et al., 2007a,b,c; Kennedy et al., under review). We have assembled a collaborative team of experienced investigators and research partners to successfully implement this proposed project."}
{"text": "The focus of this project is development and refinement of statistical procedures for the design and analysis of screening, detection, and related studies in cancer. Statistical problems under investigation include sample size determination, comparison of analysis methods, and development of monitoring techniques and stopping rules. Each of these problem areas is common to screening and prevention trials in which the Division participates, but the methods for screening studies must address the special lead time and length biases inherent in screening programs. The research includes investigation of techniques to estimate and adjust for screening biases in the analysis of survival data, and the study of the relationship between long-term mortality and short-term outcome measures, such as stage shift. Probabilistic models of disease and screening are being developed to aid in the interpretation and evaluation of data from screening programs, and a breast cancer model relating doubling time to survival is under derivation in collaboration with the Biometry Branch, DCCP."}
{"text": "Each year, 6.2 million persons are newly infected with human papillomavirus (HPV) and 26,000 new HPV- related cervical, genital, and or pharyngeal cancers are diagnosed, resulting in >$4 billion in annual medical costs. Despite US guidelines for vaccinating all adolescents starting at age 11 with 3 doses of HPV vaccine, in 2012 only 53% of 13-17y females had >1 dose and 35% had 3 doses; 21% of teen males had a vaccination. The most effective strategy for improving vaccination rates is patient reminder/recall (R/R), in which providers send reminder messages by telephone, mail or other modalities to parents of children or teens who are eligible for vaccinations, or recall messages for vaccines that are overdue. Yet few practices use reminder/recall for any vaccinations because of costs and lack of personnel time; very few use reminder/recall for HPV vaccine. Statewide immunization information systems (IISs) now exist in 49 states to track childhood vaccinations, but they have not been used for reminder/recall for HPV vaccinations. We have united two leading immunization research groups (Rochester, NY and Denver, CO) for this dissemination/implementation project to determine whether IIS-based reminder/recall is more effective/cost-effective than practice- based reminder/recall in increasing HPV vaccination rates in adolescents. Our study has 4 aims. Aim 1 is to adapt R/R procedures used for children (protocols, clinical decision support, and message content) for state IISs to conduct reminder/recall for HPV vaccinations. Aim 2 is to assess the impact and cost-effectiveness of auto dialer (phone) IIS R/R in increasing HPV#1 and HPV#3 vaccination rates in adolescents 11-17 years. We will use a population-level, pragmatic comparative effectiveness trial, and randomizing counties to IIS R/R versus practice-based R/R training. We will apply the RE- AIM framework to evaluate the reach, effectiveness/cost-effectiveness, adoption and implementation of IIS R/R. Aim 3 is to assess the additional benefit and cost-effectiveness of adding mailed IIS R/R to auto dialer R/R for those with wrong or disconnected phone numbers or if no response to phone IIS R/R. This will involve a second pragmatic RCT, performed simultaneously with Aim 2, and also assessed using the RE-AIM framework. Aim 4 is to disseminate IIS R/R across New York and Colorado, develop a toolkit, and work with 4 other IISs and a technical advisory group to develop a sustainable IIS R/R system for the US. By the end of the study we will have a feasible, sustainable, cost-effective model for HPV vaccination reminders that can be used nationally to prevent cervical cancer and other HPV-related cancers."}
{"text": "Multiple myeloma (MM) is a B-cell malignancy that remains incurable in most patients. After treatment with high-dose chemotherapy and autologous stem-cell support, complete remission rates of 50% are achieved. However, relapses are inevitable in most patients. Additional measures are needed after transplantation to eliminate minimal residual disease, and immunotherapy is an appealing option for this purpose. We hypothesize that optimized dendritic cell (DC)-based immunotherapies are able to elicit a strong myeloma-specific cytotoxic immune response in immunocompetent patients that may effectively control or even eradicate residual tumor cells. Compared to idiotype protein (Id), myeloma cells contain a multitude of tumor antigens that can stimulate an increased repertoire of anti-tumor T cells. Indeed, our preliminary results demonstrate that myeloma lysate-specific T cells are promising effector cells for immunotherapy. In this project, we propose to examine the efficacy of intranodal vaccination of myeloma patients with tumor antigen-pulsed, CD40L-conditioned mature DCs. Newly diagnosed, untreated patients (most of who have nearly intact immune systems) will be targeted, and DC vaccines will be given to the patients prior to and after high-dose therapy in order to achieve a maximal tumor reduction without compromising the effects of immunotherapy. Thus, aim 1 of this project is to evaluate anti-tumor immune and clinical responses in patients receiving Id versus tumor lysate-pulsed DCs, to determine whether tumor lysate-pulsed DCs induce stronger responses. Antigens and keyhole limpet hemocyanin (KLH)-pulsed, CD40L-matured DCs will be used as the vaccine. CD40L-conditioned/matured DCs can activate CD8+ T cells directly and break T-cell tolerance. In Aim 2, we will evaluate whether re-infusion of blood T cells collected after initial DC vaccination helps to further expand specific T cells in vivo. Aim 3 is to evaluate whether long-term booster immunization could further augment anti-tumor immune and clinical responses in patients, and aim 4 to elucidate the biological mechanisms of the immunotherapy strategies by examining the induction, number and function of myeloma-specific T cells and subsets in blood and bone marrow of patients and whether immunotherapy may induce epitope spreading and loss of tumor antigens by myeloma cells. Anti-myeloma immunological and clinical responses are the primary and secondary end points of the studies. These innovative approaches will substantially contribute to the ability of immunotherapy to induce or improve long-term survival in patients with MM."}
{"text": "The research to be performed for this application will increase understanding of bacterial cell division. Although cell division is one of the most basic biological processes, there is relatively little known about what each component of the subcellular cell division machinery does and how the entire process is regulated. For this work the model organism Escherichia coli will be used. This organism is ideal for the study of cell division because (1) much about the basic genetics and cell biology of this organism is already known;(2) excellent genetic tools are available;and (3) the basic protein components of cell division in E. coli are highly conserved in other bacterial species. When a cell is about to divide, an essential protein called FtsZ polymerizes into a ring-like structure known as the Z ring at the center of the cell. Numerous other proteins, many of them also essential, are recruited to the Z ring. One of these proteins is FtsA. The ftsZ and ftsA genes are one of the most highly conserved bacterial gene pairs. Some of the activities of FtsA are known, but the molecular roles of FtsA remain elusive. The current line of thought is that FtsA regulates the formation of the Z ring by two mechanisms. First, FtsA tethers the ring to the inner membrane of the cell, connecting the Z ring to inner membrane proteins. Second, FtsA regulates the disassembly of the Z ring, so that it will constrict and divide the cell. The current data suggest that membrane binding, self-interaction and nucleotide binding are all critical for FtsA function and that each of these properties can be correlated to each other. Mutations that inhibit any one of these FtsA activities seem to affect FtsA-FtsZ interactions, which prevents normal cell division. The research proposed here will (1) determine how FtsA membrane binding and self-association are correlated and (2) define how each of these activities determines the ability of FtsA to affect the Z ring. Because they share some of the same properties, FtsA and MinC may affect FtsZ through analogous mechanisms. The study of bacterial cell division is important because it is a basic cellular process that needs to be understood and because it is a highly relevant target for potentially new antimicrobial drugs. PUBLIC HEALTH RELEVANCE: When bacterial cells cannot divide, they cannot multiply and spread. In this time of increasing bacterial resistance to antibiotic treatments it is critical that we decipher the mechanisms that allow these populations to proliferate. Understanding the mechanism of bacterial cell division will provide more targets for new antimicrobial therapeutics."}
{"text": "The long term goal of this work is to understand the underlying error and pathophysiology of cystic fibrosis, the most common lethal genetic disease of whites. In cystic fibrosis, there are inherited abnormalities in alpha-adrenergic, beta-adrenergic and cholinergic systems. This proposal will further elucidate the biochemical bases of these abnormalities and investigate whether they contribute to the development and progression of pulmonary disease. The beta-adrenergic defect is probably at the level of receptor-cyclase coupling, so this proposal will investigate Ns (a coupling protein), phospholipid methylation, receptor desensitization, and pharmacologic modulation of receptor-cyclase coupling in leukocytes from patients with cystic fibrosis, their parents, and appropriate controls. In vitro systems will be developed to investigate the cause of increased cholinergic responses in cytic fibrosis, such as cholinergic stimulation of lysosomal enzyme release from granulocytes, or of lymphoproliferation. Changes in intracellular calcium in response to cholinergic agents will be monitored with Quin 2, and the binding properties of muscarinic receptors on lymphocytes and granulocytes will be determined. To investigate the pathophysiologic significance of these autonomic abnormalities, three strategies will be used. First, using minimally invasive tests, like the cyclic AMP response to beta agents in leukocytes and power spectral analysis of heart rate variability, this project will test autonomic function in children with cystic fibrosis prior to the onset of significant disease. Second, autonomic function will be determined in tissues affected by the cyctic fibrosis disease process using the beta-adrenergic responses of nasal biopotentials in vivo and the cyclic AMP response to beta agents in tracheocytes obtained by bronchial brushing as test systems. Third, the relation of autonomic dysfunction to airway reactivity in parents of patients with cystic fibrosis and appropriate controls will be further defined. Thus, the study will determine if autonomic dysfunction is present early in the course of the disease and if it occurs in involved tissues, and will further elucidate the relation of autonomic dysfunction and airway reactivity. Such studies have implications for the development and progression of obstructive airways disease in general, and for cystic fibrosis in particular."}
{"text": "Many patients with hypertrophic cardiomyopathy have severe symptoms in spite of medical therapy with beta adrenergic blocking agents and/or calcium channel blocking agents. Recently we have been investigating the use of amiodarone, a benzofuran derivative with potent hemodynamic and antiarrhythmic properties in this same subgroup of patients and have noted an improvement in cardiac symptoms and an increase in exercise capacity. However, there remains a subgroup of patients who are intolerant of amiodarone or who do not improve on amiodarone and continue to have marked symptomatology. In response to a compelling clinical need in this subgroup of refractory patients, we felt it appropriate to explore other potential pharmacologic modalities. We have hypothesized that the functional and structural abnormalities in HCM are related to a primary membrane disorder leading to increased cytosolic calcium levels as a result of altered calcium fluxes involving both the myocardium and the vascular smooth muscle of the small intramural coronary arteries. Lidoflazine has been shown to be a potent calcium entry blocker, and has a cellular protective effect against calcium overload in vascular smooth muscle and cardiac muscle during ischemia, preventing ischemic contraction and myonecrosis. These properties of the drug afford an ideal mechanism for testing the above hypotheses, as well as offering a potentially important therapeutic alternative. The study will consist of 3 phases. The first phase to assess the clinical efficacy of the drug; the second to characterize the hemodynamic/metabolic correlates of the drug that may determine its efficacy; and the third to compare in a double blind fashion lidoflazine versus standard therapy. We have enrolled thus far 3 patients in phase I, two of whom have had symptomatic and exercise improvements."}
{"text": "Over half of the acute respiratory tract illness in infants and young children can presently be related to respiratory syncytial virus, a parainfluenza virus, an adenovirus, an influenza virus or Mycoplasmapneumoniae. The role of other agents apparently is much less important. The epidemiology of the more common agents is becoming increasingly understood, but often has shown surprising variations. Little is known regarding the pathogenesis of infection with these agents, particularly with respect to the factors of host resistance, and the role of local, specific cellular, or serum antibody in protection or in production of disease. In order more fully to understand these infections and their possible prevention we propose: (1) to determine (a) the relationship of non-bacterial agents to pediatric respiratory tract illness, (b) the clinical relationship of these agents. (2) to determine the pathogenesis of infection and development of resistance to respiratory tract viral infection. (3) to study the natural history of viral and mycoplasma agents."}
{"text": "It is currently understood that arterial baroreceptors control sympathetic nervous system tone, while vestibular end organs provide afferent input to a vestibulo-sympathetic reflex that normally augments the compensatory baroreflex. However, the chemical anatomy, connectivity and synaptology of the neural pathways underlying this integration remain largely unknown. The overall goal of the proposed project is to identify the neurotransmitters and modulators involved in mediating vestibulo-autonomic synaptic interactions. The four aims address the chemoanatomy and synaptology of these vestibular projections in adult rats and evaluate the overall hypothesis that different transmitter/modulator signatures and distinct synaptic architectural arrangements are utilized by vestibular projections to the caudal and rostral ventrolateral medulla (CVLM and RVLM, respectively). Aim 1 is to identify the locations of vestibulo-medullary and medullo- sympathetic projection neurons, determine the relative proportions of vestibular projections to CVLM and RVLM, identify the primary amino acid neurotransmitter(s) utilized by these pathways and determine whether these projections terminate on glutamatergic and/or GABAergic neurons in CVLM and RVLM. Aim 2 is to determine whether vestibular terminals in CVLM and RVLM co-express IAA-RP and/or L-citrulline. Aim 3 is to conduct a qualitative analysis of the main neurotransmitters and modulators of vestibulo-recipient cells in RVLM and CVLM, to test the hypothesis that glutamatergic vestibular afferents terminate on interneurons and on distal dendrites of GABAergic/IAA-RP-containing CVLM neurons, GABAergic and noradrenergic dendrites in RVLM, and directly on the somata of glutamatergic/IAA-RP-containing RVLM neurons with direct projections to the spinal cord. Aim 4 is to visualize and quantify parameters of vestibulo-sympathetic synaptology, and will test the hypothesis that the synaptology of vestibular terminals in RVLM involves complex interactions among multiple transmitter systems, whereas the synaptology of vestibulo-sympathetic terminals in CVLM reflects direct axo-dendritic contacts with CVLM output neurons. Throughout the project, the vestibulo-sympathetic pathways will be identified using anterograde and retrograde tract-tracing in tissue that will be further processed using additional immunofluorescent tags to identify transmitters and modulators of interest. Ultrastructural studies will employ immunogold labels to identify and quantify terminals and synaptic contacts. These studies will characterize the neurotransmitters, modulators and synaptic articulations utilized by vestibular projections to sympathetic pre-motor medullary neuron pools and will yield qualitative analyses and quantitative estimates of parameters of their synaptic architecture. This information does not currently exist in the literature, and is important for the development of evidence-based pharmacotherapeutics for the treatment of vestibulo-autonomic disorders such as orthostatic hypotension, as well as the development of more specific anti-hypertensive medications that do not elicit disabling vestibular side-effects such as dizziness and vertigo. PUBLIC HEALTH RELEVANCE: Orthostatic hypotension and vestibular side effects of drugs targeting the sympathetic nervous system (e.g. anti-hypertensive medications) impact large populations, especially the elderly. However, little is known about the neurochemical organization of vestibulo-sympathetic pathways. This project will provide fundamental information about the chemoanatomic and synaptic organization of vestibular projections to cardiovascular neurons in the ventrolateral medulla, may suggest new drug therapies to ameliorate orthostatic hypotension and intolerance, and may lead to the development of more specific anti-hypertensive medications that do not elicit disabling vestibular side effects such as dizziness and vertigo. [unreadable] [unreadable] [unreadable]"}
{"text": "Neoplasms contain increased quantities of O-alkyl lipids. In order to detect surface membrane abnormalities which might exist in malignant versus non-malignant tumors, we have measured the O-alkyl lipid content of these structures. The surface membrane O-alkyl lipids of a series of transplantable metastasizing and non-metastasizing rat tumors was quantitated by photodensitometry. The results suggest that more rapidly growing and more malignant neoplasms contain greater quantities of O-alkyl phospholipids than less aggressive tumors. We are currently conducting a similar evaluation of a series of human mammary tumors obtained as surgical specimens and analyzed for estrogen and progesterone receptors. We are attempting to correlate the degree of malignancy of these tumors with their ether lipid content and with their concentrations of estrogen and progesterone receptors. An additional project in progress is an attempt to purify O-alkyl synthase, the enzyme responsible for the synthesis of O-alkyl dihydroxyacetone-phosphate. Although a method for purifying this enzyme has been recently published, we are taking a somewhat different approach involving novel forms of chromatography. Preliminary studies in this area involve isolation of this enzyme from Tetrahymena pyriformis. If successful, the procedure developed will be used on the enzyme found in tumors. In addition to the above, we are continuing work on the mechanism of action of O-alkyl synthase. In the past year, we have conducted experiments confirming the basic validity of our proposed mechanism and confirmed the work of others that the action may be, in part, reversible. We are also studying the stoichiometry involved in that aspect of the mechanism involving a hydrogen exchange and the release of fatty acid from acyl-dihydroxyacetonephosphate. Preliminary data suggest that the release of one proton is accompanied by the release of one fatty acid which retains both carboxyl oxygens. This unusual form of ester cleavage was recently demonstrated in this laboratory. The results have broader implications with regard to the mechanism of action of a related enzyme that may function via a similar mechanism, namely, the acylhydrolase which cleaves acyl-dihydroxyacetonephosphate. (A)"}
{"text": "3D Microscopy with Ultraviolet Surface Excitation (3D-MUSE) Summary. We will create powerful techniques for performing 3D histology-quality imaging of small to large volume specimens (e.g., entire mouse or human organ) with extraordinary contrasts to address a very large number of preclinical, biotechnology, and clinical applications. We will combine Microscopy with Ultraviolet Sur- face Excitation (MUSE) (invented at Lawrence Livermore National Laboratory and extended at University of California at Davis), with 3D serial sectioning and block face imaging technologies (developed at Case West- ern Reserve University), to implement 3D-MUSE. Briefly, MUSE fluorescence imaging uses excitation at wave- lengths shorter than 300 nm, a range at which tissue penetration is limited to a few microns deep. This allows one to image?at sub-nuclear resolution?only the very surface of a piece of tissue, making it ideal for serial block-face imaging. Many common histological stains, autofluorescence, and fluorescent proteins excite at these sub-300-nm wavelengths and emit at their familiar wavelengths in the visible range. Because the excita- tion range is distant from emitted signals, images can be captured using a color camera, without additional fil- ters, providing a broad and informative color palette, beyond what is obtained using narrow-band-filter sys- tems. With color-sensitive volume rendering, and combined color and morphology-based segmentation, 3D microanatomy and pathology will be visualized and quantified. Image-guided histology will allow 2D/3D live- time monitoring to guide optional section acquisition for molecular studies using immunohistochemistry and manual or laser-capture micro-dissection. When fully realized, potential applications, out of many, include 3D microanatomy, mouse phenotyping, embryo cell lineage tracking, monitoring therapeutic nanoparticle delivery, as well as studies of cancer physiology, cancer immunotherapy, stem cells, toxicology, and biology. In all of these cases, we anticipate that there will be great value added by the third dimension. We will build on exper- tise in MUSE and block-face imaging systems to create test beds for evaluating 3D-MUSE. We will optimize tissue immobilization, staining, and imaging, and advance MUSE-specific software for 3D segmentation, visu- alization, and analysis. If our research is successful, we will transfer 3D-MUSE technology to commercial enti- ties that can create products for researchers across the world. It will likely be possible to create inexpensive, highly informative 3D-MUSE imaging systems that will empower many applications. Ultimately, the number of researchers using 3D-MUSE around the world will define project success."}
{"text": "Nearly 5 million people in the US alone are afflicted with Alzheimer's disease, and there is currently no means to prevent or treat the disease. Though the molecular basis of the disease is unclear, the deposition of amyloid plaques in the brain is a key hallmark of the disease. These plaques are widely believed to be pathogenic, and tremendous efforts have focused on the mechanism governing their formation. Amyloid plaques are composed of the 42-residue amyloid ? peptide (A?), which is generated through proteolytic cleavage of the amyloid precursor protein (APP) by ?-secretase. This enzyme is localized within cholesterol-rich lipid raft membrane domains, while the APP substrate exists in both the fluid-phase and lipid raft domains of cellular membranes. Therefore, the efficiency with which the A? peptide is generated may be governed by the distribution of APP between the raft and non-raft membrane domains. Recent work in the Sanders lab on the 99-residue C-terminal fragment of APP (C99) has revealed a cholesterol binding pocket within the TM domain (present in APP). These studies have confirmed that C99 binds cholesterol with high affinity in bilayers, which suggests that the localization of C99 (and APP) may depend on the distribution of cholesterol in biological membranes. We recently tested this hypothesis by characterizing the localization of C99 within phase-separated giant unilamellar vesicles (GUVs), which contain both fluid phase (L?) and liquid-ordered (Lo) domains. The results show that C99 is specifically localized within raft-like Lo domains. However, C99 variants carrying mutations that abolish cholesterol binding strongly prefer the non-raft L? phase. This confirms cholesterol binding directly affects the distribution of C99 within the membrane. The most intuitive explanation for this phenomenon is that C99 is driven into the Lo domain due to the high concentration of the cholesterol ligand, which leads to favorable binding energetics. However, thermodynamic evaluations of this partitioning suggest that binding energetics cannot account for the observed differences. Thus, the physical mechanism for this coupled binding and partitioning remains unclear. In the following, I propose a series of experiments aimed at dissecting the energetic contributions of both the membrane and the protein in the coupled binding and partitioning of C99. I will first use EPR spectroscopy to assess the binding energetics of cholesterol in Lo and L? like membranes. The results will suggest whether the cholesterol binding energetics are sensitive to changes in the bilayer. Next, I will use protein engineering and confocal fluorescence microscopy to determine how differences in the length and rigidity of the TM domain affect its partitioning. These studies will reveal the structural features of C99 that are critical for its sorting within te membrane. Finally, I will examine the structural dynamics of free and cholesterol-bound C99s using solution NMR in both Lo and L? like bicelles in order to determine how bilayers affect its binding mode. Together, the results will provide novel insights into the molecular basis of Alzheimer's disease and elucidate the molecular determinants of protein sorting within the membrane."}
{"text": "PROJECT SUMMARY DNA replication is undeniably important for life and as a consequence, cells have evolved mechanisms to monitor replication fidelity and to coordinate completion of replication with other cell cycle events. The goal of this project is to understand how cells choreograph the duplication of their chromosomes, and how defects in DNA replication may contribute to some recently discovered disorders in humans. Chromosome replication in eukaryotes is a process that involves the regulation of multiple initiation sites (origins) per chromosome. Origins show variation in timing-not all origins fire at the same time in S phase-and in efficiency-not all origins necessarily firing in every cell cycle. In addition, cells appea to maintain a backup set of origins (dormant origins) for use under special circumstances and these origins may differ in their basic control properties. Understanding how origin use is regulated is therefore critical for understanding how genome integrity is maintained. This notion is underscored by three recently described genetic disorders with links to replication defects: 1) Meier-Gorlin Syndrome with its point mutations in genes essential for origin licensing; 2) a point mutation in a gene encoding part of the replicative helicase that is associated with breast cancer in mice and growth abnormalities in humans; and 3) human segmental triplications with an inverted central copy that can be explained by a replication fork error. Yeast is an ideal model organism for studying DNA replication because of its small chromosomes, well defined origin sequences, ease of altering chromosome structure, and exceptional systems for genetic and genomic analysis. This project will apply a combination of molecular, genetic and genomics approaches to: uncover modulators of origin efficiency and timing in yeast, to explore/investigate how orderly replication contributes to genome stability use point mutations in the yeast orthologs of genes involved in human disorders to understand the downstream consequences of defects in replication as they relate to human health and disease, and to identify genetic and chemical suppressors of the defects determine whether DNA replication errors are responsible for a particular class of inverted amplicons that can contribute to cancer progression and congenital copy number variants. This project will address outstanding questions regarding the biology of chromosome replication control: why do origins initiate replication at different times, what distinguishes origins in different temporal categories, what i the molecular basis for inefficient origins, and what defects in replication fork progression predispose chromosomal loci to inverted amplification? In addition, this work will give insight int human disorders resulting from errors in chromosome replication and could lead to diagnostic or therapeutic advances."}
{"text": "Huntington[unreadable]s disease (HD) is a dominant neurodegenerative disease that is caused by the expansion of a stretch of CAG triplet repeats encoding polyglutamine (polyQ) within huntingtin (htt), the protein product of the HD gene. HD is considered to be the consequence of a deleterious gain-of-function caused by the expanded polyQ stretch that is unrelated to htt[unreadable]s normal function. Recent work suggests that although gain-offunction may play an important role in HD pathogenesis, a corresponding loss of normal htt function also contributes to the disease process. Our long-term objective is to use genetic and biochemical approaches to understand the role of htt[unreadable]s normal function in HD pathogenesis, and to discover new potential therapeutic strategies for the treatment of HD based on restoring normal htt function in HD. To accomplish this objective, we propose three specific aims that are designed to help us understand how the expanded polyQ stretch can affect normal htt function, and how a version of htt that lacks its normal short stretch of polyQ (?Q-htt) is able to rescue HD phenotypes in a mouse model for HD. In Aim 1, we will test the hypothesis that ?Q-htt is able to rescue HD mouse model phenotypes by enhancing autophagic clearance of mutant htt. We will focus on two potential mechanisms that may be responsible for ?Q-htt[unreadable]s effects. First, ?Q-htt may mediate the enhanced recognition of mutant htt aggregates by p62/SQSTM1, a polyubiquitin binding protein that can target such aggregates for autophagic degradation. Second, ?Q-htt may affect autophagic degradation of mutant htt aggregates indirectly by enhancing retrograde transport of autophagosomes to lysosomes. To test these mechanisms, we will characterize brains and primary neurons derived from mice expressing ?Q-htt with or without 140Q-htt expression, and conditional knockout mice lacking neuronal htt expression, for p62/SQSTM1 function. In addition, the efficiency of retrograde transport will be characterized in primary neuronal cultures by measuring organelle and dynein complex movement. Recently, we have also observed that increasing the length of the mouse htt polyQ stretch from 7Q to the normal human average length of 20Q can accelerate interactions of normal and mutant htt. To test the hypothesis that an interaction between normal and mutant htt can affect HD pathogenesis, we will compare in Aim 2, behavioral and neuropathological phenotypes in mice expressing 7Q/140Q htt, and in mice expressing 20Q/140Q htt. Finally, in Aim 3, we will test the hypothesis that mutant htt expression can influence the interaction of wild-type or ?Q-htt with their binding partners by using mice expressing epitope-tagged htt alleles to detect differences in the repertoire of normal and ?Q-htt interacting proteins in the presence and absence of mutant htt expression."}
{"text": "The North Central Cancer Treatment Group (NCCTG) is a regional organization made up of 10 community cancer centers with the Mayo Comprehensive Cancer Center serving as Operations Office and Statistical Center. It is designed to conduct cancer treatment research at the level of the community clinic where most cancer care is delivered. It is the intent of the NCCTG to produce the most complimentary clinical research interaction of community clinic, comprehensive cancer center, and national cooperative group. By bringing cancer patients into national research programs at an early stage of their disease, the NCCTG meets an expressed need of the National Cancer Program. Particular emphasis had been placed on multidisciplinary programs with strong participation of medical oncology, surgical oncology, radiation therapy, and pathology. We believe extraordinary high quality of research data has been achieved and we will continue our efforts to maintain and enhance quality control procedures. Significant and timely clinical research protocols are being conducted in colorectal carcinoma, lung cancer, breast cancer, pancreatic cancer, gastric cancer, gynecologic cancer, and brain tumors. Protocols for urologic cancer are under active planning. Whenever pertinent, protocols are randomized in design and concurrently controlled with untreated controls used when justified. A unique characteristic of clinical research within the NCCTG is unparalleled cost effectiveness. In spite of the severe handicap of grossly inadequate funding the NCCTG has proven itself to be a strong, viable, high quality, and productive clinical research organization."}
{"text": "Hepatitis C virus (HCV) is one of medically important RNA viruses and causes a wide spectrum of liver diseases, which are also a leading indication for liver transplantation in the United States. Given its high priority in public health, an in vito model for HCV life cycle is highly demanded for both basic and translational HCV research, however, current in vitro HCV models could only mimic some steps of viral life cycle. There is so far only one particular HCV genotype 2a strain, named JFH1, is able to be propagated in vitro. The JFH1-based cell culture has been established through the rescue of infectious viruses from cloned viral genomes, so called reverse genetics approach. A well-known constraint in such an approach is the unpredictability of viral mutations introduced internally or externally during the preparation of full-length viral clones. Some of these mutations may be lethal or detrimental for successful rescue of infectious viruses, in particular in the setting of HCV due to its coordinated genome network and remarkable genetic diversity. When the number of strains/clones tested is large enough to overcome the barrier of mutations from either heterogeneous viral population or experimental introduction, HCV cell culture could be established with strains other than JFH1. However, this goal is hard to be achieved by conventional reverse genetics that is notable for its tedious process. Based on our previous work, we thus propose to develop a rapid reverse genetics platform capable of screening clinical HCV isolates in a high-throughput manner. We hypothesize that the establishment of such a platform is feasible by the integration of our long RT-PCR technique (US patent No: 7,786,294 B2) and a seamless assembly method. The proposed platform is a plasmid-based reverse genetics in which long RT-PCR and 3'UTR amplicons of HCV will be fused into the target plasmid using the Gibson assembly method, followed by direct transfection into Huh-7 cells and subsequent measurement of potential infectious viruses. We will screen a total of 61 patient-derived HCV 1a strains that have already been characterized for viral mutations at the full-length genome level. The method developed with the proposed project has general applicability in RNA viruses other than HCV."}
{"text": "The proposed research focuses on the central unsolved problem of psychiatric epidemiology, the problem of how to measure psychiatric disorders independently of treatment status in the general population. Its major aim is to test the sensitivity and specificity of the newly developed Psychiatric Epidemiology Research Interview (PERI) as a first-stage instrument to screen functional psychiatric disorders among adults in a demographically complex urban population. To this end, two field operations would be conducted. In the first, we would investigate PERI's relationship to other potential screening instruments of a sample of 600 adults from the general population and calibrate PERI on a sample of 500 psychiatric patients with diagnoses of schizophrenia, affective psychosis, neurosis, anti-social personality, and other personality disorders. In the second field operation, we would examine PERI's relationship to two diagnostic instruments, the Present State Examination (PSE) and the Schedule for Affective Disorders and Schizophrenia (SADS). In this second study, interviews would be conducted with a full probability sample of 1400 adults from the general population. The research setting for these investigations would be Washington Heights, a section of Manhattan in New York City that surrounds the Columbia Presbyterian Medical Center. It is a demographically complex urban area containing large percentages of black and Puerto Ricans as well as members of more advantaged ethnic groups. The results should provide a textbook example of the development and testing of a two stage procedure for case identification and classification. The findings should also be important for needs assessment and to the planning and evaluation of mental health service in Washington Heights."}
{"text": "The aim of this research is to understand how the circulatory system is controlled, with the ultimate objective of determining which control factors are most directly involved in the production of essential hypertension. Over the past year we phased out the baboon research. In one aspect of the work we completed the lesions of the HACER (hypothalamic area controlling emotional responses), a region of the hypothalamus. Data analyses showed a dramatic correlation between the resting level of blood pressure and the decrease in that pressure level after hypothalamic lesions. Retrospective analysis of data obtained since 1986 showed that animals with spontaneous hypertension were returned to normotensive levels. In the second aspect, we found that dominant male baboons in social situations showed significantly elevated heart rate, blood pressure, and catecholamines during the presentation of food, whereas subordinate males did not show elevations in these variables."}
{"text": "Thebroad,longtermobjectiveandspecificaimsofthefasttrackSBIRresearcharetocreatea reagent,RecombiRED,foruseinmicroinjectionofembryosforidentifyingasubsetofembryos asrecombinationcompetentinjections.Theabilitytoquicklyidentifygenomeeditingeventsin anembryoallowsresearchertoquicklymakeprecisionanimalmodelsofhumandiseasestates. Preciseanimalmodelsallowforgreaterscientificunderstandingofahumandiseaseand providesarobustplatformfordrugdiscoveryscreening.TheRecombiREDreagentrelieson inducedfluorescenceinresponsetoactivationofhomologousrecombinationrepairwithinthe embryo.DetectionofhomologousrecombinationrepairininjectedembryoswithRecombiRED allowsanimalhusbandryandfurtherscreeningeffortstoberestrictedtoasubsetofembryosby upto100xforhavingadesiredpreciseknockinofnewgeneticmaterialataspecificgenetic locus.ThephaseIresearchadaptsatheRecombiREDreagentdevelopedinC .elegansmodel organismtohavetheappropriateexpressioncontrolelementsenablingsufficientactivityin zebrafishembryo.InPhaseII,theadditionalresearchfurtheroptimizesparametersofthe RecombiREDreagenttominimizefalsepositiveandfalsenegativereadouts,whichthenallows maximalforecastingoftargetedgenomeeditevents.Finally,asetof15realworldtransgenesis projectsareusedaspremarketbetatesttodemonstratetheubiquityandrobustnessofthe RecombiREDforsimplifyingdiscoveryofgenomeeditinginthecreationofanimalsmodelsfor useindrugdiscovery."}
{"text": "ABSTRACT Stroke is one of the major medical concerns for United States military veterans. Stroke is devastating as currently no therapy is available to prevent stroke-induced neurological deficit. Prophylactic or therapeutic supplementation with omega-3 polyunsaturated fatty acids (n-3 PUFAs) has recently emerged as a highly promising neuroprotective strategy for stroke. Epidemiological studies have shown an inverse association between n-3 PUFAs intake and risk of ischemic stroke. Supplementation with n-3 PUFAs, especially DHA, effectively reduces the extent of brain damage and neurological deficits in the acute phase of ischemic injury in experimental animals. However, the long-term effect of n-3 PUFAs on brain damage and post- stroke neurological recovery is unknown. We have recently created transgenic (Tg) mice over-expressing the C elegans fat-1 gene encoding an enzyme that converts endogenous n-6 to n-3 PUFAs. Our preliminary studies with fat-1 Tg mice have suggested two-phase (acute and delayed) beneficial effects of n-3 PUFAs on ischemic stroke. The fat-1 Tg mice at either young (3-month-old) or old age (15-month-old) were remarkably resistant to focal ischemic injury compared to their age-matched wild-type (Wt) littermates, showing reduced infarct size and improved neurological functional performance up to 14 days after middle cerebral artery occlusion. We have found that angiogenesis and neurogenesis were robustly enhanced after ischemia in 3-month old fat-1 Tg mice compared to Wt littermates, suggesting that n-3 PUFAs promote post-stroke neurovascular regeneration. Strikingly, we found that post-stroke angiogenesis and neurogenesis were profoundly impaired in aged mice, but almost fully restored by transgenic expression of fat-1. This proposal attempts to further explore the long-term protective effect of n-3 PUFAs on focal cerebral ischemia. The goal is to develop n-3 PUFA supplementation as a novel, clinically feasible prophylactic and/or therapeutic strategy to promote long-term neurological recovery after stroke through stimulating the generation of functional blood vessels and new neurons. The central hypothesis to be tested in the current proposal is that prophylactic or therapeutic n-3 PUFA treatment improves long-term neurological outcomes after stroke by stimulating and enhancing post-ischemia brain repair, including augmented neurogenesis and angiogenesis. The following specific objectives are proposed: Aim 1. Test the hypothesis that n-3 PUFAs reduce long-term neurological deficits as well as brain tissue damage after focal cerebral ischemia in both young adult and aged mice. Two clinically applicable methods: 1) dietary delivery; and 2) augmented endogenous production of n-3 PUFAs via adeno-associated virus (AAV)-directed fat-1 gene expression, will be established to elevate brain levels of n-3 PUFAs. The effect of elevated n-3 PUFAs on ischemic brain injury induced by MCAO will be quantitatively evaluated. The endpoints of assessment include functional outcomes, infarct size, and white matter injury. Aim 2. Test the hypothesis that n-3 PUFA treatment enhances cerebral neurovascular regeneration, including augmented angiogenesis and neurogenesis after cerebral ischemia in both young adult and aged mice. The proposed studies will quantitatively determine the effect of n-3 PUFAs on post-stroke neovascularization and on neural stem cell proliferation, migration, differentiation and neuronal replacement following ischemic stroke. PUBLIC HEALTH RELEVANCE: NARRATIVE Stroke is one of the major medical concerns for United States military veterans. Stroke is devastating as currently no therapy is available to prevent stroke-induced neurological deficit. Prophylactic or therapeutic supplementation with omega-3 polyunsaturated fatty acids (n-3 PUFAs) has recently emerged as a highly promising neuroprotective strategy for stroke. Supplementation with n-3 PUFAs, especially DHA, effectively reduces the extent of brain damage and neurological deficits in the acute phase of ischemic injury in experimental animals. However, the long-term effect of n-3 PUFAs on brain damage and post-stroke neurological recovery is unknown. Our preliminary studies have suggested two-phase (acute and delayed) beneficial effects of n-3 PUFAs on ischemic stroke. We have found evidence that n-3 PUFAs promote post-stroke neurovascular regeneration. Thus, the objective of the current proposal is to develop n-3 PUFA supplementation as a novel, clinically feasible prophylactic and/or therapeutic strategy to promote long-term neurological recovery after stroke. First, we will test the hypothesis that n-3 PUFAs reduce long-term neurological deficits as well as brain tissue damage after focal cerebral ischemia in both young adult and aged mice. Second, we will test the hypothesis that n-3 PUFA treatment enhances cerebral neurovascular regeneration."}
{"text": "DESCRIPTION OF OVERALL CENTER (provided by applicant): The Kansas Interdisciplinary Center for PKD Research (Kansas PKD Center) was established to provide support for highly meritorious new research programs to explore the basic mechanisms causing polycystic kidney disease (PKD); to provide opportunities to bring new investigators into PKD research; and to foster opportunities for more interdisciplinary research in a dynamic, enriching, and interactive setting. The long term goal for the Kansas PKD Center is to find a treatment for PKD that will alleviate the suffering of patients and families affected by the disease. The central theme of the research projects in the Kansas PKD Center is \"cell proliferation in polycystic kidney disease.\" This theme was chosen because Center investigators have been able to identify the cell proliferation defect in PKD as one that involves a cyclic AMP- and calcium-dependent mechanism. The focus will be on using the mouse as a model organism, and on human PKD cyst cells. The Kansas PKD Center is comprised of eight components. The Administrative Core will provide administrative support for Center investigators. The Biomaterials Research Core will establish and maintain a repository of human and animal biological materials for PKD research including primary cultured cells and tissues, and DMA and RNA from ADPKD, ARPKD, and normal human kidney tissues. The Core will also establish and maintain immortalized cell lines from mice carrying unique PKD gene mutations and will provide technical assistance and training in cell culture methodology for all Center investigators. There will be four Biomedical Research Projects: Project 1, \"Calcium Regulation of cAMP-Dependent Proliferation,\" Project 2, \"Polycystin-1 Mediated Calcium and cAMP Signaling,\" Project 3, \"Role of Oxidant Stress in Progression of PKD,\" and Project 4, \"Cux-1 and Cell Cycle Regulation in PKD.\""}
{"text": "Murine-based retroviruses continue to be developed as viral vectors, despite their association with oncogenic transformation and insertional mutagenesis. This is highlighted in the past month with the association of xenotropic murine leukemia virus-related virus (XMRV) with human diseases including chronic fatigue syndrome and prostate cancer. Although comparative retroviral/lentiviral analysis of integration sites has noted differences in the positioning within the host chromosome, the mechanism for tethering the murine-based viruses to the host chromosomes is not understood. Structural analysis of proteins can provide essential insights into function. This proposal develops and extends preliminary structural analysis of the N-terminal domain (NTD) of the Moloney Murine Leukemia Virus Integrase (M-MuLV IN) protein to define the function of this domain within the preintegrative complex. The structural analysis of this domain will be extended to XMRV, which maintains structural homology with MuLV IN. The first focus of the research is structural studies of the IN NTD. The MuLV NTD is distinct from that of HIV and avian retroviruses in that it encodes an additional 50 amino acids N-terminal to the HHCC zinc-binding domain. This adds complexity to the NMR structural analysis of the 105 aa dimer. Through development of a novel growth/labeling system in E. coli utilizing condensed cultures, a preliminary NMR structure of the MuLV IN NTD monomer has been obtained. Experiments modify this system utilizing amino acid auxotroph strains to determine the dimer structure by NMR. Structural comparison with the IN NTD X-ray structure will be made, for which diffraction quality crystals have been obtained. SAXS analysis of the full-length IN ( DNA) will be performed. The IN NTD structures will be critical in defining domain localizations within the synaptic complex. Based on the structural analysis, the predictive function of the MuLV IN NTD will be analyzed. Specifically, the role of the putative winged-helix domain to interact with chromatinized DNA or associated factors will be determined. Mutagenesis studies will identify the role of specific amino acids in the dimer interface as well as interacting with viral and host factors. The ability of known DNA viral tethering domains to functionally complement the mutant IN NTD domains will be examined. Insights into the tethering of IN to chromatin has broad applications into target-site selection, but also to the requirement for mitosis for MuLV-based vectors. The safety of retroviral-based vectors is dependent on the target-site selection during retroviral integration. Through this understanding, mechanism to alter or limit integration can then be rationally designed."}
{"text": "The literature on HIV risk behavior has shown that women who are at risk for HIV sexual disease transmission tend also to be at risk for a host of other issues, including increased substance use and past and current experiences with interpersonal violence. From increasing evidence in the relatively separate literatures on sexual risk behavior, substance use, and interpersonal violence (IPV), there is a pressing need for research to evaluate the intersection, or syndemic (Singer, 1994), of these risk issues with women's HIV sexual risk, so that interventions can attend to needs in domains that are interrelated and can theoretically effect behavioral changes of greater magnitude and greater duration. Bioecological Systems Theory (BST; Bronfenbrenner, 1993) may provide a framework for a greater understanding of the context of multiple risk behaviors in women. This prospective study will examine factors corresponding to the four levels of the BST model as they relate to the interaction of sexual risk behavior, substance use, and violence in women of lower socioeconomic status. Further, we will also examine changes in their reports over a six-month interval. The specific aims are: (1)To recruit 396 inner-city women living in urban housing developments between the ages of 18 and 45 in order to retain 375 participants; (2) To perform multiple assessments on factors pertaining to the levels of the BST model, as they relate to sexual risk, substance use, and trauma/victimization history; these levels and factors include but are not restricted to: a) Individual (Ontogenic) factors, b) Interpersonal (Microsystem) factors (the immediate context of the behavior and relationships with others); c) Community (Exosystem) factors (encompassing community structures, institutions, and the social networks surrounding the individual's life); and d) Socio-cultural (Macrosystem) factors (attitudes, practices, and convictions shared throughout society at large); (3) To perform similar follow-up assessments with these participants six-months and 12-months following the first set of assessments; and (4) To analyze the above variables in relation to occurrence of occurrence and severity of sexual risk behavior, substance use, and interpersonal violence (either perpetrated or as a victim of) using structural equation modeling to examine application of variables related to Bronfenbrenner's BST (1993) model. Findings from this research will provide important new information: (1) leading to greater insight of the dynamics and complexity of sexual risk behavior, substance use and trauma history in at-risk women; (2) needed to design interventions that attend to multiple risk issues in at-risk women; (3) needed to design effective strategies to prevent these issues from occurring; and (4) to evaluate the applicability and validity of BST to the context of women's sexual risk behaviors, substance use, and trauma history. PUBLIC HEALTH RELEVANCE: The primary goals of this project are to examine individual, interpersonal, community and sociocultural factors (Bioecological Systems Theory; Bronfenbrenner, 1995) as they relate to the interaction of sexual risk behavior, substance use, and trauma history/victimization in low income African American women living in urban housing developments in Milwaukee, as measured at three points over a 12-month interval. Findings from this research will provide important new information: (1) leading to greater insight of the dynamics and complexity of sexual risk behavior, substance use and trauma history in low income African American women; (2) needed to design interventions that attend to multiple risk issues in low income African American women; (3) needed to design effective strategies to prevent these issues from occurring; and (4) to evaluate the applicability and validity of Bioecological Systems Theory to the context of low income African American women's sexual risk behaviors, substance use, and trauma history."}
{"text": "Pro-inflammatory mediators, such as IL-1 beta and TNF-alpha, are known to inhibit smooth muscle contractility in part by acting directly on smooth muscle. Our hypothesis is that these mediators inhibit contractility by inducing or suppressing the expression of critical targets in the signaling pathways mediating contraction and relaxation. In preliminary studies on control and TNBS colitis muscle strips, and on freshly dispersed and cultured muscle cells, we have identified six novel cytokine (IL-1 beta)-mediated effects reflecting changes in the expression and/or activity of specific signaling targets. These include: (i) up-regulation of RGS4 and RGS12 expression, which leads to inhibition of initial Ca2+-dependent contraction by Gq and Gi-coupled receptor agonists, respectively; (ii) down-regulation of the endogenous MLC phosphatase inhibitor, CPI-17, which leads to inhibition of sustained RhoA-dependent contraction; (iii) up-regulation of SERCA2 and IP3R-I, which regulate Ca2+ uptake and Ca2+ release, respectively; (iv) up-regulation of PDE4D5 and PDE5A expression, which leads to greater degradation of cAMP and cGMP, respectively; (v) down-regulation of soluble guanylyl cyclase (sGC) expression via a PKG/JNK-dependent post-transcriptional mechanism, which leads to inhibition of cGMP synthesis; and (vi) inactivation of adenylyl cyclase (AC) via iNOS-dependent nitrosylation, which leads to inhibition of cAMP synthesis. Where examined so far, the effects elicited in IL-1 beta-treated colonic muscle were observed also in colonic muscle from TNBS colitis. The specific aims are to: (1) characterize the changes in the expression and activity of signaling targets (RGS4/RGS12, CPI-17, SERCA2 and IP3R-I) mediating initial Ca2+-dependent and sustained Ca2+-independent contraction; (2) characterize the changes in expression and activity of signaling targets (PDE3A, PDE4D5, PDE5A; sGC and AC) that regulate relaxation; and (3) characterize the transcriptional regulation of RGS4/RGS12 and CPI-17 by NF-kappa B and modulatory kinases (p38 MAP kinase, ERK1/2 and the PI 3-kinase/Akt/GSK3beta pathway), and the post-transcriptional regulation of sGC expression via a PKG/JNK/AP-1/HuR pathway. These studies will provide a comprehensive analysis of the mechanisms by which inflammatory cytokines inhibit contractility at cellular level, both in vitro and in an established model of colonic inflammation. [unreadable] [unreadable] [unreadable]"}
{"text": "Over 2 million adult cases of hepatitis E virus (HEV) are thought to occur annually in India alone, and HEV accounts for the majority of acute viral hepatitis hospitalizations worldwide, yet accurate population-based estimates of HEV burden remain scant. The hallmark and most concerning aspect of HEV is a high mortality in infected pregnant women. There have been few rigorous, population-based, prospective studies of HEV incidence, immunopathogenesis and natural history among pregnant women. Our current understanding of the risk factors and host characteristics that lead to severe consequences in pregnant women and neonates is largely based on hospital-based convenience samples. There is a need to carefully define the burden of HEV infection and disease in terms of maternal and neonatal consequences in a typical, endemic resource-poor population. To characterize the population- based epidemiology of HEV and describe maternal morbidity and mortality, pregnancy outcomes, and neonatal survival, we will 1) enroll and follow ~10,000 newly pregnant women through 3 months postpartum in a representative, rural Bangladesh population to identify the trimester-specific incidence of HEV infection, rates of illness, and risk factors for infection and hepatitis E disease and 2) recruit and follow pregnant women hospitalized with acute HEV infection and/or disease to characterize the immunologic, nutritional and other host factors associated with various treatment outcomes, including vertical transmission and neonatal survival. We will also isolate and sequence HEV from acutely ill women, for insight into the molecular epidemiology of HEV. We have over a decade of HEV research experience in this endemic population partnering with the ICDDR,B's Matlab Research Center, and we have estimated, in a smaller study, an incidence of HEV in pregnancy of ~40 per 1000 person-years. Diagnostic support will be provided by one of the most reputable HEV reference laboratories: Dr. Robert Purcell's Hepatitis Laboratory at the National Institutes of Health (Bethesda, Maryland). Co-investigators with expertise in immunology and access to the Johns Hopkins Becton Dickinson Immune Function Laboratory further strengthen this applications. The proposed project will be the first to quantify and characterize HEV infections in a large cohort of rigorously monitored pregnant women, and provide precise estimates of the burden and consequences of HEV in pregnancy and to the neonate. We will clarify the burden of this under recognized contributor to maternal mortality and morbidity in endemic areas, based on large population- based data, providing important information for future prevention and vaccine efforts."}
{"text": "The goal of this proposal is to increase the understanding of the mechanisms contributing the exacerbation of neurological complications in Human Immunodeficiency Virus (HIV) infected drug abusers. The use of dopaminergic drugs, such as cocaine and methamphetamine, increases both the incidence and severity of HIV-induced neurological disease in HIV infected individuals. These drugs act by increasing extracellular dopamine levels in the central nervous system (CMS). Studies show that increased extracellular dopamine exacerbates HIV induced CMS pathology, but the mechanisms that mediate this effect are not fully characterized. Macrophages, the major target for HIV in the CMS, also play a central role in the neuropathology of HIV infection. The preliminary data in this proposal show that dopamine treatment increases HIV replication in macrophages, and demonstrate the presence of dopamine receptors on the surface of the eels. These data indicate that dopamine may play a direct role in the development of HIV induced CMS pathology through dopamine mediated modulation of HIV infection of macropahges. To determine the mechanism mediating this effect, studies in this proposal will fully characterize the pharmacology and kinetics of dopamine-mediated increase in HIV replication in macrophages. HIV infection in the presence of dopamine receptor agonists and antagonists will determine which dopamine receptors mediate the increase in HIV replication in macrophages. To examine the mechanism mediating this effect, studies will examine crosstalk between the active dopamine receptors and the HIV receptor CD4, and coreceptors CXCR4 and CCR5 as well as dopamine-induced alterations in the viral entry, integration, and budding processes. Experiments will also use protein kinase inhibitors during HIV infection to examine the signaling pathways involved in dopamine enhanced HIV replication. Defining the mechanisms by which dopamine increases HIV replication in macrophages will contribute to the understanding and treatment of the heightened incidence and severity of neurological disease among HIV infected drug users. PUBLIC HEALTH RELEVANCE: HIV infection can result in a variety of neurological complications that are exacerbated by drugs of abuse. Defining the mechanisms by which drug abuse increases the incidence and severity of HIV induced neurological complications will increase the understanding of HIV infection in the brain and help to develop intervention strategies to limit the devastating consequences of neurologic impairment in HIV infected drug users."}
{"text": "Approximately 470,000 Americans have end-stage renal disease and most are treated with hemodialysis. A vascular access is required for performing the hemodialysis procedure and can be provided with a native arteriovenous (AV) fistula, a synthetic AV graft;or a central venous catheter. The AV fistula is the preferred type of vascular access because thrombosis rates, infection rates, access-related expenditures, and total healthcare expenditures all are lower for patients with fistulas than for those with either synthetic AV grafts or central venous catheters. However, the advantages of fistulas are counterbalanced by the substantially higher proportion of fistulas than grafts that are never able to be used for dialysis because of failure to mature adequately to support effective hemodialysis. Maturation failure is the major barrier to increasing fistula prevalence, and, for many patients, leads to multiple surgical procedures or prolonged use of central venous catheters, the least desirable type of vascularaccess. Recent studies suggest that 20-50% of new fistulas fail to mature. This application is a response to an NIDDK RFA to establish a consortium for designing and performing an observational cohort study of patients with new fistulas. We are applying to be one of the Participating Clinical Centers of the Consortium. The overall objective of this application is to identify determinants of fistula maturation outcomes in order to enable early identification of failing fistulas, elucidate mechanisms underlying fistula maturation, and identify potential targets for maturation-enhancing interventions. Our application has five specific aims. These include 1) determining the utility of ultrasound as a method for early identification of fistulas that are failing to mature, 2) evaluating the impact of pre-existing vascular function on fistula maturation outcomes, 3) identifying surgical factors that are associated with fistula maturation outcomes, 4) creating evidence-based criteria for fistula maturation, and 5) characterizing the clinical consequences of fistula maturation failure. These aims assume an enrollment of 75-100 subjects over two years at our site for an overall study cohort of 600 subjects. Several of our aims include sub-studies that will be performed in a subset of the subjects to provide additional mechanistic information about the fistula maturation process. Our established research focus and expertise in hemodialysis vascular access, our success and leadership as a Clinical Center for the NIDDK Dialysis Access Consortium (DAC), the multidisciplinary expertise of the collaborators for this application, and the resources available at Boston University make us ideally suited to serve as a Participating Clinical Center for this NIDDK initiative."}
{"text": "Hyperbilirubinemia is probably the most frequently diagnosed and treated condition in the human newborn. Treatment is aimed at preventing the entry of bilirubin into the brain because of the risk of permanent neurologic damage. The mode by which bilirubin enters the brain, its metabolic fate after entry, and the sites of toxic action are unknown. Using osmotic stress, the blood-brain barrier was opened in rats, permitting entry of bilirubin. However, in this acute model, the bilirubin was rapidly cleared, with a half-time of l.6 hours. To provide chronic exposure, especially in newborn animals, we are now studying two inbred strains of rats which lack glucuronyl transferase."}
{"text": "This research project aims to investigate a part of the neural control pathway for reach to grasp movements in cats. These movements are an essential part of many behaviors including feeding and defense. A reach to grasp movement was most likely utilized to pick up this document. When the movement fails, life changes drastically. To repair a deficit, an understanding of the basic neural control of the movement is necessary. The proposed research will contribute to this understanding. One of the fundamental aspects of the neural control of a movement is the organization of its control pathway in the nervous system. What areas of the brain control the movement? How do the control signals change as they progress through the brain? How is the control of the movement organized within the brain structures involved? Dr. Gibson's laboratory has concentrated on the control of the reach to grasp movement. His work has identified a pathway which appears dedicated to the control of the grasping aspect of the reach to grasp movement. This pathway includes the interpositus nucleus in the deep cerebellar nuclei and the magnocellular red nucleus. Neurons in these areas discharge sharply only to reach movements with a grasp. A grasp pathway suggests that there is a reach pathway. Recently, work in Dr. Gibson's laboratory has identified an area of the mesencephalic reticular formation where neurons discharge for reaching during the reach to grasp movement. Preliminary anatomical data from the lab suggests that the dentate nucleus projects to the reach related area of the mesencephalic reticular formation. The proposed research will first investigate the discharge from neurons in the dentate nucleus during reach to grasp movements. Next, the contribution these neural areas make to the reach to grasp will be studied through inactivation experiments of the dentate nucleus and the mesencephalic reticular formation. The deficits from the inactivation of each of the areas will be compared."}
{"text": "ABSTRACT Spinal cord diseases and disorders such as spinal cord injury (SCI) and amyotrophic lateral sclerosis (ALS) are debilitating, often resulting in loss of mobility and decreased quality of life for those affected. One promising treatment involves the transplantation of neural progenitor cells (NPC) into the spinal cord, which has been shown to have neuroprotective properties. However, most NPC-based therapies fail after reaching clinical trials, and without a method to monitor the injected cells and viability, locating injected NPCs is limited to postmortem histology. The ability to track and assess the viability of transplanted cells could be crucial in understanding whether the failure is technical or biological in nature, such as whether the injected cells were delivered to and remain at the correct location or survived transplantation. We propose the development of multi-modal contrast agents and a clinical ultrasound and photoacoustic (US/PA) imaging system for image guided delivery of trans- planted cells and longitudinal monitoring. Together with clinical magnetic resonance imaging (MRI), the use of US/PA imaging can allow for pre-, intra- and post-operative visualization of the cells. The MRI and US imaging modalities are well established in the clinic, and PA can be easily integrated with existing clinical US imaging systems. We hypothesize that visualizing the location and viability of injected neural progenitor cells obtained through the labeling of NPCs with contrast agents will allow for better understanding of transplanted cell behavior and improved translation of cell based therapies. Two contrast agents will be developed: photo-magnetic nano- particles, which will allow for photoacoustic and MRI tracking, and a dye-based apoptosis reporter, which will provide photoacoustic contrast upon apoptotic activity. These will be delivered to the cytosol of the neural pro- genitor cells and assessed for labeling efficiency and toxicity. Once optimized, labeled NPCs in different ratios of live to dead will be injected into the spinal cords of rats to assess the feasibility of distinguishing live and dead cells in vivo. After injection, the NPCs will be monitored longitudinally using trimodal US/PA/MR imaging. After validating the performance of the contrast agents in vivo, a clinical US/PA imaging system will be developed to demonstrate real-time image guided-delivery, US/PA/MRI longitudinal tracking, and PA viability assessment of the labeled cells. The ability to monitor the transplanted cells at every point during and after the procedure will allow for more accurate delivery of the cells and could elucidate common issues and behaviors of injected NPCs that could lead to therapeutic improvements. Furthermore, if successful, it will validate both the contrast agents and US/PA as a valuable tool for tracking and monitoring cell-based therapies to improve clinical translation."}
{"text": "This proposal seeks to understand in detail the role of alpha-lactalbumin in lactose biosynthesis (\"lactose synthetase\"). Alpha-lactalbumin, a common milk protein both plays an important regulatory role and is itself subject to a fine regulation at the levels of organ specificity, cellular structure and hormonal control of both its biosynthesis and secretion. In more detail this work is directed at examining structure, conformation and changes therein associated with galactosyl transferase function at the molecular level. This research utilizes the extremely powerful techniques of magnetic resonance to investigate the interactions of both substrates and the two proteins (galactosyl transferase and alpha-lactalbumin) involved in the biosynthesis of lactose. BIBLIOGRAPHIC REFERENCE: Lawrence J. Berliner and Shan S. Wong, \"Mn(II) and Spin Labeled-UDP Binding to Bovine Galactosyl Transferase,\" Biochemistry 14, 4977-4982 (1975). Lawrence J. Berliner and J. Grunwald, \"Immobilized Bovine Lactose Synthetase: A Method of Topographical Analysis of the Active Site.\" FASEB Abstracts 67th ASBC Mtg. 6-10 June 1976 San Francisco, CA."}
{"text": "Despite their prevalence in nature and as drug and therapeutic targets, many membrane proteins continue to evade structure determination by X-ray crystallography and NMR. The combination of site-directed spin labeling with electron paramagnetic resonance (SDSL-EPR) is becoming an increasingly popular method for the structural characterization of membrane proteins due to the relative ease with which they can be studied. However, SDSL-EPR does not yield high-resolution structures directly. The current proposal describes a new method, ROSETTAEPR, to overcome this obstacle. ROSETTAEPR will be a toolkit in which distance and accessibility restraints determined by EPR will be combined with Monte Carlo-based computational methods for the de novo structure prediction of proteins. After developing knowledge-based potentials derived from EPR experimental data, it will be benchmarked on proteins of known structure using both simulated and real EPR data. In addition, ROSETTAEPR and EPR experimental distance and accessibility data will be used to determine the LeuT apo, Na+, and Na+/leucine bound structural intermediates involved in leucine transport. LeuT is a bacterial homolog of the neurotransmitter sodium symporter (NSS) protein family, which includes the dopamine, serotonin, and norepinephrine transporters. While there are no high-resolution structures of the NSS transporters, extracellular-facing substrate-bound conformations of LeuT have been determined by X-ray crystallography. However, the current structures are static snapshots of the LeuT transport cycle; furthermore, they are believed to have been captured in a potentially inhibited form. Therefore, EPR spectroscopy has been employed to shed light on the dynamics of the protein. It was found that Na+ binding causes an increase in protein flexibility in the extracellular loops and hydration of the substrate permeation pathway, while subsequent binding of leucine causes the extracellular vestibule to close and become rigid. ROSETTAEPR will allow for the high-resolution structural elucidation of these intermediates based on low-resolution EPR data. PUBLIC HEALTH RELEVANCE: The dysfunction of neurotransmitter sodium symporter (NSS) proteins is a common characteristic of central nervous system (CNS) diseases, such as depression, anxiety, obsessive compulsive disorder (OCD), and epilepsy. Understanding how these proteins function on a structural level will aid in the development of new, more effective therapeutic agents that specifically target neural processes underlying mood, reward, and locomotion."}
{"text": "Ischemic preconditioning (IPC) refers to a phenomenon in which a tissue is rendered resistant to the deleterious effects of prolonged ischemia and reperfusion (I/R) by prior exposure to brief periods of vascular occlusion. Although first described over a decade ago, the mechanisms underlying the beneficial effects of IPC remain uncertain. However, preliminary data from our laboratory indicates that IPC prevents I/R-induced intestinal oxidant production, leukocyte/endothelial cell interactions, and venular protein leakage. Since the production of reactive oxygen metabolites from xanthine oxidase and other sources during reperfusion of ischemic intestine initiates the formation of chemotactic stimuli, induces the expression of endothelial cell adhesion molecules, and reduces intestinal levels of the potent antiadhesive agent nitric oxide, we hypothesize that IPC-induced reductions in leukocyte adhesion are mediated by the effect of preconditioning to reduce the postischemic oxidant production. To address this central postulate, four specific aims are proposed. One aim is to determine whether IPC prevents the increase in mesenteric xanthine oxidase activity or influence of IPC on the elaboration of proinflammatory stimuli (platelet activating factor (PAF), leukotriene B4(LTB4), complement activation) in postischemic small bowel. A third specific aim is to determine whether IPC attenuates the expression of P-selectin by the endothelium in postcapillary venules exposed to I/R. Since the beneficial effects of IPC on postischemic mesenteric microvascular dysfunction are reversed by topical application of adenosine deaminase during IPC or protein kinase C (PKC) inhibitors during the period of prolonged ischemia, fa fourth aim is directed at elucidating the mechanisms that may be involved in coupling adenosine production during IPC to the postischemic reductions in oxidant production, leukocyte adhesion, and venular protein leakage. To accomplish these aims, we will utilize intravital microscopic approaches to quantitate oxidant production, leukocyte/endothelial cell interactions, and venular protein leakage in rat mesentery. Mesenteric xanthine oxidase, nitric oxide synthase, phospholipase C, and PKC activities, nitrite/nitrate production, PAF and LTB4 levels, and the extent of complement activation will also be assessed. In vivo mesenteric P- selectin expression will be quantitated using a dual-radiolabeled monoclonal antibody approach. The proposed studies should not only substantially improve our understanding of the mechanisms whereby IPC reduces postischemic microvascular dysfunction in the intestine but should also provide a rationale for the development of novel therapeutic interventions in I/R that exploit the mediators of the preconditioning phenomenon."}
{"text": "The lab has previously shown that adipsin, a mouse adipocyte-derived serine protease, is secreted into the bloodstream and is markedly deficient in several models of genetic and acquired obesity, suggesting a possible functional role for this protein in the systemic or local regulations of energy balance. The overall goal of the proposed work is two-fold: to understand the role of adipsin in adipose physiology and obesity and to use the adipsin gene as a model to understand how obesity genes regulate the function of other gene promoters. Both of these goals are novel and will increase our understanding of the biochemical and genetic basis of obesity. Adipsin's direct role in the physiology of adipose cells will be approached in vitro by treating fat cells with adipsin and measuring changes in lipogenesis and lipolysis. Since adipsin activates the alternative pathway of complement via its intrinsic Factor D catalytic activity, the ability of various components of the alternative complement pathway to regulate adipose cell physiology will be studied, including complement-derived ligands such as C3a, C5a and Ba. In addition to an in vitro approach, adipsin will be administered acutely and chronically to obese and lean animals to ascertain the role of this protein and the alternative complement pathway in systemic energy balance in vivo. Multiple measures of systemic energy balance and hormonal regulation will be measured. Preliminary pharmacokinetic studies in lean and obese mice support the feasibility of altering adipsin levels in vivo by administration of the pure protein. New data in transgenic mice shows that a rodent obesity gene (db) suppresses adipsin expression through its action (direct or indirect) on the adipsin promoter. Complementary cellular and biochemical approaches will be used to study this pathway of signal transduction and identify the cis- and trans-acting factors involved in the \"obesity-response element\". Cultured adipocytes will be treated with serum from lean and obese mice and with various hormones to model the in vivo effects of obesity on the endogenous adipsin gene. By transfection of chimeric constructions and a series of deletions and mutations from the adipsin gene promoter into these cells, sequences involved in a putative \"obesity-response element\" will be mapped. A biochemical approach will involve mapping this response element in the adipsin promoter via DNA footprinting (and gelshift) analysis of the adipsin promoter DNA using nuclear extracts from lean and obese animals. The identity of putative \"obesity-response elements\" will be critically tested in vivo using point mutagenesis in the adipsin promoter and the construction of transgenic, obese mice. The trans-acting factor(s) binding to this response element and ultimately responsible for aberrant adipsin expression in obesity will be cloned and characterized by biochemical and new molecular genetic techniques."}
{"text": "Multiple lines of evidence have suggested that viral infection may play a pathogenetic role in biliary atresia (BA). However, searches for putative viral pathogens have been restricted to a small number of selected viruses and have not focused on virus-specific immune responses. The hypothesis underlying this proposal is that viral infection plays an etiopathologic role in biliary atresia. The purpose of the present proposal is three-fold: 1) Establish a Clinical Biliary Atresia Center. Five referral centers including the Johns Hopkins Pediatric Liver Center will recruit 10-20 new BA patients/year to add to the 73 BA patients in the present database. The database will gather demographic and clinical information as well as biological specimens pertinent to the viral infection hypothesis. 2) BA Peptide Project, (3-years) will employ a powerful new technique, bacteriophage peptide display, to search for novel antigenic epitopes specific to BA. BA sera will be utilized to \"biopan\" random peptide libraries to identify epitopes not recognized by control sera. One patient/control group from each of the Clinical Centers will be studied, for the greatest possible geographic, seasonal, and ethnic diversity. \"BA peptides\" will be sequenced and synthesized. Peptides will be used to immunoaffinity purify the corresponding monospecific antibodies from BA sera. Protein databases will be searched for homologous viral proteins. To determine if the BA peptides exhibit cross reactivity as HLA-A2+ restricted T cell epitopes, the peptides will be incubated with dendritic cells, antigen-presenting cells (APC) harvested from HLA-A2+ maternal peripheral blood lymphocytes (PBL). Maternal PBL will be incubated with the peptide-pulsed APC to identify peptide-specific CTL using target cell lysis assays. 3) The BA-Dimer Project, (1-year) will employ a sensitive new \"dimer\" technique to determine if HLA-A2+ biological mothers of BA (MBA) patients exhibit unique peptide-specific CTL. The dimer technique uses immunoglobulin as a molecular scaffold to produce a divalent peptide-HLA-A2-Ig complex. Dimers will be loaded with BA or homologous viral peptides and PBL from MBA and control mothers from each Clinical Center will be tested for dimer reactivity using FACS analysis. Novel BA-specific peptides, monospecific antibodies, and BA-dimers could be used for early diagnosis, identification of cellular targets and intrahepatic T cells."}
{"text": "We propose to continue a nationally visible and responsive center focused on the development of novel fluorescent biosensor and detection technologies for investigating pathways and networks in real time and high spatial resolution in living cells. The renewed center retains the combined experience and infrastructure of two existing centers in Pittsburgh: the Molecular Biosensors and Imaging Center at Carnegie Mellon University and the Center for Biologic Imaging at the University of Pittsburgh. The Technology Development (Core 1) component of the center will create a powerful toolbox of intracellular fluorescent biosensors and reporters that can be used to study many, if not all, of the pathway proteins and activities in living cells. This fluorescent biosensor development program integrates efforts across multiple disciplines, including dye chemistry, molecular biology, biochemistry, structural biology, modeling, cell biology, image acquisition and analysis, and high throughput screening. We have made substantial progress in this effort during the previous 3 years of funding. Four exciting Driving Biology Projects (Core 2) are essential to the technology development effort in Core 1. These DBPs are focused on important and currently un-addressable biological problems, and will serve both as test-beds for the technology and compelling demonstrations of the value of the biosensors and reporters developed in this program. The Infrastructure (Core 3) is provided through the Center for Biologic Imaging at the University of Pittsburgh. The role of the CBI is to act as the application and outreach ami of the project as a whole by testing the new biosensors with challenging biological problems. During the last cycle of this proposal the clear mission of the CBI evolved to become the catalyst in probe implementation, and to strengthen and broaden the impact of the new probes developed by MBIC. This is achieved by providing facilities and expertise to test and validate the probes in the context of the driving biological projects, and ultimately, the biomedical research community at large. The program contains a significant technology transfer component to disseminate concepts, knowledge, software, materials, and resources to users in both academic research labs and industry. This is achieved in our Infrastructure, Training, and Dissemination cores (Core 3,4, 5) and through the technology transfer activities in the Management core (Core 6)."}
{"text": "Oxidants generated by activated white blood cells are critical to host defenses against microorganisms. However, overproduction of reactive species can damage host tissue. Indeed, white blood cells represent the cellular hallmark of inflammation, and oxidants have been implicated in tissue injury in inflammatory diseases ranging from atherosclerosis to neurodegenerative disorders to cancer.We have investigated four phagocyte-dependent pathways that oxidatively damage proteins in vitro. The pathways and their characteristic products are: myeloperoxidase and 3-chlorotyrosine; tyrosyl radical and o,o-dityrosine; hydroxyl radical and ortho-tyrosine; and reactive nitrogen species and 3-nitrotyrosine.Using two clinically relevant models of inflammation, we will study genetically engineered mice whose phagocytes are unable to produce specific oxidants. In the proposed research, we will ask three related questions. First, we will identify the pathways that generate chlorotyrosine, dityrosine, ortho-tyrosine, and nitrotyrosine in viva. The experiments will reveal whether a genetic deficiency of any of these oxidant-generating systems inhibits production of any of the chemical markers, thereby determining which pathway generates a particular marker in viva.Second, we will determine whether tissue, plasma, and urinary levels of the oxidized amino acids change in parallel. We will also investigate the absorption, metabolism, and urinary excretion of the oxidized amino acids. These experiments will determine whether plasma and urinary levels of these well-characterized products can be used as noninvasive markers of oxidative stress.Third, we plan to determine whether two proposed antioxidants-vitamin C or vitamin E- inhibit oxidative stress in our models of inflammation. Collectively, the proposed experiments will identify the oxidative pathways that cause phagocytes to damage tissues and will test the hypothesis that levels of oxidized amino acids in urine and plasma indicate levels of oxidative stress in vivo."}
{"text": "This integrated basic and clinical research program is directed at development of more effective therapy for malignant disease. The disciplines include pharmacology, both new drug and new drug usage principles, cell biology, transplantation biology, cellular kinetics and cell physiology. The specific research projects are: 1) Biometry Consultation Group; 2) Pharmacokinetically Designed Cancer Chemotherapy; 3) Synthesis and Biological Evaluation of 1-Aryl-3, 3-Dimethyltriazenes; 4) Prediction of Clinical Response to Arabinosyl-cytosine (Ara-C); 5) Study of Arabinosyladenine Toxicity In Vitro and In Vivo; 6) Detection of Proliferative Potential of Human Bone Marrow and the Prediction of Response and Relapse in Leukemia; 7) Cell Surface Antigens and Structure in Cancer Immunology; 8) Isolation and Characterization of Tumor-Associated Antigens Cross-reactive with BCG; 9) Clinical and Laboratory Studies on Mitogen-Induced Proliferation in Human Leukemic Cells; 10) Cellular Response to Antineoplastic Agents; 11) Cellular Chemotherapy and Kinetics."}
{"text": "This project is concerned with new understanding of the effects of molecular structure on fundamental parameters of hydrogen-bonding and the relationship of these parameters to acid-base phenomena generally. Both a physical picture of, and the energetics for, the specific solvation of organic solutes (both neutral and charged) with highly localized charges are being approached using models involving discrete equilibria for successive hydrogen bond attachments of individual solvent molecules. These equilibria are being determined in the gas phase using ion cyclotron resonance spectroscopic methods and in the condensed phase using nuclear magnetic resonance spectroscopic methods. Analysis of results by methods of linear free energy relationships and by ab initio molecular orbital calculations provides an effective complimentary approach."}
{"text": "Early nutrition can have far-reaching effects on behavior: emerging research has revealed the existence of sensitive periods during postnatal development in which the developing brain has heightened sensitivity to environmental influences, including flavors of foods. While children's innate taste likes and dislikes create obstacles to healthy eating, research in humans and other animals has revealed that early experience can affect these likes and dislikes. The challenge is to determine whether there are optimum times when experience promotes greater liking of the flavors of healthy foods. Our previous experiments have shown that, as with other mammals, what a nursing mother eats during the first months of lactation can influence her child's food preferences. We propose to conduct a detailed randomized within- and between-subject study of women and their infants during a 16-month window, varying the timing and duration of exposure to three vegetable flavors (carrot, beet, and cabbage) to determine effects on the acceptance of these flavors by children, their mothers' own liking of these foods, and the likelihood that mothers will feed these foods to their children after weaning. Based on solid, previous research in humans and other animals, the over-arching goals of the proposed research are to specify the timing (Specific Aim 1) and consequences (Specific Aim 2) of the sensitive period for flavor learning in infants via flavor variety in mother's milk, and to determine the effects of reproductive state (Specific Aim 3) and genetic variation (Specific Aim 4) on vegetable liking in newly parturient mothers and their children. The hypotheses behind this research, examining highly pervasive and under-investigated behavioral phenomena surrounding how to introduce vegetables to children's diets, focus on early life, unlike many other studies that attempt to modify food habits in older children, and focus on the maternal-infant dyad because mothers' food consumption and feeding choices during the sensitive period may have far-reaching consequences for children's food exposures and choices later in life. Knowledge regarding the most efficacious time mothers should eat foods to develop preferences can lead to intervention strategies for both infants and their mothers, and consequently their families. Furthermore, knowledge about the timing of a sensitive period for learning about flavors in breast milk will shed light on long-term effects of not being exposed to these flavors early in life. The knowledge gleaned from this basic behavioral work could be translated to effective behavioral interventions to promote vegetable acceptance that are effective and implemented widely. PUBLIC HEALTH RELEVANCE: Early experience modifies children's innate taste likes and dislikes, and promotes healthy eating behaviors. We propose a randomized trial to specify the timing and effects of the sensitive period for flavor learning in infants via flavor variety in mother's milk, and the effects of genetic variation and the mother's reproductive state on vegetable liking. This knowledge may lead to strategies for healthy eating interventions for infants, mothers, and families."}
{"text": "Project Summary Ion channels are membrane proteins that selectively regulate the movement of ions across cellular membranes. Ion channels play a role in many pathophysiological states, such as pain, epilepsy, metabolic disorders, obesity, diabetes, cardiac arrhythmias and renal disease. Studying the role of ion channels in disease is often slow because traditional patch clamp electrophysiology, although precise, is also a low throughput technique that can only be done by researchers who have specialized training and equipment. This bottleneck restricts the number of ion channel research problems that can be addressed by any lab or institution, compared to other drug targets, such as the G-protein-coupled receptors (GPCRs). Recent advances in ion channel technologies have demonstrated that studying ion channels can be done in high- throughput (HT). The first instrument that was specifically designed to study ion channels in HT was IonWorks HT. IonWorks HT was breakthrough technology because it enabled the study of ion channels in a 384-well ion channel instrument for the first time. The current market leader in HT electrophysiology (HT ephys) is Nanion. Nanion's SyncroPatch 384PE can record 384 cells in parallel, which can generate 20,000 data points per day. This gigaseal HT ephys instrument can measure the physiology and pharmacology of all classes of ion channels, including rapidly sensitizing ones such as alpha 7 nicotinic receptors. The Center for Chemical Genomics (CCG) is the well established home of HT biology and pharmacology at the University of Michigan (U-M), and has conducted over 200 high throughput screens over the last decade. The CCG's research team collectively has eight decades of pharmaceutical experience which provides them extensive knowledge of best practices in HTS assay development, validation, execution and informatics, and has the expertise to implement HT ion channel screening. However, there is currently no HT ephys instrument at U-M. Adding this highly scalable technology to the CCG, will allow the full exploration and exploitation of ion channel research and ion channel drug discovery at U-M and our collaborating partners."}
{"text": "This proposal seeks financial support for travel to Copenhagen. Denmark for the purpose of negotiating with the staff of the UCLA/University of Copenhagen Joint Center for Studies of Health Programs and the Danish National Committee for Health Information relative to the possible involvement of the Health Services Research Center of the University of North Carolina at Chapel Hill in the Danish National Health Education Project with regard to the evaluation of the Project's impact on the health promotive behaviors of the Danish people."}
{"text": "This project extends its previous work on excitation-contraction (E-C) coupling and relaxation (R) coupling in skeletal muscles of the frog and mammals to determine the mechanisms of these processess and their interactions that determine the flux of activator Ca2 ion regulating the contraction-relaxation cycle of the twitch and the tetanus. The studies of E-C coupling which depended previously on general electromechanical correlations in the twitch, now concentrate on the electromechanical \"transforms\" which specifically distinguish between the mechanical effects of the rising and falling phases of the spike, and which are used to determine possible mechanisms by which the mechanical threshold and the duration of the action potential modulate E-C coupling and thus vary the release of activator Ca2 ion and the associated active state. It is planned to determine the role in E-C coupling of Ca in T tubule action and to test further whether the latency relaxation is a sign of the release of Ca2 ion by the SR, this latter especially by D2O which, our work shows, greatly slows development of the LR. The role of the T tubules in generating the action potential, and especially their possible role in causing delayed rectification, will be investigated by studying action potentials and membrane current/voltage relations of detubulated fibers, which are treated with various agents that alter the rate of rise and fall of the spike. A new active state theory will be used for tracing the course of twitch and tetanus activity in relation to the flux of Ca2 ion as indicated by the E-C coupling electromechanical transforms, and by possible manifestations of R coupling and of regenerative Ca-release. The general procedures will be used to determine E-C and R coupling and active state course in normal and denervated slow and fast mammalian muscles. And certain special studies are planned to determine whether mouse dystrophy originates in a neurogenic or myogenic pathology."}
{"text": "Artemis has been shown recently to be involved in a human severe combined immunodeficiency syndrome (SCID) in which an absence of development of T and B cells is observed. This defect is due to a requirement for Artemis in the coding joint formation step of V(D)J recombination. In addition, patient cell lines were shown to be hypersensitive to ionizing radiation suggesting that Artemis is also involved in cellular responses to DNA damage. The goals of this application are to characterize the function of Artemis in regard to its role in mediating the cellular response to DNA damage. Specifically, we will determine the nature of proteins that Artemis interacts with, and examine its cellular localization before and exposure to genotoxic agents. Our findings show that Artemis is rapidly phosphorylated upon exposure of cells to DNA damage including that induced by both IR and UV. We have also shown that this phosphorylation is mediated by the PI3 kinases DNA-PK, ATM, and ATR. We will characterize this phosphorylation of Artemis and prepare phosphospecific antibodies that will be utilized for functional studies of Artemis. These functional studies will be conducted on cells defective in Artemis to determine its role in either DNA repair and/or cell cycle checkpoint pathways. It is expected that these studies will elucidate the function of Artemis in DNA damage response pathways, and thus provide an explanation for the radiosensitivity of the RS-SCID syndrome. Finally, we will prepare a knock-in mutant of Artemis in the mouse that is mutated at sites of phosphorylation mediated by ATM. This experiment is designed to determine if the modification of Artemis by ATM is involved in the immunodeficiency observed in AT patients."}
{"text": "Project Summary Transcriptional repressors represent the necessary counterweight to transcriptional activators in metazoan development. The mechanisms of transcriptional repression have been intensively investigated, but we lack crucial insights into how repressors act at a mechanistic level across many targets in the genome. In particular, the coordinated recruiting of transcriptional co-repressors can generate diverse effects on chromatin structure and modification, and we still lack insights on the functional significance of many changes that can be measured in `omics studies. To develop key insights into eukaryotic transcriptional regulatory mechanisms, we use the natural setting of the Drosophila embryo to identify basic biochemical processes in a developmental setting, where differential gene expression is used to drive the developmental fate of particular cells and tissues. In this proposal, 1) we will use genome-wide methods developed in our laboratory to identify direct biochemical, chromatin-based processes that are directed by a set of five endogenous transcriptional regulators that repress through short-range and long-range mechanisms. 2) We will study the in vivo activity of wild-type and mutant repression complexes to identify the contributions of distinct transcriptional co-repressors Groucho and CtBP, testing their contributions to quantitative and/or qualitative effects in chromatin modifications and gene regulation. 3) To identify the cis-regulatory context in which transcriptional repressors act on different enhancers, we will quantitatively measure and mathematically model the output of specific enhancers to uncover the fundamental quantitative properties of specific classes of repressors interacting with activators ? i.e. the common ?rules? by which these proteins interact on many target genes. The three interrelated aims will provide predictive tools for interpretation of genomic cis regulatory content of the metazoan genome, providing essential underpinnings for studies of evolution and disease in higher eukaryotes."}
{"text": "This proposal seeks STTR funding for the preliminary development of a Web-based screening tool to help elementary school health and mental health service providers quickly and accurately assess students' risk and protective factor status for adolescent substance abuse. Early identification and targeting of known predictors of adolescent substance abuse reduce its prevalence and its negative health, social, and academic consequences. The instrument will seek information from 3rd - 5th graders, and their parents and teachers. It will assess the social environment, individual characteristics, and functioning of children. Internet and computer technology will be used to increase there liability and validity of the child sell-report data, and facilitate the collection and scoring of data from children, parents, and teachers. Aims of Phase I are to: (a) create a preliminary version of the screening tool, (b)develop supplemental administrative and training materials, and (c) pre-test the child report section. Phase I tasks include developing the list of questionnaire items, computer formats, and supplemental materials. Expert consultation and cognitive testing with children will be used to evaluate the quality and feasibility of the tool. In Phase II, the preliminary instrument and administrative procedures will undergo pilot testing and large-scale psychometric testing. PROPOSED COMMERCIAL APPLICATION: The proposed screening tool has commercial applications among health and mental health practitioners working in elementary schools. Taking advantage of schools' existing investment in Internet technology, the tool will make comprehensive risk and protective factor screenings more feasible than existing options. The range of domains assessed, the child self-report component, computer scoring, and rapidly available individual and group results designed to guide intervention planning are unique and meet a market need."}
{"text": "Flavonoids constitute a group of polyphenolic compounds primarily known for their anti-oxidant properties. Studies on flavonoids show their promising use for the prevention and treatment of a variety of cancer, inflammatory and heart diseases. Quercetin is a naturally occurring bioactive flavonoid ubiquitously available in fruits, vegetables and other plants. Several studies have shown the bioactivity of Quercetin against a variety of tumor cells and angiogenesis. However, several other studies have also indicated the carcinogenic potential of Quercetin. Moreover, Quercetin has been described as one of the principal molecules of some toxic plants. Therefore, the conditions under which Quercetin is bioactive as an anti-tumor agent or as a carcinogen are not well known. Understanding how Quercetin and other flavonoids function at the molecular level is of paramount importance to public health because it will help us establish the conditions under which specific bioactive flavonoid compounds may or may not be beneficial for therapeutic purposes. [unreadable] [unreadable] In this study, we propose to determine the mechanisms of Quercetin bioactivity under conditions where specific protein regulators of cell signaling have been ablated. Our specific aims are to examine: (1) what genes are expressed or inhibited by Quercetin; (2) how Quercetin modulates cell survival or cell death through cell signaling regulatory proteins. To address these aims, firstly, we will study the global and differential gene expression profiles of cells, which are intact or null for the p53 protein, by treating them with Quercetin. We will also examine the effect of Quercetin on the expression and function of cell cycle and apoptosis proteins regulated by p53. Secondly, we will analyze the DNA-damage response pathway through examination of the ATM/ATR proteins and downstream events. Thirdly, we will examine the levels and activities of other key cell cycle and apoptosis regulatory proteins after treatment with Quercetin. To examine the role of key proteins identified through the initial treatment experiments, we will use cells knocked out for the proteins or we will knock down the proteins by short interfering RNAs. The results will help us understand if treatment with Quercetin influences cell cycle, apoptosis, and DNA-damage regulatory proteins in cells. In future studies, we will utilize knowledge from these aims to evaluate the effect that Quercetin exerts in tumor cells in vivo. [unreadable] [unreadable] [unreadable]"}
{"text": "We propose to clone the entire I regions from BALB/c and AKR mice. We will identify, sequence and determine the organization of all class II genes with the I region. We will transfer class II genes into mouse L cells and normal bone marrow derived cells by DNA indicated gene transfer. We will study the DNA elements which control the expression of class II genes in the model mouse L cell system and normal hematopoietic cells. The class II genes will be altered by in vitro mutagenesis and their expression and functions studied after appropriate gene transfer experiments."}
{"text": "HPV plays an etiologic role in the development of a distinct subset of oral cancers that arise predominantly from the tonsils. HIV-infected individuals are at increased risk for tonsillar cancer. Oral HPV infection is therefore a newly appreciated public health problem. The objective of this proposal is to perform a comprehensive molecular epidemiologic study to determine the prevalence, risk factors, mechanisms of transmission, and natural history of oral human papillomavirus (HPV) infection in HIV-infected individuals and to identify precursor lesions for HPV-associated head and neck squamous cell carcinomas (HPV associated HNSCC). Tonsillar cancers comprise approximately 15-23% of all oral and oropharyngeal cancers. There was a 2-3% increase in tonsillar cancer incidence per year from 1973-1995 in men in the United States. Concurrent HPV and HIV infection may have contributed to this unexplained increase. Preliminary data suggest that the odds of oral and tonsillar infection by a high-risk HPV type are significantly elevated in HIV infected individuals, and that oral HPV infection is associated with sexual behaviors, severity of immunosuppression and the presence of potentially premalignant oral mucosal lesions. A prospective cohort study of 800 HIV-infected men and women with 36 months of semiannual follow-up is proposed to accomplish the following specific aims: (i) to determine the prevalence and type distribution of oral mucosal HPV infection and to characterize host, environmental (tobacco and alcohol) and biological factors (e.g. CD4 count, HIV viral load) associated with oral HPV infection; (ii) to identify specific sexual practices associated with presence of oral HPV infection after adjustment for confounders; (iii) to evaluate the relationship between high-risk HPVs (presence of HPV genomic DNA, viral load, integration) and tonsillar dysplasia and other oral mucosal abnormalities, and (iv) to determine if tobacco use, immunosuppression, and HPV type (low versus high-risk) affect the incidence and/or persistence of oral HPV infection. This proposal has the potential to yield information important for future efforts at primary prevention and early detection of HPV-HNSCC."}
{"text": "The recent studies indicated that the existing smoking prevention cessation strategies have failed to produce population reductions in teen smoking prevalence. This is despite significant progress in intervention development and increasing financial investments in anti-smoking activities. The investigators believe several possible explanations merit consideration for this paradox. First, effective smoking prevention programs have not been widely disseminated. Second, when adopted, teachers often fail to adhere to program guidelines. Third, teens who smoke are reluctant to identify themselves to seek cessation assistance. Fourth, smoking prevention and cessation programs often fail to account for salient individual differences among students. Finally, most existing smoking prevention and cessation programs do not utilize modern communication technologies and do not meet the demands of today's adolescents. The goal of the proposed project is to design, implement, and evaluate an interactive CD-ROM curriculum aimed at adolescent smoking prevention and cessation intervention. By interactive, we mean assessing each student individually and channeling him or her into the most appropriate intervention. The software will assess student smoking status, stages of change for smoking acquisition and cessation, level of addiction, and symptoms of depression. In this manner, recruitment of smokers is facilitated because both smoking and nonsmoking students will be asked to participate in the program and the intervention they receive will be customized to meet their individual needs. The intervention will also be tailored to gender and ethnicity. By utilizing CD-ROM the investigators hope to facilitate subjects' adherence to intervention protocols and subsequent program dissemination. The investigators hypothesize the intervention will result in: a) lower onset of smoking; b) greater smoking cessation outcomes; and c) impact on variables that mediate the process of change compared to an alternative treatment control group. The study design is an 18-month, group-randomized, controlled trial. Study participants will include students 14-16 years of age (10\" graders at baseline) from 16 Houston area high schools."}
{"text": "During the past decade, researchers have emphasized the contribution of parent-teen relationships to adolescent adjustment and development. A general finding is that the quality of relationship with parents serves to support rather than obstruct the adolescent's growing independence and autonomy. While this finding points to the importance of parent-teen relationships, new questions have emerged. For instance, how is the quality of parent-adolescent relationships best conceptualized and assessed? What are the specific mechanisms that link the quality of the parent-teen relationship to the adolescent's adjustment and ability to function outside the home? How is the teenager's understanding of the parent-teen relationship influenced by his or her cognitive development and how does the teenager's growing cognitive understanding of the relationship influence his or her adjustment and well-being? The proposed project addresses these questions by viewing the parent-teen relationship from the perspective of attachment theory and emotion regulation. First, the study will attempt to further validate the Attachment Interview as an assessment of adolescent attachment security. More specifically, the validity of the Attachment Interview as a measure of individual differences in \"styles of affect regulation\" will be evaluated. Concurrent psychophysiological correlates of the different regulatory strategies identified in the interview will be examined as measures of inhibition and somatic activity. Second, measures of emotion regulation and attachment in the mother-teen relationship will be developed and the extent to which the ability to regulate negative affect in the Attachment interview generalizes to successful emotion modulation during parent-ten interaction will be tested. It is proposed that teenagers who are secure or who have the ability to modulate negative affect in the context of the Attachment interview will demonstrate more optimal patterns of interactions with parents in problem-solving and confiding tasks. Finally, the implications of attachment and emotion regulation for adolescents' ego-resiliency and well-being will be considered."}
{"text": "A group of current biomedical research projects which have a strong need for modern electron paramagnetic resonance equipment are outlined. These projects include studies of dipolar and exchange coupled binuclear cooper models for Type 3 biological copper, a detailed examination of the effects of ligand geometries and types on the EPR parameters of copper and manganese systems, investigations of superoxide dismutation and autoxidation mechanisms, vanadyl as a spectral probe of protein interactions and as an EPR-active phosphate analogue, and nitroxide spin-label probes of transketolase mechanism, lipid analogues, cooperative ligand binding in ferrihemoglobin, and details of cytochrome P450 action. A sophisticated EPR facility is described which will allow all of the projects presented herein to be carried out and will provide current generation EPR capability for the scientific community in northern Ohio and the surrounding area."}
{"text": "Neurodegenerative diseases are characterized by the progressive and relentless loss of neurons. One strategy to prevent or slow down neurodegeneration is to stimulate molecules that have neuroprotective activity. Using tissue culture systems we have demonstrated that FoxG1, a member of the Forkhead family of transcription factors, is necessary for the maintenance of neuronal survival. Furthermore, elevated expression of FoxG1 has strong neuroprotective effects. We propose to extend our studies in vivo and predict that elevated levels of FoxG1 will be also be protective in animal models of neurodegenerative disease. We propose to test this prediction by generating transgenic mice that express elevated levels of FoxG1 selectively in neurons. These mice will be crossed with two different mouse models of neurodegenerative disease and the effect on progression of neuropathology evaluated. The two specific aims of this proposal are - Aim 1: To generate transgenic mice overexpressing wild-type FoxG1, a constitutively-active form of FoxG1, and a dominant-negative form of FoxG1. We will use the mouse prion protein (mPrP) promoter to drive expression of the three forms of FoxG1 in mice. Aim 2: Analysis of the effect of FoxG1 overexpression on the brain and in mouse models of neurodegeneration. We will examine the effect of increasing FoxG1 activity, through expression of WT and CA FoxG1, on neuronal survival in the normal brain. In a second part of this aim we will examine whether elevated FoxG1 activity protects mice against neurodegeneration. We will cross FoxG1-overexpressing transgenic mice with R6/2 mice (a model of HD) and with p25/CDK5 inducible-transgenic mice (a model of AD). The project will result in the development of three new mouse lines expressing wild-type and mutant FoxG1 transgenic mice. FoxG1 transgenic mice do not currently exist. We feel that generating these mice and testing whether elevated FoxG1 can protect against neurodegenerative disease will shed insight into the function of FoxG1 in postmitotic neurons in vivo and could identify it as a target for the development of novel therapeutic strategies for neurodegenerative disorders. PUBLIC HEALTH RELEVANCE: Previous work in our laboratory has demonstrated that elevated activity of the FoxG1 protein protects neurons against death. The goal of our project is to extend this observation in vivo by generating transgenic mice that express higher and lower activity of FoxG1. These mice will then be bred with existing models of neurodegenerative disease."}
{"text": "The proposed investigation focuses on the mechanism by which the strongly anticoagulant phospholipases from cobra venoms interfere with hemostasis. A hypothesis that strong anticoagulant phospholipases found in cobra venoms have a non-catalytic mechanism will be tested by incubation of the strongest anticoagulant phospholipase with tissue factor deprived of lipid and reconstituted with non-cleavable phospholipid. The basic anticoagulant phospholipase from Naja nigricollis venom will be chemically and proteolytically modified in an attempt to eliminate catalytic activity while retaining the anticoagulant effect. The enzyme will also be cleavedto obtain peptides which will be tested for anticoagulation. All three phospholipase isoenzymes from N. nigricollis will be sequenced to tru to identify cationic and external hydrophobic domains which can contribute to the anticoagulant effect. Reversibility of the anitcoagulant effect with antibodies specific to the anitcoagulant phospholipase will be examined. The classes of phospholipids cleaved and the kinetics of their cleavage from tissue factor and platelets by the three phopholipase isoenzymes will be examined. The lipid analyses will be extended further by the identificaion of fatty acids released during enzyme treatments. All lipid analyses will be correlated with functional studies of coagulation and/or platelet aggregation. These studies should prove insights into the mechanism of action of the strong anticoagulant phospholipases. They should also give new information about structural requirements of lipids critical to the coagulation process and platelet aggregation. Such anticoagulant enzymes might ultimately have clinical applications and should prove to be valuable research tools for future studies on hemostasis, thrombosis and their regulation."}
{"text": "The purpose of studies is to elucidate the role of female reproductive hormones relaxin, estrogen & progesterone in the etiopathogenesis of TMJ disease in women. We plan to determine association of relaxin, b-estradiol & progesterone with symptoms of TMJ disease in women using a case control study design. We also will determine if salivary levels of relaxin, b-estradiol & progesterone correlate with serum levels these hormones. We will assess if changes in relaxin, b-estradiol & progesterone over short period of time during pregnancy predispose to TMJ disease. We will evaluate, by a longitudinal prospective study design, whether aberrations in levels of relaxin, b-estradiol & progesterone during puberty and early adulthood are assoicated with the onset of TMJ disease."}
{"text": "The proposed research brings novel data to bear on the emergence of racial and socioeconomic disparities in health over the life course. We examine the intersection and mutual influence of three factors socioeconomic status (SES), cognitive ability, and health itself. Extant research has demonstrated amply that indicators of SES predict health outcomes, that health affects socioeconomic attainment, and that cognitive ability predicts both educational attainment and health. But how do such associations between SES, cognitive ability, and health arise over the life course? And what role does racial discrimination play in influencing each of these factors to create disparities by race? A theory of dynamic interplay of these factors across the life course is posited to explain the emergence of racial and socioeconomic disparities in general health, obesity, respiratory function, and depression. A California-based cohort, the Child Health and Development Study Adolescent Sample (CHDS-A) (n=2020), born 1959-1966, provides the novel data to evaluate this theory. Detailed information was collected at multiple time points from before birth until age 15-17 years for black and white offspring and their families. Sufficient numbers of black and white offspring from both high and low SES families were included in the sample. The data already collected include assessments during childhood and adolescence of SES, cognitive ability, and the selected domains of health. This project will add assessments at age 42-49 years. Analysis of the accumulated data will elucidate the relationships among fundamental sources of health disparities and inform the construction of policies and interventions designed to reduce such inequalities. Health disparities by socioeconomic status and race represent major public health issues in need of resolution. This study will provide novel data from a bi-racial birth cohort that has been intensively followed through adolescence and is now in middle-age adulthood. The analysis of key data collected in the proposed study will create an unusual opportunity to understand when and why health disparities emerge over the life course, and further guidance for shaping the content and timing of interventions to ameliorate observed disparities."}
{"text": "The adeno-associated virus 2(AAV)-based vector system has attracted considerable attention as an alternative to the more commonly used retroviral vectors for its potential use in gene therapy primarily because AAV is a non-pathogenic virus for humans, and the wild-type (wt) AAV genome has been shown to integrate into the human chromosomal DNA in a site-specific manner. However, a number of questions related to the basic molecular biology of the wt AAV interactions with normal human diploid cells in general, and that of the recombinant AAV in particular, remain largely unexplored. We have initiated systematic studies to pursue answers to these questions, and obtained preliminary evidence that the wt AAV interacts with normal human diploid fibroblasts in a manner that is distinct from that with human aneuploid cells. The hypotheses to be tested in this subproject are that the wt AAV integrates into normal human hematopoietic cells site-specifically at a site distinct from that characterized in human aneuploid cells, and that recombinant AAV genomes lacking the viral coding sequences (rep and/or cap) integrate in diploid hematopoietic cells at sites different from that for the wt AAV genome. The following Specific Aims will be pursued: 1. Comparison of patterns of integration of the wt AAV genome in human aneuploid and diploid hematopoietic cells, including purified populations of primitive progenitor cells from normal human bone marrow and umbilical cord blood: Using Southern blot and polymerase-chain reaction (PCR) analyses, the patterns of integration of the wt AAV genome will be compared in human aneuploid and diploid hematopoietic cells. 2. Evaluation of the role of AAV-encoded proteins in the site-specific integration of the wt and the recombinant AAV genomes: Recombinant viral vectors containing either the viral rep or the cap gene sequences will be used to evaluate whether AAV-encoded proteins play a role in the site- specific integration. 3. Molecular cloning and characterization of the AAV-integration sites in diploid cells, and comparison with that from aneuploid cells: In addition to direct cloning into bacteriophage lambda and plasmid vectors, PCR-based cloning strategies will be employed to obtain DNA sequences that contain the AAV-integration sites from human hematopoietic cells. The primary structure and transcriptional potential of these sequences will also be determined. 4. Evaluation of AAV-mediated transduction in vivo, and the potential for long-term expression of the transduced genes in a murine model system: Murine hematopoietic stem and progenitor cells will be transduced ex vivo, and following in vivo reconstitution of recipient mice, the patterns of integration of the wt and the recombinant AAV genomes as well as safety and efficacy of the AAV-based vector system will be examined. These studies will provide new insights into the basic molecular biology of the AAV-normal diploid cell interactions, and also evaluate the in vivo efficacy and safety of the AAV-based vector system prior to its potential use in human gene therapy. These studies also relate to our long-term interests in parvoviruses and human disease."}
{"text": "The 3' ends of eukaryotic messenger RNAs are formed by cleavage of the primary transcript, followed by addition of a poly(A) tail. Polyadenylation is required for RNA stability and the efficiency by which a gene's transcript is polyadenylated directly influences the level to which its mRNA accumulates and is translated. This is exemplified by a case of alpha-thalassemia in which a single point mutation was found in the poly(A) signal of the alpha-globin gene. One goal of the proposed research is to define the structural requirements of poly(A) signals. The experimental approach involves mutagenesis of portions of poly(A) signals followed by functional assays involving transfected COS cells or HeLa cell nuclear extracts. The mouse beta maj globin and SV40 early poly(A) signals will be studied in detail, as well as a variety of hybrid and synthetic constructs. The concept of poly(A) signals as interrupted palindromes is used as a guide for many of the experiments. The second goal of the proposed research is to elucidate the rules that govern poly(A) site selection. How poly(A) sites are selected when other poly(A) or splicing signals are located on the transcript will be studied using both in vivo and in vitro approaches. The relationship between poly(A) signals and introns will be explored in detail. These studies will give insight into the regulation of differential RNA processing."}
{"text": "Alcohol continues to be the drug of choice among American youth and teen drinking remains one of the nation's most serious public health problems. Yet in the Surgeon General's recent \"Call to Action to Prevent and Reduce Underage Drinking\" (USDHHS, 2007), he asserted that \"underage drinking is not inevitable, and schools, parents, and other adults are not powerless to stop it.\" Accordingly, in line with scientific recommendations and NIAAA's (2006) research priorities, we propose to further develop and rigorously test two family interventions for adolescent alcohol problems, Multidimensional Family Therapy (MDFT) and a Family Motivational Interviewing Intervention (FMII). Family interventions have strong research and clinical traditions in the treatment of adult alcoholism and adolescent drug abuse, but little research has focused on family interventions for teen drinking. In this controlled trial, 250 youth ages 12 to 18 with alcohol-related crises in the ER or trauma center will be randomized to one of these two family interventions or to standard care. Both family interventions, MDFT and FMII, aim to potentiate the influence of the family to motivate and help youth to change, but they rely on different theoretical foundations and clinical techniques. Both family interventions provide 2 initial engagement sessions in the homes of participants within 72 hours of the ER visit. Youth in MDFT will then be engaged into a full course (3 months) of this family-centered treatment, and FMII youth will be enrolled into 3 months of behaviorally oriented 12-step group treatment. Standard care participants will be referred to the same group treatment as youth in FMII, but will receive no engagement or family sessions. We propose a randomized controlled trial with five aims: 1. To investigate the engagement potential and effectiveness of a family-centered intervention (MDFT) and Family Motivational Interviewing Intervention (FMII)/group for teens with alcohol-related crises;2. To explore differential treatment effects with comorbid adolescents;3. To examine the role of motivation and family factors as treatment mediators;4. To examine long-term abstinence, patterns and predictors of relapse up to 18 months follow-up;and 5. To compare the total and net monetary benefits to society of MDFT, FMII/group, and standard care. A multiple time point (intake, 3, 6, 9, 12, and 18 month follow-up), multiple domain and method assessment approach will be used to compare the interventions on their engagement and retention rates, clinical outcomes, and total and net monetary benefits to society. Latent growth curve modeling and other state-of-the-art statistical techniques will be used to test study hypotheses regarding adolescent and family change over time, differential effects of the interventions on comorbid youth, the mechanisms by which the treatments achieve their effects, and relapse patterns and predictors. The study has significant potential to advance adolescent alcohol treatment by providing new knowledge to providers and policy makers about effective and cost-beneficial family interventions for youth identified with alcohol problems in the ER and ultimately in other health care settings. PUBLIC HEALTH RELEVANCE: Adolescent alcohol abuse is one of the nation's most serious public health problems, and youth presenting for alcohol-related crises in the emergency room (ER) represent a particularly vulnerable group (Maio et al 2000). Findings from the proposed study have the potential to yield new knowledge for providers and policy makers to guide decisions about implementing the most effective and cost-beneficial alcohol interventions for youth in the ER and other public health care settings. Further, better understanding of the treatments'mechanisms of action and effects on comorbid problems, as well as youths'relapse patterns and predictors following these interventions, may guide further development of family-based interventions to halt the progression of alcohol problems among highly susceptible adolescents."}
{"text": "The first few divisions in C. elegans embryo has examples of both proliferative divisions in which both the daughters are equivalent, and differentiative divisions in which the daughters take on different fates. Studies are being undertaken of the dynamic properties of the cytoskeleton that are unique to differentiative divisions. In particular: factors are found to be segregated to one region of the cell prior to division; divisions are usually assymetrical due to an assymetry in the centrosomes of the mitotic apparatus; the mitotic apparatus is seen to align on a preformed axis of polarity. The purpose of this project is to use both in viva imaging and correlative electron microscopy to study these phenomenon so as to gain insights as to the mechanisms used in the generation of cell diversity The focus of this study is to elucidate the role of microtubules in positioning the spindle in early asymmetric cell divisions of Caenorhabditis elegans. We will use in viva fluorescence microscopy to characterize the organization and dynamics of microtubules during spindle alignment in wildtype and mutant embryos. We are especially interested in mutants that exhibit alterations in the dynamic organization of microtubules in order to identify genes involved in spindle alignment. In addition, we will examine the ultrastructure of cortical microtubule attachment sites. Results will provide insight into the basic mechanisms and molecular components underlying cleavage plane specification in determinative cell divisions."}
{"text": "Isolated rhesus monkey luteal cells offer a model system for the in vitro study of primate corpus luteum function and assessment of the direct influence of various factors on the corpus luteum during its lifespan. Initial topics of interest in this project include: 1) Morphological and functional correlations between the intact corpus luteum and subsequent isolated luteal cells, 2) Comparison of the in vitro steroidogenic potential of luteal cells obtained from corpora lutea at various stages of the menstrual cycle and pregnancy, and 3) evaluation of the influence of potential luteotropic and luteolytic agents on in vitro steroidogenesis of macaque luteal cells."}
{"text": "The past decade has seen the definition of large families and super-families of neural genes whose related but different sequences provide great opportunity if we can understand their functions and exploit their diversity. There can be no doubt that a major challenge facing modern neurobiology is the understanding and manipulation of gene function, both known and unknown. Institutions concerned with the critical issues of mental health and cognitive disability must look beyond gene identification and into the structure and function of the proteins encoded by these newly discovered sequences and the roles these proteins play in the development and behavior of the individual. While the techniques for gene discovery have become less expensive and more accessible, the most powerful techniques for the study of function have become more expensive and more technically demanding. It is increasingly difficult for any single investigator to be able to fully explore the structure of a gene and its regulation, the structure of the protein it encodes, the localization of the protein in the animal, the role of the protein in the normal animal, and the consequences of the absence or alteration of that protein in disease. However, successful exploitation of the gene discovery requires at least portions of each of these activities in part simply to set priorities for further studies. It is the purpose of the BCM-IDDRC cores to provide access to techniques and assays that will allow the investigator to make maximum progress, without undue duplication of effort. The Mouse Physiology Core is designed to provide BCM-IDDRC investigators with a battery of functional assays that will provide initial insight into the neurophysiologic consequences of a specific mutation. This core is considered a significant component of the BCM-IDDRC because it will help address the most common question following the creation of a new mouse mutant;\"What is wrong with my mouse?\" The BCM-IDDRC proposes to offer its investigators access to a battery of electrophysiologic assays that will help answer this question and direct the investigator's attention to experiments that might more directly address the role of a particular gene in generating a mental retardation or developmental disability phenotype. The BCM-IDDRC at Baylor College of Medicine is well established in studying synaptic transmission and synaptic plasticity in the central nervous system. For many years Dr. Rosenmund's laboratory has been investigating basic function and dysfunction of excitatory and inhibitory synapses as well as hippocampal electrophysiology and plasticity. Dr. Jeff Noebels has been a pioneer in the use of EEG techniques to understand the genetic and molecular basis of epilepsy, and specifically in the use of mouse models to understand epilepsy. The Mouse Physiology Core will be divided into two components. The Synaptic Physiology component of the Core will allow investigators to determine the basic attributes of synaptic function from cultured neurons as well as from acute slices from hippocampus. These preparations will allow for detailed examination of synaptic properties, circuitry function and synaptic plasticity. This information is particulariy germane to the mission of the BCM-IDDRC, given the well-documented role of the hippocampus in learning and memory, and the newly arising notion that autism and related diseases have their etiology (at least in part) at dysfunctional synapses. The procedures established will allow the assessment of several parameters related to normal synaptic physiology. For the presynaptic site, this includes determination of quantal content, readily releasable vesicle pool size, vesicular release probability, synaptic release probability, and several forms of short time facilitation and depression. For the postsynaptic site, this includes mlPSC and mEPSC amplitude and kinetics, GABAA, AMPA and NMDA receptor function, as well as the determination of synaptic and extrasynaptic receptor population. These measurements will be based on patch clamp whole cell recording techniques. Morphological analysis of dendritic structure, synapse formation and synapse activity are provided using quantitative light microscopy analysis. In slices, additional analysis of input-output relationships for various intensities of presynaptic stimulation as well as several short- and long-term forms of synaptic plasticity will be assessed, including: paired-pulse facilitation, post-tetanic potentiation, long-term potentiation (LTP), and longterm depression (LTD). Latter procedures will utilize extracellular recording in the hippocampal slice preparation, using ongoing standard protocols already used here. The Electroencephalography component of the Core will enable BCM-IDDRC investigators to evaluate the development of cortical excitability and brain function over prolonged periods in behaving animal models of mental retardation produced by genetic engineering techniques. Depressed excitability or abnormal brain rhythms are among the eariiest objective phenotypes of genetic human mental retardation syndromes. A high incidence of epilepsy is also associated with mental retardation, and the facility specializes in state of the art seizure detection techniques and assessment of seizure threshold. The ability to correlate spontaneous EEG activity with behavioral analysis by use of synchronized video/EEG monitoring is critical to the interpretation of the mutant nervous system phenotypes studied by the BCM-IDDRC."}
{"text": "Many elderly people complain of significant sleep disturbance. Complaints of multiple and prolonged awakenings and fragmented nocturnal sleep indicate that elderly individuals have difficulties in maintaining, rather than initiating sleep. Age-related changes in the circadian system underlie, at least in part, sleep disturbance. Timed exposure to bright light can alleviate such symptoms, although compliance to long-term treatments is poor. As such, alternatives and/or supplements to bright light are needed to relieve sleep disturbance. In humans, light phase shifts circadian rhythms according to a phase response curve. Nonphotic stimuli, such as social interaction, and sensory stimuli including auditory and olfactory cues, can also phase shift circadian rhythms in mammals and birds. Few studies have investigated rigorously whether nonphotic stimuli can affect human circadian rhythms. This study will determine whether an auditory stimulus, with an arousing component, can phase shift circadian rhythms in a sample of healthy male and female elderly (60+ yr old) subjects. An ancillary question will assess gender differences in response to auditory stimuli in the elderly. Subjects will spend two 4-day sessions in a sleep laboratory under constant dim light (less than 20 lux), receiving either a 2-h auditory stimulus or a control stimulus (absence of auditory cue) from 0100 h-0300 h on the second and third nights. Subjects will participate in both sessions, with presentation order counterbalanced among individuals. Polysomnographical sleep and core body temperature will be collected continuously and salivary melatonin samples will be obtained once each hour from 1800 h to 2400 h on Night 1 (baseline night) and Night 4 (post-stimulus night). To measure putative phase shifts, circadian phase of core body temperature minimum and the dim light melatonin onset (DLMO), before and after sensory stimulation, will be assessed in the control and experimental sessions. This study serves as the first essential step toward describing rigorously the effects of sensory stimuli on circadian rhythms in males and females. Completion of this project will allow for further exploration regarding the significance of nonphotic entrainers for the human circadian system, in particular as alternatives for relieving sleep disturbance in older subjects."}
{"text": "Cancer studies are limited by the availability of the types of data routinely collected by cancer registries. This project will collect data on comorbidity and hospital characteristics to evaluate survival effects in registry based studies. The objectives of this study are: 1) Link case records on rare cancer sites from central cancer registries with records from appropriate state health offices to obtain patient comorbidity information and hospital treatment information. Types of data from the state offices can include public datasets of inpatients data collected from state licensed hospitals. Examples would include datasets with a record for each inpatient discharged from a state-licensed hospital. Licensed hospitals could include general acute care, acute psychiatric, chemical dependency recovery, and psychiatric health facilities. 2) Covariate information will be merged into a database with case information to evaluate the rare cancer survival. 3) Linked data will permit the ability to construct follow-up of rare cancer patients from more detailed examination of treatment details and prognosis with facility information to uncover rare cancer outcomes and subsequent diagnosis and treatment of other health conditions, resulting in a more comprehensive understanding of rare cancer survival and treatment."}
{"text": "Although many studies have addressed the issue of the cellular components present in PHF none has defined the relationship of these components to PHF structure nor to each other. This lack of knowledge has resulted from a focus on components rather than their relationship. Further it has suffered from the necessity of a hypothesis with regard to likely components before a putative PHF component is examined. In the present proposal, an in vitro model system is developed utilizing the properties of polymers to define the relationship of identified PHF components as well as opening the possibility for identification of new PHF components. The utility of this approach is the possibility of assigning PHF components to a minimum of two classes, core or associated proteins, by non-immunochemical methods. PHF components detected by this protocol, but not identifiable by immunochemical approaches, will be defined by amino acid sequencing. These studies will provide an under standing of PHF as polymers current to that known for the most prevalent cellular linear polymer, the cytoskeleton."}
{"text": "Dystonia is a neurological disorder characterized by involuntary twisting movements or abnormal postures caused by excessive activation of specific muscle groups or abnormal co-activation of agonist and antagonist muscles. The neurobiological basis for this disorder is not well understood, and even the neuroanatomical substrates remain uncertain. While clinical studies of affected patients have traditionally associated dystonia with damage or dysfunction of the basal ganglia and its connections, laboratory studies of rodents have more consistently associated the disorder with dysfunction of the cerebellum and its connections. The overall goal of this proposal is to bridge our understanding of the gaps between the basal ganglia and the cerebellum in the expression of dystonia. Our hypothesis is that dystonia is not simply the reflection of dysfunction of a single motor control system, but may result from dysfunction in either the basal ganglia or cerebellum, or abnormal interactions between these two regions. In Aim 1 we will investigate the contributions of the basal ganglia in two well-characterized mouse models where dystonia is known to be triggered by dysfunction of the cerebellum. These studies will be valuable for demonstrating the involvement of the basal ganglia in these models as well as important interactions between these two motor control systems in the expression of rodent dystonia. In Aim 2 we will perform manipulations of the cerebellum in rhesus monkeys analogous to those that have been demonstrated to provoke dystonia in rodents. These studies will establish a role for abnormal cerebellar output in the genesis of dystonia in monkeys and have the potential to result in a novel monkey model for dystonia. Aim 3 is devoted to a careful reassessment of autopsy material from the cerebellum of dystonia patients. These studies will be valuable because prior autopsy studies, which traditionally focused almost entirely on the basal ganglia, have generally failed to disclose any consistent neuropathological changes in most forms of idiopathic dystonia. Overall, the studies described in this proposal have the potential to stimulate a major revision of current concepts concerning the neuroanatomical basis for dystonia. A more complete understanding of the neuroanatomical substrates for dystonia is of elemental importance for any future studies of dystonia addressing relevant pathogenetic changes at the molecular, cellular, and physiological levels. In addition, the studies will have direct relevance for modern neurosurgical approaches to the management of dystonia that involve focal brain stimulation or lesion techniques. [unreadable] [unreadable]"}
{"text": "[unreadable] The HIV Evolution and Dynamics Meeting is a small annual HIV meeting with a 15 year history. This meeting has given people who perform data analysis, mathematician modeling, and HIV evolutionary analysis a specific opportunity to come together to discuss new research, both in terms of new studies and their implications, and new analysis tools and methods that can be applied to HIV. Mathematical and statistical aspects of the work can be discussed in greater depth than is possible in the larger HIV meetings, which primarily focus on experienial and clinical presentations. Historically this meeting has alternated venues between Europe and the US, to allow greater access to the meeting for researchers on both sides of the Atlantic. It can be one of the most important meetings of the year for HIV Scientists directly involved in the analysis of data. The meeting draws individuals who model in vivo dynamics and disease progression, global variation of HIV with considerations of the epidemic history, immunology and vaccine strategies, and individuals that study the emergence of drug resistance. The talks and posters range from important new studies that have complex aspects that may be presented more from a traditional view, but are discussed from an analysis perspective; mathematical oriented talks from scientists outside the HIV field who develop new analysis methods that might usefully be brought into the sphere of HIV research. On the order of 100 to 150 scientists attend, this number has depended on the venue in the past. There is ample time for discussion and developing contacts to facilitate and extend ongoing studies, and to initiate new collaborative efforts. The meeting has traditionally been supported by the OAR and the CDC with supplemental funding coming in through industry and the fund raising efforts of the primary organizer in any given year. We are requesting a continuation of the invaluable support received this grant for past 5 years which has permitted the attendance of junior investigators and participants from under developed countries. The UCSD Office of Continuing Medical Education has offered to support the financial and organizational infrastructure of the conference for the 3 year period for which we are requesting funding, and will work directly with the primary scientific organizer regardless of his or her home institution. The HIV Evolution and Dynamics Meeting is a small, intensive, annual HIV meeting with a 15 year history. This meeting has given people who perform data analysis, mathematical modeling, and HIV evolutionary analysis an opportunity to come together to discuss new research, both in terms of new studies and their implications, and new analysis tools and methods that can be applied to HIV. The information presented and generated by this meeting has potential important implications regarding HIV transmission, prevention, disease progression and treatment. [unreadable] [unreadable] [unreadable] [unreadable]"}
{"text": "DESCRIPTION (Adapted from applicants abstract): An important part of the defense against parasites is the specific immune response of the host. The stimulus initiating the response is the binding of parasite-derived antigenic fragments to major histocompatibility (MHC) molecules and their presentation by these molecules to T lymphocytes. The MHC molecules vary from one individual to another and this variability is encoded in polymorphic Mhc genes. Some of the variant molecules are apparently incapable of presenting certain peptides to T lymphocytes and in such situations the capability of the host to respond to a parasitic infestation could be impaired. It is therefore important to understand the nature of the polymorphic variation in the Mhc genes. The main objective of the proposed research is to explain the origins, persistence, and significance of the Mhc polymorphism. The specific aims of this research are to determine what proportion of the allelic variation pre-and postdates the formation of a species; to define the mechanisms of Mhc diversification; to estimate the age of human allelic and haplotype polymorphisms; to identify the mechanisms possibly maintaining the Mhc polymorphism in natural populations; to provide evidence that parasites are indeed the main driving force behind the Mhc diversification; to establish the part played by population structure in the long-term persistence of Mhc polymorphism; and to formulate an all-encompassing theory of Mhc polymorphism. The research will be carried out on 3 model systems: the primates, the mouse, and the zebra fish. It will include both molecular analyses of the Mhc genes and the study of the Mhc in natural and model populations."}
{"text": "Ethanol is a teratogenic agent responsible for the fetal alcohol syndrom (FAS) that is characterized by mental retardation in children born to chronic alcoholic women. While the FAS invovles central nervous system (CNS) dysfunction, the cellular basis for understanding the defect related to central catecholaminergic (CA) neurons is not known. CA neurons in their CNS could be implicated in the FAS for the following reasons: (1) CA terminals provide a major input to the immature neocortex and neostriatum, and (ii) some biochemical evidence suggests that central CA neurons are susceptible to ethanol. CA terminals can be quantitatively assessed by fluorescence microscopic (FM) and electron microscopic (EM) morphometrics. The present proposal will use an FM/EM morphometric analysis to examine effects of ethanol on CA neurons in the neocortex and meostriatum of developing rats. By using glyoxylic acid-induced FM and 5-OH-DA EM techniques, parameters such as CA terminal density and CA synaptogenesis will be studied in neonates which have been exposed to ethanol through their meternal blood and breast milk from pregnant/lactating rats fed with 35% ethanol-containing liquid diets. This part of the study is to examine an early effect of ethanol on central CA neurons. These ethanol-exposed neonates will also be studied when they become adults, in order to examine long-term effects of ethanol on central CA neurons. Similarly, CA terminals will be evaluated by using glyoxylic acid FM method, and CA boutons in the adults will be studied by suing a potassium permanganate EM method. Since dendritic spine abnormalities of cortical neurons have been described in mentally retarded children, we will use the rapid Golgi method to evaluate effects of ethanol on dendritic spines of cortical neurons in adult rats exposed to ethanol during their prenatal and perinatal development. The goal of this proposal is to provide us a fundamental and ultrastructural basis for better understanding mechanisms by which ethanol affects CNS function."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Grant Number: R21-CA111988 Project Start: 22-FEB-2005 Project Original End Date: 31-JAN-2007 Project extended to January 31, 2008 Project Summary Surgical resection has been shown to be an effective treatment for secondary liver tumors, with five-year survival rates between 20-30%. Unfortunately, only select patients are candidates for resection, and it has a relatively high complication rate. About 35% of patients suffer minor complications, 15% have more serious complications, and the mortality rate is 2-5%. In addition, the average hospital stay is about two weeks, even without complications. Minimally invasive methods to thermally coagulate liver tumors have eliminated some of these disadvantages and have been shown to be an effective alternative treatment. The number of patients who receive such thermal ablation treatments is growing rapidly, and this technology is poised to enter widespread use. A way to substantially improve the safety and effectiveness of these treatments would be to have a method to easily and reliably quantify the tissue online that has reached a sufficient thermal exposure to induce thermal coagulation. Our hypothesis is that a novel magnetic resonance imaging (MRI) contrast agent that is activated at a threshold temperature level can be used to reliably successfully monitor thermal ablation of liver tumors. This agent consists of paramagnetic material encapsulated in a liposome shell. When encapsulated, the material has a minimal effect on the MR signal intensity. The liposome shell undergoes a phase transition at a temperature (Tc) that can be precisely chosen, and MR signal enhancement occurs which can be easily detected with standard imaging methods. With a Tc of 57[unreadable]C, which is the approximate threshold for thermal coagulation for heating of at least a few seconds, the signal intensity enhancement thus indicates the regions that are thermally ablated. Since perfusion is halted after thermal coagulation, any released paramagnetic agent will remain in the tissue for an extended period of time, clearly marking the ablation progression. Our preliminary tests of this agent in vivo have demonstrated its basic functionally. Here, we propose in vivo animal experiments to extensively characterize its performance and to test its effectiveness in monitoring thermal ablation of liver tumors. This research will lead to more effective and safer thermal ablation treatments in liver, and perhaps allow more difficult cases to be feasibly treated with thermal ablation methods, since it would provide straightforward and reliable online feedback of the treatment progression. Thesaurus Terms: contrast media, liver neoplasm, magnetic resonance imaging, neoplasm /cancer thermotherapy, technology /technique development image enhancement, liposome, noninvasive diagnosis bioimaging /biomedical imaging, histology, laboratory rabbit ICD: NATIONAL CANCER INSTITUTE IRG: RTB"}
{"text": "The specific aims of this proposal are to a) purify melanoma-associated antigens of defined specificities, b) develop radio-immunoassay (RIA) and enzyme immunoassay (EIA), c) apply these sensitive and specific serologic assays to detect antibody and free circulating antigen levels in sera and other body fluids of patients with malignant melanoma, and correlate the results of RIA and EIA with clinical course of disease. It is expected that by developing the sensitive serologic assays and improving their specificities, these assays will become potentially useful tools for prognosis and diagnosis of human cancer. Cancer patients who are rendered disease-free by surgical removal of tumor can be monitored for the levels of free circulating tumor-associated antigens and their corresponding antibodies in the body fluids. Such information will be valuable to the physician for the management of human cancer. Besides being applicable as an immunodiagnostic aid, such information will expand the understanding and knowledge of tumor-host relationship. The isolation and purification of specific melanoma-associated antigens and their characterization will provide an insight into the immunobiology of human malignant melanoma."}
{"text": "Growth factors signaling through the cell-surface receptor tyrosine kinases (RTKs) represent one of the most critical pathways in the cellular and inter-cellular regulation of vertebrate biological systems. Defects/malfunctions of the class III RTKs, such as the Stem Cell Factor (SCF) receptor KIT, the Macrophage Colony Stimulating Factor (MCSF) receptor FMS, and the FLT3 Ligand (FLT3L) receptor FLT3, contribute to the genesis and development of many types of cancers such as leukemia, gastrointestinal stromal tumor (GIST), and others. These receptors are ideal targets for cancer therapies, but the mechanistic basis underlying their ligand recognition and activation is incompletely understood. Our long-term goal is to elucidate the structural mechanisms used by these receptors in cellular regulation and malignancies. The project will perform a thorough analysis of the whole activation process of class III RTKs: how these receptors maintain static states when the ligands are not present, how their extra cellular domains recognize ligands, and what are the conformational changes required for receptor activation. The specific aims are: (1) Biochemical reconstitution and structural analysis of the complex between KIT and its ligand SCF. Crystals of the SCF/KIT complex that diffract X-rays to 3.2 Angstroms resolution have been obtained. The structure will be solved by multi-wavelength anomalous diffraction (MAD). (2) Biochemical reconstitution and structural analysis of the complex between FMS and its ligand MCSF. Crystals of the MCSF/FMS complex that diffract to 2.4 Angstroms resolution have been obtained. The structure was determined by a combination of partial molecular replacement and single isomorphous replacement with anomalous scattering (SIRAS);refinement is in progress. (3) Biochemical characterization and structural analysis of the complex between FLT3 and its ligand FLT3L. Small crystals of the FLT3L/FLT3 complex have been obtained recently. (4) Investigation of the binding specificity and energetics of the receptor/ligand interactions through protein engineering of the receptor/ligand interfaces. These studies will elucidate novel structural mechanisms that will lay the groundwork for therapeutic development to treat class III-RTK-related cancers."}
{"text": "The retinoblastoma family of proteins (also designated as pocket proteins) comprises the product of the retinoblastoma tumor suppressor gene and the structurally and functionally related proteins p107 and p130. This project focuses on the regulation of the retinoblastoma family member p130 by CDKs. This project seeks to determine those phosphorylation events that modulate p 130 function in a unique manner, which obviously is not shared by pRB and/or p107. The project leader's goal is to link phosphorylation of certain clusters of phosphorylatable sites with changes in p130 protein stability, which deplete p130 from cells as they enter S phase. It is important to detemaine how this occurs and its potential role in maintaining cell cycle check-point activity and suppressing tumorigenesis. The phosphorylation status of the three pocket proteins is regulated in a cell cycle-dependent manner. Among the pocket proteins, p130 is the one that exhibits the most conserved pattern of phosphorylated forms in normal cells. While a variety of differently phosphorylated pRB forms are detected when comparing different cell types under similar physiological conditions, the patterns of p 130 forms detected by SDS/PAGE followed by Western blot analysis are coupled to cell cycle phases as well as to the quiescent stage. In addition, differently from pRB and p107, the levels of p130 appear to be linked also to the phosphorylation status of p 130 and thus, to the cell cycle stage. Dr. Grafia's team has studied in some detail how particular changes in the phosphorylation status and protein levels of p130 modulate its association with members of the E2F family of transcription factors. Their studies show that while certain changes on p130 phosphorylation status are catalyzed by the concerted action of at least two different types of G1/S cyclin/CDK complexes, other phosphorylation events do not involve these kinase complexes. Their recent data demonstrate that E1A blocks hyperphosphorylation of p130 and p107 without affecting phosphorylation of pRB. These studies have shed light on the regulation of p130 by phosphorylation during the cell cycle in normal and E1Aexpressing cells. The aims of this project are as follow: Aim 1. To identify the regulatory mechanism(s) that tag p130 for cellular depletion as cells progress through the mid-late G1 and S phases of the cell cycle. Aim 2. To determine the biochemical pathway that mediates cell cycle dependent degradation of the p130 protein. Aim 3. To characterize p130 /unctions other than its ability to repress E2F-dependent transcription by using a genetic annmach. This project will utilize core B for Aims 1-3. and core A for Aims 3."}
{"text": "(Supported by NIH GMS 43264 to R. Palazzo and GMS 40198 to C. Rieder) This is an ongoing project in which IVEM and HVEM tomography are being used to analyze the structure of centrosomes isolated from Spisula (surf clam) oocytes prior to and after various experimental manipulations. This year we characterized the high-resolution 3-D structure of control and KI extracted centrosomes, and also extracted centrosomes that had regained their function by incubation in high speed extracts from oocytes. We found that heavily KI extracted centrosomes are non-functional and consist of an insoluble 15-nm filamentous \"centromatrix\" material. When this centromatrix is incubated in high speed extracts of activated egg oocytes, the extracted centrosome regains its functional abilities. The loss of centrosome function is correlated, at the IVEM tomographic level, with the loss of gamma tubulin containing 20-nm diameter ring-shaped structures, while regain of function is correla ted with the reappearance of these structures. The data reveal that the centromatrix serves as a scaffold for binding those proteins involved in microtubule nucleation. Because centrosomes are extremely small (0.25 by 0.5 um) and complex, the only way their morphology and replication can be studied is with 3-D EM. Over the past 12 years we have shown that IVEM/HVEM tomography of serial thick sections greatly facilitates such studies, and have published numerous papers on this topic-many recently with the Palazzo laboratory. We are now exploring the utility of combining high resolution antibody labeling with IVEM tomography of high pressure frozen and freeze substituted samples. Schnackenberg, B.J., A. Khodjakov, C. L. Rieder and R. E. Palazzo (1998). The disassembly and reassembly of functional centrosomes in vitro. Proc. Natl. Acad. Sci. USA 95:9295-9300."}
{"text": "Aim: Acute rejection remains a major problem for pancreas transplant recipients and accounts for 30 percent of graft failures in the first year after transplantation, in spite of improved surgical techniques and advances in immuno suppression. There are no universally accepted clinical indicators of rejection for pancreas allografts. Biopsy proven rejection is the standard for diagnosis; however, pancreas biopsies may be technically difficult to perform and associated with risks to the recipient. Recently, increased cytotoxic lymphocytic expression of perforin, granzyme B, and Fas ligand in peripheral blood lymphocytes (PBL) has been associated with renal allograft rejection and may be indicative of subclinical acute rejection. Because of the increased incidence of acute rejection and the difficulty in establishing a clinical diagnosis, a similar technique to evaluate early markers of acute rejection is uniquely important in pancreas transplantation. We hypothesize that by measuring expression levels of 3 genes, perform, granzyme B, and Fas ligand, using a newly developed molecular technique (real-time reverse transcriptase polymerase chain reaction [RT-PCR]), we can noninvasively detect acute allograft rejection at the time or prior to development of biopsy proven rejection. The ultimate goal is to develop a reliable, non-invasive method to detect acute rejection, thus allowing for earlier therapeutic intervention and better outcomes in solitary pancreas recipients. Long-term, we aim to expand the use of this technology to identify other genetic markers for acute rejection and develop microarray chip technology to clinically monitor the immunological status of transplant recipients. Methodology: Over a 2-year period, 25 solitary pancreas transplant recipients will be entered into the study. At least 40 milliliters of blood will be obtained pretranspiant and at time of surveillance biopsies or clinic visits (10 time points). Peripheral blood lymphocytes (PBL) will be separated for mRNA extraction. State-of-the-art real-time RT-PCR (ABI PRISM 7700) will be used to quantify gene expression levels of perforin, granzyme B, and Fas ligand. T-tests will be used to examine differences in gene expression levels between those patients with and without hiopsv proven acute reiection."}
{"text": "This project is concerned with (a) the genetic diversity of Trypanosoma cruzi and its implications to the epidemiology, course and diagnosis of Chagas' disease, (b) the development of high resolution flow cytometry instrumentation for analyses of cell populations and, (c) the utilization of flow cytometry and low light level video microscopy for the analyses of infectious agents. Flow cytometry was used to analyze the population dynamics of T. cruzi clone mixtures. The relative numbers of each clone in the population changed rapidly and the results are in quantitative agreement with mathematical models of competitive population growth; no dynamic equilibrium was found. These data emphasize the importance of working with well-defined clones in the laboratory and stress the importance of rapid isolation of single clones from clinical specimens. A similar situation occurs in Dipetalogaster maximus except that clones can coexist for longer periods of time in the bug. Electrocardiographic analyses of inbred mice infected with T. cruzi clones demonstrate that several known aspects of chronic Chagas' disease can be mimiced with the model. Electron bean x-ray microchemical analyses of T. cruzi clones demonstrate marked inter-clonal differences in Fe, Zn, S, Mg, K, Ca and P."}
{"text": "The murine lymphocyte receptor for IgE, with regard to both structure and function is the focus of this application. Intact FceR levels increase during B cell activation in the presence of interleukin 4 (IL-4) suggesting a role for the FceR in B cell activation. Anti-FceR both in the soluble and sepharose bound form will be tested for the capacity to either directly activate B lymphocytes or to influence B cell activation by anti- Immunoqlobulin. Parameters to be measured include calcium influx, cell size changes, phosphatidyl inositol levels and Ig production. The FceR spontaneously releases lower molecular fragments via proteolysis at the cell surface and preliminary evidence suggests that these fragments enhance the level of IgE synthesis in the presence of IL-4. This phenomena will be further studied with regard to isotype specificity, role of FceR carbohydrate in the biologic activity and molecular size of the FceR fragment involved. In addition, it will be determined if the fragment interacts with a specific component on the B cell membrane. The site of interaction of IgE with the FceR is important with regard to receptor degradation and the location of this site will be explored in detail, both by using anti-peptide antibodies, mimicking specific areas of the FceR and by using crosslinking reagents that will label the site(s) of close proximity with IgE. Antibodies against the site of the FceR that interacts with IgE will also be tested for their capacity to induce FceR upregulation, analogous to IgE and their effect on IgE synthesis will be determined. The low affinity FceR on macrophages and T cells will be further investigated with regard to physiochemical relationship to the B cell FceR, association with other membrane components and regulation at the cell surface. The overall aim is to gain further insight into the structure and regulation of the low affinity FceR and to determine if FceR/anti-FceR related components are useful in the regulation of IgE synthesis."}
{"text": "Summary Osteoblasts are emerging as a key component of tumor microenvironment in breast cancer bone metastasis. It has been demonstrated recently that disseminated breast cancer cells interact with the osteoblastic niche to survive and colonize the bone. However, the detailed mechanism for the interaction between tumor cells and osteoblastic niche in bone colonization remains mostly uncharacterized. Furthermore, almost no studies have been done to elucidate the functions of osteoblasts in the resistance of bone metastasis to traditional therapies (i.e., chemotherapy and radiation therapy). In our preliminary studies, we discovered that chemotherapy significantly increases Jagged1 expression level in osteoblasts and in mesenchymal stem/stromal cells (MSCs), the precursors of osteoblasts. Transgenic expression of Jagged1 in osteoblasts in our newly created Col1a1-Jagged1 transgenic mouse model significantly increases the formation of bone metastasis. Importantly, we have developed a humanized monoclonal antibody against Jagged1 (15D11), which demonstrated promising therapeutic effects against bone metastasis in preliminary studies. We hypothesize that the increased expression of Jagged1 in MSCs and osteoblasts promote bone colonization of breast cancer and contribute to chemoresistance and radioresistance by providing survival signaling to metastatic tumor cells. Neutralizing antibodies against Jagged1 may block bone metastasis formation and outgrowth, and sensitize them to traditional therapy. Col1a1-Jagged1 and a series of well establish mouse models will be used for bone metastasis allograft and xenograft studies to evaluate the functional importance of osteoblast Jagged1 in bone metastasis formation and treatment resistance. We will use Jagged1 neutralizing antibody 15D11 in vivo to test its synergistic effect with chemotherapy, radiation therapy and osteoclast-targeting therapy. Furthermore, we will perform experiments to understand the exact molecular pathways leading to elevated expression of Jagged1 in osteoblast in response to chemotherapy, as well as Jagged1-dependent osteoblast-tumor interaction in promoting metastatic colonization and treatment resistance in bone. Our proposed studies will confirm the importance of osteoblast Jagged1 in bone metastasis colonization and treatment resistance. We will also reveal molecular signaling/pathways responsible for the Jagged1-dependent function of the osteoblast niche in bone colonization and treatment resistance. Successful pre-clinical testing of Jagged1 neutralizing antibody will also pave the way toward their application in human patients in the near future. Therefore, we believe our study will likely have a significant impact on improving the treatment of metastatic breast cancer."}
{"text": "Mitochondria from young and mature BHE and Wistar rats were studied. Strain and age affected respiration, shuttel activity, ATP production, ATPase activity, and adenylate kinase activity. Mitochondria were less active in BHE rats than in Wistar rats and with age became even less active. The slower shuttle activities and Ca ions Mg ions ATPase activity in BHE rats could be made more active with thyroid hormone treatment suggesting that the genetic aberration in metabolic control which results in maturity onset lipemia and glycemia, and in a shortened lifespan, is not due to an aberration in the mitochondrial shuttle systems or of the ATPases. Studies of glucose production by isolated hepatocytes showed that gluconeogenesis was more active in BHE rats than in Wistar rats but that in BHE rats, thyroid treatment was without effect as a stimulant of glucose production."}
{"text": "DESCRIPTION: (Provided by the Applicant) Most patient safety improvement occurs incrementally within single institutions. A new method to develop comprehensive statewide risk models that could be exported to large segments of the health care industry is evaluated. Working hypothesis: Sociotechnical probabilistic risk assessment (ST-PRA) can create risk models identifying common medication system and behavioral elements that raise the risk of serious errors and these risk models can be used to design statewide risk reduction programs for nursing and community based care (CBD) long term care facilities. These facilities need robust, well-designed medication systems because they serve a growing and often frail population, administer highly toxic drugs, and must perform to high standards using an unstable and sometimes minimally skilled labor force. Design: Developmental study. Methods: This project uses four tools--process mapping, control system mapping, failure modes and effects analysis (FMEA), and socio-technical probabilistic risk assessment (ST-PRA)--to create two comprehensive probability risk assessment (ST-PRA) models, one for nursing facilities (NFs) and one for CBC (residential care/assisted living) facilities, to identify processes and behaviors that increase the risk of wrong drug, wrong dose, wrong patient medication delivery errors in LTC facilities. The NF risk assessment model is created by focus groups of staff, pharmacists, and physicians from nine randomly selected facilities in a stratified sample of three large, volunteer LTC chains. The CBC process is similar, with nine randomly drawn CBC facilities. Focus groups of CBC residents and their families will be invited to provide input into the CBC model. Appropriate human subjects and privacy protections will be in place. Models are validated in stratified, random samples of nursing and community-based care facilities to determine whether each model is representative of medication delivery systems in the respective types of facilities, using a combination of structured focus groups and direct observation. Recommendations for interventions to address the systems and behavioral risks identified will be made and lessons learned while undertaking this large-scale, multi-facility ST-PRA project will be reported."}
{"text": "The melanotropic neuropeptide, alpha-MSH, is known to inhibit interleukin 1 (IL1)-induced fever in the brain and systemic immune responses. The long-term objectives of this proposal, in continuation of studies underway, are to determine the mechanism of alpha-MSH modulation of IL1 action within the brain and to characterize the melanotropin receptor(s) of the brain and of lacrimal gland, a peripheral melanotropin target organ and potential tissue source for purification of brain-type melanotropin receptors. Two separate hypotheses will be tested that may explain the mechanism of action of alpha-MSH-mediated inhibition of IL1 action within the brain: A) First, that alpha-MSH inhibits IL1-induced release of prostaglandins E and/or F2- alpha, and/or interleukin-6 (IL-6), in hypothalamic cells; B) Second, that alpha-MSH inhibits IL1-induced secretion of corticotropin releasing factor (CRF), recently implicated as a mediator of fever and other IL1 actions in brain. This will be tested in vivo, by measuring febrile responses to IL1 and alpha-MSH in normal and CRF-deficient Lewis rats, and in vitro, by measuring the effects of alpha-MSH on IL1-induced CRF secretion in cultured brain cells. Based on the observed biphasic nature of alpha-MSH actions including modulation of IL1 effects, the hypothesis will also be tested that multiple melanotropin receptor forms exist in brain and in lacrimal glands, which may mediate different aspects of melanotropin-IL1 interactions: A) By determining whether different structure-function relationships exist for binding of a series of melanotropins in the septal- preoptic area, hypothalamus, midbrain and lacrimal gland; B) by determining the relationship between melanotropin receptor occupancy and stimulation of adenylate cyclase in diencephalic cells; C) by determining whether melanotropin receptors are differentially down-regulated by exposure to alpha-MSH, ACTH, or melanotropin receptor class-selective ligands; D) by determining whether melanotropin receptors exhibit differential binding kinetics or regulation by cations and guanine nucleotides; and E) by isolating and characterizing the melanotropin receptor(s) of the brain and lacrimal gland. Endogenous melanotropic neuropeptides may form part of an endogenous counterregulatory system, protecting against the damaging effects of excessive cytokine activity. By determining the mechanisms of neuropeptide-cytokine interactions and characterizing the receptors for this model class of peptides, the proposed research will advance our understanding of the coordinated response to infection and trauma, and the basis of autoimmune and inflammatory diseases."}
{"text": "A study of electrical activity, how it is affected by ischemia and edema, and how recovery of activity is related to temporary ischemic insults of graded durations. During this period, instrumentation has been procured and or constructed, optimized and familiarized. Preliminary experiments have begun."}
{"text": "Abstract LSUHSC CARC Administrative Core The purpose of the Louisiana State University Health Sciences Center (LSUHSC) New Orleans Comprehensive Alcohol-HIV/AIDS Research Center (CARC) is to conduct cutting-edge translational research on the causes and biomedical consequences of alcohol use and abuse and their impact on biomedical and psychosocial comorbid conditions of persons living with HIV/AIDS. The goal of the CARC is to generate evidence-based knowledge on the interaction of alcohol and HIV disease that will inform health care providers and will lead to effective primary care-based interventions to decrease risky alcohol use, HIV transmission and improve health outcomes. The Administrative Core of the CARC will provide oversight and the organizational framework for the direction, management, and coordination of all activities. Specifically, the Administrative Core will (1) monitor the performance and productivity of the individual research components and cores; (2) manage the budget and fiscal activities of the CARC; (3) supervise the CARC personnel; (4) provide data collection, management, and analysis support, and (5) provide oversight for the educational, enrichment and dissemination functions of the CARC. In essence, the Administrative Core is responsible for centralizing, coordinating, and integrating research and administrative components of the CARC. Dr. Molina, Principal Investigator and Director, and Dr. Nelson, Co- Director, will direct all CARC-related activities in consultation with the Intramural Center Committee. The Administrative Core will facilitate logistics for synergistic, productive, and seamless interaction among the participants to substantially enhance the success of CARC activities. The Administrative Core will foster formal and informal exchanges among members of the CARC and the intra- and extramural scientific community to allow for peer-review, idea cross-fertilization, and development of new collaborative projects focused on the biomedical health consequences of alcohol abuse. The Administrative Core is a mature, integrated component of our CARC with a successful record in supervising, promoting, and developing comprehensive, thematically focused research programs that are both productive and cost-effective in an area of great clinical relevance."}
{"text": "In eukaryotic cells, heterochromatin governs diverse cellular processes such as silencing transcription and maintaining genomic integrity. Once formed, heterochromatin must be transmitted to daughter cells during cell division. Inappropriate gain or loss of heterochromatic structure plays a causal role in cancer. Therefore, studies on how heterochromatin is inherited during S phase or epigenetic inheritance will not only increase our understanding of this basic biological process, but also shed light on how this process goes awry in cancer cells. The yeast S. cerevisiae offers an excellent model system to study epigenetic inheritance. Yeast heterochromatin is marked by the presence of hypoacetylated histories and four silent information regulator (Sir) proteins. SirSp and Sir4p associate with hypoacetylated histones H3 and H4 for establishment and maintenance of yeast silent chromatin. During DNA synthesis, much of the epigenetic information such as modifications on histones as well as Sir proteins is temporarily disrupted. Immediately after DNA synthesis, the epigenetic information must be reestablished. Chromatin assembly factor 1 (CAF-1) is one of the factors involved in inheritance of epigenetic information. However, the molecular mechanisms by which CAF-1 functions in this process is not clear. Our long-term goal is to understand how chromatin structures are inherited during the S phase of the cell cycle. The objective of this proposal is to study molecular mechanisms whereby CAF-1 functions with its modulator and effector proteins to inherit silent chromatin structure in yeast. These studies will address how heterochromatin is inherited in yeast. Moreover, knowledge gained from these studies will also lead to a better understanding of epigenetic inheritance in mammalian cells and cancer caused by malfunction of this process."}
{"text": "To study the safety, effectiveness and pharmacokinetics of temozolomide in patients with advanced cancer."}
{"text": "Project Summary Approximately 85-90% of those with serious mental illness are unemployed (9, 10). This level of unemployment exists despite the finding that, among people who have a psychiatric disability and are unemployed, 55-61% desire employment, with the total reaching 75% when concern about losing benefits was taken into account (1, 2). The rewards of employment for adults with SMI are greater than simply an increase in financial resources. Studies have found that people with serious mental illness who worked competitively scored higher on measures of self-esteem, satisfaction with finances, leisure, and overall life satisfaction compared to those worked little or not at all (11). Supported employment (SE) has been more effective than any other vocational rehabilitation approach for this population (3, 4) and the VA has mandated that SE be integrated into all current VHA Compensated Work Therapy (CWT) programs (VHA Directive 2007-005). Two areas of concern include (a) national utilization of SE services is low (13), and (b) the percentage of SE participants who obtain at least one job during the studies averages 56% even though everyone who enters the program expressed an interest in working (13). Motivational interviewing (MI) is an evidence-based practice that has been effective in enhancing a range of clinical services (6), but has just recently been applied to employment and vocational services by Drebing et al. (7) and Glynn et al. (8). In the Drebing et al. study, a single session of motivational interviewing was associated with a 43% increase in entry and doubling the length of participation in VR, raising the likelihood that motivational enhancement interventions may be effective for addressing a range of VR outcomes. The proposed project is a randomized trial of Adapted Motivational Interviewing for Supported Employment (AMI-SE) provided to veterans and their significant others (family members or key friends) in order to address the internal and external barriers to the veteran enrolling in supported employment and returning to work. The intervention consists of 6 sessions that (a) identify and resolve a veteran's and the significant other's ambivalence about working, (b) provide information about the SE program to both parties, and (c) assist the dyad to develop a change plan to address the veteran's and significant other's emotional and practical concerns regarding SE enrollment and employment. One hundred and fifty veterans from the Bedford VA Hospital will be randomly assigned to AMI-SE or a control condition. The groups will be compared on SE entry rate, CE entry rate, number of weeks working in CE, and SE participation. In addition, the study will examine whether variables related to the Health Belief Model are predictive of vocational rehabilitation entry and outcome. PUBLIC HEALTH RELEVANCE: Project Narrative At completion, results will provide VHA policymakers, investigators, and vocational rehabilitation specialist with findings and recommendations regarding (a) the usefulness of an extended motivational interviewing protocol for veterans and their significant others, and (b) topic areas for development of additional strategies to improve access and outcomes in supported employment. If effective, the motivational intervention could be conducted by case managers of clinicians of veterans with serious mental illness to increase rates of employment. The proposed study seeks to ensure full utilization of SE, including the provision of services specifically designed for those who are unsure about work and employment, moving VA SE services into the cutting edge of access, utilization, and success."}
{"text": "This proposal was prepared in response to the announcement of a Research Program on Breastfeeding and Human Milk. We are specifically addressing th section of the program announcement headed Processes Involved in Milk Formation and Secretion, proposing studies which will determine how choline influences milk formation and secretion. Choline is necessary for normal growth and development. It is a precursor for the biosynthesis of phospholipids - essential components of all membranes, it is an important methyl donor, and it is needed to make acetylcholine. Most mammals derive much of their daily choline requirements from their diets, and milk is an important dietary source of choline. Choline moiety is needed to make the phosphatidylcholine which coats the secretory vesicles and milk fat globule made within the mammary epithelial cell. In hepatocytes, choline is absolutely required for the export of a fat globule (VLDL). It is possible that varying the availability of choline might alter the excretion of casei and lipid by the mammary epithelial cell. Our laboratory has made the following observations: 1) Neonatal rats, ferrets and humans have extremely high blood choline concentrations - making more choline available to tissues; 2) The above is, in part, due to ingestion of milk which is high in choline content; 3) There is a large increase (6-fold) in the choline concentration of human milk just after the start of lactation, followed by an equally large decrease in choline content 4-5 days later; 4) Mammary epithelial cells are capable of accumulating choline via mediated transport so that milk choline concentration can be 70-fold higher than is maternal blood choline concentration; 5) Mammary epithelial cells are capable of de novo biosynthesis of choline moiety. We propose to culture rat and human mammary epithelial cells on a matrix. We will characterize choline transport and metabolism, de novo biosynthesis of choline, as well as formation and secretion of milk fat globules and casein using this cell culture system. We will examine the changes that occur during differentiation of the cells, and during exposure to varying levels of prolactin, insulin hydrocortisone, progesterone, estradiol-17beta or oxytocin. We will also examine the effects of varying the availability of choline upon each of these parameters. In some tissues, carnitine transpor depends upon choline availability, we plan to determine whether this is tru in the mammary epithelial cell."}
{"text": "Summary of Work: The purpose of this agreement was to support the collection and analysis of data on cause of death and characteristics of the last year of life in the planning of the 1992 Pretest and 1993 Main Survey of the 1993 National Mortality Followback Survey (NMFS), conducted by NCHS, CDC. This survey supplements information from death certificates in the vital statistics file with information on characteristics of the decedent. The pretest examined approximately 800 deaths of individuals aged 15 years and over who died in 1992. The main survey examined approximately 22,951 deaths of individuals aged 15 years and over who died in 1993. This includes 884 deaths to centenarians. A revised death certificate/informant(DC/I)file was delivered to the NIA/EDB by the NCHS in 1998 and has been extensively examined by EDB staff and the SYTEL computing support contractor. This file contains information keyed from the death certificate file and the informant interviews. Data from the final 1993 mortality file has been merged with the DC/I file and stripped of personal identifiers to form a working file. Post-stratification adjustment factors and non-response adjustments have been developed to form the final sample weights for each file record.Some additional problems remain with the revised data set, which will be reissued by NCHS in the fall of 1999. Meanwhile, data analyses have been initiated by EDB staff.At the Kentucky conference on Statistical Methods in Alzheimers Disease Research in 1998, EDB staff held discussions with a neurologist who had completed an analysis of data from the 1986 NMFS on dementia in the last year of life. A preliminary analysis of the 1993 data to explore dementia trends over the 7-year period between the two surveys has shown an almost three-fold increase in the recognition of dementia mortality both by physicians completing the death certificates and by informants responding for the decedents in the survey. Another preliminary analysis has shown a high rate of receipt of surgeries for the implantation of hip and knee joints and other devices related to lower limb mobility problems in the decedents. Future analytic plans will look at trends in reported lifetime history of other conditions between the 1986 and 1993 NMFS, examination of lifestyle factors related to death at very old ages, hypotheses regarding decreased use of health services in the last years of life among the very old, and possible studies of compression ofmorbidity among the oldest old. - death certificate, morbidity, oldest old - Human Subjects & Human Subjects: Interview, Questionaires, or Surveys Only"}
{"text": "A new X-ray facility will enable the full potential of 19 NIH sponsored grants to be realized. An existing X-ray generator is 22 years old and no longer functional;the existing area detector and low temperature device is 10 years old and no longer cost-effective to maintain. The equipment requested is: a Rigaku MSC, Inc., MicroMax-002+ sealed tube microfocus X-ray source coupled with a high performance Confocal Max-Flux optic that produces a monochromatic, focused X-ray beam with flux 3X greater than the existing RU200 rotating anode generator;a Saturn 944+ CCD X-ray area detector with 94 mm x 94 mm active image area that provides 8 MHz readout, 15100:1 dynamic range, and high sensitivity;a AFC-11 computer controlled 4-axis goniometer;and a X-STREAM-2000 low temperature system. This equipment will provide high throughput instrumental capacity for all crystallographic experiments with biological macromolecules, including screening for crystal quality and cryoprotection conditions with membrane proteins and protein-DNA complexes, and high resolution data collection from many hundreds of protein crystals. A reliable, in-house X-ray facility will accelerate prescreening of many crystals, optimize the use of synchrotron beam time for membrane proteins, and enable structure determination for drug discovery and protein engineering experiments. Equipment items are also requested to upgrade a specially designed single crystal microspectrophotometer for evaluation of protein oxidation states and active site chemistry in experiments that probe mechanism. The microspectrophotometer is essential to correlate biochemical properties with crystal structures, and will be used in direct conjunction with the new X-ray facility. The new equipment will benefit the projects of all seven principle investigators, who have a long and productive track record of collaboration and shared use of the existing X-ray equipment. The new equipment is cost-effective to maintain, and is manufactured in the United States. The new X-ray facility will provide hands-on training opportunities for graduate students and postdoctoral fellows in crystallographic methods, and will stimulate scientific interactions, enhance the quality of learning and research, and help to retain and create jobs in the academic and biomedical sectors. The new equipment is a long term investment, given that the existing X-ray generator was in use over 20 years, and the existing detector and low temperature device have been in use for 10 years. HEALTH RELEVANCE: Crystallographic experiments require a reliable in-house X-ray facility for high throughput screening of crystal quality and cryoprotection conditions, and for high resolution data collection from many hundreds of protein crystals. The new equipment is required to support these experiments for 19 active NIH grants being directed by seven principle investigators."}
{"text": "According to NCI's Strategic Plan, community-engaged research is vital for understanding cancer-related health disparifies and devising effective, culturally-appropriate strategies for reducing these disparities.^ In the first funding cycle (2001-2006), the MMC-VICC Cancer Partnership lacked a community outreach component and primarily focused on basic and clinical cancer research, with only one clinic-based behavioral research project. In 2006, the Partnership created a new Community Outreach Core (COC) guided by a framework of CBPR'*'^ to further the aims ofthe Cancer Partnership in reducing cancer disparities. Given its strong history of community-based research with minority communifies, TSU was brought in as a subcontractor to co-lead the new COC, under the leadership of Dr. Baqar Husaini (TSU), Dr. Elizabeth Williams (VICC) and Dr. Paul Juarez (MMC), and with Dr. Pamela Hull (TSU). In August 2010, Dr. Wujcik took over as VICC Co-Leader when Dr. Williams left VICC to join the TSU public health faculty."}
{"text": "The long-term objective of this program is to develop an automatic, non- contacting method of irrigating dry eyes. The device will be mounted on eyeglass frames, cosmetically acceptable, and capable of replacing the human tear volume. The specific aims of this first year project are to construct a working prototype with sufficient functionality to test its effectiveness on a small number of severe dry eye patients for up to 30 days. The system components consist of a miniaturized pump, a reservoir for solutions, and an electronic control circuit and battery for programming wetting solution applications."}
{"text": "UCLA and UCSD and their affiliated campuses at Cedars-Sinai and Salk, respectfully, have integrated, highly productive and well-funded programs in diabetes and its complications and in endocrinology. Because of growing interaction and collaboration between Los Angeles-based and San Diego-based investigators, the Southern California Diabetes Endocrinology Consortium (SCDEC) was conceived as a means to formalyze, enhance, and continue to fertilize these relationships. By joining forces and aligning common interests, basic and clinical scientists at both Centers have recognized the enormous potential to strengthen and expand their research opportunities and resources. Thus, the SCDEC DERC represents a critical foundation to consolidate diabetes and endocrinology investigation at two premier academic centers. The objectives of the SCDEC DERC are to: 1) Unite investigators from relevant disciplines in a manner that will enhance and extend the effectiveness of their research in diabetes/endocrinology and the complications of diabetes, 2) Foster collaborations between diabetes/endocrinology researchers at UCLA and UCSD so the whole is greater than the sum of its parts, 3) Enhance, support and develop Cores to facilitate diabetes/endocrinology research at UCLA and UCSD, and 4) Develop new areas of research and foster young investigators focused in diabetes and its complications through pilot and feasibility grants. Herein, the SCDEC DERC presents six exceptionally strong Research Bases comprised of 93 talented investigators supported by six Research Cores. The Research Bases include 1. Nuclear (n=10), 2. Signaling (16), 3. Metabolism (21), 4. Macrovascular (23), 5. Microvascular (14), and 6. Beta Cell (10). The Cores are: A. Transgenic and Knockout Mouse, B. Mouse Phenotyping, C. Transcriptional Genomics, d. Human Genetics, and E. Biochemistry and Molecular Assay. These Cores are designed to specifically meet the greatest needs of DERC investigators. They represent high quality, state-of-the-art and novel technology, which if provided to the SCDEC DERC will bring diabetes and endocrinology research in Southern California to a new level."}
{"text": "Whole body hyperthermia is being studied at NIH as a possible means of treatment for cancer. This project includes development of an instrumentation and control system based on utilization of a Tektronix 31 programmable calculator, digital plotter, and interface for data acquisition. The esophageal temperature of the patient is regulated to 0.1 degree C accuracy by feedback control of the temperature of water circulating in a set of hyperthermia blankets. This project was completed August 1980, however additional publications have been made."}
{"text": "As a result of the advocacy of the benefits of vegetable oil/polyunsaturated fatty acid rich diets in the prevention and amelioration of coronary heart disease and atherosclerosis the consumption of processed fats and oils has increased significantly. These hydrogenated fats contain unusual fatty acid isomers which may, over the long term have deleterious effects on normal lipid metabolism. The primary objective of this initial study is to determine if mixtures of trans isomers of linoleic acid when fed to male rats over a long (6 month) period results in an imbalance of prostaglandins and of their precursors in specific tissues. Initially the effects of linoleic isomers on the relative concentrations of different prostaglandin species, gamma-homolinolenic, and arachidonic acid in serum, stomach and heart tissue will be determined. The stomach is selected because the levels of prostalglandins are higher, which facilitates quantification. It also is sensitive to precursor availability which is helpful in this proposed dietary study. In addition, heart tissue will be carefully monitored to obtain more information on its prostaglandin complement, the effects of dietary fat on the precursors, the activities of prostaglandin synthetase and phospholipase A which may regulate the availability of prostaglandin precursors. Serum will be monitored for PGF2 levels. Male weanling rats (10 per treatment) will be fed increasing levels (0,25,50,100 percent) isomerized safflower oil, replacement in a normal diet containing 20 percent fresh safflower oil. Tissue will be removed at 2,4 and 6 months and analyzed for prostaglandins, their precursors, prostaglandin synthetase and phospholipase A activity to determine the effects of the trans isomers."}
{"text": "Pain remains an important cause of morbidity for frail, older persons in nursing homes. As high as 83% of nursing home residents voice complaints of pain. Often, nursing home residents' complaints of pain are not recognized or not appropriately treated. A substantial scientific evidence base attests to readily available pharmacological and non-pharmacological modalities to treat pain. Research utilizing continuous quality improvement, including a recently completed study by Center for Gerontology and Health Care Research in cooperation with RI Quality Partners, has impacted on nurses' behavior in the assessment, documentation, and the use of non-pharmacological treatment of pain. This study resulted in 33% reduction in pain. However, our intervention did not significantly impact on physician proscribing behavior. Based on debriefing of nurse coordinators and others research, nurse-physician communication and physicians' lack of knowledge are two important barriers to appropriate pain management. In this R21 application, we propose to develop and pilot test a Personal Digital Assistant (PDA) based decision support intervention for pain management in long term care. This decision support will assist the nurses in conducting a pain assessment, provide treatment recommendation(s), and print a summary report to be fixed to the physician office. Existing guide lines from the American Geriatric Society, American Medical Director Association, and AHRQ pain management guidelines as well as the advice of expert panel will be utilized to formulate decision support tool. In the development of this intervention, we will conduct focus groups with end users (long term care nurses and physicians), utilize cognitive testing, and conduct a six month pilot test that will examine the use of the decision support application in two nursing homes in RI (one academic base NH and one community base NH). An evaluation will collect information on the time to decision recommendation, acceptability of the PDA application, whether recommendations were utilized, and the reported level of pain prior to and after the use of decision support. If we are successful in development of this intervention, a R01 research application will be prepared to conduct a block randomized trial of whether this intervention when combined with the rapid cycle continuous quality improvement intervention improves quality of pain management. [unreadable] [unreadable]"}
{"text": "The ocular lens provides an ideal system to study cell cycle regulation during terminal differentiation. The lens is composed of two cell types: the epithelial cells, which are capable of cellular proliferation, and the fiber cells, which are post-mitotic. I hypothesize that the differentiation of epithelial cells into fiber cells will cause alterations in activity of the genes that regulate the cell cycle. In order to help test this hypothesis, the alphaA-crystallin promoter will be used to direct lens-specific alterations in gene expression in transgenic mice. Transgenic and non-transgenic mice will be used to study two general classes of genes that are likely to be critical for cell cycle control during differentiation: tumor suppressors and cyclin-dependent kinases. The Specific Aims of this grant application are: l) to assess the role of the retinoblastoma (rb) protein in cell cycle control and terminal differentiation of lens fiber cells; 2) to assess the role of p53 in the induction of lens tumors by SV4O large T antigen; 3) to assay for changes in cyclin dependent kinases that accompany fiber cell differentiation; 4) to characterize the cell death that is induced by rb inactivation in fiber cells; and 5) to genetically reverse lens tumorigenesis induced by full-length T antigen. Our preliminary experiments have shown that expression of viral proteins that bind to rb and/or p53 can induce lens cell tumorigenesis or programmed cell death. Therefore, the cell cycle can be altered in lens cells with fascinating and unexpected consequences. The proposed studies should provide insights into cell cycle regulation in vivo, not only for the lens, but for other cells undergoing terminal differentiation or neoplastic transformation."}
{"text": "This work is designed to detect changes in gene expression in mouse fibroblasts or lymphocytes transfected with human leukemia cell DNA. Using a combination of 2-D electrophoresis, differential labeling and immunochemical techniques, we will detect gene products in the transfectants that differ qualitatively or quantitatively from products in the parental mouse cells. We will attempt to determine the relationship between these changes and the transforming event - i.e., Are they a direct effect of the incorporation of \"oncogenic DNA\" into the cells, or do they represent changes secondary to the initial transforming event? Are they produced by the human DNA transfected into the mouse cells or by the mouse genome? Do any of these \"new\" gene products produce pleiotropic effects which can account for the variety of phenotypic changes in the transformed cells? What is the relationship of these changes to the differentiated state of the transformed cell? Does the same DNA transfect fibroblasts and lymphocytes, and are the effects the same?"}
{"text": "The long-term objective of this project is to study the components of the ternary complex of antigen, T cell receptor (TCR) and MHC class I molecule whose interaction leads to T helper cell activation structure/function relationships between components of this ternary complex are currently being analyzed using murine T cell clones specific for well defined epitopes on the model protein antigen sperm whale myoglobin (Spw Mb). A panel of T cell clones has been generated from DBA/2 mice, reactive with Spw mb epitopes characterized with overlapping sets of synthetic peptides. The specific aims are as follows: 1) To further characterize the antigen and MHC specificity of these clones and of additional mb clones generated in the mouse strains B10.D2 BALB/c and D2.GD. These studies will include the use of horse myoglobin and substituted peptides to identify residues within the epitope important of interaction with the TCR or restricting Class II molecules 2) To sequences alpha and beta chains from matched sets of clones that shave either epitope specificity or MHC restriction in common, and to compare these sequences with those already derived from independent clones that share epitope specificity and MHC restriction. The relationship between TCR and antigen specificity/MHC restriction will be further addressed by transfection of chosen TCR alpha or beta chain genes into Mb- reactive T cell hybridomas expressing endogenous TCR genes of known sequence; 3) To make use of class II (I-Ed) variants generated by site-directed mutagenesis to analyze the interaction of TCR and antigen with the restricting class II molecule. These experiments should determine whether the same set of TCR genes are used to respond to a particular epitope in association with an MHC class II gene product regardless of the non-MHC background if they do, then this result would support the concept that HLA associated diseases might result form the formation of a particular ternary complex involving a shared MHC restriction molecule, a common \"disease-associated\" epitope, and a shared T cell receptor."}
{"text": "Developmental funds have played an important role in the development of the Cancer Center, particularly in recruiting new investigators and in establishing new shared resources. During the current funding period developmental funds were instrumental in the recruitment of six new investigators and in the development of two new shared resources (Bioinformatics and Proteomics). We propose to continue our recruitment efforts to strengthen the scientific programs of the Cancer Center, and to develop a new shared resource devoted to Chemical and Systems Biology."}
{"text": "The normal function of the brain relies on precise patterns of neuronal connections, and aberrant connectivity leads to human neurological and psychiatric disorders. The human brain consists of approximately100 billion neurons with trillions of synapses. Because of the enormous neuronal diversity and staggering synaptic complexity, very little is known about the molecular mechanisms that lead to the assembly of specific synapses in different neuronal types. Protocadherin (Pcdh) genes (14 Pcdh-a, 22 Pcdh-fi and 22 Pcdh-y in mouse) are attractive candidates for such a role because they can potentially generate a significant number of cell-surface \"codes\" through a combination of cell-specific promoter activation andcis- alternative splicing. It has been suggested that the distinct combinatorial Pcdh expression patterns might specify neuronal types and their connectivity. To evaluate their roles in neural development, we initiated functional analyses of these genes using genetically modified mice. Our analyses on Pcdh-j mutant mice provide the first in vivo evidence that protocadherins are essential for vertebrate CNS development and play an important role in establishing neuronal connectivity. However, Pcdh-y's function during synaptic development is not well defined and its molecular mechanisms of action are completely unknown. We plan to combine molecular and genetic approaches to further our understandingof Pcdh-y's functions.Specifically, we propose 1) to investigate the rules of expression for individual isoforms of Pcdh-y;2) to define the role of Pcdh-y's diversity;and 3) to identify and characterize the signaling components of Pcdh-y. The attainment of these goals will shed light on our understandingof the molecular basis for the precision and complexity of neuronal circuitry in the brain."}
{"text": "The effects of normal aging on cerebral metabolic processes have been studied with methods developed in this Laboratory: the [C-14] leucine method for local rates of protein synthesis and the [ C-14] deoxyglucose method for local rates of glucose utilization. We have found that rates of glucose utilization and protein synthesis both decrease with age in the components of the primary auditory and visual pathways. These changes may be the consequences of a chronic lack of sensory input due to age-related degenerative changes which are known to occur in both retina and inner ear. Our studies also show that glucose utilization is particularly and significantly decreased in the striatum in aged rats. In order to examine the functional consequences of the senescent changes in the striatum we are studying the effects of age on the metabolic responsiveness to apomorphine, a dopaminergic agonist. Aluminum toxicity has been proposed as an etiological factor in Alzheimer's Disease. Aluminum has been shown to affect numerous biochemical processes including several enzymes in the metabolic pathway for glucose. Experiments have been carried out on the effects of prolonged (2 years) intake of low levels of aluminum in drinking water on local cerebral glucose utilization in aged rats. The results of these studies show decreased glucose utilization in the treated rats in 2 out of 26 regions, ventral pallidum and temporal cortex."}
{"text": "The purpose of this study is to identify factors associated with maintenance and change of mental health in the poor, urban, Black elderly using a short-term longitudinal approach. A panel of 150 Black men and women, over 60 years of age, will be contacted at short, regular intervals for repeated psychological, social physiological measurements for a period of three years. The project will be carried out in Newark, N.J. In order to optimize the longitudinal nature of the project, the sample will partly consist of individuals who took part in a recently terminated study on the mental health of the Black aged by the authors of this proposal. Personal and social data on this group are available for 1972 and 1974, and these contain some of the measurements expected to be used with the current population. Variables considered will include physical and mental health, life styles, self concept, life satisfaction and morale, depression, social interaction and isolation, use of available services, environmental conditions, family and friendship networks, need and use of short term and long term care facilities. Analysis of the data will pay particular attention to changes that occur with time. A review panel consisting of both expert and lay members will be consulted with regard to interpretation and health care implications."}
{"text": "The human peptidase clan CE is defined by seven proteases with a common fold and catalytic mechanism, commonly known as SENPs (sentrin specific proteases - sentrin being an alternative name for SUMO). Current information suggests that most SENPs are specific for processing of SUMO precursors and removing SUMO conjugated to protein substrates. Small-molecule compounds that target SENPs in a selective manner will provide an important toolset to study the biology of these enzymes, to aid in the discovery of their role in disease etiology and progression, and to facilitate the subsequent development of therapeutics against disease-relevant targets. The availability of simple kinetic assays that are applicable the SENPs would significantly enhance the discovery of chemical probes for members of the family, and for .orphan. SENPs with lesser known natural substrate specificity. Our application squarely addresses this [unreadable] important unmet need for a development of high-throughput substrate-based assays. We propose to develop small peptide based substrates with broad specificity for all seven human members of the SENP family, and generate simple assay procedures. The assays are expected to provide a tool for the rapid development of chemical probes for members of the family. [unreadable] [unreadable] [unreadable]"}
{"text": "There is a general concern that many environmental chemicals to which humans are exposed to are mutagenic. Several metal compounds have been identified through occupational exposure and laboratory testing as potential carcinogens. Therefore, screening of these agents is of paramount importance. In the proposed study, the ongoing investigations on the influence of heavy metal salts on the induction of chromosome aberrations, sister-chromatid exchange and micronuclei in CHO cells will be performed. Additionally, a detailed study will be conducted on the effect of various arsenic compounds on the above parameters. the student participants will be involved in all phases of the investigation. They will be introduced to the techniques of mammalian cell culture, cytogenetics, and genetic toxicology. Through involvement in MBRS research, the student participants will be motivated to pursue advanced degrees in health-related sciences."}
{"text": "The goal of this study is to elucidate the molecular mechanisms by which lac repressor interacts with lac operator. The lactose operon of E. coli is chosen as a system in which the general principles of regulation of transcription can most easily be studied. Genetic methods will be used to isolate and characterize specific repressor mutants (a) which specifically overcome oc mutations and (b) which are defective in subunit aggregation. One class of repressor mutations which overcome oc mutations and of which some have a largely increased affinity for the operator has been obtained. These repressors will be further investigated. Operator mutations which increase the symmetry of the operator will also be selected, and the effect of these mutations on the repressor-operator interaction will be studied. A secondary binding site for the repressor will be mutated to increase its affinity for the repressor and make it function as an effective operator. My long-range concern is to identify the amino aicd residues of the lac repressor and the base pairs of the lac operator which contribute to the repressor-operator interaction."}
{"text": "Determine the relative importance of different T cell activation components in the induction of HIV replication, and the influence of viral early gene product, Nef, on these required activation events."}
{"text": "Marburg virus (MARV) belongs to the filovirus family and is highly pathogenic in humans. Despite being classified as Category A Priority Pathogen by NIAID, and its potential to cause large-scale outbreaks, similar to the recent Ebola virus outbreak, research on MARV lags significantly behind that on other non-segmented negative sense (NNS) RNA viruses. Here, we propose to perform in-depth analyses of MARV transcription and gene expression. Dissecting the mechanisms of MARV gene expression will not only be instrumental for the targeted development of antiviral drugs, it will also reveal unifying paradigms and distinctions between the NNS RNA viruses. The filovirus genome is transcribed by a virally encoded RNA-dependent RNA polymerase complex, which is capable of generating capped and polyadenylated mRNAs. This process occurs in the cell cytoplasm, close to ribosomes and cellular RNA binding proteins. This project will examine three different stages of MARV gene expression. In Aim 1, we will elucidate the mechanism of transcription initiation at the MARV promoter and investigate the role of structural features of the polymerase in this process. Notably, the MARV promoter sequence has some unusual features, and we intend to explore the functional relevance of these characteristics. In Aim 2, we will determine the function of conserved hairpin loops that are formed at the 5 end of each MARV mRNA. We will explore the effect of these structures on transcription, RNA stability, trafficking and translation. In Aim 3, we will focus on mRNA polyadenylation and release. The mechanism of mRNA release is not well understood for any NNS RNA virus, and the results obtained in this aim will help to broaden our understanding of NNS RNA virus transcription strategies. This proposal brings together expertise in studying NNS RNA polymerases, MARV molecular biology, and mRNA-protein interactions. Together, the research team has established a unique tool set to achieve the goals of this proposal, including a MARV in vitro polymerase assay, various MARV reverse genetics systems, and highly innovative single molecule mRNA-protein binding assays. These studies will shine new light on a crucial aspect of MARV infection and enhance our understanding of NNS RNA virus biology."}
{"text": "This project is being proposed in light of the continuing increase in reported AIDS cases among the Latino population of the United States. In the past two years the number of reported AIDS cases among Latinos has nearly doubled, going from 15,271 in June of 1989 to 29,586 in June of 1991. As HIV/AIDS continues to impact this population it is imperative that we develop a national AIDS research agenda to deal with this particular population. The goal of this project is to establish an organization and planning framework for the development of a national Latino focused HIV/AIDS research consortium. The objectives of this project are: 1) to begin to develop both national and regional networks of Latino AIDS researchers to facilitate networking, information sharing and research collaboration; 2) to identify potential institutions with the capabilities to implement the research activities of the consortium; and 3) to identify individuals to serve on a National Latino AIDS Advisory Committee to provide direction in planning the research consortium. To achieve these objectives the project will establish a Central Planning and Coordination office responsible for: 1) establishing an Executive Committee of Latino AIDS Researchers, 2) identifying institutions, 3) establishing regional linkages in areas with the highest concentration of Latino AIDS cases, 4) producing a national directory of Latino AIDS researchers, 5) hold regional meetings to establish regional networks of Latino AIDS researchers and to provide input on the development of a P20 planning grant proposal for Latino focused HIV/AIDS research."}
{"text": "The mechanism by which cell therapy improves cardiac function remains unclear. Although injected cells minimally differentiate into cardiomyocytes or vessels, and only a small fraction survive long term in the recipient, common beneficial effects are observed regardless of injected cell type: improvement in pump function, reduction of fibrosis, and enhanced angiogenesis. These results are consistent with the idea that cell therapy recruits endogenous repair mechanisms which, however, have not been identified. The immune system has been implicated in a variety of sterile diseases, including processes regulating myocardial damage and repair. Although unresolved inflammatory processes controlled by infiltrating and tissue-resident immune cells worsen heart failure, depletion of macrophages in the infarcted myocardium leads to LV rupture and death. Thus, immune cells appear to play diametrically opposite roles in the heart. Macrophages have been shown to be required for spontaneous regeneration of neonatal mammalian myocardium after injury, and recent findings implicate them as direct contributors to cell therapy-mediated myocardial repair. Nevertheless, how immune cells regulate myocardial repair and what determines their harmful versus salutary actions remains unknown. Furthermore, the impact of cell therapy on reparative immune cells has not yet been studied in the heart. Our preliminary data show that injection of cardiac mesenchymal cells (CMCs) into the infarcted heart promotes accumulation of reparative macrophages. Thus, the central hypothesis of this proposal is that CMCs facilitate recruitment of monocytes and activation of reparative macrophages, which are essential endogenous mediators of repair. By generating detailed flow cytometric analyses of immune cell populations following CMC administration, we will not only resolve the time course of immune cell recruitment, but also determine how inflammation is eventually extinguished in the heart after cell therapy. To elucidate the mechanism whereby CMCs regulate inflammatory processes in monocyte-derived macrophages, we will determine how these cells regulate NF?B-p65 subunit expression, with emphasis on horizontal transfer of miRNAs to macrophages through CMC-derived EVs. Finally, using macrophage genetic fate mapping and genetically modified CMCs, we will elucidate the role of monocyte-derived macrophages in CMC-induced myocardial repair. This project will be the first systematic analysis of how cell therapy modulates immune cells ? a mechanism that has been relatively understudied. The results will provide novel insights not only into the mechanisms regulating cell therapy-mediated myocardial repair, but also into how endogenous reparative activities of macrophages are recruited. We will also determine whether EVs recapitulate the salutary effects of CMCs on immune cells. Thus, these studies have far-reaching implications for our understanding of how the immune system regulates myocardial homeostasis in general."}
{"text": "This revised proposal to develop a mathematical model for the stability of pulmonary airways is a basic inquiry into the mechanisms that lead to airway closure of the lung in health, disease, and its modifications by therapies. Closure results from a surface tension instability that tends to pull the airway to a smaller diameter while potentially driving the liquid lining into the formation of a plug, given the right conditions. A plug blocks the lumen, stopping gas exchange and delivery of aerosol medications. It can be the terminal event in an asthmatic attack, for example. From purely the fluid mechanical perspective, the plug may or may not form depending on the liquid physical properties. Significant non-Newtonian fluid properties in disease (asthma, emphysema, cystic fibrosis) include viscoelasticity, shear-thinning viscosity, and a yield stress, all of which can alter the fluid movement. Surfactants at the air-liquid interface also influence the instability by reducing surface tension and creating surface tension gradients. The lung volume at which closure occurs is the closing volume, and it is a common pulmonary function test that is often difficult to interpret, due largely to inadequate models. This surface-tension/fluid system couples to the airway and lung elastic properties. New to this revision is the inclusion of non-linear airway elasticity, airway smooth muscle dynamics, and effects of a surrounding parenchymal tethering. This novel combination of effects will allow the development of a theory which can incorporate the simultaneous issues presented by diseases such as asthma and emphysema where bronchoconstriction, parenchymal tissue elasticity changes, and fluid property modifications (non-Newtonian fluids) acting together present a complex mechanical situation; The proposed model will provide a platform for predicting the impact of these interactions on closing volumes, as well as predict the effectiveness of therapies which may include those aimed at surfactants, or liquid material properties, or bronchoconstriction or the parenchyma. This proposal outlines a set of mathematical models, building from the simplest to the more complex, that examine the stability of a liquid-lined airway tube as it depends on (1) Newtonian vs non-Newtonian fluids, (2) rigid vs flexible tubes with parenchyma and smooth muscle tone, (3) axisymmetric vs non-axisymmetric deformations, (4) effects of surfactants, (5) effects of a forced, oscillatory core flow. [unreadable] [unreadable] [unreadable]"}
{"text": "We and others have demonstrated that induction of antibodies to the beta-amyloid (AB) peptide endows therapeutic effects in Alzheimer's disease both in pre-clinical and clinical settings. This approach is currently hampered by elicitation of pathological autoreactivity. In order to overcome this problem we have developed several strategies to limit pathological immunity. For example, we generated an epitope vaccine composed of small immunodominant self B cell peptide of AB42 fused with a foreign CD4+Th cell epitope and demonstrated that such vaccine induced high titers of anti-AB antibodies without generation of potentially harmful autoreactive T cells specific to AB. Importantly, these antibodies were therapeutically active, as we showed in two different mouse models of AD (APP/Tg 2576 & 3xTg-AD). After having demonstrated feasibility of selectively inducing a beneficial antibody response to AB in absence of pathological autoreactivity, we decided to expand these studies to a more clinically applicable system. In collaboration with our co-investigator, we have used the influenza virus platform for delivery of immunodominant B cell epitopes of AB42 (AB1-10) into the host. In our preliminary data we have generated a flu-AB1-10 vaccine that induces robust anti-AB and anti-influenza antibodies and reduces AB-deposits in the brains of immune 3xTg-AD mice. Thus, in the first three translational Aims of this proposal we plan to learn about (i) immunogenicity and efficacy of recombinant flu- AB1-10 vaccine in 3xTg-AD mice without AD-like pathology (Aim 1), as well as with early (Aim 2) and late (Aim 3) AD-like pathology. The last Aim 4 of this study is designed to explore immunological mechanism/s of generation of anti-AB antibodies by this vaccine and identify specificity of anti-viral memory Th cells involved in this process. Thus, at the end of this study we will learn about cellular and molecular mechanisms governing the generation of antibodies specific to both AB1-10 and influenza. The long-term goal of this proposal will be a generation of the safe and effective dual (flu-AB) vaccine that may prevent development of AD pathology in pre-symptomatic people, protecting them from the flu infection at the same time. PUBLIC HEALTH RELEVANCE: Alzheimer's Disease is the major cause of dementia in the US and is characterized by an insidious onset and progressive cognitive decline that impacts memory, language, judgment, orientation to time and place, etc. Pathologically there is an increase in the presence in amyloid plaques, neurofibrillary tangles, dystrophic neurites and a general loss of neurons. It was demonstrated that induction of antibodies to the beta-amyloid peptide endows therapeutic effects in Alzheimer's disease both in pre-clinical and clinical settings. This approach is currently hampered by elicitation of pathological autoreactive T helper cells. In order to overcome this problem we and others are developing several strategies to limit pathological immunity. In current project we are proposing to generate the safe and effective dual vaccine, based on influenza viral vector and immunodominant B cell epitope from beta-amyloid peptide that may prevent/reduce development of Alzheimer's disease pathology in pre-symptomatic people diagnosed with early-stage AD, protecting them from the flu infection at the same time."}
{"text": "The longer-term goal of our research effort is to understand why persons with addictions change their social networks in either positive or negative ways, and how important health outcomes and health behaviors affect are influenced by social changes. Our research focuses on persons with addictive disorders because addiction can have strong, even catastrophic, effects on our strongest social ties. On the other hand, there is substantial evidence, including new analyses we present, that social relationships are a strong determinant of addictions outcome. Since addictions are chronic, relapsing disorders, if we were able to promote durable healthy relationships for addicts, these relationships might reduce the need for costly and not always accessible professional services. Unfortunately, we lack a basic understanding of why people change their social relationships, for better or for worse. Our research will illuminate the processes of change in addicts' social networks. We will recruit 300 persons from addictions treatment facilities and follow them naturalistically for two years, gathering intensive longitudinal data on social relationships, substance use, and other variables. Our study has three aims: (1) To determine how a person's relationships are affected by other relationships, and by substance use, co-varying for relationship type and history; (2) To replicate and extend prior research on the effects of social network characteristics on substance use outcome; and (3) To determine social network influences on entry into substance abuse treatment, and mutual-help participation. Cox proportional hazards regressions and hierarchical linear models with time varying covariates will be used to test the a priori predictions laid out i the proposal."}
{"text": "Veterans are disproportionately affected by peripheral artery disease (PAD) and coronary artery disease (CAD) because of high rates of tobacco use in the veteran population. Up to one-third of the saphenous vein bypass grafts performed for either PAD or CAD fail secondary to a process called neointimal hyperplasia. We have established a new model to study saphenous vein grafts by implanting human saphenous veins into immunodeficient nude rats. This model develops significant neointimal hyperplasia, unlike prior animal models using animal vein grafts. Our previous work on human saphenous vein grafts suggest that adventitial cells may prevent neointimal hyperplasia and graft failure. We have also been testing a novel therapeutic agent SB-030, which when administered luminally, decreases the neointimal hyperplasia reaction to angioplasty-induced arterial injury in humans presumably by inhibiting smooth muscle cell proliferation. This proposal will test whether adventitial cells can be used therapeutically to decrease neointimal hyperplasia. In addition, we will test whether inhibiting adventitial cell proliferation by applying SB-030 to the adventitia causes increased neointimal hyperplasia. In contrast, we expect luminal application of SB-030 will decrease neointimal hyperplasia by inhibiting smooth muscle cells. These results will be relevant for future treatment of vein grafts with SB- 030 or other therapeutics that target both adventitial cells and medial smooth muscle cells. Successful completion of this proposal will better establish the new model, demonstrate whether adventitial cells reduce neointimal hyperplasia, and determine whether adventitial or luminal application of a novel therapeutic will best inhibit neointimal hyperplasia. This will lead to new therapies to improve the success of vein bypass grafts for patients."}
{"text": "Osteoporosis is a leading cause of mortality and morbidity in aging women. The mechanisms of phosphate and calcium balance were studied as part of a major effort to understand the pathophysiological bases of senile osteoporosis and osteomalacia. This project represents a continuation and extension of previous projects - Z01 AG 00041-07 LMA and Z01 AG 00042-07 LMA. Notable scientific achievements include: (1) The active form of Vitamin D, 1, 25-dihydroxy Vitamin D3, was found to enhance specifically the sodium gradient-dependent phosphate uptake system in the kidney; (2) Vitamin D was found to have a direct effect on renal membrane phospholipids, resulting in an altered permeability towards calcium; (3) Evidence for a novel parathyroid hormone-independent mechanisms for regulating phosphate reabsorption by the kidney was obtained; and (4) The mechanism by which calcium is actively transported in the kidney was described."}
{"text": "The etiology of atrophic gastritis and the reason for the increased prevalence of gastric carcinoma with atrophic gastritis are not known. It is also not known why certain patients with atrophic gastritis, but not other, develop gastric carcinoma. There is a high prevalence of autoantibody and cell-mediated autoimmunity in patients with pernicious anemia (PA) suggesting that immunologic perturbation may contribute to the pathogenesis of the underlying atrophic gastritis. This possibility has been challenged, at least as it applies to gastric autoantibody, by our earlier work which showed that patients with severe impairment of humoral immunity have a greatly increased prevalence of PA. In the present study, tests for humoral and cell-mediated autoimmunity against gastric antigens will be performed on patients with classical PA, gastric carcinoma and controls with peptic duodenal ulcers. A group of at least 5 patients permits us a unique opportunity to study cell-mediated autoimmunity in patients with severe impairment of antibody responsiveness. Both homologous and hog gastric antigens will be used. Results using normal human gastric juice and gastric juice from patients with PA will be compared. The objectives are to compare manifestations of humoral and cell- mediated gastric autoimmunity. We wish to see if findings recorded with atrophic gastritis differ from those recorded with gastric carcinoma. We also wish to see if severe immunoglobulin deficiency and lack of gastric autoantibody changes results recorded with PA."}
{"text": "The objective of this project is to improve the clinical management of cancer patients in North Carolina. To accomplish this objective, the proposal combines basic research on the patterns of care provided by physicians in different type practice settings for selected disease sites (breast, cervical and endometrial) and the use of that data to encourage the acceptance of current technology in the clinical care provided cancer patients. The project is considered a demonstration cancer control effort involving a limited geographic area in North Carolina and involving only three disease sites. The project will provide the basis for requesting state funding to develop and maintain an onocologic medical education program. Moreover, the project will provide the basis for developing further studies of the patterns of care involving community physicians throughout the state in cooperation with the Cancer Centers at Duke, Bowman Gray and East Carolina University."}
{"text": "Functional inactivation of menin, encoded by the MEN1 gene, causes the inherited multiple endocrine neoplasia type 1 (MEN1) syndrome and some but not all sporadic pancreatic endocrine tumors. Therefore, unraveling molecular events upstream or downstream of menin could point to other causative genes and/or regulatory events responsible for such tumor types. Menin resides in a histone methylating protein complex that trimethylates histone H3 at lysine-4 (H3K4me3), an epigenetic mark for active gene expression. Therefore, we have determined a genome-wide map of menin-dependent H3K4me3 (using ChIP-Seq) and menin-dependent gene-expression program in wild-type (WT) and menin-null mouse embryonic stem cells (ESCs) and in pancreatic islet-like endocrine cells (PILECs), which we derived from WT and menin-null mouse ESCs through in vitro differentiation. We found menin-dependent H3K4me3 specifically targeting the Meg3 gene in mouse ESCs, and all four Hox loci in differentiated PILECs. Gene expression from the Meg3 locus and from all of the four Hox loci was abolished in menin-null cells. Both Meg3 and Hox loci have been implicated in MEN1-like sporadic tumors: MEG3 in pituitary tumors, and HOX in parathyroid tumors. Our data suggest that these genes with menin-dependent H3K4me3 could be relevant players in the tumorigenesis of endocrine cell types associated with MEN1. Furthermore, our work shows that menin-null mouse ESCs could also be differentiated in vitro into islet-like endocrine cells, underscoring the utility of menin-null ESC-derived specialized cell types for genome-wide analyses studies. Our current efforts are directed towards understanding the regulation and activity of genes at the MEG3 and HOX loci. We have shown that cyclin-dependent kinase inhibitors (CDKIs) of the INK4 family (4 genes) and the Cip/Kip family (3 genes) that negatively regulate cell cycle progression and cell proliferation have rare germline or somatic mutations in endocrine tumor states related to MEN1. Also, mouse models show an endocrine neoplasia phenotype in 'knock-in' mice homozygous for the CDK4-R24C mutation, or by the combined loss of two different CDKIs, p18 and p27. Therefore, understanding the molecular basis of CDK and CDKI regulation could provide insights into their contribution to endocrine tumorigenesis. We have investigated the contribution of cell cycle regulators in endocrine tumorigenesis, particularly mutations in CDKI genes. We are interested in investigating the molecular basis of cell cycle regulation in endocrine cells."}
{"text": "The long-term objective of this project is to characterize the molecular machinery responsible for eliminating misfolded, neurotoxic proteins so that they may be exploited for therapeutic intervention for neurodegenerative diseases such as spinobulbar muscular atrophy (SBMA). SBMA is a hereditary disease characterized by progressive loss of motor neurons in the brainstem and spinal cord. SBMA is caused by trinucleotide (CAG) repeat expansion in the first exon of the androgen receptor gene leading to polyglutamine expansion in AR protein. Like other polyglutamine diseases, SBMA is characterized by accumulation of misfolded protein aggregates in degenerating neurons. Histone deacetylase 6 (HDAC6) is a microtubule-associated deacetylase with intrinsic polyubiquitin-binding activity. We recently determined that over-expression of HDAC6 rescues degeneration in SBMA flies, flies with proteasome mutations, and other fly models of neurodegenerative disease. HDAC6 rescues degeneration by facilitating the degradation of aberrant, ubiquitinated protein by the autophagy-lysosomal system. In this application, we will (1) test specific hypotheses regarding the molecular mechanism whereby HDAC6 facilitates autophagic degradation of aberrant protein, (2) determine the therapeutic potential of HDAC6 in mammals, and (3) use the power of fly genetics to gain unanticipated insights into the role of HDAC6 in cellular management of misfolded protein stress. PUBLIC HEALTH RELEVANCE: Histone deacetylase 6 contributes to elimination of misfolded proteins from neurons and protects against neurodegeneration. It is not known how HDAC6 functions. This application seeks to understand the mechanism of HDAC6 function so that it may be exploited for therapeutics development. [unreadable] [unreadable]"}
{"text": "Dementia, with its most prevalent subforms Alzheimer's disease (AD), dementia with Lewy bodies (DLB), and vascular dementia, is a very substantial medical and societal problem. The emergence of many drugs and drug candidates that interfere with the disease mechanism of specific types of dementia makes it crucial to distinguish early between the various forms. Useful diagnostic tools will make it possible to direct patients to the most appropriate drug regimen, to avoid exposure to potentially harmful drugs, and to monitor the efficacy of disease-modifying drugs. Brain imaging techniques provide a non-invasive tool for differential diagnosis and monitoring of disease progression of dementia. We and others have been developing radiopharmaceuticals that allow detection of A amyloid deposits typical for AD. Our efforts have focused on 18F-labeled compounds for PET imaging, with the goal that the imaging diagnostics can be made widely available at minimal costs. We now propose the clinical development of a new radioligand for the differential diagnosis of DLB from AD as well as the monitoring of DLB disease progression. 18F-AV-133 is a highly selective ligand for the vesicular monoamine transporter VMAT2, believed to be the best marker for the functional integrity of dopaminergic neurons which are known to degenerate in DLB. Our proposal tests that hypothesis that imaging of VMAT2 provides a useful biomarker for dopaminergic neuron degeneration, which can be synergistically combined with amyloid plaque imaging to generate a powerful diagnostic work up for classification of dementia patients. We propose to conduct two clinical studies to establish safety and proof-of-concept for 18F-AV-133 as a diagnostic imaging agent for DLB. PUBLIC HEALTH RELEVANCE: Successful completion of the proposed phase I studies will provide a key step towards the provision of diagnostic tools for the clinical diagnosis and monitoring of disease progression of dementia with Lewy bodies (DLB), Alzheimer's disease (AD) and dementias in general. The studies will identify a diagnostic tool to allow the physicians to direct patients towards the most appropriate drug regimen and prevent that the patients are exposed to potentially harmful drugs. Furthermore, the resulting diagnostic tools will help significantly in the development of disease-modifying drugs, by making it possible to monitor treatment efficacy at the molecular level in vivo. Such a diagnostic tool will represent a significant technological and medical benefit to society."}
{"text": "The candidate has a primary interest in investigating the associations between neighborhood socioeconomic (SES) environments, behavioral and psychosocial risk factors, and cardiovascular disease (CVD). The mentored phase will include coursework in Geographic Information Systems (GIS) and complex multilevel modeling, as well as data linkages prior to the independent phase project. The independent phase will establish the candidate's independence while enabling skill development in GIS and multilevel modeling. Data on nearly 90,000 women from the Harvard-based Nurses'Health Study, a well-established cohort, and nationally-representative data on approximately 262,000 men and 314,000 women from the National Health Interview Survey, will be analyzed using multilevel discrete-time survival analysis models to estimate the relations between neighborhood SES and risks of non-fatal and fatal coronary heart disease (CHD), and to test for the presence of behavioral and psychosocial mediators. Furthermore, GIS methods will be used to assess whether some of these mediators may be determined by particular neighborhood services and amenities. By contributing to the knowledge base on the neighborhood determinants of and pathways to CHD, the project's efforts may ultimately translate through interventions into more effective reductions in CVD burden among Americans. This award should enable the candidate to pursue a successful career in the study of the neighborhood determinants of CVD."}
{"text": "The Center for Botanical Lipids and Inflammatory Disease Prevention is a new version of a P50 at Wake Forest University Health Sciences that has been continuously funded since 2005. Research over the past 50 years has shown that both systemic and localized inflammation play an important role in the onset and progression of destructive diseases such as cardiovascular disease, metabolic syndrome/diabetes and allergic asthma. The overall goal of this Center is to delineate the molecular mechanisms by which botanical oils prevent or impact these diseases with a particular focus on immunity and inflammation. The central hypothesis of the Center is that medium chain poly-unsaturated fatty acids (PUFAs) in botanical oils and their metabolites impact several key mechanisms (including altering pro-and anti-inflammatory mediator production and blocking inflammatory gene expression) that inhibit inflammatory processes. A secondary hypothesis is that that the metabolism and effectiveness of PUFA-based botanical dietary supplements is strongly associated with genetic polymorphisms in the fatty acid desaturase (FADS 1-3) cluster on chromosome 11 in a region known as 11q12-q13. This chromosomal region has repeatedly been linked to pro-inflammatory conditions. This Center brings together investigators from five internationally-recognized lipid groups and a world-renowned human genomics center to examine cellular and molecular mechanisms by which PUFAs within botanical oils impact human health. Selected botanical oils and combinations (including olive, flax seed, borage seed and echium seed oils) found in supplements are used to test key hypotheses. Projects 1 and 2 examine the mechanisms leading to the pleiotropic effects of botanical PUFAs on macrophage/ monocyte activation, inflammatory states and eicosanoid generation related to atherosclerosis and asthmatic inflammation, respectively. Project 3 examines the critical role genetic variations in the FADS cluster plays in determining PUFA levels and investigates how specific variations in that cluster are associated with the effectiveness of PUFA-based botanicals supplements in metabolic syndrome/diabetes. The interactive and synergistic Projects and Cores have a strong, contemporary and translational scientific basis and should allow this scientific team to identify additional mechanisms and identify human individuals and populations that are most likely to be affected by PUFA- based botanical supplements. PUBLIC HEALTH RELEVANCE: Thirty-eight percent of adult Americans are using complementary and alternative medicine modalities. The most commonly used modalities are natural products enriched with PUFAs (37 % of natural products). The key purpose of this Center is determine the molecular mechanisms by which medium chain PUFAs in botanical oils prevent of complex diseases such as CVD, asthma or metabolic syndrome and to determine the populations where they are most likely to be effective."}
{"text": "The contact of whole mammalian blood with polymeric material results in rapid deposition of a film of plasma proteins. Protein deposition is followed by a complex series of events involving platelet activation and aggregation and blood transport of clotting factors and additional cellular elements to growing thrombi. Recently we have developed a sensitive in-vivo technique by which dynamic initial deposition profiles of radio-labeled platelets and protein fractions on polymeric arteriovenous shunt surfaces can be measured. Small amounts of platelets and proteins of interest are tagged with radio-isotopes and injected into canine subjects. The radiolabeled platelets and proteins adsorbed to the walls of femoral A-V shunts are detected in order to determine the platelet deposition profile and the composition of the adsorbed protein layer. Experiments using electron microscopy and x-ray photoelectron spectroscopy and immunolabeling of proteins in the adsorbed layer are planned to characterize the nature of protein-formed element interactions in thrombosis. We propose to study conventional medical polymers and additional polymeric substances thought to be superior in thromboresistance. In addition to the major protein fractions of blood consisting of albumin, gamma-globulin and fibrinogen, the thrombogenic characteristics of fibronectin, von Willebrand factor, alpha2 macroglobulin and high molecular weight kininogen pre-adsorbed on test surfaces will be studied. Adsorption profiles of each protein fraction will be determined. These measurements will provide insight into the mechanism of blood response to a variety of surfaces in-vivo, how the adsorbed protein layer interacts with the formed elements in blood and whether other minor protein fractions play a role in the blood-material interaction."}
{"text": "Protein drugs have been approved for many therapeutic indications and represent a rapidly growing segment of the pharmaceutical industry. However, many approved protein drugs and candidates in development fail to reach their potential efficacy due to suboptimal pharmacokinetic properties and immunogenicity concerns. These properties include short circulating half-lives, short shelf lives, low solubility, rapid kidney clearance and susceptibility to proteolytic degradation. The modification of proteins with hydrophilic chemical polymers like polyethylene glycol (PEG) is a clinically validated approach to addressing these limitations. However, the challenges associated with chemical modification procedures required for the attachment of these polymers present significant challenges. Our ultimate goal is to generate amino acid sequences that mimic the physiochemical properties of hydrophilic chemical polymers like PEG. Our proposal is based on the observation that glycine rich sequences (GRS) which contain few hydrophobic amino acids will not fold into compact 3-dimensional structures but will adopt random conformations with large hydrodynamic radii similar to PEG. We hypothesize that they will confer similar pharmacokinetic improvements when attached to therapeutic proteins. These sequences can be attached to proteins using conventional recombinant technology and thus completely obviates the need for chemical modifications steps. We have synthesized a 198 amino acid glycine rich sequence based on sequences that occur in human proteins. We aim to express this protein and systematically test its physiochemical and biological properties relevant to pharmacokinetic enhancement. Our specific aims are: 1) Produce 5 mg of a purified GRS protein in E. coli expression system for downstream characterization and studies. 2) Characterize serum stability, protease resistance and biophysical properties of the GRS protein. 3) Characterize plasma pharmacokinetics and immunogenic potential of the GRS protein in animal models. In Phase II, we will perform detailed optimization of GRS for high-level expression, plasma half-life extension and reduced immunogenicity. We aim to advance optimized GRS which are fused to pharmaceutically active proteins like interferon-alpha or G-CSF into animal and clinical studies. Our ultimate goal is to validate and make this method readily applicable to therapeutic proteins. Our project aims to generate glycine rich sequences that can be attached recombinantly to therapeutic proteins to improve their pharmacokinetic properties. This approach would circumvent the difficulties associated with the modifying proteins with hydrophilic polymers like PEG. [unreadable] [unreadable] [unreadable]"}
{"text": "This research proposal, submitted in response to NIH RFA HG-03-004 (Technologies to Find Functional Elements in Genomic DNA), describes the development of a novel assay technology to begin to unravel the complexities of alternative RNA splicing. The proposal seeks to develop a means of measuring, in parallel, all predicted exon-specific mRNA sequences coded by the 30MB ENCODE targeted genomic sequences for their presence in a given population of mRNA and their contextual linkage to other exons. These studies are critical to understanding how the human genome is transcribed and translated into an enormous repertoire of molecular diversity from an unexpectedly small set of identified human genes. [unreadable] [unreadable] The proposed assay, termed an \"Exon-Linkage Assay\" will combine a variety of technological innovations to the high-throughput characterization of the human spliceome through the use of a strategy of dual, internal-primer mediated double-stranded cDNA synthesis on a spatially resolved solid phase. The assay leverages a highly adaptable DNA microarray synthesis technology to provide an RNA splicing analysis tool that would be labor or cost-prohibitive by other means. [unreadable] [unreadable] The described method has the potential to quickly identify differential pre-mRNA splicing patterns in different tissues as well as shifts in splicing (caused either by regulatory defects or genomic mutations) associated with human disease in a rapid, scalable assay platform. [unreadable] [unreadable]"}
{"text": "The broad objective of this proposal is to identify effective culturally sensitive behavioral interventions by nurses to reduce the risk of sexually transmitted HIV infection and AIDS among inner-city Black women. AIDS is the leading cause of death among Black women of childbearing age in New Jersey. A related problem is pediatric AIDS cases. More than one-half of the reported cases of pediatric AIDS in New Jersey have occurred among Black children. To reduce the spread of HIV infection among women of childbearing age and the associated perinatally acquired infections f children, efficient and cost-effective theoretically based behavioral interventions are needed. Nurses are key health professionals in the effort to combat AIDS, and interventions by nurses in the health-care setting may maximize resources. There are no published studies that tested AIDS risk-reduction interventions on women of childbearing age. The subjects in the proposed experiment will be 720 Black women of childbearing age attending the Family Planning Clinic of a Women's Health Center serving a low-income community in Newark, New Jersey. The approach draws on Bandura's social cognitive theory, Fishbein and Ajzen's theory of reasoned action, and the applicant's previous AIDS risk-reduction research with inner-city Black populations. The subjects in the proposed research will be randomly assigned to a social cognitive intervention designed to increase perceived self- efficacy and favorable outcome expectancies regarding condom use or to one of two control conditions: an informational intervention designed to increase AIDS knowledge or the standard clinic treatment of brief one- to-one AIDS prevention counseling. All interventions will be implemented by nurses. Repeated measures ANOVA, planned contrasts, and multiple regression will be used to test experimental effects immediately postintervention and at 3, 6, and 12 month follow-ups. The outcome variables include self-reported sexual behavior, condom-coupon redemption, and clinically documented sexually transmitted infections. Other measures include theoretically relevant variables hypothesized to mediate intervention effects, including AIDS knowledge, self-efficacy, outcome expectancies, and intentions. Analyses on these variables will address the important theoretical question of why the interventions have effects on outcome measures. Analyses will also examine intervention effects are systematically different depending on participant characteristics, including age, relationship status, and self-esteem. The results of this project will contribute to the development of efficient and effective risk reduction programs for inner-city Black women of childbearing age."}
{"text": "Protein purification is a vital and expensive step in biomedical research and in the development and manufacturing of therapeutic proteins. Unfortunately, affinity methods, which are at the heart of most protein purifications, often present a bottleneck in the separation process because of slow diffusion of proteins in the pores of chromatographic gels. Protein-absorbing membranes can overcome this challenge because convective flow through membrane pores provides rapid mass transport to binding sites. Such flow can also effectively remove undesired proteins to increase purity. However, membrane absorbers are not widely used for protein isolation because they have low protein-binding capacities. The aim of this work is to modify membranes with functional polymer brushes to increase protein-binding capacities by an order of magnitude and enable rapid, selective protein purification. Additionally, properly designed brushes will be resistant to nonspecific adsorption and provide new methods for purification of \"sticky\" proteins that are not amenable to column-based purification. This research will involve synthesis and characterization of polymer brush-modified membranes that bind histidine6- and glutathione S-transferrase-tagged proteins with minimal nonspecific adsorption. Preliminary results demonstrated purification of a histidine6-tagged protein in a cell extract, with a purity that greatly exceeds similar resin-based purification. Future work aims at developing brush-modified membranes in polymeric supports with pore sizes that will allow large increases in permeability. This will permit the use of lower pressures and thicker membranes with much higher capacities. Formation of such membranes will require both development of new synthetic methods that are compatible with polymer supports and growth of thicker brushes that rapidly bind more protein. Hence, protein binding and non-specific adsorption will be examined as a function of brush thickness, composition, density, and functionalization, and membrane composition and geometry. With new membranes in hand, a variety of tagged proteins expressed in E. coli will be purified including SNAP-50, human MIP synthase, and SNAPc complex. MIP synthase is vital for inositol biosynthesis, while SNAP proteins are critical in gene expression. Additionally, SNAP-50 provides an example of a sticky protein that cannot be purified with typical affinity gels. PUBLIC HEALTH RELEVANCE: This research will yield rapid, inexpensive methods for isolating remarkably pure proteins. Such techniques will be crucial in production of therapeutic proteins as well as research studies aimed at isolating proteins to understand their structure and health- related function. [unreadable] [unreadable] [unreadable]"}
{"text": "Unlike most chronic renal diseases, in which the female gender is a protective factor, this \"female advantage\" is lost in diabetes as diabetic females develop more renal disease than non-diabetics;we propose that this is due to low levels of plasma estradiol (E2) in diabetic females. The findings suggest that estrogens (including E2 and its metabolites, such as 2-methoxyestradiol (2-ME)) contribute to the pathophysiology of diabetic renal disease. Based on our preliminary data, we hypothesize that: Specific Aim 1: E2 regulates the renal renin-angiotensin system by reducing the expression and activity of the RAS: specifically by 1. tonically downregulating AT1Rs, 2. decreasing Ang II and renin levels, 3. increasing AT2Rs and consequently attenuating the renal functional and structural changes (in particular vascular changes) associated with diabetic nephropathy. Specific Aim 2: E2 and an estrogen metabolite 2-methoxyestradiol (2-ME) attenuate oxidative stress associated with diabetic nephropathy by reducing NADPH oxidase activity and NADPH oxidase-induced O2.- generation in the diabetic kidney. Specific Aim 3: E2 and 2-ME attenuate inflammation associated with diabetic nephropathy by reducing 1. Acute inflammation and markers of the acute phase inflammatory reaction: IL-6, MCP-1 and M-CSF;2. Chronic inflammation: vascular permeability, migration and infiltration of inflammatory cells in target tissues;3. glucose and AT1R-mediated Ang ll-induced NF-kB expression in target cells and resultant 4. NF-kB- induced TGF-b protein expression and subsequent activation of the Smad signaling pathway. Specific Aim 4: E2 and 2-ME attenuate glomerulosclerosis and tubulointerstitial fibrosis associated with diabetic nephropathy by 1. Reducing hyperglycemia/Ang ll-induced cell growth and ECM protein synthesis, namely collagen type I and IV, laminin and fibronectin, 2. Increasing ECM protein degradation, by increasing the activity and expression of matrix metalloproteinases, MMP-2 and MMP-9. The findings from these studies will provide insight into the mechanisms by which estrogens contribute to the pathophysiology of diabetic nephropathy and may stimulate novel ideas for developing gender-specific treatment modalities for diabetic nephropathy."}
{"text": "Helicobacter pylori (Hp) gastric infection is a common and serious infectious disease of humans. Current therapeutic regimens, while successful in 80 percent of the instances of infection, are complicated by expense, duration of treatment, emergence of resistant strains of Hp and lack of patient compliance. An ideal alternative to antimicrobial therapy is prophylactic or therapeutic vaccination for Hp. In this phase I SBIR, we will test the hypothesis that proteolytic digests of Hp incorporated into a novel adjuvant formulation will prevent manifestations of bacterial gastritis and promote gastric bacterial clearance in Hp-infected gnotobiotic piglets, an established animal model of this disease. Piglets will be parenterally immunized with Hp digests prior to and after established gastric infection. Evidence for protection (reduced/absent bacterial cfu, local and systemic immunity, etc) will be sought. Protection will be correlated to patterns of Western blot immunoreactivity of convalescent immune sera and isolated leukocyte in vitro responses to putative bacterial antigen(s). Reactive peptides will be isolated by HPLC, sequenced and identified. From these data, the feasibility of parenteral vaccination for Hp will be determined in this nonrodent model of bacterial gastritis and a phase II SBIR will be prepared if the protection data are positive. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE"}
{"text": "This competitive renewal investigates the mechanisms and significance of dopaminergic (DA) and serotonergic (5HT) neurotransmission systems in Tourette's syndrome (TS) using PET neuroreceptor imaging which should provide a more complete understanding of pathophysiology and possible treatments. Our recruitment strategies have focused on completing our original DA and 5HT measure sample size, originally funded for only 45% at Year 1 of this R01. We now request funds to complete this sample size given that we have substantial progress including (1) sustained elevations of dopamine release (DArei) with a new PET scanner which is a reproduction of our published paper, (2) demonstrate reductions in SERT in our TS subject, (3) uniquely carrying out amphetamine induced DArei in TS but also pre synaptic DA transporters (DAT) and receptor density (D2Bmax) in the same individuals, (4) similarly also studying both serotonin transporters (SERT) and 5HT2A receptors in the same subjects. Completion of the sample will determine what the inter-relationships are between these pre, post and intra synaptic measures for dopamine and for serotonin transporters and postsynaptic receptors in TS. In addition to our findings in TS alone, we have found an interesting contrast between TS subjects with and without co-morbid OCD (TS + OCD). In particular we found that TS + OCD had elevated DArei compared to TS - OCD and both elevated compared to controls. We found a significant decline in SERT in midbrain in 9 TS + OCD vs. 2 TS - OCD with trends for elevations for TS + OCD vs. TS - OCD for 5HT2A. In 3 subjects who had both DA and 5HT measures, we found correlations between SERT, DArei and 5HT2A which along with the DA and 5HT group differences were suggestive of the co-morbid effect of OCD in TS. This suggested that lower intrasynaptic 5HT resulted in elevated DArei and 5HT2A and lower SERT in these subjects. Further evaluation in AIM 4 with both DA and 5HT measures will help us directly test the hypothesis about the role of 5HT in modulating our tonic/phasic DA model of TS by additionally incorporating features of OCD. At the end of this renewal, we will have completed our originally funded AIMS and examined new possibilities of 5HT and DA interrelationships and role of co morbid OCD and TS. [unreadable] [unreadable] [unreadable]"}
{"text": "With the recent availability of radioimmunoassays for both angiotensin I and angiotensin II, as well as for plasma aldosterone, the problem of screening large numbers of patients with essential hypertension to determine the prevalence of normokalemic primary aldosteronism should be considerably easier. We have proposed that, because of these advances, it may become possible to screen hypertensives on the basis of a single blood specimen upon which determinations for plasma renin activity and aldosterone would be carried out. Since such values are greatly influenced by (1) salt intake, (2) recumbent versus upright posture, and a circadian rhythm, we have been attempting to determine the optimum time and conditions in which a single blood specimen from patients with primary aldosteronism will give the greatest variation from healthy subjects studied under the same conditions. Nineteen patients with subsequently proven primary aldosteronism have now been studied in detail to determine these variables. The results will be applied to unselected patients with essential hypertension. With the use of I131-19- iodocholesterol intravenously, we have developed an adrenal photo-scanning procedure which has detected, preoperatively, aldosterone-producing tumors as small as 1 cm in diameter. With a dexamethasone-modified radiocholesterol scanning procedure, we have been able to distinguish aldosterone-producing tumor from 'idiopathic' aldosteronism (bilateral hyperplasia). Much more work is required before we will know the accuracy with which this distinction can be made. This is important because, to date, a preoperative distinction of these two groups has been virtually impossible. An in-depth study of a case of 'primary reninism' (renin-producing juxtaglomerular cell tumor) has provided new insights into some aspects of the physiology of the renin-angiotensin- aldosterone system in man and provides new directions for further research in our area of interest. We continue to study the relationships between serum electrolyte abnormalities, carbohydrate tolerance, plasma insulin and plasma proinsulin."}
{"text": "DESCRIPTION (provided by candidate): The mammalian olfactory bulb is an ideal system to study general principles of sensory coding. Olfactory bulb circuits convert simple monotonic sensory input from olfactory receptor neurons into complex spatiotemporal patterns of mitral cell activation that are relayed to higher order brain centers. Very little is known about how the olfactory bulb produces this transformation. This application seeks to investigate the intrinsic electrophysiological properties of mitral cells-the principal output neurons of the olfactory bulb-and analyze how these properties pattern mitral cell responses. Using electrophysiological approaches, we will address two key questions: 1) what are the kinetic properties and molecular identity of slowly inactivating K+currents in mitral cells; and 2) how do recurrent and lateral synaptic inhibition interact with intrinsic currents in mitral cells to modulate spike timing and action potential propagation? We expect that the results from these experiments will shed important light on how the olfactory bulb processes sensory information, unveil general principles of how local circuits in the brain interact to perform computational tasks, and aid in the development of paradigms for therapeutic electrical stimulation to treat neurological diseases."}
{"text": "In March 2007, professional societies representing primary care disciplines endorsed the \"Joint Principles of the Patient-Centered Medical Home (PCMH)\". Consequently, the National Committee for Quality Assurance developed a tool to recognize practices as PCMHs. The tool emphasizes practice structures with a strong emphasis on information technology. It is unclear that these practice structures are associated with the actual provision of primary care characteristics, the theoretical foundation of a PCMH, or patient outcomes. PURPOSE: This career development proposal will 1) examine the correlation between the PCMH recognition tool and a tool measuring the evidence-based features of primary care, and 2) identify the factors underlying each of the tools, and 3) assess the correlation between the PCMH recognition tool and a tool measuring the evidence-based features of primary care with clinical outcomes. This project will lead to an R01 application to develop an improved tool for PCMH recognition, incorporating elements of the original tool correlated with primary care and with improved clinical outcomes. CANDIDATE: The candidate is an assistant professor in a research position jointly appointed in the Department of Family Medicine and the Department of Pediatrics and Human Development at the Michigan State University College of Human Medicine. TRAINING GOALS: The goals of the training are to 1) gain didactic training in health services systems theories and practice and 2) apply the theories to applications of health services research to identify features of the current PCMH recognition tool correlated with evidence-based features of primary care and improved clinical outcomes. RESEARCH METHODS: The research methods include a correlation analysis of total scores of and domains in the NCQA PPC-PCMH and the PCAT tools, a factor analysis of each tool, and correlation of each tool with clinical outcomes at the practice level. OUTCOMES: The outcomes include a greater understanding of the correlation of PCMH recognition tool with primary care attributes and the relationship of each to improved clinical outcomes. BENEFIT: The results will be used to develop policy to appropriately classify practices as PCMHs and to inform reimbursement strategies for PCMHs based on patient outcomes. The work resulting from this study will provide an understanding of the correlation between the current tool intended to recognize clinical practices at patient-centered medical homes (PCMHs) and the evidenced-based features of primary care and patient outcomes. The results will inform policymakers on the utility of the current PCMH recognition tool in appropriately classifying practices as PCMHs and inform payers on the utility of the current tool in identifying practices with better patient outcomes"}
{"text": "Toxicity is a major problem in the chemotherapy of cancer. The existence of circadian fluctuation in host toxicity is well established from work in chronopharmacology and chronotoxicology. There are significantly different responses to drugs at different times along the 24-hour day, i.e., hours of changing responsiveness or susceptibility. Can knowledge of rhythmicity in over-all host toxicity and of the rhythmic fluctuation in cell division of normal and tumor tissues be effectively used to treat tumor-bearing hosts with maximal toxicity to the tumor and minimal toxicity to the host's \"normal\" tissues? Our main objectives have been and still are: (1) To identify susceptibility-resistance rhythms to four diverse chemotherapeutic agents (Adriamycin, actinomycin-D, cyclophosphamide and cytosine arabinoside); and (2) To quantify in three groups of mice the rhythmic fluctuation in 3H-thymidine incorporation into the DNA of bone marrow, thymus, spleen, gut and liver, and to similarly analyze the mitotic index of liver and corneal epithelium. One group will have adenocarcinoma of the breast, another the Lewis lung carcinoma and the third will have L1210 leukemia; each group will be compared with non-tumor-bearing controls. The rhythmicity of DNA will be expolred for all tumors, and the effects that tumors of different ages (for each of the three types) have on the variables under study are being determined. Using the data obtained from objectives 1 and 2 above, we have as objective 3: To use chronotherapy to attack tumors by employing the different agents either singly or in combination. To date we have been able to demonstrate significant advantages of timed treatment by utilizing different protocols designed to give the highest levels of drug when the host is least susceptible and the lowest levels when most susceptible. The advantages demonstrated for mice include an increase in survival time, and in several studies it was shown that chronotherapy could significantly increase cure rate in L1210 leukemia in mice over conventional therapy. The potential for further increasing these advantages is promising."}
{"text": "Actin filaments and microtubules are dynamic cytoskeletal polymers that have important roles in cell physiology and determine cell morphology. The complex cellular morphology of neurons is directly related to the function of neurons in the nervous system. Thus, neurons provide an excellent model system for investigating the dynamics of the cytoskeleton as it relates to cellular functions. Actin filaments are of fundamental importance to the development and maintenance of connectivity patterns between neurons, and the formation and function of synapses. Neurons constantly deliver new cytoskeletal proteins synthesized in the cell body to the distal axon through axonal transport. The process of axonal transport is required for maintenance of a functional nervous system and is impaired in a variety of disease states. Filopodia are slender finger-like cellular projections that are strictly dependent on actin filaments and are required for axon guidance and synapse formation. Understanding the mechanisms responsible for filopodial initiation is thus of direct relevance to the understanding of regeneration and the maintenance of nervous system function. This proposal presents studies aimed at (1) determining the form (e.g., filaments versus monomers) of actin transport in axons, and (2) determining the actin-based mechanism of filopodia initiation. The form in which cytoskeletal proteins are transported is controversial. From in vitro studies using neurons transfected with EGFP-actin, we provide preliminary descriptive evidence suggestive of the transport of actin filaments and experimental evidence in favor of the transport of monomeric actin. A series of studies is detailed to experimentally test the hypothesis that actin filaments are the form of actin transport. Additionally, based on live imaging of EGFP-actin in axons and growth cones we have identified the presence of spontaneously formed actin puncta that serve as precursors to filopodial formation. These observations provide a model system for investigating the dynamic cytoskeletal basis of filopodial formation in living neurons. We present studies aimed at determining the role of extra-cellular signals, signaling pathways, and microtubules in regulating the earliest events in filopodial initiation."}
{"text": "These studies are designed to elucidate how changes in extracellular pH influence contractile activity in porcine coronary and carotid arterial smooth muscle. Changes in extracellular pH will be produced by simulating respiratory acidosis and alkalosis and by simulating metabolic acidosis and alkalosis. The effects of changes in extracellular pH on fast and slow components of contractile events elicited by a variety of stimuli (norepinephrine, serotonin, angiotensin, KCl and electric field stimulation) will be analyzed using kinetic approaches. Changes in intracellular pH, calculated by the DMO technique, will be evaluated as a function of extracellular pH, passive stretch, and active generation of force. Studies at each extracellular pH will be performed at different concentrations of Ca ions in the external medium. Ca ions-sensitive actomyosin will be isolated from the arterial smooth muscle preparations, and the extent to which pH modifies Ca ions-sensitive actomyosin-ATPase activity and superprecipitation of actomyosin will be assessed. Thus the effects of extracellular pH on arterial smooth-muscle will be evaluated at the tissue (mechanical responsiveness), cellular (intracellular pH) and molecular levels (actomyosin interactions). These basic studies, integrated at three levels, will reveal how changes in H ion may modulate contractile activity of arterial smooth muscle. In addition to the intrinsic interest regarding the regulation of arterial smooth muscle, the studies bear directly on important clinical settings associated with acid-base imbalances such as circulatory shock and myocardial infarction. Thus, information gained through these studies could be useful in defining how local myocardial acidosis associated with localized cellular damage will influence the responsiveness of the coronary vasculature to therapeutic interventions."}
{"text": "DESCRIPTION: Occupational asthma (OA) is currently the most frequently reported diagnosis of work-related respiratory disease in developed nations. The prevalence of work-related asthma and work-related wheezing in the U.S. is 3.7% and 11.5% respectively and the proportion of asthma attributed to occupational exposures varies [unreadable] from 28.5% to 36.5%. In the U.S. approximately 3% of the working population reportedly works in the cleaning occupation. Several studies, primarily from the European countries, have found an association between occupational exposures and cleaners. However, the major limitations of these studies were non-inclusion of the work aspect in formulating the definition of asthma as the outcome variable, and poor characterization of exposures. We propose to conduct a focus group study to 1) identify an appropriate [unreadable] population of domestic and industrial cleaners; 2) determine the feasibility of contacting and tracking representative groups of domestic and industrial cleaners, for use in future studies of asthma in this worker group; 3) characterize occupational exposures among domestic and industrial cleaners; 4) use information obtained from the focus groups to develop a Job Exposure Matrix that could be used in future studies of asthma in this worker group; and 5) develop and pilot test an English and Spanish version of a survey instrument for use in future studies of asthma among domestic and industrial cleaners. A total of twelve focus group interview sessions (six sessions involving domestic cleaners and six involving industrial cleaners) are proposed for this study. Approximately 12 people (with the aim of 6-10 people attending), between the ages of 18 and 70 and working as a cleaner for at least one year, will be invited to participate in each session. Each session will be moderated by a bi-lingual moderator, who will then use the open ended semi-structured questionnaire to ask questions regarding occupational task, occupational exposures, and asthma and allergy symptoms. At the end of each session, the research team will meet for debriefing. The focus group interview sessions will be audio taped. The interview session audiotapes will be transcribed and analyzed using the content analysis model using \"Ethnograph\" qualitative analysis software. The information from the focus group interviews will be used to develop JEM and a draft survey instrument for use among cleaners. [unreadable] [unreadable]"}
{"text": "Binding of benzodiazepine recognition sites by various ligands can elicit opposite types of responses, such as proconvulsant/anticonvulsant, or anxiogenic/anxiolytic. In order to answer whether a new class of drug belongs to benzodiazepine (anxiolytic), or beta-carboline (anxiogenic) type of ligand we developed a behavioral animal model that predicts the anxiogenic and anxiolytic potency of a drug. The inhibition of the response by the pure antagonist (partial agonist?) of the benzodiazepine recognition site assumes that proconflict and anticonflict action depends on the binding of the drug to the benzodiazepine recognition site."}
{"text": "Development of endometriosis appears to involve shedding of endometrial cells from the uterine cavity and implantation of the cells on ectopic pelvic loci. It is not known to what extend shedding of endometrial cells occurs in normal women at various stages of the menstural cycle or what factors may be involved in determining the biologic fate of these cells. Methodology used in the past has not allowed quantitative characterization of endometrial cells in the pelvic environment. The purpose of the present proposal is to test various biochemical cell markers for utility in parallel characterization of endometrial epithelial cells and inflammatory macrophages. This will be done by applying both conventional and newly developed marker techiques to endometrial and pelvic fluid aspirates and tissue samples of ectopic pelvic endometrial implants. The cell populations from endometrial aspirates, tissue samples and peritineal fluid will be characterized for a variety of enzyme, antigen and receptor markers using conventional cytochemical analysis and a flow cytofluorometric approach in parallel. The comparative analyiss of endometrial cells and inflammatory cells is important because preliminary studies suggest that pelvic macrophages are different in patients with endometriosis as compared to naormal women. The results of these studies will eventually show: 1. If any characteristic marker pattern distinguishable from normal can be found in aspirates of uterine endometrial cells or pelvic endometriotic implants in women with endometriosis. 2. If free endometrial cells in the peritoneal fluid are more closely related to cells in ectopic peritoneal implants or to cells in uterine endometrium in the same individual. 3. If physiologic cyclic changes exist in endometrial cells in peritoneal fluid in normal women or in patients with endometriosis during the menstrual cycle. 4. If biochemcial indicators of macrophage activation follow a physiologic or rhythmic pattern in each group of patients. 5. If pelvic macrophages from endometriosis patients are capable of inducing changes in adherence, cell growth, biochemical marker expression of normal endometrial cells when cocultured in vivo."}
{"text": "This project will evaluate the translation of the Gold (the Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease) COPD guidelines into primary care practice. This study is based upon the premise that an informed, activated patient will interact with a prepared, proactive team to improve COPD screening, diagnosis and management. Testing a similar hypothesis and using a similar design, the project team has demonstrated a successful translation of the NHLBI ATP III cholesterol guidelines into clinical practice. During phase 1, a needs assessment will evaluate barriers and facilitators to implementation of COPD guidelines into clinical practice through focus groups of primary care patients and providers. Using formative evaluation and feedback from the focus groups, three tools will be developed, refined and pilot tested. These include: a computerized patient activation tool that will be placed in each primary care office's waiting room for patient use; a [web-based], interactive COPD guidelines tool to be used by primary care providers as a decision support tool at the point of care and a COPD patient education toolbox to be used by the practice team. During phase 2, a block, randomized design cluster trial will be performed with one year of intervention within non-academic primary care practices (30 practices) throughout the state of Rhode Island and southeastern MA. The effectiveness of the materials developed in phase I will be tested in phase II regarding physician performance of COPD guideline implementation and the improvement in the clinically relevant outcomes (appropriate screening, diagnosis and management of COPD) compared to usual care. We will also examine the use of a patient activation tool - 'MyLungAge' to prompt patients to talk with their health care provider regarding their lung health and risk for COPD. Products for dissemination from this grant will include the results of the focus groups barriers and facilitators to implementation and adherence to COPD guidelines, a computerized data collection module for quality of care assessment regarding COPD guidelines, a refined computerized patient activation tool, an enhanced [web-based] COPD interactive guideline tool, tailored academic detailing materials, and the results of the randomized clinical trial on the effectiveness of these tools to improve COPD guideline adherence in primary care practice. Public Health Relevance Statement: This project will evaluate the translation of the Gold (the Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease) COPD guidelines into primary care practice. During phase 1, a needs assessment will evaluate barriers and facilitators to implementation of COPD guidelines into clinical practice through focus groups of primary care patients and providers. Using formative evaluation and feedback from the focus groups, three tools will be developed, refined and pilot tested. The effectiveness of the materials developed in phase I will be tested in phase II (an RCT conducted with one year of intervention within non-academic primary care practices) regarding physician performance of COPD guideline implementation and the improvement in the clinically relevant outcomes (appropriate screening, diagnosis and management of COPD) compared to usual care."}
{"text": "The primary goal of this research is to being to common availability the precise, sensitive, nondestructive viscosity and density measurements by J.W. Beams on small (approximately 0.2 microliters) samples of biomacromolecular solutions employing only tiny (less than approximately 10 to the minus 3rd power dynes/sq. cm) stresses. In the coming year improvements will be aimed at allowing these measurements to be made simultaneously, without interference from each other. A new \"oscillating buoy\" method which has been developed, will be evaluated in addition to the improved rotary machine of Beam's design. Larger numbers of actual experiments will be made, using the recently automated datataking means. Mechanical improvements, improved buoy design, and refinement of the new support and rotation electronics are in latter stages, and will be the primary means of achieving the independence of the two measured variables."}
{"text": "Substance abuse in adolescence is an epidemic which takes a significant toll on society and has deleterious physical, psychological, and social effects on adolescents and their families. As such, a great deal of research in recent years has focused broadly on finding patterns among social and behavioral determinants of substance abuse in adolescence, and specifically on the goal of finding clinically relevant subgroups of adolescents according to substance abuse behaviors. This goal is reflected by a recent NIDA program announcement, PA-15-003, entitled Epidemiology of Drug Abuse; this announcement lists trajectories of drug use and patterns of comorbidity with substance use disorders, two areas frequently explored using subgrouping strategies, as areas of interest. The goal of forming meaningful behavioral subtypes has largely been pursued using mixture models, a broad class of models which seeks to divide the sample into meaningful subgroups, known as latent classes, of subjects. Mixture models have seen extensive use in substance abuse studies in the past decade, with behavioral groupings being linked to imaging data, biomarkers, and genetic information. However, despite the their widespread use, virtually all current applications of mixture models have failed to take into account one critical aspect of behavioral measurement known as differential item functioning (DIF). Given an item intended to measure some underlying construct, DIF occurs when subjects with the same level of that underlying construct differ systematically in their responses to an item measuring that construct due to their gender, race, age, or any other individual characteristic. When DIF occurs and is not accounted for, it may render estimates of the level of the underlying construct biased, and inferences about differences between groups invalid. Though techniques for measuring continuous latent variables such as item response theory (IRT) and confirmatory factor analysis (CFA) have given serious attention to DIF as a fundamental threat to the validity of inferences, there has not yet been systematic study of DIF in the measurement of latent classes in mixture models, and there are no known applications of DIF analyses to mixture model results in the substance abuse literature. The proposed research seeks to rectify this shortcoming by rigorously investigating DIF in mixture models with a focus on behavioral studies of substance abuse. The four aims of the proposed project are to: (1) analytically define the ways that DIF can manifest in mixture models; (2) develop a flexible test of DIF in mixture models; (3) use a computer simulation to test (a) the consequences of un-modeled DIF in mixture models and (b) the efficacy of the test developed in Aim 2; and (4) to use DIF modeling techniques in an empirical study of substance abuse in the transition to adulthood. Through the pursuit of these aims, this project will allow fo mixture models to better consider individual differences, thereby enhancing the generalizability of these results in behavioral substance abuse research."}
{"text": "[unreadable] The FASEB Summer Conference on the Molecular Biology of Lipid Transport and Metabolism will bring [unreadable] together scientists in a variety of related fields with the purpose of defining, describing and analyzing [unreadable] critical scientific problems and recent advances in an intimate and interactive environment. A key feature of this meeting is the opportunity to facilitate the interactions of students and postdoctoral fellows with senior established figures in the field. Such interactions have historically been successful in defining new research agendas and fostering scientific discourse and collaborations. This application requests partial funding for a Conference to be held July 15 to 20, 2006 at the Omni Resort in Tucson, Arizona. This will be the sixth such conference on this theme; the last one was held in the summer of 2003. This conference is unique in its highly multidisciplinary approach to study a key problem of mammalian physiology. Thus, we will bring together a diverse group of senior and junior investigators with expertise in the areas of nutrition, physiology, biochemistry, protein chemistry, cell and molecular biology, and developmental biology, to address recent advances in lipid metabolism and its regulation. The format will consist of seven major oral sessions scheduled over four days (four AM and three PM), an evening Keynote Address and two poster sessions. To date 26 highly noteworthy invited speakers have confirmed their willingness to participate. The seven formal sessions focus on active research areas: 1) Intestinal Sterol Trafficking; 2) Lipid Transport and Bile acid Metabolism; 3) Dietary Lipids in Extra-hepatic Tissues; 4) Fat Metabolism in Model Organisms; 5) Lipid Disorders with Multi-organ effects; 6) Nuclear Receptors with Lipid Ligands; and 7) Cell biology and Genetics of Lipid Metabolism. The ultimate session will be a key note address from Jeffrey Gordon on Intrinsic and extrinsic regulators of intestinal lipid metabolism. All participants will be invited to submit an abstract to one of two poster sessions that will be held during the conference in order to allow all attendees, especially students and junior investigators to present data and participate in discussions. Additional speakers will be chosen from the abstract submissions, and all participants will be encouraged to submit particularly novel late-breaking developments for brief oral presentation in the form of \"hot topics\". These hot topics sessions will be targeted specifically to accommodate junior scientists. In the past this conference has promoted interactions and scientific discussions among basic and clinical investigators from academia and industry. The limited attendance, state of the art scientific sessions, \"hot topics\" additions, and novel poster presentations combine to provide an essential forumthat will foster exchange of ideas and further scientific advances in a field where advances in basic research have historically been directly translated into improved therapeutic options for a large fraction of the population. [unreadable] [unreadable] [unreadable] [unreadable]"}
{"text": "This research involves an electron microscopic examination and lipid and fatty acid analysis of ground squirrel hearts in the active and hibernating condition and a comparison to the heart of non-hibernating mammals, the white rat and the monkey. There are known physiological differences between the hearts of hibernating and non-hibernating mammals, such as maintenance of a rhythmic heart beat at 1 degrees C in the hibernator and ventricular fibrillation and heart failure in the non-hibernator at 15. degrees C. The electron microscope will be used to continue the critical scrutiny of heart ultrastructure to further corroborate the previous findings relating to the anatomical differences with are sufficient to account for the observed physiological differences. The lipids of the ground squirrel heart in the active and hibernating condition will continue to be analyzed by column and thin layer chromatography to corroborate initial findings of modifications in lipid content during the hibernating state. Additionally, the fatty acids, particularly those bonded to the lipids with are modified during hibernation, will be analyzed to determine shifts in the degree and pattern of unsaturation during the hibernating state. Results from these analyses may further elucidate the factors responsible for the continued automaticity of the ground squirrel heart at near- freezing temperatures. This may in turn further enhance medical knowledge and facilitate the use of hypothermia in various aspects of surgery. Since hibernator hearts do not fibrillate at any temperature from 0 degrees C, to 37 degrees C, this knowledge may eliminate, or reduce the frequency of, the extremely dangerous ventricular fibrillation which occurs in most human hearts at about 25-28 degrees C."}
{"text": "Despite advances in intraaortic balloon pumping and emergency myocardial revascularization, salvage of ischemic myocardium has been limited in man by the lack of effective collateral circulation into an area of acute infarction. We are currently carrying out experimental trials of perfusing ischemic myocardium by retrograde diastolic pulsation of oxygenated blood into the coronary veins draining an area of ischemia via a balloon tipped catheter that can be introduced transvenously. Results to date indicate correction of EKG changes, reversal of dyskinetic areas and improvement in myocardial performance in acutely ischemic myocardium following institution of retroperfusion."}
{"text": "Daniel Mueller will direct the transplantation core. He will be responsible for the training and supervision of the Junior Scientist (to be named) on the project. He will assure the quality of the core?s surgical expertise, and will also manage the financial aspects of the core. This Junior Scientist, to be hired, will have significant expertise in animal husbandry and small animal surgery, and will have a background in a biological discipline such as physiology or immunology. This individual will be trained by the Core leader and his associates to harvest mouse tracheas for implantation under anesthesia, to transplant the tissue under anesthesia (survival surgery), to monitor the transplanted animals during the recovery period following the transplantation, and to sacrifice the animals at the end of the experiment. He/she will then be responsible for the recovery of tracheal graft tissues as well as relevant lymphoid organs (lymph node, spleen) at the end of the experiment, and processing of the tissue per the request of the investigator. This will entail the freezing of tissue blocks in OCT medium and frozen sectioning, as well as the preparation of cellular suspensions from collagenase-treated tracheal tissue. This individual will make his or her expertise available to all scientists on this program project on a pre-arranged schedule, and free of charge. This investigator will also be responsible for purchasing of the relevant expendable supplies and for the maintenance of the small equipment (e.g., tissue forceps) and the cryostat."}
{"text": "[unreadable] [unreadable] About 40% of the women diagnosed with breast cancer will progress to metastatic disease. However, metastases cannot be removed by surgery or radiation, and most metastases are chemoresistant. Therefore, therapies that specifically prevent or eliminate metastases offer great promise in the outcome. Recent studies indicate that enhancement of specific helper or cytotoxic T lymphocyte (CTL) responses to breast tumors through vaccination could potentially lead to the specific elimination of micro-metastases. However, poor vaccine-induced immune and clinical responses underscore the need for improved vaccine strategies. This research proposal is focused on the improvement of vaccine therapy against metastatic breast cancer by combining vaccination with the IL-6 inhibitor curcumin, using the preclinical metastatic mouse breast tumor model 4T1. We previously demonstrated that Mage-b DNA vaccination provides significant protection against breast cancer metastases in this model, albeit not completely. Interleukin (IL)-6 may contribute to decreased vaccine efficacy. It is highly up regulated in the 4T1 primary tumors and metastases, probably via the Nuclear Factor Kappa B (NFkB), and is a potent regulator of dendritic cells (DC) differentiation in vivo. IL-6 activates the expression of transducer and activator of transcription (STAT)3 in immature DC, preventing the DC from maturation and subsequent presentation of antigens to T cells. This may lead to T cell unresponsiveness. Agents that are able to inhibit IL-6 may lead to enhanced vaccine efficacy. Curcumin could be such an agent, since it inhibits IL-6 production. Both Mage-b and IL-6 are frequently detected in human breast cancer, which makes the 4T1 model clinically relevant. The long-term objective of this proposal is to develop a novel non- toxic combination therapy of Mage-b vaccination and curcumin in the 4T1 model, with a focus on its translation into human clinical trials, if successful. The hypothesis is that curcumin improves T cell activation and subsequent immune responses upon Mage-b DNA vaccination, resulting in further reduction of the frequency of breast cancer metastases. The specific aims are as follows:(1) Testing the effect of Mage-b vaccination with and without curcumin on tumors and metastases. For this purpose, mice will be immunized preventively or therapeutically with Mage-b DNA vaccine and challenged with 4T1 tumor cells. As soon as the primary tumor can be felt curcumin will be administered daily. Frequency of metastases, tumor size, and survival times will be determined; (2) Testing the direct effect(s) of curcumin on vaccine- and tumor-induced immune responses, and on proliferation and apoptosis of tumor cells. For this purpose, spleen, lymph nodes, tumors and metastases of treated and control mice will be analyzed for the presence of activated CD4 and CD8 T cells, regulatory T cells and mature DC. In addition, tumors and metastases of treated and control mice will also be analyzed for the expression of NFkB and NFkB-regulated genes that are directly involved in immune responses such as IL-6, TNF1, and STAT3, and for proliferation and apoptosis. PUBLIC HEALTH RELEVANCE: Current treatment of cancer is ineffective against metastases. In this research proposal a novel non-toxic combination therapy of Mage-b vaccination and curcumin will be developed that is effective against breast cancer metastases, using a preclinical mouse model that reflects metastatic breast cancer in humans. [unreadable] [unreadable] [unreadable]"}
{"text": "DESCRIPTION (applicant's abstract): The long term goal of this project is to elucidate the mechanisms that regulate functions of the noradrenergic locus coeruleus (LC) system. Our previous results defined afferents to the LC nucleus, and characterized LC dendrites that extend into specific extranuclear zones. However, these findings also generated new questions: (i) what are the inputs to extranuclear LC dendrites, (ii) what circuits are LC afferents a part of, and (iii) what LC output functions are regulated by these afferent circuits? The proposed experiments will answer these difficult but important questions using new anatomical technology and electrophysiology. LC neurons have extensive extranuclear dendrites, and the zones containing these distal dendrites receive numerous inputs that do not innervate the LC nuclear core. However, it has been difficult to identify which of these afferents terminate on LC dendrites vs. other elements in this region. We will retrogradely label afferents that specifically innervate extranuclear LC dendrites using the recently developed transsynaptic tract-tracer, pseudorabies virus (PRV). Dendritic afferents will be confirmed by ultrastructural analyses, and their impact on LC neuronal activity will be determined. In addition to direct afferents to LC neurons, it is important to determine inputs to these direct afferents, and thereby identify circuits that regulate LC function. We will map indirect afferents to the LC in a detailed time-course study of transsynaptic transport of PRV. For prominent indirect afferents, the relays to the LC will be identified and the influence of these afferent circuits on LC activity will be determined. Our preliminary results indicate that the suprachiasmatic nucleus (SCN) is a prominent indirect input to the LC. The SCN is the brain's circadian pacemaker, and controls among other rhythms circadian properties of sleep and waking. As the LC has long been implicated in arousal, we hypothesize that the SCN-LC circuit is a key neural substrate linking circadian and sleep-waking processes. We will test this hypothesis by manipulating SCN activity, recording the effects on EEG arousal, and testing the role of the LC and associated relay nuclei in the effects obtained. This will be the first analysis of a neural link between circadian and arousal processes. These studies will extend our analysis of afferent control of LC function to identify inputs to distal LC dendrites and circuits that regulate LC activity. They will also provide the first demonstration of afferent circuit regulation of an important LC output function, cortical arousal."}
{"text": "Objectives: 1. The total number of primary malignant melanomas studied by our group now approaches 570 cases. Of this group, approximately 250 to 300 cases have complete histological data and clinical follow ups of greater than 5 years. From the investigation of these cases, we hope to determine the parameters for high and low risk categories. These studies include cellular parameters of the primary lesions and a variety of epidemiologic data from the patients. 2. We are attempting to study comparatively the immunologic status of patients in high and low risk categories using percentage of circulating T and B cells, percentage of B cells, T cells and histiocytes in the host cellular response to the tumor, determination of cell mediated cytoxicity to three reference melanoma cell types and determination of host blastogenic response to melanoma antigens. 3. After establishing over the past three years malignant melanoma in guinea pigs, we are in the process now of studying its developmental biology. 4. An ultrastructural study to delineate the different between the fine structure Spitz tumor and malignant melanoma is nearly complete. 5. Additional studies include: Fine Structure of Host Tumor Cells and Host Responding Cells, Fine Structure of Nuclear Tubular Bodies in Human Malignant Melanoma-Paramyxovirus-like Structure, and the Study of Significance of Polyclonism in the Vertical Growth Phase or Intralesional Transformation as Related to the Subsequent Development of Metastases. Bibliographic references: Suffin, S. C., Waisman, J., Clark, W.H.: Congruence of Diagnosis of Malignant Melanoma, Lab. Inv., 32 (3):436, 1975. Leone, P.G., Leone, C., Clark, W. H.: Acquired and Congenital Nevi, Lab. Inv. 32(3):464, 1975."}
{"text": "Diabetes accounts for nearly 22 million physician visits annually and is the third leading cause of repeat outpatient visits in the United States. Depression is also strongly related to increased medical (nonpsychiatric) health care utilization, although its specific effect in diabetes has not been studied. Depression is 304 times more prevalent in diabetes than in the general US population. Depression in diabetes has been associated with several factors (e.g., poor glucose regulation, poor compliance with the diabetes regimen) that could lead to increased health care use. We proved during the last period of NIH support, in a double-blind, placebo- controlled trial, that antidepressants were significantly more effective than placebo in managing depression; 65% of treated patients remitted. We also have shown that in the absence of treatment, diabetic depressions are chronic. We plan to recruit 60 depressed patients with NIDDM from a metropolitan primary care practice, and treat them with antidepressant pharmacotherapy for a period sufficient to detect an effect on health care utilization (1 year). Because of the extended treatment interval, the chronicity of untreated depression in diabetes, and the known response/ nonresponse to pharmacotherapy, it is not ethical, practical, or methodologically necessary to include a depressed, no-treatment control group. A comparison sample of 60 nondepressed NIDDM patients matched for age, sex, other comorbid medical illnesses, treating physician, and diabetes type, severity, and method of treatment is included. Health care utilization will be assessed in both depressed and control patients for the year prior to study enrollment and during the year of treatment. Measurements of psychiatric disease activity, family history of depression, personality characteristics, life stress, diabetes symptoms, glucose regulation, and functional status and well-being, will be taken at specified points during the year of depression treatment. We hypothesize that: 1) depressed diabetic patients will make greater demands for medical health care than the nondepressed matched control sample; 20 medical health care utilization will be greater in depressed diabetic patients whose depression persists compared to patients who become nondepressed during treatment; and 3) utilization in the depression improvement group will be similar to that measured in the nondepressed controls. Failure to recognize and treat active depression in diabetes may fuel higher health care utilization. A component study is described that aims to develop a brief psychometric screening tool with positive and negative predictive values for major depression in diabetes greater than 0.80. Witnessing that improvement in depression is associated with a favorable reduction in health care utilization by NIDDM patients would suggest the potential benefits of depression recognition and treatment to society in general, over and above the recognized patient benefits in mood and quality of life."}
{"text": "The studies outlined in this proposal will focus on cyclin D1, a key cell cycle control protein that is one of the most prevalent oncogenes in human cancers. Studies from this laboratory have identified cyclin D1 as an important mediator of hepatocyte proliferation in the regenerating liver. However, the biochemical and cellular functions of cyclin D1 have not been fully characterized despite extensive study. The purpose of these studies is to characterize a novel effect of cyclin D1 in hepatocytes. Based on preliminary evidence, the primary hypothesis is that cyclin D1 regulates sex steroid hormone metabolism and signaling at the cellular and tissue level, and that these effects play a biologically significant role in the liver. It is also hypothesized that the C-terminus of cyclin D1 plays an important role in modulating sex steroid signaling as well as other biologically relevant effects in the liver. Additional studies will identify novel cyclin D1 binding partners. Identification of novel roles for cyclin D1 in the liver are expected to increase our understanding of an important liver oncogene. [unreadable] [unreadable] [unreadable]"}
{"text": "We are examining the three-dimensional (3-D) fine structure of the Golgi complex in rapidly-frozen pancreatic beta cells to provide new data on the sites of formation and sorting for secretory granules, constitutive secretory vesicles, and vesicles destined for the endo-lysosomal pathway. Insulinoma cell lines (HIT-T15 and MIN-6) and isolated rodent islets of Langerhans are being fixed by high-pressure freezing and freeze-substitution. 250-400 nm thick sections of plastic-embedded samples are imaged in a high-voltage electron microscope (HVEM) and serially tilted at 1.5x intervals (q60x) about two orthogonal axes. These digital images are used to generate high resolution (5-7 nm) tomographic reconstructions which can then be analyzed and modeled in 3-D. These reconstructions will be used to examine the relationship between trans-cisternae and the TGN, to resolve novel coat structures on nascent vesicles and granules, and to address hypotheses relating to cisternal progression-maturation and protein trafficking via membranous tubules. Our preliminary data show that small dense-core granules (average diameter 95 nm) form from cis-, medial- and trans-cisternae, not just from the trans-most cisterna or TGN. This suggests that secretory product condenses at many, if not all, levels of the Golgi complex. These dense- core granules appear to be connected to Golgi cisternae and to each other by tubular projections of uniform diameter and density, arranged like \"beads on a string\". The result is a continuum of developing granules, connected with Golgi cisternae at one end and projecting away from the Golgi stack at the other. C3"}
{"text": "The purpose of this project is to understand the molecular mechanisms responsible for replication of picornaviruses in susceptible target cells. This virus family includes numerous human pathogens (poliovirus, coxsackievirus, echovirus, enteroviruses, rhinoviruses, hepatitis A virus). Infection of cells with these viruses leads to major changes in the host cell's structure and metabolic activity. Cellular protein and RNA synthesis are inhibited; the intracellular membrane network becomes rearranged into vesicles that surround and provide a scaffold for viral RNA replication complexes; cellular proteins are subverted into facilitating viral protein and RNA synthesis. The unique combination of viral and cellular proteins together accomplish a highly efficient production of viral RNA, proteins, and particles. We are studying the activities of individual viral gene products, expressed alone or in combination, in cultured human cells, to determine their individual roles in the replication process. Interactions of each viral protein with each other and with cellular proteins are being characterized. The biochemical reactions required for translation of the viral RNA and replication of the viral genome are being defined. Understanding the precise biochemical activities of viral proteins in the replication process will allow the development of new strategies for vaccine development and the design of antiviral drugs."}
{"text": "The Administrative Office, under the direction of the Chairman, Dr. George Lewis, in submission of the annual progress report will state the objectives, goals, and progress of the Gynecologic Oncology Group. Our institution continues to be a major contributor to the group."}
{"text": "This is an ACTG sponsored four center safety, pharmacokinetic, and dose finding trial of AZT for infants one day to 3 months of age who are born to women with HIV infection. Dosing and intravenous and oral AZT pharmacokinetic studies are done on three occasions during the 6 week protocol. Participants are monitored closely for evidence of AZT toxicity and for development and progression of virologic and clinical evidence of HIV infection."}
{"text": "Alcohol misuse is a significant public health problem, not only in the US but around the world. Just in the US, alcohol misuse is estimated to be the second cause of preventable death at a cost of more than $220 billion in a single year. Further, it is also estimated that it reduces the lives of those who died by 30 years. Screening and brief interventions have proved effective in promoting behavior changes that lead to increased awareness and decreased consumption when properly conducted at the primary care setting. However, there remain four main barriers that have curtailed broad adoption of screening and brief interventions by primary care: lack of access to validated tools, lack of training, reluctanc to raise the subject to the patient, and lack of face time. Overcoming these barriers creates a vastly significant clinical, public health, and commercial opportunity. VisionQuest Biomedical and the University of New Mexico have formed a team to develop and test the feasibility of PA?MSTM, a mobile platform for the delivery of screening and brief interventions for alcohol misuse. PA?MSTM technical innovation is that it shifts the delivery paradigm from paper and in-person delivery to software-as-a-service. PA?MSTM is not an electronic form only but a complete set of tools that allow electronic communication between patients, providers, and support groups from screening to brief interventions to follow up care. By enabling on-demand, up-to-date patient-centered screening and brief interventions PA?MSTM clinical innovation is that it extends the reach of primary care and facilitates care coordination and decision making. In Phase I, a prototype PA?MSTM will be developed and tested in a primary care setting on 40 patients who will be followed for two months. Standard metrics of usability, acceptability, and user satisfaction will be assessed in order to prove the feasibility of the prototype and plan for an improved version that will be tested in a multi-center clinical study in Phase II."}
{"text": "Characterization of Platelet Function in Patients with Bleeding Disorders: In FY2015 (the fourth year of this project) we have studied several patients with suspected or documented disorders of platelet function and have identified patients with deficiency of dense granules (storage pool disease), and characterized platelet function in people with congenital or acquired platelet dysfunction. The ability to make a reliable, detailed characterization of platelet function may help investigators who are studying genetics of inherited platelet disorders. One family with abnormal platelet function and other abnormalities has been described and submitted to the journal Blood for consideration to be published (York Platelet Syndrome, T. Markello et al, authors and collaborators). Another patient has been identified who has a novel platelet disorder that may be a variant of Glanzmann's thrombasthenia. He has been referred to the Undiagnosed Diseases Program (NIH/NHGRI) for further analysis and possible genome sequencing to identify the basis of this disorder. Characterization of D-Dimer and Related Coagulation Proteins in HIV-Infected Research Subjects Undergoing IL-2 Therapy: During the fifth year of this project, in support of intramural NIH research protocols, we have measured D-dimer levels and numerous coagulation parameters in research subjects who are infected with HIV or who are normal volunteers. We seek to correlate the D-dimer levels with these various parameters to gain insights into the mechanism for the observed elevations in D-dimer levels in HIV infected patients that correlate with increased morbidity and mortality. Effect of Factor VIII Haplotypes on Inhibitor Development. We have identified a set of 800 DNA samples from patients with severe hemophilia A in an NHLBI repository and have IRB approval to determine their factor VIII protein-coding polymorphisms to determine the effect of factor VIII haplotype on inhibitor development. We expect to complete the DNA sequence determinations in the coming fiscal year, and will see if the effect of protein coding polymorphisms on inhibitor development that was seen in a minority population (African-Americans) can be seen in the population as a whole. We have observed two cases of acquired factor VIII inhibitor in the setting of non-myeloablative stem cell transplantation for sickle cell disease in African-Americans, a report on one of which has now been published."}
{"text": "The long-term goal of this proposal is to provide insight into mechanisms critical for stress-facilitated memory formation, or in other words, the processing of memories under stressful conditions. Two stress- and memory-associated disorders, posttraumatic stress disorder and major depressive disorder, are prime examples of disorders with abnormal stress-facilitated memory processing, and they both have an estimated lifetime prevalence of over 5% among Americans. A key signaling pathway implicated in stress-induced memory processing is the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. [unreadable] Activation of this pathway leads to downstream activation of cAMP response element-binding protein, which results in transcriptional changes needed to form new memories. The activator of the MAPK/ERK pathway that modulates stress-facilitated memory is still not well defined. However, evidence suggests the second messenger, cAMP, may be the upstream initiator as cAMP production can activate this pathway to cause changes in memory. Moreover, calcium-stimulated adenylyl cyclases (AC), AC1 and AC8, couple neuronal activity and intracellular calcium increases to the production of cAMP, thus, implicating calcium-stimulated AC activity in modulating stress-facilitated memory changes. As a paradigm for stress-facilitated memory, we will use a classical conditioning test, which serves as a good model to investigate our hypothesis as both the MAPK/ERK signaling pathway and calcium-stimulated AC activity have been shown to effect learning on this paradigm. Therefore, we aim to examine how this activity may activate the MAPK/ERK pathway to modulate stress-facilitated memory. Through use of a novel transgenic mouse model, which uses a tetracycline-inducible system to replace AC8 expression in AC1 and AC8 double knock-out mice, we are able to assess the time-specific importance of this activity. Moreover, through the use of gene therapy techniques, we can assess the region-specific importance of this activity via lentivirus administration that turns on AC8 expression. Relevance: The research we are proposing will give insight into a mechanism of memory formation under stressful conditions and may reveal a primary role of memory-related symptoms in psychiatric disorders. More specifically, it will look at the time- and region-specific manner in which this stress-facilitated memory mechanism modulates memory formation. This work may eventually lead to the development of therapeutic interventions for people with abnormal memory changes from stress-related disorders as global, non-specific treatments can facilitate a host of unwanted side-effects.[unreadable] [unreadable] [unreadable] PUBLIC HEALTH RELVENCE The research we are proposing will give insight into a mechanism of memory formation under stressful conditions and may reveal a primary role of memory-related symptoms in psychiatric disorders. More specifically, it will look at the time- and region-specific manner in which this stress-facilitated memory mechanism modulates memory formation. This work may eventually lead to the development of therapeutic interventions for people with abnormal memory changes from stress-related disorders as global, non- specific treatments can facilitate a host of unwanted side-effects. [unreadable] [unreadable] [unreadable]"}
{"text": "The purpose of the proposed research is to examine developmental changes in children's understanding of attention (\"meta-attention\"). It is expected that there are changes in concepts concerning the nature of attention and distraction, strategies for attending to stimuli, and variables which facilitate or hinder attention. Meta-attention is important because it is believed to influence attentional behavior, especially the selection and execution of appropriate strategies for problem-solving. Since there is almost no research on meta-attention, the proposed research would fill an important gap. Although the emphasis will be on developing valid nonverbal procedures for assessing meta-attention, a variety of response measures will be used--the child's selection of stimuli to expose, prediction of one's performance, construction of \"easy\" versus \"hard\" attentional tasks, selection of pictures or other stimuli from several alternatives, rating pictured situations according to a scale, reaction time, and responses to questions concerning strategies. Although children from age 3 to 14 will be used, the emphasis will be on the grade school years. When possible, the tasks will be adapted for use with preschool children in order to examine the beginnings of meta-attention. The studies will examine meta-attention in several tasks commonly used to measure attention in children--\"incidental learning,\" visual search, dimensional sorting of stimuli, stimulus matching, and same-different judgments."}
{"text": "The influence of parenteral nutrition on bone mineralization and metabolism was evaluated. Four children, partially ad fully dependent on parenteral nutrition were evaluated over 24 hours with dietary assessments, and timed urine and blood collections. Mediators of calcium and bone metabolism were monitored."}
{"text": "EVP 1001 is intended to be a unique, intracellular, cardiac specific magnetic resonance imaging (MRI) agent which specifically enhances both the myocardium and the vasculature. It will satisfy currently-unmet needs for the rapid, non-invasive diagnosis and treatment planning of ischemic heart disease, including simultaneous assessment of both structure and function (stenosis, occlusion, wall motion, myocardial perfusion and delineation of viable versus non-viable myocardium). The preclinical research proposed for Phase 1 will augment the cardiac safety of EVP 1001 and will define the relationship between its pharmacokinetics and its MR enhancement. Does regimens which provide effective cardiac and vascular MR enhancements will be delineated from these data. The unique magnetic properties of EVP 1001 are expected to provide enhancement at low doses compared to current gadolinium agents (3-15 versus 100-200 mumol/kg). Phase 2 will assess the safety and efficacy of EVP 1001 in models of cardiac ischemia, laying the foundation for human trials. When approved, this contrast agent will make optimal use of the powerful diagnostic potential of MRI and its rapid technical evolution, becoming the \"one-stop shop\" for diagnosis of coronary artery disease and replacing more costly and invasive tests. PROPOSED COMMERCIAL APPLICATIONS: The long-term goal of this research is to register EVP 1001 in the U.S. and elsewhere as a contrast agent for cardiovascular MRI. By combining the noninvasive assessment of both cardiac structure and function, this agent should provide more cost-effective diagnosis and treatment planning than echocardiography, nuclear perfusion/functional imaging, and x-ray angiography. In addition, its use as a vascular enhancement agent may be extended to other regions of the body."}
{"text": "Deposition of amyloid in cerebral blood vessels (cerebral amyloid angiopathy, CAA) is a common condition in the elderly associated with both hemorrhagic and ischemic strokes. CAA is also a potentially important model for understanding the pathogenesis of Alzheimer' s disease, the other major beta-amyloidosis of the central nervous system. At the Alzheimer Research Unit at Massachusetts General Hospital, we have used clinical, neuropathological, and genetic approaches to define the pathogenic steps involved in CAA. These studies have been limited, however, by the static nature of post-mortem brain tissue and the two-dimensional nature of single brain sections. The current proposal seeks to move beyond these limitations by using multi-photon confocal microscopy to observe the development of CAA in transgenic mice over time, and by using multi-photon microscopy and computer-aided image reconstruction to analyze the three-dimensional structure of affected blood vessels. We will use these techniques to examine the processes of 1) amyloid deposition in vessels; 2) breakdown of the amyloid-laden vessel wall; and 3) response to anti-amyloid immunotherapy. We will test whether Abeta initially deposits near vessel branchpoints, whether vessel amyloid affects the deposition of parenchymal amyloid, and whether amyloid deposits preferentially on specific vessels. We will generate a timeline of CAA, from the first deposition of A13 species through formation of amyloid, loss of smooth muscle cells and subsequent vasculopathic changes. We will determine whether there are potential detrimental effects to vessels by anti-Abeta therapies. Studies will be performed on mice doubly transgenic for mutant amyloid precursor protein and presenilin- 1, mice that demonstrate detectable CAA at ages as early as six months. Parallel studies on the three-dimensional structure of amyloid deposition and vessel breakdown will be performed on human tissue taken from collected cases of CAA, including the unique Iowa form of familial CAA. Successful completion of these studies will delineate the pathways involved in CAA initiation, propagation, and vessel damage, as well as determine the feasibility of specific immunotherapy for this currently untreatable disorder."}
{"text": "Endothelial dysfunction is associated with corresponding changes in vascular tone and increases in contractility, a condition that may cause hypertension in susceptible individuals which may have allelic variants in cyp2j-epoxygenases, soluble epoxide hydrolase (sEH), A2A, and A1 adenosine receptors (A2A AR & A1 AR) genes. These allelic variants may have similarities to our transgenic mice which may regulate vascular tone and blood pressure (BP). Our preliminary data have suggested a possible link between adenosine-induced relaxation and opening of KATP channels through A2A AR-cyp2j-PKA-PPAR? pathway. Also, it is possible that a link may exist between adenosine-induced contraction and closing of KATP channels through A1 AR-sEH-cyp4a- PPAR? pathway. A combination of pharmacological tools and transgenic mice would allow us to identify the possible targets as a long term goal to treat population which may have allelic variants leading to hypertension. Therefore, there is a critical need to explore the possible mechanism involving cyp2j2-epoxygenases, sEH/cyp4a, A1 AR/A2A AR, PPAR?/?, PKA/PKC?/? and KATP channels in adenosine-induced vascular responses. Our central hypothesis is that adenosine induces vascular relaxation and decreases in BP through cyp2j-epoxygenases via A2A AR-cyp2j-PKA-PPAR? signaling leading to opening of KATP channels. On the other hand, adenosine induces vascular contraction and increases in BP through sEH via A1 AR-sEH-cyp4a-PPAR? pathway leading to closing of KATP channels. To test this hypothesis, we propose to explore in depth mechanism(s) using A2A AR-/-, A1 AR-/-, cyp2j5-/-, sEH-/-, Tie2-cyp2j2Tr (endothelial-cyp2j2 overexpressed), Tie2-sEHTr (endothelial-sEH overexpressed), wild-type mice, immortal renal endothelial cell line from H-2Kb- tsA58 mouse, mouse aortic endothelial cells (MAEC) and mouse aortic smooth muscle cells (MASMC). Further, we will also explore the possible treatment with sEH inhibitors (AUDA/t-AUCB) in drinking water (or gavage) for A2A AR-/-, cyp2j5-/- and Tie2-sEHTr mice which may have high BP. We will measure BP, and we will use aortas/renal arteries (organ bath/DMT-wire myograph) with treatments (adenosine-receptors agonists & antagonists), cyp-epoxygenases, sEH, adenylyl cyclase, PKC?/?, MAPK and PKA inhibitors, PPAR?/?, EETs and KATP channel (activators & inhibitors). EETs & DHETs will be analyzed (UPLC-MS/MS). Western blot & RT-PCR will be used for proteins & mRNA expression. We propose 3 specific aims to determine: (1) whether the cyp2j-epxygenases or sEH affects BP, adenosine-induced vascular response and EETs/DHETs; (2) whether the presence or absence of A2A AR affects adenosine-induced vascular response through PPARs via cyp2j-epoxygenases, sEH (3) whether the presence /overexpression of cyp2j-epoxygenases or sEH regulate KATP channels through A2A AR-cyp2j-PKA-PPAR?/A1 AR-sEH-cyp4a-PPAR? pathway in adenosine-induced vascular response. Such results can have a positive impact, as the identified components are expected to provide new targets to curb clinical problems linked with dysfunctional endothelium leading to hypertension."}
{"text": "Bridging Interventional Development Gaps (BrIDGs) makes available, on a competitive basis, certain critical resources needed for the development of new therapeutic agents. Investigators do not receive grant funds through this program. Instead, successful applicants receive access to NIH contractors who conduct preclinical studies at no cost to the investigator. In general, synthesis, formulation, pharmacokinetic, and toxicology services in support of investigator-held Investigational New Drug (IND) applications to the Food and Drug Administration are available. Within the fiscal year, three projects were completed. These projects involved therapies for Diabetic Retinopathy, Parkinsons Disease and Radioisotope Contamination. The retinopathy project was discontinued due to milestone failure. An IND was filed for the Parkinsons therapy and the first clinical trial is expected to begin in Fall 2012. NIAID is responsible for supporting additional studies related to radioisotope contamination product. An IND is not expected until late FY13 or FY14. In FY 2012, five new BrIDGs projects were approved for potential therapies for rheumatoid arthritis, metabolic disorder, epilepsy, multiple sclerosis and pancreatic cancer. Other ongoing projects support the development of potential therapies for Fibrodysplasia Ossificans Progressiva, Anemia of Inflammation, Epilepsy, Pancreatic Cancer, Niemann-Pick C Disease, Aromatic L-Amino Acid Decarboxylase Deficiency, Alzheimers Disease, Hyperinsulinism, Chronic Dry Eye, Osteoarthritis, Hypoparathyroidism, and Atherosclerosis. In spring 2012, BrIDGs solicited new applications for the first time under NCATS. 44 applications were received and awards will be made in FY13. Products are being developed for the treatment of Rheumatoid Arthritis, Metabolic Disorder, Multiple Sclerosis, Fibrodysplasia Ossificans Progressiva, Anemia of Inflammation, Epilepsy, Pancreatic Cancer, Niemann-Pick C Disease, Aromatic L-Amino Acid Decarboxylase Deficiency, Alzheimers Disease, Hyperinsulinism, Chronic Dry Eye, Osteoarthritis, Hypoparathyroidism, and Atherosclerosis."}
{"text": "Thermo acoustic tomography (TAT) is a rapidly emerging ultrasound-mediated hybrid imaging modality that promises to have a major impact on diagnostic imaging. TAT combines high ultrasonic resolution and strong microwave contrast in a single hybrid modality. Human brain imaging represents an important imaging application that can benefit tremendously by the development of TAT methods. Existing high-resolution human brain imaging modalities such as X-ray computed tomography (CT) and magnetic resonance imaging (MRI) are expensive and employ bulky and generally non-portable imaging equipment. Moreover, X-ray CT employs ionizing radiation and is unsafe for patients who need long time monitoring of brain diseases or injuries. Alternatively, ultrasonography is an established portable pediatric brain imaging modality, but its image quality degrades severely when employed after the closure of the fontanels and therefore is not effective for imaging adults. The development of TAT brain imaging methods would circumvent these limitations and result a powerful new brain imaging modality that would fill an important void left by the available techniques. The major technical challenge in TAT brain imaging is to compensate for the distortion introduced into the TAT measurement data by the skull. The broad objective of this proposal is to make TAT brain imaging a practical, useful, and highly effective brain imaging modality by developing robust image reconstruction methods that can account for skull-induced signal distortion and other physical factors. By use of information regarding the skull morphology and composition obtained from previously acquired adjunct image data, or alternatively from the TAT measurement data themselves, we will develop and investigate robust imaging models for TAT that account for the effects of skull-induced wave front aberrations and other physical factors related to the measurement process. We will develop reconstruction algorithms for obtaining accurate images from incomplete data sets that correspond to clinically useful measurement configurations. The developed methods will be systematically evaluated in computer-simulation and experimental studies. The first in vivo study of human TAT brain imaging will also be conducted. The specific aims of the project are: (1) To develop imaging methodologies that incorporate the effects of skull-induced phase aberrations; (2) To develop image reconstruction algorithms for practical brain imaging scanning configurations; (3) To validate the reconstruction methods for TAT brain imaging in computer-simulation studies; and (4) To assess image quality in phantom and in-vivo studies."}
{"text": "Phylogenetic analysis is crucial to a wide range of biological and medical research. A new type of data based on gene order and gene content within whole genomes has attracted increasing interest from researchers in the past several years. [unreadable] [unreadable] Specific Aims: The complexity of genome evolution poses many exciting challenges to developers of mathematical models and algorithms. However, the current tools can only be applied to small genomes (such as organelle genomes) evolving via very simple rearrangements events, hence their breadth of usage is limited. We will address these problems by: (1) mathematical modeling and theoretical analysis of complex evolutionary events such as gene duplication and loss; (2) algorithm design and implementation for phylogenetics and gene order reconstruction (3) performance assessment of these new algorithms through extensive testing on simulated and biological datasets; (4) high-performance implementation of the algorithms using algorithm engineering techniques and a flexible approach to parallelization. [unreadable] [unreadable] Contributions and Broader Impact: The broader impacts of the proposed project are several. (1) The development of new theories and algorithms for the efficient reconstruction of phylogenies and inference of ancestral genomes based on complex genome rearrangements will considerably enlarge the scope of research in the field and give rise to interesting new problems in mathematical and computational biology. (2) Efficient and accurate software for phylogenetic analysis and genome comparison, tested on a large variety of real datasets and on an extensive range of simulations, is expected to reveal new evolutionary patterns and to enable the investigation of novel biological questions. (3) A web server hosted by our group (or by our collaborators) will enable biologists to submit their datasets through a user-friendly web interface and get results back within reasonable amount of time, without the burden of installation and learning parallel computation. (4) The project team combines expertise in mathematic modeling, algorithm design, high-performance computing, comparative genomics, and phylogenetics. Students (both undergraduate and graduate) and postdocs on this project will receive valuable interdisciplinary training experience. (5) Both universities have established programs to boost research in computational biology. This project will enable the PIs to establish close interdisciplinary collaborations among departments from both universities and recruit graduate students (especially minorities) to this fast-growing research field. [unreadable] [unreadable] [unreadable] [unreadable]"}
{"text": "Stroke prevention may be achieved through lifestyle changes on a variety of issues such as physical activities and medication adherence. It is therefore difficult to overstate the importance of developing and disseminating behavioral intervention programs as a public health measure to prevent strokes. For the same reason, a behavioral intervention program naturally involves multiple components addressing the various issues; and a successful multi-component program is likely a direct result of administering each interventional component in an optimal sequence, based on the intermediate health outcomes, so as to maximize the eventual health outcome such as blood pressure reduction over 12 months. This type of treatment program tailors the intervention sequence according to an individual's own characteristics, and is sometimes called dynamic treatment regime (DTR). This research aims to develop, validate, and disseminate statistical methods to identify optimal DTR through carefully designed randomized community-based studies. We plan to achieve this research goal in four steps. First, we will develop a data analytical technique, called Q-learning, that will enable us to identify an optimal DTR in an unbiased fashion using data from community- based studies. Q-learning is a cutting-edge technique originating from the computer science literature; this research will adapt this innovative idea to clinical applications where data are observed with high level of variability (noise). Second, we will develop statistical designs that facilitate the discovery of optimal DTR through Q-learning while benefiting the trial participants. This will involve novel synthesis of two clinical trial design concepts: sequential multiple assignment randomized trial (SMART) and adaptive randomization (AR). Third, we will validate the proposed theory and methods by using computer simulation and analyzing data from an actual behavioral intervention study. Fourth, we will disseminate the methods by building software with public access and employ the methods in the planning of the next stage of intervention study; this step is intended to close the lag time between novel methods and its clinical applications. Our long-term public health goal is to enhance the capability of developing optimal behavioral intervention curriculums."}
{"text": "The abuse and misuse of opiods is already a significant healthcare problem that is expected to escalate dramatically in the next decade. Traditionally, the treatment of opioid-dependence has been provided in specialty care settings, such as residential therapeutic communities, methadone maintenance clinics, and dedicated inpatient or outpatient treatment programs, yet the availability of these specialty care facilities has not kept pace with demand. As a result, an astounding 80% of opioid-dependent persons currently remain untreated in the U.S. One way policy makers have tried to address this treatment gap is by sanctioning delivery of Buprenorphine therapies by physicians working in office-based environments. Although shown to be generally safe and effective, the potential of this treatment modality remains unfulfilled, as only a small percentage of physicians have taken advantage of this opportunity. The reasons cited for this include concern about possible patient abuse and/or misuse of the drug, the intensive demands associated with providing treatment to a population of addicted patients, and the additional effort and legal liabilities involved in complying with burdensome DEA requirements for prescribing controlled substances. To address this problem, we propose the development and testing of BupreCare, a comprehensive treatment management solution for delivering Medication Assisted Therapy (MAT) to opioid-dependent persons in an office based setting. BupreCare will be centered around three primary components - a specialized drug dispensing device (DDD) pre-loaded with a generic formulation of buprenorphine/naloxone that controls access to medications via limited-use pass codes, a web/phone (IVR) patient portal patients use to obtain these pass codes and report on treatment status, and a web-based administrative portal providers use to setup treatment parameters and monitor patient progress. Phase I development efforts will focus on the development and testing of the BupreCare Induction Kit (BIK), which patients and providers can use to facilitate a safe and convenient induction experience. To this end, a high fidelity looks-like, works-like physical prototype of the BIK will be iteratively refined during usability testing, and then used in a pilot study in which the induction experience of patients using BIK is compared to those receiving treatment as usual (TAU). Outcome measures of interest will include opioid usage at 1-week, as well as treatment cost, opioid cravings, symptom severity, and treatment satisfaction. Patient and provider acceptance will also be measured as part of an acceptance study. Finally, the remaining research and development tasks, commercialization plan, and field trial design proposed for Phase II will also be completed. PUBLIC HEALTH RELEVANCE: The use and abuse of opioids is a significant healthcare problem costing the U.S. upwards of $300 billion annually. However, the vast majority of opiod dependent persons remain untreated for the disorder, even though effective medication therapies, such as Buprenorphine, could be made available to them through their own personal physician. This research will develop and test a low-cost treatment management solution designed to increase the practicality, safety, and effectiveness of medication therapies for opioid dependence when delivered by physicians working in an office-based environment, thereby making the treatment more readily available to larger segments of the patient population."}
{"text": "Retroviral proteases are enzymes that cleave gag and gag-pol precursor polyproteins into functional proteins of mature viral particles, an event necessary for infectivity of virus particles. Therefore, characterization of these proteases and development of specific inhibitors should lead to control of infectivity. Recent evidence suggests that the HIV protease belongs to the aspartic proteinase family and may be a dimmer. The specific aims of this project are: (1) to isolate HIV protease, (2) to characterize its substrate specificity as an aspartic proteinase, (3) to test its interaction with known aspartic proteinase inhibitors, (4) to develop screening methods for tests of candidate de novo inhibitors, and (5) to characterize its structural properties, in order to investigate additional means of inhibiting the protease. The protease will be isolated from infected cell lysates and from an E. coli recombinant expression system using affinity to pepstatin-Sepharose, or to antibody resin, or by conventional means. The amino- and carboxyl-terminal sequences of the isolated protease will be determined to establish the precise boundaries of the protease gene in the viral genome. An assay based on proteolysis of the gag precursor will be developed. Subsequently, substrate specificity toward gag sequences and to aspartic proteinase substrate and inhibitors will be tested in order to determine how related the catalytic mechanism of the protease may be to other proteases. The active site subsite specificity will be mapped by chemically mutating the known gag protein sequence and determining affinities of these analogs to the protease. Physical properties such as the effect of various conditions on enzymatic activity and stability will be tested. These studies will allow us to gain a better understanding of the substrate specificity and inhibitory spectrum of the HIV protease and should allow us to ultimately design even more efficient inhibitors which could possibly be used in vivo to inactivate viral action."}
{"text": "Almost 200,000 new cases of epilepsy are diagnosed every year in the United States. Many of these are caused by an initial precipitating injury (IPI) (e.g status epilepticus, febrile seizures or traumatic brain injury). There is a need to develop interventions that could prevent the occurrence of epilepsy in these patients. The clinical challenge for testing and applying antiepileptogenic therapy is in identifying the subset of those who eventually became epileptic out of approximately 2 million individuals experiencing an IPI each year. The NINDS, in association with the AES, recently published a report identifying the most important research directions that should be undertaken to ultimately find cures for epilepsy. One of the 3 benchmarks considered as a top priority for the near future is the identification of biomarkers for epileptogenesis. At the present time, no biomarkers predictive of the likelihood of developing epilepsy after an IPI are available and this is an important reason why no clinical trials have identified an intervention during the latent period that clearly prevents the occurrence of epilepsy. The main goal of this proposal is to determine whether a new noninvasive electrographic putative biomarker of epileptogenesis in an animal model of chronic epilepsy can be consistently recorded during the latent period, and whether it can be used to reliably identify which animals later develop recurrent spontaneous seizures. The results of the proposed research could be used to facilitate assessment of antiepileptogenic interventions in patients. The putative biomarker we wish to study is an abnormality of the UP-DOWN State (UDS) EEG pattern. The features of the normal UDS pattern consist of an UP-phase and a Down-phase as a slow oscillation with a frequency of less than 1 Hz. The UP-phase is associated with prominent beta-gamma oscillations. The features of the pathological UDS pattern, which are seen only in epileptic animals include: 1- the occurrence of epileptiform events we have termed UPspikes during the UP-phase, and 2 - a prolongation of the UP-phase duration. We hypothesize that this pathological UDS pattern could be a valuable predictor of future seizure occurrence. We also propose to evaluate mechanisms of pathological change in the UDS pattern by analyzing the activity of principal cells and interneurons identified by juxtacellular labeling. We will focus our efforts on the analysis of the UDS electrographic pattern prior to pilocarpine induced status epilepticus and compare it to activity recorded during the latent period before spontaneous seizures occur. We anticipate that the identification of pathological features in the UDS EEG pattern will make them a valuable early diagnostic biomarker of epileptogenesis and predictor of later seizure occurrence. An understanding of the neuronal mechanisms underlying the pathological UDS EEG patterns will provide novel targets for future approaches to the treatment of epilepsy in its earlier stages, and help pave new ways to prevent epilepsy. PUBLIC HEALTH RELEVANCE: At the present time, no biomarkers predictive of the likelihood of developing epilepsy after a traumatic brain injury are available and this is an important reason why no clinical trials have identified an intervention during the latent period that clearly prevents the occurrence of epilepsy. Early diagnosis of progressive epileptogenesis with early intervention is important for more effective treatment options and disease management strategies. The goal of this proposal is to identify cellular mechanisms responsible for generation of UPspikes, which is a new biomarker of epileptogenesis."}
{"text": "PROJECT SUMMARY/ABSTRACT Individual birth cohort studies have identified risk factors for developing childhood asthma, including environmental exposures in early life such as allergens, pollutants, patterns of infection and colonization with viruses and bacteria, and psychosocial stress. Despite such advances, further progress in understanding the root causes of asthma have been hampered by at least two factors. First, procedures and scientific methods are not standardized across cohorts, making it difficult to compare and validate findings. Second, asthma definitions across cohorts vary considerably. In fact, asthma is a syndrome; there are different subtypes of asthma with distinct clinical features (?asthma phenotypes?) and likely different etiologies (?asthma endotypes?). We hypothesize that host factors (genetics, epigenetics) interact with environmental exposures during the prenatal period and early childhood to cause specific endotypes of childhood asthma. We further propose that identification of endotypes and associated molecular biomarkers in early life can provide a new paradigm for asthma prevention. Unfortunately, single cohorts have limited ability to identify asthma endotypes due to small sample size and unique population characteristics. To overcome shortcomings of individual cohorts, investigators leading 12 asthma birth cohorts across the U.S. now propose the establishment of the Children's Respiratory Research and Environment Workgroup (?CREW?) consortium. This consortium proposes to identify asthma endotypes and overcome shortcomings of individual cohorts by: 1) providing a large (nearly 9000 births and long-term follow-up of 6000-7000 children and young adults) and diverse national data set, 2) harmonizing data related to asthma clinical indicators and early life environmental exposures, 3) developing standardized measures for prospective data collection across CREW cohorts and other ECHO studies, and 4) conducting targeted enrollment of additional subjects into existing cohorts. This approach will enable collection of samples that are optimized for a systems approach to understanding how environmental and host factors in early life promote the development of specific asthma endotypes. Collectively, the results of this comprehensive research to identify the root causes of asthma vs. resilience and health will go far beyond what can be accomplished by individual cohorts, and thus provide a foundation for future efforts aimed at personalized prevention of chronic childhood asthma."}
{"text": "Protocadherins are new members of the cadherin superfamily. Despite the great number of protocadherin genes identified in vertebrates, little is known about their biochemical properties, functions or working mechanisms. Xenopus paraxial protocadcadherin (PAPC) was recently shown to play a role in morphogenetic cell movements during early embryonic development. However, the adhesive properties of PAPC still remain ambiguous and poorly understood, and whether its morphogenetic role is realized through its adhesive function or by other mechanisms is unclear. The long-term goals of this proposal are to elucidate the adhesive functions and mechanisms of PAPC and to understand the mechanisms of its morphogenetic activity by identifying the cytoplasmic PAPC-binding proteins. First, the adhesive properties and mechanisms, including adhesive strength, specificity, lateral dimerization and clustering, will be examined and analyzed with the cell aggregation, bead aggregation and cell attachment assays; second, the cellular localization of PAPC will be monitored to assess its role in polarized cell movements; third, the function of PAPC cytoplasmic domain, especially a highly conserved region, on the adhesive and morphogenetic activity of PAPC will be analyzed using deletion mutants; and finally, the proteins that bind PAPC cytoplasmic tail will be identified as candidates for regulation of PAPC adhesive function or PAPC-mediated signaling by means of affinity chromatography and/or the yeast two-hybrid method. The proposed research will shed light on the role of protocadherins in embryonic development and should provide important suggestions to the treatment of birth defects and other developmental diseases."}
{"text": "In this study we will take advantage of the ease of manipulation of yeast for the study of the regulation of mitochondrial biogenesis as influenced by the determinants of lipid synthesis, especially phospholipid synthesis. (A) The purification of CTP phosphatidic acid cytidyl transferase is to be pursued with the hope of preparing specific antibodies to this enzyme, thought to be located mainly in the inner mitochondrial membrane. Such antibodies are to be used to study the synthesis of this enzyme and its regulation. (B) Attempts are being made to isolate lipid mutants, including temperature sensitive mutants for the CTP phosphatidic acid cytidyl transferase to further elucidate the role of lipids in the control of mitochondrial biogenesis. (C) We will continue to probe the mechanism that determines the differential synthesis and assembly of cytochrome oxidase in unsaturated fatty acid auxotrophs of yeast grown in cis or trans fatty acids."}
{"text": "The astroglial transporter GLT-1/EAAT2 is responsible for the largest percentage of glutamate transport in forebrain. Abnormal expression/function of EAAT2 is common to sporadic ALS, to transgenic models of the disease, as well as other neurological injuries. The mechanism that underlies transporter deregulation is not fully understood, but may involve altered promoter activation. We hypothesize that regulating expression of these proteins could provide powerful therapies to retard disease progression. Initial studies provide exciting evidence that increasing EAAT2 protein/activity can retard neurodegeneration in ALS animal models;diminish seizures associated with epilepsy, and retard brain tumor growth. In this proposal we will use recently generated GLT1 and GLAST-promoter reporter mice to evaluate the regulation of transporters. These rodents will be used to study the dysregulation of transporters in neurodegenerative disease. We propose to alter transporter gene activation as a novel means to delay neurodegenerative disease. The completion of these studies will provide a comprehensive understanding of transporter regulation in normal and abnormal CNS and their potential as neuroprotectants. Specifically we propose: 1) To understand the normal regional and temporal regulation of astroglial glutamate transporter gene expression, thru analysis of GLAST-BAC and GLT1-BAC promoter reporter expressing transgenic mice;2) To test the hypothesis that altered glutamate transporter expression in neurological injury is the result of promoter deregulation;3) To determine if altered cellular expression of astroglial glutamate transporters can prevent neuronal degeneration. Using drugs capable of increasing GLT1 and GLAST promoter expression we will determine if increased expression of the astroglial transporter(s) can prevent neural injury in vivo and we will determine if cell specific expression of glutamate transport alters chronic neurodegeneration using an animal model of amyotrophic lateral sclerosis. We hypothesize that over expression of GLT1 or GLAST will be neuroprotective in disease models. Over all Significance: In this proposal, we will develop a basic understanding of expression of GLAST and GLT I, discover reagents that can alter glutamate transporter activity, and determine if these approaches are useful for chronic neurological insults."}
{"text": "This program tests molecular imaging variables, how they predict response to therapy, and how they change over the course of different treatments. We propose 5 interactive projects: 1. Proliferation studies to validate how well FLT compares to TdR in patients with a variety of tumors and explores ways to simplify the PET procedure and still distinguish flux from transport. Our hypothesis is that FLT with appropriate data analysis can be as accurate as TdR for reporting tumor proliferation. We will test the hypothesis that FLT PET plus MRI can distinguish between tumor progression and pseudoprogression in patients with GBM that finish initial therapy with external beam RT plus concurrent TMZ. 2. Tumors have variable levels of multi-drug resistance that reduces effectiveness of drug treatments. We will quantify transporter activity of P-glycoprotein as a suspected cause of treatment resistance and failure by analyzing [11C]-verapamil kinetics. We will test the hypothesis that pts who up-regulate their tumor Pgp activity after neoadjuvant chemotherapy will have shorter survival and time to progression. 3. Hypoxia is an important resistance factor that is imaged using [18F]-FMISO. In pts with brain tumors we will test the hypothesis that FMISO images following initial surgery define an appropriate target for focal boost RT that will improve time to tumor progression and survival. In pts with ER-neg metastatic breast cancer, we will compare the extent of hypoxia to response to systemic therapy and progression-free survival. Other experiments will compare FMISO and [64Cu]-ATSM and we will measure temporal changes using an FMISO test-retest protocol and BOLD-MRI to look for short term changes in regional oxygenation of tumors. 4. This project will test the use of 18F-FES PET to choose therapy in pts with a history of ER+ breast cancer who have failed prior regimens and are being considered for salvage endocrine therapy and use FES PET to measure regional ER as a pharmacodynamic response to endocrine therapy and FDG PET to localize active tumor and identify early effects of drug therapy, including HER2 and ER. The project will also image androgen receptor function in prostate cancer to identify heterogeneous AR expression. 5. Preclinical radiopharmaceutical development and research. New methods will be developed to image important aspects of the tumor phenotype. Some are new molecules;some are new applications for old molecules;all to develop promising imaging agents for characterizing the tumor phenotype. These include ligands for HER2 as a factor in treating breast cancer, new agents for imaging of androgen biology, a monoamine oxidase A ligand to select patients for a new clinical trial for prostate adenocarcinoma and studies comparing Cu-ATSM and FMISO for different mechanisms of uptake."}
{"text": "The purpose of this investigation is to improve understanding of the effects of children's direct exposure to 9/11 and future mental health disorders, substance use or abuse and other risky behaviors as they now enter into adolescence and emerging adulthood. Based on the behavioral development literature, our city-wide assessment of children 6 months after 9/11 and the numerous findings of post- 9/11 consequences amidst exposed adults, there is strong evidence to suggest that exposure to 9/11 early in life would increase directly exposed children's vulnerability within the domains of interet in this investigation. Unfortunately, to date, the science-base of post-disaster behavioral consequences for youth, including those who were 9/11 exposed as children, lacks a longitudinal assessment, that includes in-depth measurement of the multiple, complex relevant domains, from type and severity of exposure to well-defined behavioral outcomes and intrapersonal, familial, academic and environmental factors. The proposed study will assess 1,000 children directly exposed to 9/11 at ages 0-12 and currently ages 12- 24, drawn from the World Trade Center Health Registry (WTCHR) Pediatric sample, who will be compared to unexposed Controls (N=500). The study involves a synergistic collaboration between the Child Psychiatric Epidemiology Group (CPEG) at Columbia University-Research Foundation for Mental Hygiene and the WTCHR. The WTCHR has longitudinal data on this population since 2002-2003, including excellent participant tracking information. CPEG has conducted many large-scale assessments of vulnerable youth, including 9/11 exposed children and their parents, addressing themes of high"}
{"text": "This project is concerned with 8 major areas of neuropsychopharmacologic drug investigation: 1) early rapid-screening activity studies of new short-acting and long-acting psychoparmacologic compounds in severely ill and chronically hospitalized schizophrenic patients (phase I and II); 2) intensive double-blind studies in this same population; 3) improvement and development of blood extraction procedures, e.g., chromatographic analyses, to correlate those blood concentrations; red blood cell concentrations as well as plasma and serum levels of psychopharmacologic agents required to produce specific therapeutic changes in man (since recent measures of plasma levels show no correlation with therapeutic response--see supporting data concerning long-acting haloperidol); 4) attempts to correlate these blood concentrations required to produce CNS side effects in man; 4) six month evaluations of those long-acting oral and intramuscular compounds that appear to be especially promising therapeutic agents for evaluation of toxicity as well as ability to show sustained antipsychotic activity in the above patient groups; 6) controlled inpatient studies in adolescent schizophrenic patients as well as in acute adult schizophrenic subjects; 7) early rapid screening studies of new antidepressants (including comparison studies with lithium) and antianxiety compounds in nonpsychotic alcoholic inpatents in order to determine if one or more specific psychopharmacologic-responsive subgroups exist within this population; 8) double-blind studies of new active antianxiety and antidepressant agents in the same patient population (see supporting data fo drug-placebo differences in this population)."}
{"text": "SUMMARY Intestinal failure is caused by a variety of enteropathies ranging from short bowel syndrome due to conditions such as necrotizing enterocolitis to congenital intestinal disorders. The mainstay therapy for patients with intestinal failure is parenteral nutrition (PN) but there are significant clinical complications associated with PN including infections, hepatic dysfunction and sepsis. Thus, a major challenge for intestinal research is to successfully develop new concepts and therapeutic approaches for the treatment of patients with intestinal failure that are dependent on PN. Advances in intestinal stem cell (ISC) biology, obtained through analysis of genetically-modified mice and cell lineage tracing studies, have garnered much interest in ISCs as a therapeutic target. Our long-term goal is to understand the extracellular signaling pathways that regulate human ISC niche, and how these pathways can be utilized for regenerative medicine or modulated in disease states such as intestinal failure. The recent development of directed differentiation of human pluripotent stem cells into intestinal tissue has afforded new opportunities to study human gastrointestinal development, tissue homeostasis and disease processes in vitro. Human intestinal organoids (HIOs) provide an in vitro model to study the direct interactions of epithelial and mesenchymal signals involved in maintenance of the ISC niche. Significantly, for patients with severe enteropathies where access to intestinal tissue is restricted, iPSC-derived HIOs provide a novel approach to study the intestinal biology of these patients. ADAM17 (TNF? converting enzyme) is a ubiquitously expressed sheddase responsible for shedding and activation of several growth factor/cytokine receptor families. In the mouse intestine, ADAM17 is implicated in complex intercellular communication involving EGFR/ErbB and TNFR signaling within multiple cell types of the ISC niche to maintain intestinal homeostasis, especially when faced with intestinal injury and inflammation. Recently, several patients from two different families were identified with distinct homozygous autosomal recessive ADAM17 null mutations defining a new rare congenital intestinal disorder termed neonatal inflammation skin and bowel syndrome. Our central hypothesis is that ADAM17 regulates signaling cross-talk between epithelium and mesenchyme of the human ISC niche. We will address this hypothesis in 3 aims: 1. generation of ADAM17 mutant and ADAM17 mutation corrected iPSC lines from dermal fibrobalsts obtained from an ADAM17 null patient. 2. Analysis of intestinal programming from ADAM17 mutant and ADAM17 mutation corrected iPSCs using HIOs and HIO xenograft transplantation and 3. Study the contribution of ADAM17 signaling from epithelial and mesenchymal layers from these HIOs. At the conclusion of this project, we should have gained detailed understanding of the role for ADAM17 signaling in maintaining the human ISC niche and defined ADAM17 or its substrates as potential new molecular targets, that may result in new approaches for stem cell therapies in regenerative medicine and for the treatment of intestinal failure."}
{"text": "We are interested in how high frequency (30+ Hz) brains oscillations are generated using the mouse olfactory bulb as our model system. These oscillations are a prominent feature of neural activity in many brain areas including the olfactory system. The mechanisms that underlie these oscillations, as well as the functional role they play are poorly understood, but they have recently been implicated in many aspects of brain function and disease. In the previous funding period, we have described a novel mechanism, called stochastic synchrony, by which correlated fast fluctuating inputs can generate synchronous gamma-frequency (30-80Hz) oscillations in populations of neurons. While much of our previous work used olfactory bulb neurons as a model system for analysis of this novel mechanism of synchronization, this prior work ignored many details of olfactory bulb neurons and circuits in order to describe the general features of this phenomenon. In this application we propose to extend our previous work by considering how key features of olfactory bulb circuitry and physiology modulate stochastic synchrony. We are particularly interested in whether the observed heterogeneity of neural properties is a useful feature of neural networks or is a \"bug\" that results from the intrinsic imprecision of biological systems. Homogeneity should enhance synchrony but recent data suggests that heterogeneity across neurons of the same type may provide certain functional advantages. We are also interested in how the synaptic connectivity of the olfactory bulb may facilitate or disrupt synchrony. Exploring these mechanisms will improve our understanding of the function and disorders of synchrony, especially in the context of the vertebrate olfactory system PUBLIC HEALTH RELEVANCE: Activity of neurons in the olfactory system represents information about sensory stimuli in the environment. One of our long term goals is to understand how to interpret features of neuronal activity as representing features of olfactory stimuli. To accomplish this goal we believe that it is critical to develop our understanding of the dynamics of neuronal activity through the use of experiments and computational models. In this proposal we describe an integrated experimental and computational approach to analysis of the mechanisms of patterns of periodic synchronized activity in the olfactory system. In particular we want to understand how the detailed and diverse biophysical and synaptic properties of olfactory bulb neurons contribute to the generation of patterned activity."}
{"text": "The broad, long-term objective of this proposal is to understand which tissues and genes respond to caloric restriction, and to explain how such changes affect the overall physiology, health and longevity of animals, including humans. The hypotheses to be tested are: (1) An H1 linker histone, HIS-41, is a direct target of DAF-16 and is one key transcriptional output of scenarios that increase longevity. (2) HIS-41 is necessary, but not sufficient, for the metabolic shift and increased longevity conferred by reduced insulin-like signaling or caloric restriction. (3) Distinct longevity mutants induce different HIS-41 accumulation patterns appropriate for the affected target tissue. (4) Caloric restriction extends life span by activating two synergistic processes: HIS-41 dependent silencing that reduces metabolic rate and reactive oxygen species levels; and increased production of factors that are co-regulated with HIS-41, such as superoxide dismutase. The specific aims are: (1) Identify the sequence elements required for DAF-l6- dependent induction of his-41 in vivo (hypothesis 1). (2) Assay fat accumulation and life span in calorie restricted his-41 mutants or his-41- gerontogene double mutants; and determine whether forced HIS-41 expression is sufficient to phenocopy longevity mutants (hypothesis 2). (3) Identify genes that regulate HIS-41 levels and genes that are coordinately regulated with his-41 (hypotheses 1,2). (4) Determine the cell type specificity of HIS-41 accumulation in calorie restricted vs. single and double mutant combinations of distinct longevity pathways (hypothesis 3). (5) Measure the life span of transgenic animals that constitutively express HIS-41, SOD-3, and genes that are co-regulated with HIS-41 (hypothesis 4). A central view of this proposal is that widespread genomic silencing, mediated by an H1 linker histone, is a common feature of many genetic pathways that extend life span and increase stress resistance. Environmental factors, including caloric restriction, increase longevity by turning on the H1- mediated repression of genes for rapid growth and glucose-based metabolism, thereby reducing the overall rate of metabolism and the production of reactive oxygen species. The activation of the H1 repression system is done by the same transcription factor (DAF-l6/forkhead protein) known to up regulate enzymes for fat storage and utilization, as well as enhanced life maintenance (e.g. superoxide dismutase)."}
{"text": "Susan Steck-Scott, PhD, MPH, RD is a research assistant professor in the Department of Nutrition at the University of North Carolina at Chapel Hill. Through this Career Development Award, she is seeking training in molecular epidemiology and cancer survivorship to complement her strong background in the nutritional sciences and cancer prevention. Dr. Steck-Scott's primary mentor for this proposal, Dr. Marilie Gammon, is a renowned breast cancer epidemiologist with extensive experience in mentoring young investigators. In addition, Drs. Lisa Carey, Robert Millikan, Michael Schell and Fred Wright are well established investigators in their fields and have agreed to be co-mentors on this project. Training will include coursework and workshops in ethics, molecular epidemiology, carcinogenesis, pharmacology, and biostatistics, as well as a laboratory experience to learn the techniques of genotyping. This training will help the candidate to develop a unique niche in the cancer research community and establish an independent research program in the area of gene-nutrient interactions in cancer prevention and control. The primary focus of the award is a research project to examine the interaction between cruciferous vegetable intake and polymorphisms in genes encoding metabolizing enzymes in breast cancer prevention, recurrence and survival. The proposed research capitalizes on dietary data, urine and blood samples available from an already conducted population-based, case-control study, the Long Island Breast Cancer Study Project of which Dr. Gammon is the Principal Investigator. Breast cancer subjects are currently being followed up for recurrence and survival status. The specific aims of the research are 1) to examine the associations between cruciferous vegetable intake, urinary isothiocyanate (ITC) excretion, and polymorphisms in GSTM1, GSTT1 and GSTP1 in control subjects to determine whether having the non-null or active genotype for GSTM1, GSTT1 and/or GSTP 1 results in increased urinary excretion of ITC; and 2) to examine the interaction between cruciferous vegetable consumption and polymorphisms in GSTM 1, GSTT 1, GSTP 1 and CYP 1A 1 in relation to breast cancer risk in a large population-based case-control study; and 3) to examine the interaction in relation to breast cancer recurrence and survival in a follow-up study of the cases. This study represents a unique opportunity to examine whether cruciferous vegetables are modifying the effect of genetic polymorphisms in breast cancer prevention, recurrence and survival. By identifying those individuals who may be genetically more susceptible to the benefits of cruciferous vegetable intake, future and ongoing interventions may become more effective by being targeted to those people who will receive the most benefit."}
{"text": "The goal of the University of Pittsburgh Global Health Research and Research Training eCapacity Initiative is to enhance evidence-based, data-driven decision-making in India through innovations in information and communication technology using modeling and simulation to assess the health and economic impacts of alternative disease control strategies. To globalize the advantages of methods and curricula developed in the Public Health Dynamics Laboratory (PHDL) at the University of Pittsburgh, we believe it is essential to have local public health experts take the lead in developing, running and analyzing the models. Our objective is to enhance the capacity of researchers to use computational modeling, not only at our Fogarty training site, SHARE INIDA (D43 TW009078), but also at multiple Fogarty training sites in India. In the introductory Phase 1, a two-day symposium (35 participants), SHARE INDIA, Hyderabad, will introduce the concept of the integrated decision support pathway, and basic modeling software and tools. This symposium will showcase research that enables data-driven decision making using cutting-edge methods including mathematical, network, and agent-based modeling. Five Indian investigators plus one modeling support staff will receive in-depth, hands- on training in computational modeling during a week-long workshop at the University of Pittsburgh PHDL. This workshop on complex agent-based modeling will use our open-source modeling platform, FRED (Framework for Reconstructing Epidemic Dynamics). This experience will foster establishment of a new collaborative network for modeling studies among Indian researchers and with PHDL researchers. Two small modeling developmental grants will be awarded. Phase 2 will focus on sustainability. Two small research grants will help sustain newly developed, collaborative modeling projects. Two PHDL modelers will return to India to visit the recipients of the small grants and solidify collaboratios. Two week-long short courses on computational modeling in public health, incorporating educational materials and free software from the Pittsburgh Workshop, will be presented by Indian faculty at CR Rao Institute targeting 25 participants each. A second introductory symposium for 35 new participants, led by newly trained Indian modelers, will stimulate a new wave of interest in modeling. Our aims address an unmet need in Indiai.e. building capacity of researchers to use cutting-edge, yet appropriate technology to address policy-relevant questions. The education program will result in increased knowledge among Fogarty-affiliated researchers in India of the kinds of data needed to inform models and basic methods in modeling. Trainees who attend the workshops in Pittsburgh and sustain their use of modeling through small research grants are expected to continue to apply computational modeling methods to address Public Health questions of importance to India. These trainees are also expected to lead computational modeling collaborations within India and with PHDL faculty. In the future, the program is expected to result in funded research that aids data-driven decision-making in Public Health in India."}
{"text": "The goal of the proposed research is to characterize the amino acid regulatory interactions which occur in Saccharomyces cerevisiae. Growth of this organism in medium supplemented with a variety of amino acids results in a decrease in the intracellular arginine pool, but concomitant induction of arginine catabolic enzymes and repression of arginine anabolic enzymes. The specificity and magnitude of these interactions will be determined. The contribution of changes in enzyme levels to the observed decline in the arginine pool will be investigated. The kinetics of enzyme induction and repression will be compared to that observed when arginine is added to the growth medium. The role of the intracellular arginine pool in enzyme induction and repression will be determined. The physiological significance of these interactions will be examined by characterizing the behavior of mutants impaired in: (1) specific arginine metabolic regulation; (2) cross pathway regulation; and (3) nitrogen catabolite repression. The role of amino acid compartmentation in the regulatory interactions will be determined by measuring the distribution of arginine between vacuole and cytosol in cells growing in supplemented and unsupplemented medium."}
{"text": "To assess long-term symptomatic and objective relief in patients who underwent successful coronary angioplasty performed in 1980, 1981, and 1982."}
{"text": "A. Summary. We have preliminary data showing that in addition to its role in promoting osteoblast (OB) function and bone formation, fibroblast growth factor 2 (FGF2) is a negative regulator of mesenchymal stem cell differentiation into mature adipocytes (AD). We hypothesize that loss of FGF2 expression results in a shift of stromal mesenchymal progenitors from OB differentiation towards adipogenesis. The proposed studies will increase our understanding of the molecular mechanism (s) by which FGF2 affects aging bone as well as the role of FGF2 in the osteogenic and antiadipogenic effects of PTH in bone. Specific Aim 1: Determine how FGF2 modulates the adipocyte phenotype using Fgf2+/+ and Fgf2-/- mice in Col3.6-GFP or aP2-GFP genetic backgrounds. We will test the hypothesis that in the absence of FGF2, marrow progenitors have a reduced ability to choose the osteogenic pathway. To assess the age-dependence of the phenotype, we will examine young adult mice at 6-8 weeks of age and compare them to 4-5 month old adult mice that already exhibit reduced bone mass. Aim 1A: i) determine the temporal and quantitative onset of GFP expression in primary bone marrow stromal cultures (BMSC) from Fgf2+/+ and Fgf2-/- mice harboring transgene reporters for the OB (Col3.6-GFP) or AD (aP2-GFP or -Cyan) lineage; ii) characterize the AD and OB potential of GFP positive and GFP negative cells isolated via FACS analysis and then cultured in the absence and presence of exogenous FGF2 and PTH; and iii) examine changes in gene and protein expression. Aim 1B: Define the function of FGF2 during osteogenic versus adipogenic differentiation in vivo. Using mice developed in Aim 1A, we will i) assess whether there is a correlation of changes in bone mineral density and whole body and bone fat content. ii) examine the expression of key adipogenic and osteoblast signaling molecules from whole bones and from freshly isolated marrow; and iii) assess the effects of PTH, administered to mice alone or in combination with FGF2 on adipogenesis in ex vivo BMSC cultures. Specific AIM 2: Determine whether FGF2 is a necessary factor for PTH-mediated pro-osteogenic and anti-adipogeneic effect on mesenchymal progenitor cells. We hypothesize that FGF2 inhibits adipogenesis through modulation of Wnt 10b and PPARg in mesenchymal progenitors. We also hypothesize that in the absence of FGF2, PTH is unable to inhibit mesenchymal progenitor cell differentiation towards adipogenesis and this is mediated through regulation of PPARg by Runx2 and Wnt 10b downstream effects. Aim 2A: Examine the mechanisms by which FGF2 deficiency modulates the development of the OB or AD phenotype. We will determine whether FGF2 modulates Wnt 10b and PPARg2 activity and what signaling pathways mediate this in CFU-OB and CFU-AD from young and adult mice in vitro. Aim 2B: Define the transcriptional mechanisms underlying PPARg regulation by PTH and FGF2 signaling. We will test the hypothesis that one possible mechanism through which FGF2 and PTH crosstalk may regulate adipogenesis is through Runx2 and Lef-1/-catenin mediated control of the PPARg 2 promoter."}
{"text": "This is the second and final revision of a research grant to characterize nicotine withdrawal in pregnant smokers. Maternal cigarette smoking is the leading preventable cause of poor pregnancy outcomes and pediatric morbidity and mortality in the U.S. While 80% of pregnant cigarette smokers report seriously thinking about quitting while pregnant, most are unable to quit for even one week. Efficacious interventions have been developed for smoking cessation during pregnancy, but cessation rates are low, typically under 20%. One factor thought to play a substantial role in early smoking relapse and extensively investigated in non- pregnant smokers is nicotine withdrawal. To our knowledge, there are only two reports on nicotine withdrawal during pregnancy: a retrospective study in pregnant adolescents by Albrecht and colleagues (1999) and our group's recent prospective pilot study (Heil et al., 2006). Given nearly 30 years of research on smoking cessation during pregnancy, this represents a striking gap in this literature. Our initial pilot study resulted in two seminal findings. First, we observed that pregnant smokers report substantially elevated levels of withdrawal symptomatology prior to the cessation attempt. Second, even with this pre-cessation elevation, we were able to validate a number of withdrawal symptoms in pregnant smokers who sustained abstinence for five days and establish their incidence, magnitude, and acute time course. While a significant step forward, our pilot study raised additional questions about nicotine withdrawal symptomatology in pregnant smokers. The two aims that make up the proposed study are designed to follow up and extend the literature in this area. First, we propose performing a thorough examination of pre- cessation elevated withdrawal symptomatology, comparing levels in pregnant smokers to those in pregnant non-smokers and non-pregnant female smokers in an effort to elucidate the source of the increased symptomatology. Second, we propose performing a more comprehensive and rigorous characterization of nicotine withdrawal during pregnancy, increasing the sample size and lengthening the time course from our pilot study. The proposed study will partner with an ongoing clinical trial testing the efficacy of voucher-based incentives to promote smoking cessation during pregnancy. We will analyze nicotine withdrawal data collected from pregnant women who abstain as part of this trial and compare results to those of abstinent non-pregnant female smokers and pregnant non-smokers. PUBLIC HEALTH RELEVANCE: The proposed study has the potential to contribute important new information about nicotine withdrawal among pregnant women. Such new knowledge may contribute to the development of more effective treatments for one of our nation's most daunting drug abuse problems."}
{"text": "The Prevention & Control Research Core has been developed with Prevention & Control and Clinical Researchers of the DRTC to provide a range of consultation and direct services to support behavioral, patient centered/clinical, educational, and prevention research. Seven core services are: (1) assistance in recruitment of clinical and non-patient samples and recruitment of research settings for community oriented research; (2) assistance regarding clinical aspects of diabetes and assessment of clinical status and diabetes risk; (3) assistance regarding epidemiological aspects of research and analyses and accessing administrative databases; (4) assistance in planning, developing and evaluating health education interventions, including addressing cultural and psychological factors, diet and nutrition; (5) measuring psychological factors and quality of life among those with diabetes; (6) data gathering, administration of population-based and clinicbased surveys, and data management; and (7) statistical analysis of data, including biostatistical consultation and analysis using geographic information systems. Because the core is newly developed following NIDDK's changed DRTC guidelines, plans are in place to monitor the use and utility of the varied core services, eliminate underutilized services, add promising services, and reallocate funds accordingly. Not including research support for projects not using the Prevention & Control Core, users include six investigators with independent NIDDK funding (Brownson, Klein, Lustman, Racette, Sinacore, White), five investigators with other NIH funding for diabetes-related research (Haire-Joshu, Holloszy, Mueller, Newcomer, Wilfley), three investigators with non-NIH, externally funded diabetes-related projects (Fisher, McGill, Polonsky), and five investigators with prior or current Pilot & Feasibility funding and/or diabetesrelated projects planned (Auslander, Hams, Hershey, Perkinson, Schootman)."}
{"text": "We have run two subjects in the past 12-month period. The co-investigator on the grant, Dr. Thomas Schlaepfer, has temporarily returned to Switzerland in order to oversee the running of his own Neuroimaging Laboratory at the University of Bern. Pilot results so far are encouraging, and show activation in expected regions of basal ganglia, frontal lobe, and mesial temporal areas, which we hypothesized would be involved in cocaine's cerebral activity. Dr. Schlaepfer is returning to the United States for a visiting academic appointment in the 1999-2000 period and more subjects will be run in the study during this time."}
{"text": "The objective of this project is to develop a package of brief, safe and reliable measures of exercise tolerance that, in total, is broadly applicable and highly discriminating in population studies of older persons. The first phase of testing and evaluation has been completed at the Baltimore Veteran's Administration Medical Center through an interagency agreement (Y01-AG-4-0260). The following tests were administered on two occasions 7 to 10 days apart to a volunteer sample of 50 men and women age 54-80 years, 24 of whom had peripheral arterial disease: (1) timed usual and fast pace 4-meter walks; (2) timed usual and fast pace 20-meter walks; (3) 6-minute usual pace corridor walk; (4) seated step-test; and (5) treadmill walk. Heart rate at work, recovery heart rate, and blood pressure were measured. Two questionnaires were also piloted; one is a refinement of Taylor's Leisure Time Physical Activity questionnaire and the other measures physical function including ease of performance and level of fatigue as well as difficulty in performing higher order functional tasks. Preliminary results on test-retest reliability and associations among the walking and seated step tests were presented at GSA, November 1995. Analyses of the reliability and validity of questionnaires and final analyses of the exercise tolerance measures is proceeding. The original scope of work was completed in March 1996, approximately $15,000 under budget. A second phase of testing has been planned and will commence September 1996. To increase the measurement ceiling and improve the reproducibility of the 6-minute corridor walk, several consultants have suggested the walk be performed as fast as possible for the entire 6-minutes or for at least some extended portion. None of the recommended modifications have been tested against the more accepted treadmill walk protocol nor is there data or test-retest reliability. This will be accomplished in the second phase of the pilot study in which we will recruit 20 to 25 older healthy adults and test them twice 7 to 10 days apart. The following tests will be administered: (1) 400m corridor walk, with the first 100m serving as a warmup and the last 300m performed as fast as possible with encouragement and (2) treadmill walk. Heart rate will be continuously monitored and the Borg perceived exertion scale will be presented every 50m."}
{"text": "Little is known of the transduction mechanisms used by human taste cells. It is the purpose of this application to enhance techniques for taste cell isolation from fungiform papillae of human volunteers and to then investigate the physiological characteristics of these cells. Successful completion of this feasibility study will open new important routes for studying normal human taste response and give impetus to determining the course of human chemosensory disease. The three aims of this study are: to develop protocols to maximize viability of isolated human taste cells; to characterize stimulus-regulated changes in intracellular calcium ion activities and membrane potential in these cells; and to perform loose patch analyses on these cells to identify stimulus-regulated currents in these isolated cells. Procedures developed to date by the investigators on this proposal for isolated human taste cells have yet to be optimized, resulting in unacceptable variations among preparations. In order to allow characterization studies to proceed efficiently, isolation protocols need to be better defined. Experimentation with enzyme type/amount, trituration, media and holding conditions will be performed. Imaging studies of intracellular calcium and membrane potential using the dyes Fura 2 & di-8-ANEPPS will permit basic characterization studies to be performed. These will emphasize characterization of ion channels and normal responses of taste cells to pharmacological challenges. Responses to primarily sweet and bitter stimuli will be evaluated. Loose patch studies will be used to characterize individual cells as to taste modality and chemical specificity. Human taste cells have not been the subject of study for physiological experiments. The unique specificities of human taste can be used to test individual cells. Volunteers can be taste tested prior to have fungiform papillae removed to determine their response to particular stimuli. This research is of high impact since it deals directly with the receptor cells of humans, thereby strengthening the likelihood that therapy regimens can be developed to treat chemosensory disorders. Successful completion of this study will allow correlations to be made between psychophysical measures of taste and biophysical characteristics of the receptor cells. A more complete understanding of peripheral versus central processes of taste will be a likely outcome."}
{"text": "Acetylcholine (ACh) is a neurotransmitter widely distributed throughout the mammalian central and peripheral nervous systems. In the central nervous system the predominant physiological effect of ACh is a slow depolarization mediated by muscarinic receptors (slow m-ACh potential). Slow postsynaptic potentials (psps) may underlie some of the long-term changes in the nervous system. Long-lasting synaptic effects such as facilitation or potentiation may account for the synaptic plasticity that underlies the more complex psychobiological phenomena of learning and memory. Thus, better insight into mechanisms of specific slow psps may lead to better general understanding of synaptic plasticity and neuronal integration. Cholinergic synapses, in particular, are thought to be of prime importance in cortical memory processes, and disruption of cortical cholinergic pathways is involved in the development of certain dementias. A prevailing hypothesis of the pathogenesis of Alzheimer's disease is that the primary pathology is degeneration of a discreet group of cholinergic neurons in the basal forebrain. Because these neurons are the sole source of cholinergic afferents to the entire cortex, their loss leads to widespread cholinergic denervation of the cortex. The precise ionic mechanisms involved in the slow m-ACh potential in mammalian neurons are not known. Furthermore, the role of second messengers in mediating the m-ACh response has not been defined. Thus, compared with the detailed knowledge of nicotinic ACh receptors, the present state of knowledge of neuronal muscarinic ACh receptors lags far behind. These studies will use an in vitro system, sympathetic neurons in tissue culture. Voltage-clamp and patch-clamp techniques will be used to gain insight into the ionic mechanisms and the metabolic determinants that underlie the slow m-ACh response. Experiments will focus on specific ion conductance mechanisms and how they are controlled by biochemical second messengers. The specific aims are: 1) to establish the ionic mechanisms underlying the slow m-ACh depolarization in rat sympathetic neurons in tissue culture; 2) to determine the pharmacological sensitivity of the m-ACh response to various muscarinic agonists and antagonists; and 3) to ascertain if biochemical second messengers mediate the slow m-ACh potential."}
{"text": "Despite the significant reduction in HIV burden associated with HAART, reservoirs of latent virus remain in treated individuals. These reservoirs serve to provide a source for new virus when treatment is interrupted, and a site for creating drug resistant variants. In some individuals, discontinuation of HAART stimulates virus-specific T cell responses that appear to control virus. This phenomenon suggests that immunization with an effective vaccine during therapy may augment the effect of HAART. Two criteria are critical in the design of such a strategy: 1. Identification of the reservoir(s) so that immunization can be specifically targeted to it, and 2. The vaccine must induce responses that clear virus infected cells in the reservoir. We hypothesize that the intestinal lamina propria serve as a major reservoir of virus during HAART. Thus a mucosal vaccine is needed. An additional advantage is served by this approach because the GALT serves as an extrathymic site for T cell maturation, therapy overcoming the thymic destruction induced by chronic HIV infection. We propose to use a novel vaccine utilizing skin immunization with viral DNA co-delivered with vectors expressing genes for potent mucosal adjuvants, bacterial enterotoxin genes (CT or LT). Using the SIV: macaque model for AIDS this project will implement in vivo primate studies designed to compare the immunogenicity of SIV DNA with or Without vectors encoding CT or LT (project 1). Test vaccines encoding DC-specific chemokines and chemokine receptors (projects 3 and 4), and evaluate the ability of the optimized vaccine adjuvant formulation to serve as an effective adjunct to HAART in monkeys treated with PMPA (projects 1-4). Monkeys will be immunized, infected with titered stocks of SIV/DeltaB670 to determine the efficacy of treatment. The role of the GALT as a reservoir, and the ability of DNA immunization+/- PMPA to stimulate recovery (HAART +/- vaccine therapy) of immune responses in the GALT will be determined by analysis of antibody in mucosal secretions, function of CD4+ and CD8+ T cells, and quantitation of SIV-specific CD8+ cells by tetramer staining. T cells from the GALT will also be phenotyped to determine changes in lymphocyte subpopulations, the source of repopulating cells (naive versus memory subsets) and functional capability. The presence of SIV tetramer- binding CD8+ cells expressing alpha4beta7, the receptor mechanism whereby priming in the skin can induce effect responses in the mucosa, an ddetermine the relative magnitude of these responsres induced in the GALT by the different vaccine adjuvant formulations. The demonstration of this approach in identify the induction of mucosal immune response in the periphery of monkeys immunized during therapy will encourage similar studies in the analogous human trials described in project 5."}
{"text": "Biochemical, immunological, genetic and transgenetic experiments all indicate that the prion protein (PrP) is central to prion replication and disease susceptibility. Infectious prions are composed of a disease-specific PrP isoform designated PrPsc, and allelic forms of the PrP gene (Prnp) determine scrapie incubation time in mice. Mutant alleles of the human PrP gene (PRNP) are linked to familial prion diseases, and mice homozygous for a null allele of Prnp are resistant to prion disease and fail to support prion replication. However, additional factors impinge on prion replication and prion disease. Studies exploiting chimeric PrP transgenes and Prnp null mice suggest that efficient prion replication involves at least one auxiliary molecule, designated protein X. Both scrapie incubation time and the areas of the brain that are affected can differ dramatically among inbred strains of mice that express identical PrP molecules following infection with a single strain of scrapie. Treating prion incubation time as a quantitative trait led to the identification of at least two modifier genes. To identify the genes underlying these quantitative trait loci (QTLs), the QTL intervals will be isolated in congenic strains and the interval narrowed though high resolution mapping. The congenic strains also will be used to characterize the effects of each QTL on incubation time, pathology, distribution of PrPsc in the brain, PrP biochemistry, and concentration of PrPsc at onset of illness. Collaborative efforts with Dr. Hood (Project II) will take full advantage of advances in the Human Genome Project for candidate gene prioritization using oligonucleotide arrays and other technologies. BAC transgenesis and gene targeting will be used for identification of these and other prion disease modifiers that will be identified. QTL analysis is dependent on naturally occurring polymorphisms between mouse strains and thus samples only a small subset of potential modifier genes. Therefore, a screen will be established for chemically induced mutations that alter prion incubation time in transgenic mice so that all genes potentially are targeted. Finally, in collaboration with Drs. Hood (Project 11) and Prusiner (Project III), we will identify networks that are perturbed by misexpression of the PrP-related protein Dpl and screen for modifiers of Dpl-induced phenotypes. The combination of approaches will help define the genetic bases for susceptibility to prion disease and apply this new genetic knowledge to understand the underlying molecular processes."}
{"text": "During this period, emphasis of the work shifted from head restraint methods to methods of patient repositioning and to methods for reducing head motion not entirely eliminated by the thermoplastic mask restraint system now adopted as \"standard\" across the PET program. Testing revealed that the thermoplastic mask cannot, itself, be reliably used to reposition a subject from one imaging session to the next even though the mask is custom-made to each individual and can be attached to the imaging table in only one way. As a result, we used the spatial tracking system described in previous reports to measure (in normal volunteers) the repositioning accuracy of a method based on wall-mounted laser line projectors. Testing of this widely used technique revealed that although portions of the same tomographic plane can usually be replaced in the same imaging slice and within a spatial resolution element, exact repositioning of the entire slice is an uncommon event. Given this pair of results, work on the interactive repositioning system scheme using the spatial tracking device was resumed with testing ex- in the near future. Measurements have revealed that even with aggressive head restraint, residual motions within the restraint continue to compromise image quality. Independent knowledge of head position during the scan procedure can be used to correct these image data for patient motion. Feasibility studies of two such correction schemes have been carried out for both PET and conventional scintigraphic imaging studies with favorable results in both cases. Methods for efficient implementation of these computationally intensive techniques are now under development."}
{"text": "Graves? disease (GD) is the most common cause of hyperthyroidism, accounting for 60 to 80 percent of more than 250 million cases of hyperthyroidism globally. It is an autoimmune disorder caused by thyroid stimulating antibodies (TSAbs), which bind to the thyrotropin (or thyroid-stimulating hormone (TSH)) receptor on the surface of a thyroid cell. This leads to stimulation of the thyroid gland and subsequent synthesis and secretion of excess thyroid hormone. One crucial diagnostic tool is the use of assays for the quantification of TSAb, which can provide important guidance for the diagnosis and management of patients with both thyroidal GD and extrathyroidal manifestation. In addition, detection of TSAb is critical in predicting relapse or remission after initial anti-thyroid drug treatment or disease progression. Although many assays have been developed to detect autoantibodies against TSHR (TRAbs), it has been shown that among all commercially available assays, only cell-based bioassays can differentiate between stimulatory and inhibitory activity (TBAb) of circulating TRAb. However, cell-based bioassays are time-consuming, laborious, and relatively expensive. Therefore, there is a need for the development of simple, cost effective, and sensitive immunoassays that can differentiate between TSAb and TBAb in patient blood samples. Mediomics has developed a novel assay platform (PINCER assays) that allows simple homogenous mix-and-read detection of a variety of targets. These rapid-result assays can reach high specificity while remaining easy to use and at a low cost. In addition, our long-term collaborator, Dr. Tomasz Heyduk?s laboratory, developed a novel screening platform by combining both ribosome display and Next Generation Sequencing (NGS) technologies to quickly identify linear and conformational epitopes of antibodies from patient samples. Here, we propose to take advantage of Mediomics? innovative assay technology and the novel screening platform to develop highly specific immunoassays to detect stimulating and blocking autoantibodies (TSAbs and TBAbs) against the thyrotropin receptor. We have two specific aims in our Phase I project. Aim 1. Explore the entire protein sequence space of TSHR to identify peptide ligands specifically recognizing isolated human TSAbs or TBAbs. Aim 2. Prepare, optimize, and evaluate the performance of peptide-based immunoassays. If this project is successful, we would have validated the commercial potential of our assay, and will thus lay a foundation for transition to Phase II, where we will embark on the development of a commercialization-ready product and its clinical validation with well-defined clinical samples."}
{"text": "One important question about effects of media violence on anti-social behavior is whether exposure is likely to produce delayed or long term effects (Liebert & Baron, 1972). The proposed research is designed to assess whether \"memory\" variables influencing delayed recall of previous aggressive stimulation will strengthen delayed aggressive responses. Previous research has focused on the short term effects of exposure to \"external\" aggressive stimulation, but delayed responses to aggressive stimuli may occur if recalled from earlier exposures (internally-generated stimuli). The present interactive memory model predicts that an individual is likely to be aggressive if he is aroused and uninhibited when he recalls aggressive stimuli (e.g., media violence). Thus, aroused but uninhibited subjects who recall aggressive words are more likely to produce aggressive reactions than subjects who do not recall aggressive words. Three experiments are proposed to test the hypothesized interaction of recall, arousal, and inhibitions. These experiments are designed to manipulate accessibility (likelihood of recall); arousal; aggressive connotation of words; and evaluation apprehension. Subjects will serve in the \"Buss procedure\" (Buss, 1961), and shock intensity will be the measure of aggression. If the experiments support the proposed model, then it might be extended to important research questions about delayed effects of exposure to mass media violence."}
{"text": "The intestinal epithelium (IEC) with its luminal microflora serves as an ideal experimental and medical model that merges innate immune recognition in the context of a tissue regeneration environment. Genetic predispositions that cause imbalanced microbe-host interaction may contribute to the pathogenesis of inflammatory bowel disease (IBD). Various susceptibility loci for Crohn's disease and ulcerative colitis have been identified; however, their contributions to the disease mechanism remain poorly defined. A Crohn's disease susceptibility locus at chromosome 15q22 is immediately linked to RAB11A. This gene encodes a small guanosine triphosphatase (GTPase) that regulates the recycling endosome function. The applicant's laboratory has genetically targeted the mouse Rab11a, and analyzed IEC-specific Rab11a knockout mice. Preliminary data suggested that Rab11a controls epithelial-cell-intrinsic inflammatory cytokine response to enteric microbiota. Enterocytes deficient in Rab11a in Drosophila and mouse intestines overproduced proinflammatory cytokines, and caused early-onset enteritis, higher susceptibility to inflammatory neoplasia, and premature mortality. The long- term goal is to understand how the endosomal sorting of microbial receptors in IECs influences innate mucosal immunity and microbe-host homeostasis. The objective of this proposal is to establish the molecular mechanisms, by which Rab11a endosome-mediated sorting of Toll-like receptors (TLRs), in particular TLR9, support intestinal mucosal tolerance to microbiota. The central hypothesis is that Rab11a sequesters TLR9 to an inactive endosomeal compartment and dampens unwanted immune response to enteric microbiota at steady state conditions. Information learned about IEC-mediated modulation of inflammatory cytokine production and immune response may help reduce the adverse inflammatory response in IBD. This hypothesis will be tested with 2 specific aims: (1) to establish Rab11a endosomal compartment as an epithelial-cell-intrinsic modulator of cytokine response to microbiota; and (2) to determine the mechanism of Rab11a-controlled TLR9 transport and activation in response to microbial agonists. The experiments will use the novel Rab11a conditional mouse model that facilitates inducible Rab11a deletion, IEC-specific Drosophila Rab11 RNA interference (RNAi) lines, RAB11A-depleted human colon epithelial cell lines, and in vivo perfusion of microbial agonists. The combination of these genetic models with in vivo physiologic analyses in the study of endocytic control of microbe-host homeostasis is innovative and a major step toward understanding IBD."}
{"text": "The Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) is a devastating consequence of systemic inflammatory conditions such as sepsis that afflicts almost 200,000 people a year in the US with 75,000 deaths. The hallmark of ALI is inflammation-induced disruption of the endothelial cell (EC) barrier that lines the pulmonary vasculature, resulting in leakage of fluid, protein, and cells into the airspaces of the lung. Our laboratory has extensively studied the mechanisms involved in maintaining and enhancing EC barrier function as a tool for identifying possible therapeutic targets. The current paradigm of EC barrier regulation suggests a balance exists between barrier-disrupting cellular contractile forces and barrier-protective cell-cell and cell-matrix tethering forces. Both competing forces in this model are intimately linked to the actin-based endothelial cytoskeleton by a variety of actin-binding proteins. Our work has defined an essential role for the actin-binding protein, cortactin, in the resolution phase of vascular permeability with this critical function occurring via EC cytoskeletal rearrangement. Very little is known about the mechanisms governing recovery of EC barrier function following injury. Thus, cortactin is an attractive molecular target for novel therapies and warrants the intense structure/function studies we propose in this application. With this background, the PI proposes to investigate the hypothesis that cortactin regulates EC cytoskeletal rearrangements that result in altered barrier function during ALI syndromes. In SA#1 we will mechanistically characterize the key portions of the cortactin molecule involved in barrier regulation through the use of molecular biology and proteomic techniques utilizing in vitro models of barrier disruption (e.g., thrombin, TGFpl) to focus on cortactin's role during the barrier recovery phase. Transgenic animal models of ALI will extend these studies in vivo. In SA#2 we will examine the role of cortactin in cortical actin and junctional protein rearrangements that regulate pulmonary endothelial barrier function using novel atomic force microscopy (AFM) and other techniques to functionally characterize cortactin's role in peripheral cytoskeletal rearrangements involved in barrier recovery, focusing on cortical actin structures, junctional complex formation, and lipid raft signaling. In SA#3 we will characterize the functional consequences of an ALI- associated coding single nucleotide polymorphism (SNP) we have identified in the cortactin gene through a combination of molecular biology and proteomic techniques. This aim will determine the mechanistic effects of this ALI-associated SNP on cortactin function as it pertains to endothelial permeability and barrier recovery using the in vitro and transgenic animal techniques described above. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]"}
{"text": "PROJECT SUMMARY\\ABSTRACT Predisposition to develop Rheumatoid arthritis (RA) is associated with the presence of certain HLA alleles while others offer resistance. HLA-DQ8 and DRB1*0401 molecules render humans and mice susceptible to develop arthritis while DQ6/DR2 and DRB1*0402 provide protection. Collagen-induced arthritis (CIA) susceptible HLA transgenic mice produce rheumatoid factor and anti-cyclic citrullinated peptide (CCP) antibodies similar to that in RA patients. Similar to humans, the HLA-DR4 transgenic mice develop CIA more often in females than males. Our hypothesis is that DR4 has a role in gender-bias of arthritis, where DR4 expression, presentation of antigen by various APCs and ensuing responses are different in males and females. Female sex hormone, estrogen, is involved in pathogenesis by enhancing production of peptidyl arginine deiminase (PAD) enzyme that leads to citrullination of synovial proteins. These citrullinated proteins are presented with greater efficiency by HLA-DR4 leading to an autoreactive T and B cell response and disease. Smoking and aging can result in higher amounts of citrullination. On the other hand, DQ8 can present native and de-amidated peptides thus generating a robust immune response to an antigen. In this proposal we will investigate the mechanism of association of DR/DQ with arthritis by studying its interaction with receptors involved in innate and adaptive immunity via 1) binding to calreticulin, an established innate immunity receptor, and triggering signaling, 2) by selection of regulatory cells and 3) by selection/ deletion of autoreactive T cells. In addition, we will use DRB1*0401 and DRB1*0402 mice to determine the mechanism of protection to understand how 3 amino acids difference in both molecules lead to protection by the latter. In aim1, we will define the mechanism of predisposition by using mice expressing RA susceptible and resistant HLA class II molecules. In aim 2 we will test role of shared epitope in protection versus susceptibility by studying interaction of *0401 and *0402 with calreticulin, and role of T regulatory cells and Th17 cytokines. As a corollary, we will study mechanism of remission during pregnancy by studying function of T regulatory cells and antigen presenting cells. Recent literature has shown a protective/ susceptible effect of non-inherited maternal antigen (NIMA) on the development of arthritis. In aim3, we will use various matings to generate mice that have been exposed in utero to one NIMA. These mice will be evaluated for in vivo arthritis, autoantibodies and function of T and B cells. The proposed experiments will test several new, original and innovative theories on the role of MHC genes in predisposition, onset, progression, severity and modulation in arthritis. The findings would shed light on the role of immune system in health and disease and may identify new therapeutic approaches for arthritis."}
{"text": "DESCRIPTION: Environmental exposure to polychlorinated biphenyls (PCBs) occurs worldwide, and most individuals in developed nations have measurable levels of organochlorines in adipose tissues. This project proposes to estimate the risks associated with perinatal exposure to PCBs for developmental deficits or delays on various measures of neurologic, neurobehavioral, and intellectual outcomes. The investigators, with support of the European Commission, have established a birth cohort of 200 children recruited consecuttively since March, 1994 from a relatively homogenous population in the Fazroe Islands. The northeast Atlantic Ocean where the Faroes reside is known to be one of the largest environmental reservoirs of PCBs. Due to consumption of pilot whale blubber, the average PCB exposure in this cohort is believed to be substantially higher than established averages in Northern Europe and is expected to vary 10 to 20 fold. Developmental exposure to PCBs will be determined prenatally by maternal and cord serum and breast mile PCB concentrations. This cohort has already been evaluated at 10 to 15 days of age with the Touwen neonatal neurologic scale. Currently, the revised Bayley Scales of Infant Development, the Fagan Test, and measures of infant linguistic performance are being administered at 7 months of age. Follow-up assessments include at 18 months the Bayley-II, and a neurologic examination at 30 months, with more extensive test batteries at ages 42, 54, and 66 months."}
{"text": "We have found that T4 tail fibers have a myosin-like activity at their proximal half and a rare cross-beta folded protein structure at their distal half. We propose to clone the T4 tail fiber genes and sequence their primary structures. We will then look at the mechanism of their folding and dimerization into mature tail fiber halves by conventional methods as well as in clones containing plasmids with genes expressing only the tail fiber genes. We have developed rapid and sensitive methods for selecting these clones and assaying the immature and mature tail fiber parts. Our overall goal is to explain in molecular terms the rigid structure of the tail fiber parts, the springlike angle in the middle and the mechanism of action of both termini with respect to attachment to the cell surface (LPS) and the phage baseplate."}
{"text": "When challenged with inhibitors of steroidogenesis, the cultured vascular smooth muscle cell showed depressed synthesis of cholesterol and of dolichyl(pyro)phosphate and an accompanying loss of cellular cholesterol. It was deduced that a regulatory site for the synthesis of both substances might occur at the HMG-CoA reductase. As a consequence of this inhibition cellular glycoprotein synthesis was diminished, and it was concluded that the availability of dolichyl(pyro)phosphates which mediate glycoprotein synthesis may also contribute to the regulation of their assembly. Cellular response to these changes may involve redistribution of subcellular membrane systems. This redistribution, perhaps necessary as a defense response, may present the cell with a new set of problems. Such redistribution will be assessed in a variety of conditions using subcellular fractionation schemes and marker enzymes assays. It is now clear that loss of cellular cholesterol may also contribute to defective glycoprotein assembly and membrane biogenesis. Such an effect points to the importance of a metabolically active cholesterol pool which the cell synthesizes to make certain that proper assembly is maintained. In view of this, contribution of exogenous cholesterol, supplied in a variety of forms, to such a pool will also be investigated."}
{"text": "Neurotransmitter release is a key step in synaptic transmission. However, the molecular events that trigger fusion of synaptic vesicles and neurotransmitter release are unknown. One attractive hypothesis is that Ca2+-activated channels in the vesicle membrane control fusion of vesicles. If true, such a channel would provide a simple molecular link between Ca2+ entry and vesicle fusion. Previous experiments have lead to the identification of several kinds of ion channels that are credited to the synaptic vesicle membrane. The objective of this project is to study these ion channels to determine: 1) which, if any, are from the synaptic vesicle membrane; 2) the biophysical properties of these channels; and 3) regulators of the channels (such as voltage and Ca2+). These goals are directed toward elucidation of the mechanism(s) of fusion of synaptic vesicles to the nerve terminal membrane and the possible role of vesicular ion channels in this process. Plan: Planar bilayer experiments will be used to identify and characterize ion channels from synaptic vesicles purified from the electric organ of the electric fish, Torpedo californica. Vesicle membrane will be induced to fuse with bilayers by using a new technique that both enhances fusion of all vesicles and makes it possible to estimate the prevalence of any observed channels. That is, it will be possible to determine if a particular channel is representative of the population of synaptic vesicles or if it is from a minor contaminant. Preliminary Results: Synaptic vesicles from Torpedo californica have been isolated and treated so that they fuse with planar bilayers. Two kinds of ion channels have been detected from these vesicles having estimated single channel conductances of 180 pS and 14 pS."}
{"text": "Development of a miniature orally inserted blower with embedded sensing for the diagnosis and treatment of sleep disordered breathing is proposed. This Oral Positive Airway Pressure (OPAP) device represents a potentially radical change in the interface, machine, and patient adherence of sleep disordered breathing treatment. The ergonomic design of the device will facilitate oral and nasal breathing during sleep, eliminate external machines and tubing, and will be retained in the oral cavity during sleep. Inter-individual differences in airway response to sleep disordered breathing will be automatically accounted for by innovative real-time sensing of airway collapse and by the closed-loop coupling between respiratory effort and machine delivery. Ease of use and improved adherence to therapy is anticipated. This research will utilize monitoring technology embedded in the device to objectively document adherence. Information obtained will determine how the nightly duration of use and long term adherence to OPAP can affect morbidity, mortality, healthcare utilization, and quality of life issues. Phase I of this project investigates the feasibility of several candidate OPAP device designs, and evaluates their therapeutic (including air blowing and ergonomics) and diagnostic capacities in airway simulation models and oral cast models. Phase II of the project will obtain clinical trial data of this device and sensor in a clinical trial of sleep disordered breathing patients with the subsequent intention to market and sell this product. Phase II research will also support the investigation of cardiovascular outcomes for those adherent and non-adherent to OPAP treatment for OSA. [unreadable] [unreadable] [unreadable]"}
{"text": "The overall objective of this proposal is to understand the mechanism and regulation of transplacental calcium transport during mammalian embryonic development. Based on our previous identification of specific calcium-binding proteins (CaBP) in the human and mouse placentae, experiments are proposed here that directly examine the functional involvement of these proteins in the placental calcium transport process. Specifically, these projects aim to study how the CaBPs interac with Ca++, where they are located within the placental cells, how they participate in transmembrane calcium transport, and what are the cellular and molecular steps regulating their specific, temporal expression during development. These projects will be approached by first preparing purified placental CaBPs which may be used to raise specific antibodies. Detailed physicochemical analyses will be carried out to reveal conformational changes that may accompany Ca++ CaBP interactions. The specific antibodies will be used to localize the ultrastructural distribution of the CaBPs and will also be used to probe the nature of their functional involvement in placental calcium transport using cellular and subcellular methodologies. Finally, the developmental regulation of CaBP expression will be explored by 1) identifying putative regulatory factors and 2) characterizing the molecular processes leading to gene expression. The integrated approach taken here should lead to a fuller understanding of how a developing embryo obtains its calcium supply and futhermore provide general insights to the regulation of embryonic development."}
{"text": "This research proposal constitutes an attempt to develop biological-biophysical data about specific patterns of ribonucleic acid biosynthesis and those enzyme systems involved in their processing, in particular the transfer RNA-methylases, as they relate to the leukemogenic process. It is intended that this information will afford an expanded level of knowledge concerning the biochemical responsiveness of leukemia cells found to be sensitive or resistant to various antineoplastic agents (e.g. vincristine, cystosine arabinoside, cytoxan, methotrexate) administered alone or in combinations. In this connection, information will also be sought as to how such agents can modulate the biosynthesis of particular species of RNA in leukemic cells. The basic techniquesto be employd entail the isolation and purification of mouse ascites tumor cells (e.g. L1210, P383, L5178Y) and certain resistant sublines as well as human peripheral blood leukocytes, cell-culturing where appropriate and variable-interval pulsing with radioactive precursors of ribonucleic acid (uridine, L-methionine). In vitro evaluation of tRNA-methylase activities in various leukemic cells will be conducted using either a homologous or heterologous assay system in the presence of unfractionated tRNA plus partially purified enzyme preparations. Special techniques to be used for characterizing individual RNA species and ribonucleic acid components (nucleoside level) include thermal-detergent extractions, sucrose-gradient centrifugation, polyacrylamide gel electrophoresis and computer analyses of \"RNA-fingerprint\" data."}
{"text": "Elucidating childhood precursors of osteoporosis may lead to interventions that prevent or mitigate osteoporosis later in life. The central focus of this application is to investigate bone mass as measured by DEXA, including bone mineral content (BMC), bone area (BA) and bone mineral density (BMD) in relation to a wide range of putative precursors of osteoporosis. We will study a large prospective twin cohort (-2,000 twin pairs) of children and adolescents, enrolled previously in 1998-2000 in Anqing, China, with an age range of 6-21 years. The twins are currently followed by a NIH funded study to identify precursors of metabolic syndrome (MS). The specific aims are: (1) To measure biomarkers that are known or suspected to affect bone health in 1,500 twin pairs at baseline and follow-up study, including bone modeling and remodeling markers: osteoprotegerin (OPG), soluble receptor activator of NFKB ligand (sRANKL), parathyroid hormone (PTH), osteocalcin (OC), tartrate-resistant acid phosphatase 5b (TRAP-5b); nutritional markers: magnesium, vitamin K1, and 25(OH) vitamin D; and also examine other relevant biomarkers covered by the funded MS study, including steroid hormones: androgen, estrogen, testosterons, LH, and FSH; metabolic markers: fasting and 2-hr post OGTT insulin, glucose, HOMA-IR, fasting lipids; growth hormones: insulin-like growth factor (IGF-I); adipocyte markers: leptin, adiponectin; and inflammatory markers: C-reactive protein (CRP), lnterleukin-6 (IL-6), and tumor necrosis factor (TNF); (2) To conduct cross-sectional and longitudinal co-twin analyses to test the following hypotheses: (a) BMC, BA and BMD attained levels are associated with biomarker levels at the baseline and follow-up survey, respectively; (b) BMC, BA, and BMD changes between the baseline and follow-up survey are associated with biomarker levels at the baseline survey; (c) BMC, BA, and BMD changes are associated with biomarker changes between the baseline and follow-up surveys. We will further examine if the above relationships are independent of other important covariates, including age, gender, pubertal stage, body weight and composition, nutritional intake, and physical activity, and if there are interactions between the biomarkers and the covariates. The identification of potential important precursors of osteoporosis in children and adolescents would represent a huge step forward in our understanding of the biological basis of bone mass and should have important implications for the detection of individuals at high risk for osteoporosis. [unreadable] [unreadable] [unreadable] [unreadable]"}
{"text": "PROJECT 1: ENCODING AND RETRIEVAL PROCESSES IN DECLARATIVE MEMORY In the prior funding period, patients with and at risk for schizophrenia were found to show a differential deficit in recollection that scaled with phase of illness, while familiarity was intact. This deficit was correlated with reduced hippocampal and parahippocampal activity at retrieval on items for which associative memory was evident. There are two (not mutually exclusive) possible sources ofthese deficits: 1) disrupted structural connectivity, resulting from aberrant developmental changes in gray and/or white matter during peri-adolescence;and 2) failure of patients to disengage default mode network (DMN) activity during memory encoding and/or retrieval. The first hypothesis is suggested by our preliminary work indicating a steeper rate of gray matter reduction in prefrontal regions and a lack of normal age-related increase in medial temporal white matter tracks in FE and prodromal patients who convert to psychosis. The second hypothesis is based on recent evidence of reduced suppression of DMN activity and less task-related activity and functional connectivity during a working memory task in schizophrenia patients and their siblings. Increased DMN activation appears to accompany attention to internal thoughts and self-referential thinking;reduced suppression ofthe DMN may thus interfere with patients'ability to activate the episodic and associative memory circuitry at encoding and/or retrieval. In the next phase of the study we will pursue these two hypotheses in cross-sectional and short-term longitudinal studies of prodromal, FE and chronic patients and controls, using MRI and DTI to index structural connectivity and fMRI during an associative memory task and resting scans to index functional connectivity between memory-related and DMN regions. The findings will help to clarify the nature and neural underpinnings of declarative memory deficits in schizophrenia, whether behavioral and physiologic deficits predict the onset of schizophrenia over and above behavioral indicators of risk and are differentially predictive of poor functional outcome. Findings maybe used in prodromal research to identify those at greatest risk for schizophrenia."}
{"text": "Initially, this Phase II study investigated the effects of daily administration of DMP 754, a IIb/IIIa platelet inhibitor, versus aspirin on the inhibition of platelet aggregation, the incidenceof bleeding and thrombocytopenia in patients with coronary artery disease over a 6 month period of time. The study was amended in March 1998 to include combination therapy arms of DMP 754 and aspirin. Preliminary data suggested increased risk of bleeding so the protocol was amended in July 1998 to remove the 2.0 mg DMP 754 + Aspirin arm and its comparitor (2.0mg DMP 754 alone). We enrolled 8 patients between May and October 1998. Of those eight patients, two were discontinued, one due to decrease in platelet count and the other because of a positive hemoccult. DuPont closed enrollent December 1998 after meeting enrollment goals. The six-month study follow-up will conclude in May 1999. Clinical endpoints include death, progression to myocardial infarction, CABG, PTCA for recurrent ischemia, hospitalization for ischemia or unstable angina. We expect to generate an abstract for presentation, on which Mayo investigators will be co-authors, in late 1999 followed by a manuscript."}
{"text": "Pancreatic cancer is one of the most lethal malignancies in the US. The primary curative treatment is surgery and is only appropriate for approx. 20% of presenting patients because most will present with locally invasive or metastatic disease. Although Gemcitabine has been proven superior to 5-Fluoracil in advanced pancreatic cancer, long term survival is rarely seen. Other agents are clearly needed to treat this refractory disease. Strong evidence points to the importance of raf kinases in the development and maintenance of human cancers. For example, raf proteins have been demonstrated to be direct downstream effectors of ras protein functions within the MAP kinase signaling pathway. Since 75% of patients with pancreatic cancer have ras mutations, novel therapy directed against raf kinases are particularly exciting in this malignancy. ISIS 5232 is a phosphorothioate oligonucleoide antisense inhibitor of human c-raf kinase or are in an expression in-vivo and in-vitro. This is a limited institution ECOG phase II trial which will look at the response rates and toxicity of ISIS 5132 administered by continuous infusion via portable pump (provided free of charge). Numerous bug samples for pharmocokinetics and measuring of downstream effects will be needed during the course of this trial. This novel therapy has numerous avenues for future therapeutic directions, including adjuvant therapy or perhaps combination therapy with other standard chemotheraputic agents."}
{"text": "This proposal requests funding to assist in upgrading of laboratory animal facilities at The Rockefeller University, with the goal of providing research scientists with pathogen free rodent colonies. In a previously funded 1987 facilities improvement grant from the NIH we upgraded equipment, investigator safety, sanitation, surgery, water and environment components of The Rockefeller University Laboratory Animal Research Center and Field Research Center. These improvements have helped us remain a regional center for training and excellence in laboratory animal science. Matching funds are now being requested for a modification in our basic rodent husbandry care system which will enable our scientists to maintain and work with mice and rats free of common pathogens. We propose, with the assistance of this grant, to expand a limited 1200 sq ft of room with rodent microisolator housing by 2800 sq ft of additional pathogen free experimental animals. our existing limited pathogen free rodent housing was established by Howard Hughes Institute and NIH funding in addition to supplemental Rockefeller University funding. This proposal will enable us to maintain virtually all rodents which originate as pathogen free strains or stocks as pathogen free experimental animals, thus greatly enhancing the biomedical research of this institution. Approximately $45,000,000 in federal research funding is utilized at this university. Nearly two thirds of the laboratories conducting this research require laboratory animals for their studies and utilize the facilities of the Laboratory Animal Research Center."}
{"text": "Pseudomonas aeruginosa is an important cause of keratitis. The results of antibiotic treatment are unsatisfactory. We have shown that physiologic concentrations of calcium in vitro antagonize the effects of polymyxin B, colistin, and gentamicin on P. aeruginosa. These antibiotics may have less activity in vivo than one would predict on the basis of in vitro tests. We have also found that chelation of calcium significantly enhances the activity of these antibiotics in vitro. The combination of antibiotic and chelating agent may also have greater activity in vivo than the antibiotic alone. We propose to determine the relative potency of polymyxin B, gentamicin, and carbenicillin, with and without chelating agents, in the treatment of experimental Pseudomonas keratitis. Chelation may sharply increase the efficacy of treatment of Pseudomonas keratitis. Recent studies suggest that collagenase may play an important role in the pathogenesis of corneal ulcer and that chelation may block collagenolytic activity. We also propose to clarify the relationship between cations, chelation, and corneal ulcer."}
{"text": "Low and variable efficiency is a major problem in targeted gene alteration, which is used as a primary tool in gene therapy and animal model studies. We tested several types of constructs, alone, or in combination with other factors, to introduce a point mutation into the alphaB-crystallin gene in one-celled mouse embryos. We found that co-injection of single stranded DNA (ssDNA) along with antibodies against Ku70/86, or supplementing the system with hRad51/hRad54, increases efficiency of targeted mutagenesis. These findings suggest that proteins in the homologous recombination DNA repair pathway contribute, and that proteins involved in the alternative non-homologous end joining pathway inhibit, ssDNA-mediated targeted mutagenesis. This is the first successful demonstration of targeted mutation in early mouse embryos. This novel methodology of supplying protein factors to stimulate gene modification in the nucleus has not been reported previously. For this initial study we have been using an extremely time consuming PCR-based restriction fragment length polymorphism (RFLP) assay to detect point mutations introduced into genomic DNA. To improve the detection method, we have developed transgenic mouse lines in which correction of a mutation in a fluorescent protein gives an instant read-out, and many embryos can be screened simultaneously. We engineered a cassette encoding a bicistronic mRNA (mutated red fluorescent protein DsRed / an IRES element / green fluorescent protein). All mouse embryos expressing the transgene should fluoresce green (endogenous control for transgene expression), but only embryos in which the point mutation in DsRed has been corrected should also fluoresce red. We have used several enhancer/promoter elements, which have been used successfully in preimplantation stage mouse embryos or mouse ES cells, to drive expression of the cassette. A successful reporter line would exhibit easily detectable levels of fluorescent protein expression at the earliest stages of embryo development. All promoter/fluorescent protein transgenes were constructed and tested for expression of EGFP and intact DsRed in both transient assays in mouse embryos and in F1 transgenic mouse embryos. Several promoters/enhancers were used in attempts attain early embryonic gene expression. A construct, whose expression is driven by the EF1alpha promoter, was able to generate green fluorescent protein in eight cell stage embryos (48 hr pf) and red fluorescent protein at 72 hr pf. A line of transgenic mice with the EF1alpha promoter / mutated red fluorescent protein / an IRES element / green fluorescent protein construct appears to be extremely promising at this time. Four transgenic mice lines with mutated DsRed protein were established. All four lines show expression of EGFP at 48 hr pf. Sequence analysis of a PCR-amplified region of the DsRed gene, from mouse genomic DNA, confirmed presence of the nonsense mutation in codon 15 of DsRed. RT-PCR analysis shows that three transgenic mouse lines have low copy number of the construct (2 copy) and one has a high copy number (26 copies of the construct). One line of mice (with 2 gene copies) is being bred for further experiments. In addition to our work with ssDNA as a targeting vector, we are also working, in single celled mouse embryos, on another type of gene targeting which we have named Forced Homologous Recombination (FHR). In this case, we use a significantly larger targeting vector, similar to those currently used in ES cell targeting procedures, with regions of homology to a selected gene or chromosomal locus at both 5'and 3'ends. Co-microinjection of the targeting construct with key factors involved in homologous recombination (for example Rad51/Rad54 proteins, which has been shown be able to form a filament-complex with nucleotides in vitro) may increase gene targeting efficiency. We are planning to use a nucleoprotein filament complex instead of the naked DNA vector to further increase alteration efficiency. The presence of long homology arms on both ends should increase specificity of gene modification and also dramatically decrease any random integration of the targeting vector, compared to our ssDNA strategies. Two genes were selected for in vivo testing of this FHR system. The first being the alphaB-crystallin gene. This gene had previously been knocked out, however another neighboring gene, Hspb2, was also knocked out in that mouse line. If successful, the FHR system will enable elimination of only one gene with precise accuracy. The second gene is caspase-6, a knock-out of which was also developed earlier, by eliminating the first exon. However our preliminary data suggests positive identification of caspase-6 by western blot in some tissues, which could possibly arise by alternative splicing or an alternative translation start site. We are working on a construct for FHR study that should eliminate the active center and dimerization domain from caspase-6 with precise accuracy. Generation of these two vectors is presently underway."}
{"text": "This is a request for an ADAMHA Research Scientist Award. Sleep changes in late-life depression may be permanent or may recover only slowly. Hence, to clarify the relation of persistent sleep abnormalities, pathogenesis, and illness course, we propose three related longitudinal studies of: Study 1 - 200 elderly (60-80 yo) patients with recurrent unipolar depression participating in an already funded maintenance therapies research protocol (MH43832-01, C.F. Reynolds, P.I.); Study 2 - 80 elderly subjects (60-80 yo) with spousal bereavement (n=40) and bereavement-related depression (n=40); and Study 3 - 60 healthy elderly (60-80yo) non- depressed, non-bereaved controls. Participants in all three studies will have repeated (four to seven) sleep EEG assessments over a two to three year period to determine if, as hypothesized: 1a) abnormal pretreatment (T1) measures of REM sleep cyclicity/density predict elevated risk of relapse and recurrence of major depression; 1b) EEG sleep abnormalities persisting into continuation therapy (T2) and into maintenance therapy (T3-T7) predict elevated risk of relapse and recurrence, respectively; 2a) depressed bereaved differ from non-depressed bereaved elderly in baseline (T1) and followup (T2-T5) measures of REM sleep cyclicity/density; 2b) baseline (T1) and followup (T2-T5) abnormalities of REM sleep in bereavement predict a more protracted course (and need for psychiatric intervention); and 3) longitudinal changes in sleep/sleep quality (T1-T4) of healthy elderly controls are related to subclinical depressed mood. Multiple logistic regression and survival analyses will model the hypothesized relation of REM sleep abnormalities (independent variable) to outcome: recurrence/non-recurrence of depression (Study 1); or presence/absence of RDC-defined major depression at 1 and 2 years after spousal bereavement. A repeated-measures MANOVA will contrasts the long- term evolution of selected sleep variables (sensitive to aging, depressed mood, or both) in recurrent depression, bereavement, bereavement-related depression and controls. Exploratory data analyses will assess possible interrelations among biological controls. Exploratory data analyses will assess possible interrelations among biological REM sleep abnormalities, severity of psychopathology, and disruption in social rhythms."}
{"text": "The goal of this training program is to prepare physicians and basic scientists for careers focused on the development and use of radionucleotides in the diagnosis and treatment of cancer. Support is requested for five trainees, who may be either physicians or basic scientists. Physician trainees will have completed the major portion of requirements for board certification in nuclear medicine. Basic science trainees will hold the Ph.D. degree in chemistry, physics, biology, or a related science.. Each will be aiming for a career in research in a academic setting. The training program will be two to three years in duration. A broad based didactic curriculum will cover fully the field of nuclear medicine as applied to cancer. Each trainee will give focused concentration in selected tracks coordinated by a senior faculty mentor. Specific research tracks are: (1) Tumor antibodies/radiopharmaceutical, (2) Endocrine tumors, (3) Brain tumors, (4) Radiopharmaceutical therapy, (5) Quantitative radionuclide imaging, and (6) Radiopharmaceutical development. Research in one or a combination of these tracks will involve at least 50-75% of trainee time. The program will be conducted in the Division of Nuclear Medicine and Comprehensive Cancer Center facilities. Training will be supervised by members of the large, multi- disciplinary nuclear medicine faculty. This group publishes over 90 papers per year and receives over 6 million dollars per year in research grant support. Nuclear Medicine facilities include 90,000 square feet of clinic space, 17,000 square feet of laboratory space, a dedicated cyclotron, and 3 PET scanners. This program is integrated with University of Michigan Comprehensive Cancer Center activities. The combination of faculty motivation, structured curricula, excellent facilities, and superb intellectual environment is expected to prepare individuals to be tomorrow's leaders in cancer research."}
{"text": "Arthritis has reached epidemic proportions in the United States. It is the leading cause of disability, affecting about one-third of adults. Osteoarthritis (OA) is the most common form of arthritis with a prevalence conservatively estimated at 21 million Americans, or 12.1% of the adult population. We recently showed that a 5% weight loss in obese adults with knee OA had a modest effect on clinical outcomes; however, these participants remained obese at the conclusion of the intervention, so their risk of disease progression was not altered. Indeed, the nature of the relationships between weight loss and the mechanisms associated with disease progression are undetermined. We hypothesize that a more intensive weight loss (2 to 3 times greater than previously achieved in this population) would provide the stimulus necessary to reduce inflammation and joint loads and alter disease progression. The study population will consist of 450 overweight and obese, older (age > 60 yrs) adults with tibiofemoral osteoarthritis of one or both knees. Participants will be randomized to 1 of 3, 24-month interventions: intensive dietary restriction-plus-exercise; exercise-only; or intensive dietary restriction-only. The primary aim is to compare the effects of these interventions on inflammatory biomarkers and knee joint loads in overweight and obese adults with knee OA. Secondary aims are: to compare the effects of these interventions on self-reported function and pain, and mobility; to determine the dose response to weight loss on disease progression and the mechanisms associated with the OA disease pathway; to determine if inflammatory biomarkers and knee joint loads are mediators of the interventions, with significant effects on function, pain, and disease progression; and to determine the association between quadriceps strength and disease progression as a function of knee alignment. Weight loss is the most modifiable risk factor for knee OA; however, no study has determined the extent of weight loss that is beneficial for this population. This study could make intensive weight loss the standard-of- care for overweight and obese adults with knee OA, as it enhances our understanding of the OA disease process and weight-loss recommendations for older people generally. [unreadable] [unreadable] [unreadable] [unreadable]"}
{"text": "With the recognition that cancer is a genetic disease, many cancer researchers, including scientists in the Mayo Clinic Cancer Center (MCCC), require access to routine and advanced cytogenetics services. Such services can aid in the identification of genetic regions important for the development of specific malignancies. Such services can also be used to characterize the clonality of tumor specimens and determine the origin (e.g., mouse vs. human) of a specific tumor culture. Thus, the Cytogenetics Shared Resource will provide cytogenetic and molecular cytogenetic services to MCCC members. In addition, this Shared Resource will prepare EBV-transformed B-lymphocyte cell lines for MCCC members."}
{"text": "Recognizing objects is a fundamental cognitive ability, e.g., for reading or social communication, and its loss or impairment is associated with a number of neural disorders. For example, many studies have shown that individuals with Autism Spectrum Disorder (ASD) are impaired in face recognition, possibly contributing to their difficulty in social interaction. Differences in social cognition are one of the defining features of autism. However, the precise nature of the differences in the neural mechanisms of face processing seen in autism has been difficult to study and its relationship to social behavior is unknown. While our understanding of the network of brain areas underlying human face perception has advanced considerably, current models are still qualitative and treat processing within specific brain areas as \"black boxes\". To understand the graded (rather than all-or-none) recognition deficits and to identify targets for therapeutic intervention, a quantitative and mechanistic model of the neural computations underlying face perception is required that rigorously links visual stimuli to neural activation and behavior. The purpose of the proposed project is to explore the neural mechanisms of face processing in typically developing and autistic individuals using an integrated approach based on a tight interaction of computational modeling of neural processing in the brain areas underlying human face recognition and of model-driven behavioral and fMRI experiments. In particular, the model motivates experiments to probe the face representation in typically developing and ASD subjects, and to mechanistically link differences in behavior and fMRI to differences at the level of neural processing -providing insight into the neural causes of the observed face recognition deficits along with an opportunity to evaluate current theories of neural processing differences in ASD. In a second part of the study, the model will drive experiments to study high-level plasticity and perceptual learning in typically developing and autistic individuals. This will not only leverage the insights from face perception to explore more general differences between the two subject groups, but is also expected to provide a foundation for remediation efforts. The relevance of this research to public health lies in its direct application of the results of this research program to achieve a better understanding of impairments found within and outside the autism spectrum."}
{"text": "The objective of this protocol is to measure the effect of six months treatment with human growth hormone at a physiologic dose on the muscle strength and endurance and on the functional capabilities of subjects with post poliomyelitis syndrome."}
{"text": "An unexplained decrease in muscle strength occurs with increasing age. Persons over seventy years old average about 50 percent of the strength of those less than 35 years old. As strength declines with increasing age, mobility, functional capacity and independence are lost, and the risk of injury as a result of falling increases. Decreased muscle strength is also a well-documented symptom of vitamin D deficiency and particularly of 1,25 D deficiency. The myopathy associated with 1,25 D deficiency, like that seen with aging, is characterized by proximal muscle atrophy and a selective loss of type II muscle fibers. Administration of 1,25 D to patients with 1,25 D deficiency restores muscle strength. Serum concentration of 1,25 D decreases with increasing age. Serum 1,25 D levels in persons over 70 years old average about 55 percent of those in persons in their mid thirties. In uncontrolled studies, administration of 1,25 D to elderly subjects has improved muscle strength. In a pilot study, we showed a 27.5 percent increase in muscle strength in healthy elderly men after one month of oral treatment with 1,25 D, and a 35 percent improvement in those over 70 years old. In the pilot study the percent increase in strength was strongly correlated with the percent increase in serum 1,25 D level (correlation coefficient = .9). Based on these observations and biochemical evidence linking 1,25 D to muscle function, we hypothesize that the weakness associated with aging is, in part, due to inadequate serum concentrations of 1,25 D in the elderly. To test this hypothesis, we proposed to perform a randomized, controlled, double-blinded trial of the effects of oral treatment with 1,25 D in 100 persons over 70 years old using the isokinetic dynamometer as a precise measure of strength. Our study will have a 90 percent power to show a 20 percent difference between the treated and placebo groups. A positive study will establish a metabolic etiology for the weakness presently considered a \"normal\" part of the aging process and open a new area for biochemical research in aging. If effective, treatment with vitamin D may make it possible to improve mobility, reduce functional dependence and decrease the risk of falling in the elderly."}
{"text": "We have prepared highly purified tetanus toxin by both the cell extract and culture filtrate procedures. The amino acid composition of toxin prepared by both procedures was completed and agrees with that reported by other laboratories. The ordered structure of toxin prepared by both methods was estimated from their ultraviolet circular dichroic spectra and are similar. We are now preparing several large peptides from the toxin molecule. Digestion of toxin, cell extract or culture filtrate preparations with papain followed by gel filtration allows the separation of at least one large atoxic peptide and a fraction containing small peptides. We have separated filtrate toxin, by gel electrophoresis in the presence of appropriate reducing agents, into two large peptides referred to as heavy and light chains. Cell extract toxin must first be \"nicked\" by trypsin before it can be completely separated into similar peptides. These large peptides will now be treated with cyanogen bromide which should produce about 8 peptides and 5 peptides from the heavy and light chains respectively. Fragmentation with trypsin should yield an appreciably larger number of peptides. Peptides from each fragmentation procedure will be separated and sequenced. If a peptide is too large for overlap other enzymes will be used. The amino acid composition of peptides, prepared by cyanogen bromide and trypsin cleavage from both cell extract and culture filtrate toxin will be compared. This should detect any difference in the two toxins. There is no apparent reason to sequence toxin prepared by both methods. These experiments should allow us to compare the amino acid sequence of this unusual protein with its secondary structure and that of other nontoxic soluble proteins. It should also enhance our understanding of the molecular mechanism of the pathology of tetanus and ultimately aid in the prevention and care of this deadly disease. BIBLIOGRAPHIC REFERENCES: Robinson, John P., Pieklesimer, John B. and Puett, David. Tetanus Toxin: The Effect of Chemical Modifications on Toxicity, Immunogenicity and Conformation. JBC 250, 7435-7442, 1975. Wahba, Nagi, Felch, James W., Shih, J. Wai-Kuo and Hash, John H. The N,O-Diacetylmuramidase of Chalaropsis Species IV. Tryptic Peptides. J. Biol. Chem. 250, 3709-3712, 1975."}
{"text": "To further define potential abnormalities in parathyroid (PTH) secretion that may occur with aging."}
{"text": "These studies aim to identify new methods of gene transfer into hematopoietic stem cells using the genetic disease canine leukocyte adhesion deficiency (CLAD) as a model. We are using the canine model to identify new vectors for gene transfer and conditioning regimens to enable sufficient numbers of gene modified hematopoietic stem cells to engraft and reverse the disease phenotype. The canine form of this disease is an optimal model for these studies since: 1) the defect involves a membrane receptor on the surface of leukocytes, flow cytometry allows fascile detection and analysis of the number of gene corrected cells;2) low levels of gene-corrected cells result in reversal of the disease phenotype;and 3) studies in the canine model have been predictive of success in humans in the field of hematopoietic stem cell biology. The presence of a human counterpart to the canine disease allows the results from the animal model to be directly extrapolated to humans. The long-term objective of these studies is to develop strategies that will allow levels of expression of CD18 in hematopoietic cells of children with leukocyte adhesion deficiency (LAD) that are sufficient to reverse the clinical phenotype. We have utilized this model to test retroviral and foamy viral gene transfer into the bone marrrow cells of dogs. The foamy viral vector demonstrated greater efficacy and a more favorable integration profile than the conventional retroviral vectors. The foamy viral vector-treated dogs are being followed for the durability of the correction, and for any possible genotoxicity from the vector. To date, there has been no genotoxicity. These results represent the first successful use of a foamy virus (FV) vector to treat a genetic disease, and they suggest that foamy virus vectors will be effective in treating human hematopoietic diseases. We are currently testing third-generation foamy viral vectors and lentiviral vectors with cellular promoters and lentiviral vectors with cellular rather than viral promoters in our canine model."}
{"text": "Variation in bitter taste perception may play a key role in diet selection, which has an uncontestable effect on human health. Although bitter compounds are often associated with toxic substances a large number of nutritionally significant food sources contain bitter phytochemicals (e.g., broccoli [1], spinach [2]) many of which can have beneficial value [1, 3] Understanding how bitter taste is perceived and detected is of great importance to understanding diet selection [4] and increasing the acceptability of pediatric medicines [19]. Under normal feeding and drinking conditions taste compounds must inescapably mix with saliva before reaching their receptor targets but very little work has been done examining how salivary proteins modulate taste stimuli and alter bitter taste perception. Seventy percent of all salivary proteins make up a class of proteins referred to as proline-rich proteins (PRPs). PRP production is induced in rats by dietary exposure to tannic acid, a class of plant compounds that animals, including humans, regularly consume. It has been hypothesized that PRPs alter the acceptability of tannic acid by binding to tannins and thereby reduce the perceived intensity of the tannic acid solution [10, 11]. There is evidence to suggest that these proteins may have a similar relationship with other bitters. They are produced in close proximity to bitter taste receptors [13]. Genes for PRPs and bitter taste receptors are interspersed on the same chromosome [5] and lastly, gene linkage studies have implicated a role for PRPs bitter acceptance in humans and mice [15-18]. Specific Aim 1 will establish if long term exposure to bitter compounds can increase the expression of PRPs and establish whether oral exposure is necessary and sufficient to cause induction. Saliva will be analyzed for PRP content before and after exposure to diets adulterated with bitter compounds, including quinine and tannic acid. To isolate the site of exposure necessary for PRP induction rats will also be given tannic acid via either oral or gastric exposure. The ability or inability to induce PRP production is not sufficient to predict interactios between the proteins and the bitter tastants. Therefore, Specific Aim 2 will establish if PRP induction can alter the detection threshold for and superthreshold responsiveness to multiple bitter stimuli. Rats will be used to derive psychophysical detection curves of several bitter tastants in the presence and absence of PRPs. Likewise, responsiveness in the suprathreshold range will be assessed in the presence and absence of PRPs with a series of brief-access tests. Collectively, these experiments will test the hypothesis that PRPs alter taste sensitivity for bittr taste stimuli and ultimately blunt unconditioned taste avoidance responses to these compounds. Together these aims could inform methods for increasing the palatability of healthy phytochemicals and pediatric medicines."}
{"text": "The Florida Pediatric Community Clinical Oncology Program is a consortium of four Florida hospitals (All Children's Hospital, Jacksonville Wolfson Children's Hospital, Orlando Regional Medical Center, and Sacred Heart Children's Hospital) which seeks to affiliate with the Pediatric Oncology Group as its research base. These four hospitals complement the three major university teaching hospitals which are also Pediatric Oncology Group members and, in total, comprise the Florida Association of Pediatric Tumor Programs. The goal of the Florida Pediatric CCOP is to make available the latest advances in cancer care to patients in Florida through participation in clinical trials. The CCOP participants will utilize an existing Statewide Patient Information Reporting System as a log of all patients seen in the state. Patients will be registered on POG protocols through the POG Statistical Office which is co-located with the Central Office of the Florida Pediatric CCOP. This unique arrangement affords the opportunity to ensure that the majority of eligible patients are registered on appropriate POG protocols. An excess of 45 patient registrations are anticipated per year through the four hospitals which comprise the Florida Pediatric CCOP. POG quality control procedures will be extended to include the Florida CCOP, ensuring compliance with protocols and external review of pathology, radiotherapy, surgery, and chemotherapy. The Florida Pediatric CCOP extends the latest treatment advances through the opportunity to participate in clinical trials to a significant segment of the Florida population."}
{"text": "DESCRIPTION (appended verbatim from investigator's abstract): The guidance of axons to their targets during embryonic development is an intricate process requiring remarkable accuracy. The Eph family of receptor tyrosine kinases, and their ligands, the ephrins, proscribe many of these early axon guidance events. It is our hypothesis that oligomerization of these molecules controls both their signaling and their posttranslational modification, and in doing so confers upon them the accuracy and specificity of function necessary to shape the nervous system. Aim 1 will determine the mechanism of ephrin B2 oligomerization, and its importance for receptor activation. Analytical ultracentrifugation will be used to determine whether ephrin B2 undergoes homophilic binding reactions. Further, an ephrin B2 binding protein, ABP, will be assayed for its ability to oligomerize ephrin B2. We will also use our newly developed system, fusing ephrin B2 to the lac repressor, to produce dimeric and tetrameric forms of the ephrin, which will be directly compared for their capacity to activate receptors. Aim 2 and aim 3 will determine whether oligomerization of ephrin B2 drives its phosphorylation and degradation, respectively. The phosphorylation state of ephrin B2 will be measured after being clustered by mechanisms independent of the receptors. Ephrin B2 undergoes proteolysis in chick embryo fibroblasts. The proteolysis appears to be dependent upon clustering. We will determine whether dimers or tetramers undergo proteolysis. We will also use mutagenesis of ephrin B2 to determine which sequences are required for proteolysis. The fourth aim will determine the importance of the biochemical interactions elucidated in the first three aims to the regulation of axon guidance. Derivatives of ephrin B2, designed to be resistant to posttranslation modifications, will be compared for their capacity to repel motor axons. In vitro studies will use the stripe assay, which tests the preference of an axon to extend over alternating stripes, containing, or lacking the appropriate chemorepellant. In vivo studies, will use the assay we have established to observe any deviations from normal of the motor nerve pathways, following ectopic expression of the ephrin B2 derivative from a retroviral vector in chick embryos. Through these comparative studies of ephrin function we will determine the post translation mechanisms which regulate their axon guidance activity."}
{"text": "The University of Kentucky, being a land grant and comprehensive institution, has a tradition of excellence in interdisciplinary research. Such an academic environment will allow study for the overall theme of our SBRP research, which focuses on the toxicology of Superfund chemicals and how health effects of exposure can be modulated by both intrinsic and extrinsic factors, namely genetics and nutrition, respectively. The investigators will integrate five biomedical and two non-biomedical projects to concentrate on chlorinated organics (e.g., polychlorinated biphenyls, trichloroethylene) as model toxins. Chlorinated organics are prevalent in most Superfund sites, including those found in Kentucky. The preliminary findings suggest that nutrition and dietary habits can markedly influence mechanisms of toxicity of the above-mentioned Superfund chemicals. Thus, a major objective of our SBRP is to explore the paradigm that nutrition can modify Superfund chemical toxicity. The investigators hypothesize that highly refined diets, i.e., diets high in fats or calories and low in fruits and vegetables (antioxidants), are associated with an observed national epidemic in chronic diseases, and that populations associated with such dietary habits are more prone to Superfund chemical insult. Biomedical projects will focus on chronic diseases such as cardiovascular disease, cancer, obesity, hypertension, and diabetes. In addition, non-biomedical projects will explore novel techniques for both remediation (detoxification) and biosensors associated with PCBs and other chlorinated organics. Results from the interdisciplinary research will be utilized for informative/educational, technology transfer, training, policy and translational purposes as part of the objectives of the Research Translation, Community Outreach, and Training Cores. Nutrition may be the most sensible means to develop primary prevention strategies of diseases associated with many environmental toxic insults. Thus, the research may lead to novel dietary recommendations at the national level for populations at risk, i.e., people residing near Superfund sites."}
{"text": "Support through the K23 Award will provide the Candidate the necessary time, resources, and mentorship to integrate his clinical interests in substance use disorders with his experience in patient-oriented emergency medicine research. His long-term career plan involves using the knowledge gained from the tobacco smoking and nicotine dependence studies outlined in this proposal as a model for future studies of other substances that are commonly abused by emergency department patients, such as alcohol, cocaine, opiates, and \"club\" drugs. The emergency department (ED) represents a significant portion of all healthcare delivered in the United States, with 100 million ED visits annually. Approximately 50% of the patients seen in the ED have no source of regular outpatient care, and the ED acts as a healthcare \"safety net\" for people with inadequate healthcare insurance. Healthy People 2010 and other government publications have encouraged screening and interventions for tobacco smoking to be conducted during all healthcare encounters, regardless of the setting. However, the extant literature, as well as the Candidate's own pilot studies, indicate that such practices are rare in ED settings. This is true despite the fact that many of the populations shown to be most recalcitrant in their smoking, such as the economically disadvantaged and minorities, are more likely to use the ED for their acute and primary healthcare. If smoking is not addressed with these patients in the ED, it may not get addressed at all. Numerous studies have shown that brief, ED-based interventions for problem behaviors, like alcohol and substance abuse, are both feasible and effective. The noted lack of ED-based screening and intervention for tobacco smoking and nicotine dependence appears due mainly to a lack of relevant research and advocacy rather than a lack of need or barriers prohibiting such practices. The Candidate's career development plan will incorporate needed course work, field experience, and mentorship in data management, study design, statistics, and tobacco research methodology. The research plan concentrates on ED-based tobacco research, beginning with a surveillance study and culminating in a Stage 1A/B behavioral therapy development study of a Behavioral Change Counseling intervention based on principles of Motivational Interviewing. The research plan includes preparing and submitting an R01 in Year 2."}
{"text": "Two new and unusual products of V(D)J recombination in lymphoid cells have been identified. Normal recombination joins the two signal sequences that identify the rearrangement site and, in the complementary reaction, joins the fragments of coding sequences that flank the signals. In extrachromosomal plasmid substrates, these products are found, but there is also a class of \"hybrid joints\" that link one signal to the coding flank of its partner signal. A second class of \"open and shut\" events rejoins each signal to its own flank, and is identifiable because of nucleotides lost and/or added at the joint. Both these products supply evidence that DNA strand alignment is not precisely specified in V(D)J recombination. A specific defect of V(D)J recombination has been identified in lymphoid cell lines derived from mice with the severe combined immune deficiency (scid) mutation. In extrachromosomal substrates, these cells make signal joints (though with greater imprecision than normal) but are unable to make coding joints. This failure appears sufficient to explain the absence of functional B and T cells in scid mice."}
{"text": "An initial objective of the proposed research is the elucidation of the complete genomic sequences of the retinoid-binding proteins (RBPs), cellular retinol-binding protein (cRBP), cellular retinol-binding protein (cRBP), cellular retinoic acid-binding protein (cRABP) and membrane-associated retinoic acid-binding protein (mRABP). RBPs will be purified to homogeneity and their amino-terminal ends sequenced. The amino acid sequences of these proteins and a codon frequency derived from a known, highly homologous protein will be the basis for the synthesis of several oligonucleotides. These oligonucleotides will be used as primary probes in screening complete mouse cDNA libraries; the derived cDNAs will be used to identify the complete genomic clone of cRBP, cRABP, and mRABP. Retroviruses will be constructed that contain either a normal or inverted (anti-sense) genomic sequence (from or near the amino terminal end extending 3 prime to include any primary intron) of the resolved RBP genes. These retroviruses will be infected into cell lines that have been selected for (i) their expression of RBPs cRABP+, cRBP or cRABP+/cRBP+ and (ii) their ability to respond to relatively low concentrations of retinoids retinoic acid+, or retinol+. Changes in cell characteristics (e.g., proliferation, transformation, terminal differentiation) in the presence and absence of (i) an additionally expressed RBP genomic sequence or anti-sense message; (ii) retinoids, and (iii) the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, will also be evaluated. These experiments will (i) provide the complete amono acid as well as genomic sequence for cRBP, cRABP and MRABP; (ii) establish the requirement for RBPs in the control of cytodifferentiation, (iii) allow characterization of RBP secondary structures; and (iv) provide data for the prospective development of anti-retinoid and anti-RBP compounds that may be of significant value in chemoprevention."}
{"text": "Multiple system atrophy (MSA) is a progressive, degenerative neurological disorder characterized by parkinsonism, ataxia & dysautonomia. The cardinal pathological feature of MSA is the presence of glial cytoplasmic inclusions composed of alpha-synuclein (SYN) in oligodendrocytes. Recent studies suggest that abnormal SYN accumulation in neurons & glia leads to cellular dysfunction & neurodegeneration. During the previous funding period we developed in vitro & in vivo models of MSA showing that mitochondrial damage & hyperphosphorylated SYN aggregate generation may contribute to the pathogensis of MSA. However, mechanisms by which these pathways promote oligodendrogial dysfunction & neurodegeneration are unclear. In this renewal we will investigate the role of mitochondrial dysfunction in SYN phosphorylation & toxicity. Our central hypothesis is that oxidative stress due to mitochondrial dysfuntion may promote G-protein coupled receptor kinase (GRK) activation & toxic SYN phosphorylation. The main objective is to investigate neurodegeneration in MSA-like SYN transgenic (tg) models to determine if reducing SYN accumulation represents a therapeutic strategy for MSA. Aim 1. In order to determine the role of hyperphosphorylated SYN accumulation in oligodendrocytes in the mechanisms of neurotoxicity, we will analyze SYN accumulation & neurodegeneration in myelin basic protein (MBP)-SYN tg mice expressing wild-type (wt) human SYN or a nonphosphorylatable SYN mutant (S129A). MBP-SYNwt tg mice will be crossed with GRK2- or GRKS-deficient mice & MBP-SYNwt tg mice & MBP-SYN(S129A) tg mice will receive intra-cerebral infections with lentivirus expressing GRK2 or GRK5 under a oligodendroglial specific promoter (MBP). Aim 2. In order to determine the role of mitochondrial dysfunction & oxidative stress on GRK activation & SYN phosphorylation, MBP-SYN (wt and S129A) mice & oligodendroglial cells will be challenged with 3- nitropropionic acid. Aim 3. In order to determine if neuronal impairments in MSA models can be ameliorated by reducing SYN aggregation or inhibiting GRKs, MBP-SYN wt tg mice will be treated with rifampicin or GRK blockers. Behavioral performance, neurodegeneration, SYN oligomerization & phosphorylation & GRK activity will be assessed."}
{"text": "Apoptosis now is recognized to be an important mechanism of cell death during development and after injury. In neurons, it is well known that growth factor withdrawal can produce apoptotic cell death, both in vivo, and in vitro. Recently, our lab has shown that ionizing radiation (IR) induces apoptosis in post-mitotic dorsal root ganglion (DRG) neurons in vitro. The ability of IR to induce apoptosis dramatically declined over the first 3 weeks in culture to the point where 21 day old DRG neurons did not undergo radiation-induced apoptosis. Previous work in non-neuronal cells has shown that apoptosis occurring as a result of IR-produced DNA damage requires induction of p53. Increased expression of p53 results in activation of a number of downstream genes, including p21, GADD45, and mdm-2, but recent evidence suggests that it is the interaction between p53 and members of the bc1-2 gene family (including bc1-2, bc1-x(L), and bax) that determines whether apoptotic cell death will occur. The experiments described in this proposal will specifically address the individual roles of, and interactions between, p53, bax, bc1-2, and bc1- x(L) in regulating the apoptosis program in post-mitotic DRG neurons exposed to ionizing radiation. We will also examine the roles that these proteins play in producing the temporal change in sensitivity of DRG neurons to IR. By identifying the interactions critical for determining whether these neurons survive or die following exposure to IR, we may be able to design rational strategies for protecting neurons from other potentially lethal insults."}
{"text": "We propose to investigate the interface between EcoRI endonuclease and its cognate DNA recognition sequence by solution biochemical methods. The research is divided into two complementary efforts which will investigate the enzyme-DNA interface from both sides, i.e., we propose to: 1) Localize the points on the DNA which interact with the enzyme by determining the effects of base substitutions within and immediately surrounding the cannonical hexanucleotide, GAATTC. We will measure the effects of sequence changes on the enzyme kinetics, dissociation constants and the ability of the endonuclease to protect bases from Maxam-Gilbert reagents. 2) Localize points on the endonuclease which interact with the DNA by determining which amino acid residues can be: (A) Crosslinked to synthetic oligonucleotides containing the photo-activatable base analog 5-bromodeoxyuridine at unique points in the recognition sequence. (B) Protected by the DNA from reacting with reagents which modify exposed amino acid residues. We will also seek to determine whether or not the polypeptide chain is comprised of domains, one of which retains most of the recognition specificity. These experiments make extensive use of techniques developed in this laboratory for specifically binding DNA containing the recognition site to the enzyme under conditions which abolish catalysis. The ultimate goal of this research is to elucidate molecular mechanisms of sequence specific DNA-protein interactions."}
{"text": "Project Summary/Abstract Despite intensive national efforts to address childhood obesity, the epidemic persists. Recognizing the challenges, the National Institutes of Health (NIH) and the Institute of Medicine (IOM) have called for a systems-oriented approach to address childhood obesity. We hypothesize that among low-income, ethnically diverse overweight and obese children, aged 2-12 years, a systems approach to child obesity incorporating secondary prevention programs embedded within primary prevention will reduce body mass index (BMI) z-scores compared to primary prevention alone. Intervention strategies will be adapted from the portfolio of evidence-based programs developed by study investigators. An assets-based assessment will be conducted with research and community partners in disadvantaged neighborhood catchment areas in Austin and Houston; data will inform integration of intervention strategies and provide a structure for program sustainability. Our specific study aims are: Aim 1: To implement and evaluate a primary prevention obesity program in low-income, ethnically diverse catchment areas in Austin and Houston. Baseline and 2-year follow-up data on the prevalence of child overweight, risk factors, and the availability of healthcare services and community programs will be collected in 2 demonstration (n=1614) and 2 control catchment areas (n=1614). Parent, child and community data will provide matched comparison data to assess the efficacy of the primary prevention intervention. Aim 2: To implement and evaluate the efficacy of a systems approach to child obesity on reducing obesity by embedding a 12-month family-based secondary prevention program within the community primary prevention program. The secondary prevention weight management program will be conducted with overweight/obese children and their families in the primary prevention catchment areas. Families of overweight/obese children (total n=576), aged 2-12 years, will be randomly assigned to either the 12-month secondary prevention program (experimental) or prevention program alone (control), stratified by age (2-5, 6-8, and 9-12 y). Outcomes include child BMI z-score, quality of life measures, and program use indicators. Aim 3: To quantify the incremental cost-effectiveness of the 12-month family-based secondary prevention program relative to primary prevention alone for child obesity. Activity Based Costing methods will be used to quantify the incremental cost of delivering the secondary prevention program relative to primary prevention. Public Health Relevance: If this research trial is proven efficacious and cost-effective, the demonstration project could be disseminated widely to address obesity among underserved, at risk children. Program sustainability will be ensured through active involvement of community partners within healthcare, childcare, schools and community sectors."}
{"text": "The viral Gag proteins control many aspects of the HIV-1 replication cycle. The Gag precursor drives the assembly of virus particles in the infected cell, and, through putative interactions with the transmembrane envelope (Env) glycoprotein gp41, directs Env incorporation into virus particles. Following infection, the Gag proteins play a central role in uncoating and assist in the reverse transcription process. The assembly of type C retroviruses and lentiviruses, including HIV-1, takes place at the plasma membrane (PM). It is well established that this process is promoted by Gag proteins; however, the mechanisms by which assembly is specifically targeted to the PM remain to be determined. Recent studies have suggested that the PM contains microdomains with distinct protein and lipid compositions. One type of microdomain, the lipid raft, is enriched in sphingolipids and cholesterol and can be isolated as detergent-resistant membrane (DRM). Rafts have been shown to play essential roles in a variety of biological processes, often by acting as a target site for proteins involved in a common pathway. Last year, we reported that a large portion of membrane-bound Gag was recovered in DRM (Ono and Freed, PNAS, 2001). To address the specificity of Gag-raft association, we examined the kinetics of this association. We found that DRM association of Gag occurred more slowly than binding of Gag to membranes, suggesting that HIV-1 Gag is specifically targeted to PM rafts after it binds membrane. We observed that the determinant for raft association maps to the N-terminus of Gag, but that Gag-Gag interactions facilitate or stabilize raft association. To address the physiological relevance of Gag-raft association, we analyzed the impact of cholesterol depletion, which disrupts raft structure, in HIV-1-producing cells. We found that cholesterol depletion markedly and specifically reduces virus release. Moreover, virus released from cholesterol-depleted cells displays reduced infectivity. Interestingly, cells expressing a mutant Gag with substitutions in the p6 late domain motif show no reduction in virus release. These results identify the association of Gag with PM rafts as an important step in HIV-1 production, and suggest possible links between rafts and late steps in virus assembly. Recent work has been aimed at defining the mechanism by which raft disruption impairs virus production. These results also suggest that cholesterol depletion prevents the efficient binding of Gag to membrane. The p6 domain of HIV-1 is located at the C-terminus of the Gag precursor protein Pr55Gag. Previous studies indicated that p6 plays a critical role in HIV-1 particle budding from virus-expressing HeLa cells. We performed a detailed mutational analysis of the N-terminus of p6 to define sequences required for efficient virus release. By examining the effects of p6 mutation in biological and biochemical analyses and by electron microscopy, we determined the role of p6 in particle release in a panel of cell lines as well as in peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM). The results (Demirov et al., J. Virol. 2002) indicate that: i) the highly conserved P-T/S-A-P motif located near the N-terminus of p6 is remarkably sensitive to change; even conservative mutations in this sequence induce profound virus-release defects in HeLa cells. ii) Single and double amino acid substitutions outside the P-T/S-A-P motif have no significant effect on particle release. iii) The introduction of stop codons one or two residues beyond the P-T-/S-A-P motif blocks virion release, whereas truncation four residues beyond P-T/S-A-P has no effect on particle production in HeLa cells. iv) Though the effects of p6 mutation on virus replication in T-cell lines are cell-type dependent, even in T-cell lines in which p6 mutations block virus replication, these changes have little or no effect on particle release. v) p6-mutant particles produced in T-cell lines exhibit a defect in virion-virion detachment, resulting in the production of tethered chains of virions. vi) Transient heterokaryons produced between HeLa cells and a T-cell line display a T cell-like phenotype with respect to the requirement for p6 in particle release, indicating that the activity that suppresses the release defect phenotype is dominant in this cell system. vii) Finally, in primary MDM, mutation of p6 results in marked defects in both particle release and virus replication. These results reveal a strong cell-type dependent requirement for p6 in virus particle release. A variety of lines of evidence suggest a connection between retroviral L domains function and the host ubiquitination machinery. Recently, it was demonstrated that the product of tumor susceptibility gene 101 (TSG101), which contains at its N-terminus a domain highly related to ubiquitin conjugating (E2) enzymes, binds HIV-1 Gag in a p6-dependent fashion. We examined the impact of overexpressing the N-terminal region of TSG101 on HIV-1 particle production. Intriguingly, we observe that this domain (referred to as TSG-5') potently inhibits virus production (Demirov et al., PNAS 2002). Examination of cells coexpressing HIV-1 Gag and TSG-5' by electron microscopy reveals a defect in virus budding very reminiscent of that observed with p6 L domain mutants. In addition, the effect of TSG-5' is dependent upon an intact p6 L domain; the assembly and release of virus-like particles produced by Gag mutants lacking a functional p6 PTAP motif is not significantly affected by TSG-5'. Furthermore, assembly and release of murine leukemia virus and Mason-Pfizer monkey virus are insensitive to TSG-5'. TSG-5' is incorporated into virions, confirming the Gag/TSG101 interaction in virus-producing cells. Mutations that inactivate the p6 L domain block TSG-5' incorporation. These data demonstrate a link between the E2-like domain of TSG101 and HIV-1 L domain function, and raise the possibility that TSG101 derivatives could serve as potent and specific inhibitors of HIV-1 replication by blocking virus budding. To define the mechanism by which TSG-5' inhibits virus release, we investigated the importance of TSG-5'/Gag interaction in the virus release inhibition. We observed that a mutation in TSG-5' that prevents Gag from interacting with this truncated protein eliminates its ability to disrupt particle release. These results suggest that direct binding to Gag, rather than disruption of cellular sorting machinery, is responsible for the ability of TSG-5' to block budding. We also investigated the effect of overexpressing longer forms of TSG101, including the full-length protein, on virus budding. We observe that overexpressing other truncated mutants as well as the full-length protein, disrupt budding. Interestingly, the ability of full-legth TSG101 to inhibit virus release when overexpressed does not require binding to Gag. Rather, as indicated by confocal microscopy experiments, overexpressing the full-length protein intereferes with the cellular endosomal sorting machinery. To complement our studies on HIV-1, we are investigating the role that TSG101 and other host factors play in the budding of a diverse array of retroviruses, including murine leukemia virus, human T cell leukemia virus, bovine leukosis virus, Mason-Pfizer monkey virus, and equine infectious anemia virus."}
{"text": "We have documented age-related and degeneration-related changes in the metabolism of collagens,[unreadable] proteoglycans, and matrix-degrading enzymes in the human intervertebral disc. Our studies (i) confirmed the[unreadable] pivotal roles that cytokines and growth factors play in intervertebral disc degeneration and repair, (ii)[unreadable] demonstrated that certain growth factors inhibit the autocrine production of cytokines and that cytokine[unreadable] inhibitors can change basal levels of synthesis of matrix molecules and metalloproteinases, and (iii) found[unreadable] that biglycan was expressed in greater amounts than other small proteoglycans during late stages of disc[unreadable] degeneration and in the discs of old donors and plays an important regulatory role in matrix homeostasis.[unreadable] Our data suggest that an intricate balance of growth factors, cytokines and regulatory molecules is important[unreadable] for the maintenance of tissue homeostasis. Over the next five years, we propose to investigate the[unreadable] mechanisms of action and the interplay between growth factors, cytokines and various regulatory molecules[unreadable] in order to determine their respective roles in tissue homeostasis and their potential use in promoting tissue[unreadable] repair. Hypothesis 1: The effects of growth factors on the intervertebral disc are regulated by the production[unreadable] of cytokines by intervertebral disc cells and by the presence of inhibitory factors; growth factors have a[unreadable] negative feed back on the production of these cytokines. Hypothesis 2: The regulatory mechanisms of[unreadable] matrix homeostasis by cytokines and growth factors vary at different stages of disc degeneration because of[unreadable] changes in the cellular microenvironment in situ within the disc. The response to cytokines and growth[unreadable] factors and the production and activation of matrix-degrading enzymes will be different at advanced stages[unreadable] of disc degeneration. Hypothesis 3: The inhibition of cytokine action by cytokine blockers and the inhibition[unreadable] of cytokine production by growth factors can delay or reverse the degenerative status of intervertebral disc[unreadable] cells in the cellular microenvironment that can be observed in the early and middle stages of disc[unreadable] degeneration. Low back pain is responsible for enormous human suffering, high health care costs and[unreadable] significant socioeconomic losses. Although the etiology of back pain is often unknown, the intervertebral[unreadable] disc is a significant source of back problems. The results from this study will advance the field of biological[unreadable] treatment and tissue engineering for intervertebral disc degeneration."}
{"text": "Project Summary/Abstract The overall goal of this proposal is to generate, maintain, and distribute a new collection of 1,500 custom engineered Drosophila melanogaster stocks that will allow versatile manipulation of genes that are highly conserved in humans but currently lack an available mutant allele. Utilizing cutting edge CRISPR technology, this collection or 'kit' will represent a powerful new set of tools that will benefit virtually all Drosophila geneticists; as such it is likely that there will be a high demand for this resource for many years to come. Many of the most important advances in our understanding of human development have come from studies using the fruit fly as an animal model system. Since many parallels exist between Drosophila and mammals in terms of the underlying molecular mechanisms controlling biological processes, knowledge gained from research in Drosophila can be either directly applied or readily adapted to understanding human biology and disease. During Phase I of this project, we optimized the conditions for CRISPR/Cas9 mediated insertion of a custom CRIMIC cassette and correctly targeted 55 out of 63 genes attempted. This high rate of success (87%), indicates that we are perfectly poised to scale up our efforts to a larger set of genes. In this Phase II proposal, we aim to generate CRIMIC targeted alleles for an additional 1,500 conserved genes that currently lack an available mutant allele. The primary benefit of such a collection is that the CRIMIC insertion not only generates a strong loss-of-function or null allele of the targeted gene, but can also serve as a docking site for further engineering, such as transcript or protein tagging or creating point mutations that can be used to model human disease pathology. In coordination with other existing collections and the ongoing Gene Disruption Project (GDP), our Specific Aims are to: Aim 1. Select 1,800 gene targets and construct 1,800 gRNA/donor pairs Aim 2. Generate and validate a collection of 1,500 targeted gene stocks Aim 3. Create and maintain an online searchable database for targeted genes Harnessing the benefits of new technologies, this collection will fully complement existing collections and the ongoing GDP effort. Once generated, these combined resources will cover the vast majority genes in the Drosophila genome that are directly relevant to human disease."}
{"text": "1) Evolutionary classification of P-loop NTPases As a part of my ongoing research on the P-loop NTPases, which constitute the largest set of monophyletic protein domains in most proteomes, we completed the evolutionary classification of the AAA+ ATPases and the P-loop kinases. These studies helped us to identify the early diversification events in the history of these proteins. We provide evidence that the P-loop NTPases first differentiated into two classes, namely the KG class (Kinase, GTPase class) and the ASCE class (Additional strand conserved glutamate) that includes the AAA+, RecA-like, PilT/VirD4-like and ABC ATPases. These classes had several representatives that could be traced back to the last universal common ancestor of all life forms suggesting that they had undergone a vast radiation even before that stage. Hence they provide insights into some of the earliest aspects of protein evolution. 2) Analysis of the novel protease families A multi-pronged strategy including extensive sequence searches, structural modeling, and analysis of contextual information extracted from domain architectures, genetic screens, and large-scale protein-protein interaction analyses was employed to predict previously undetected components of the eukaryotic ubiquitin (Ub) signaling system. Two novel groups of proteins that are likely to function as de-ubiquitinating and de-SUMOylating peptidases (DUBs) were identified. The first group of putative DUBs, designated PPPDE superfamily (after Permuted Papain fold Peptidases of DsRNA viruses and Eukaryotes), consists of predicted thiol peptidases with a circularly permuted papain-like fold. In addition to eukaryotic proteins, the PPPDE superfamily includes predicted proteases from several groups of double-stranded RNA viruses and one single-stranded DNA virus. The apparent recruitment of DUBs for viral polyprotein processing seems to represent a common theme in evolution of viruses. The second group of putative DUBs identified in this study is the WLM (Wss1p-like metalloproteases) family of the Zincin-like superfamily of Zn-dependent peptidases, which are linked to the Ub-system by virtue of fusions with the UB-binding PUG (PUB), Ub-like, and Little Finger domains. More specifically, genetic evidence implicates the WLM family in de-SUMOylation. If validated experimentally, the WLM family proteins will represent the first case of a Zincin-like metalloprotease involvement in Ub-signaling. 3) Evolution of the nuclear membrane and nuclear pore complex The presence of a distinct nucleus, the compartment for confining the genome, transcription and RNA maturation, is a central (and eponymous) feature that distinguishes eukaryotes from prokaryotes. Structural integrity of the nucleus is maintained by the nuclear envelope (NE). A crucial element of this structure is the nuclear pore complex (NPC), a macromolecular machine with over 90 protein components, which mediates nucleo-cytoplasmic communication. Given the indispensability of these structures for nuclear function, the natural history of the nucleus can only be understood in terms of the origin and subsequent evolution of NE and NPC components. We investigated the provenance of the conserved domains found in these perinuclear proteins and reconstructed a parsimonious scenario for NE and NPC evolution by means of comparative-genomic analysis of their components from the available sequences of 28 sequenced eukaryotic genomes. We show that the NE and NPC proteins were tinkered together from diverse domains, which evolved from prokaryotic precursors at different points in eukaryotic evolution, divergence from pre-existing eukaryotic paralogs performing other functions, and de novo. It is shown that several central components of the NPC, in particular, the RanGDP import factor NTF2, the HEH domain of Src1p-Man1, and, probably, also the key domains of karyopherins and nucleoporins, the HEAT/ARM and WD40 repeats, have a bacterial, most likely, endosymbiotic origin. The specialized immunoglobulin (Ig) domain in the globular tail of the animal lamins, and the Ig domains in the nuclear membrane protein GP210 are shown to be related to distinct prokaryotic families of Ig domains. This suggests that independent, late horizontal gene transfer events from bacterial sources might have contributed to the evolution of perinuclear proteins in some of the major eukaryotic lineages. Snurportin 1, one of the highly conserved karyopherins, contains a cap-binding domain which is shown to be an inactive paralog of the guanylyl transferase domain of the mRNA-capping enzyme, exemplifying recruitment of paralogs of pre-exsiting proteins for perinuclear functions. We infer an autogenous scenario of nuclear evolution in which the nucleus emerged in the primitive eukaryotic ancestor (the ?prekaryote?) as part of cell compartmentalization triggered by archaeo-bacterial symbiosis. A pivotal event in this process was the radiation of Ras-superfamily GTPases yielding Ran, the key regulator of nuclear transport. A primitive NPC with approximately 20 proteins and a Src1p-Man1-like membrane protein with a DNA-tethering HEH domain are inferred to have been integral perinuclear components in the las common ancestor of modern eukaryotes."}
{"text": "Male and female 10-12 year old children of men with Alcohol Use Disorder (AUD) (high risk) and control fathers (low risk) will be studied at baseline and on two follow-ups (ages 12-14 and 16) to developmentally clarify liability for an Alcohol Use Disorder. In addition to obtaining behavioral measures, the project will measure cognitive, electrophysiological and eye movement processes using a multitrait multimethod (MTMM) paradigm. High risk subjects (N=400) are hypothesized to perform deficiently compared to low risk subjects (N=400) on measures that differentially reflect prefrontal cortex functioning and to be comparable on non-prefrontal measures. Within the developmental framework encompassing transition from late childhood to mid-adolescence, it is hypothesized that the manifest behavioral dysregulation (due to prefrontal cortex impairment) is associated with disruptive parent-child interactions and affiliation with deviant peers. The resulting maladjustment, namely, an externalizing disposition, predisposes to precocious alcohol exposure, early age intoxication and, by mid-adolescence, heavier alcohol involvement. In effect, individual liability (dysregulation explicable by prefrontal cortex dysfunction), via interaction with the family and peer environments, biases the developmental trajectory toward an externalizing adjustment style (proximal outcome) and problem alcohol consumption (distal outcome). Confirmation of the individual basis of AUD liability would point to innovative approaches to prevention by identifying the specific components of risk which are subsumed within well-known functional neuroanatomical systems. Furthermore, this project will afford the opportunity to elucidate AUD etiology as the culmination of person- environment interactions."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have proposed a role for anxiety in the initiation and maintenance of SIB. To test this hypothesis directly, we investigated hormonal and behavioral responses of adult male rhesus macaques to various doses of the benzodiazepine (BDZ) receptor inverse agonist FG 7142."}
{"text": "It is proposed to develop a sensitive and specific method of quantitative radioimmunoassay to measure gut-related autoimmune phenomena in Lewis-strain rats induced by highly purified syngeneic and xenogeneic intestinal mucins. Tritiated ovine salivary mucin (OSM) will be studied first in its reaction with antibodies raised in Lewis-strain rats to Lewis rat intestinal sialylmucins. After parameters of the reaction have been delineated, the technique will be used to study the time-course of antibody development in Lewis rats immunized with xenogeneic and syngeneic sialylmucins. Differential radioimmunoassays will be used to determine the various classes and subclasses involved in the immune reactions. Adsorption techniques coupled with radioimmunoassays will be used to determine the various end groups of mucins represented in the syngeneic rat antiserum reactions. Dose-response characteristics of the antibody responses will be studied by dosage manipulation one at a time, or antigen, of adjuvant (Mycobacterium butyricum), and of ancillary adjuvant (Bordetella pertussis) at fixed doses of the other two components. The requirements of T-cell dependency will be assessed in Lewis rats that have been thymectomized as adults, irradiated with 900 rads whole-body, reconstituted with 10 to the 7th power bone-marrow cells from normal Lewis rat donors, and challenged with sialylmucin preparation. Finally, it will be determined, by the method of passive transfer into normal recipients of donor lymphocytes, sensitized to sialylmucin, whether an experimental inflammatory bowel disease can be induced and whether coproantibodies and/or humoral antibodies to sialylmucins can be detected in the recipients by the proposed method of radioimmunoassay."}
{"text": "We will study the metabolism and action of methyl-n-amylnitrosamine (MNAN) in the esophagus as follows: (1) We will examine the production of hydroxy and keto metabolites of MNAN in freshly removed esophagus, forestomach and other tissues of rodents. We will complete studies of changes with age of this metabolism in key tissues of suckling rats and hamsters, determine whether these changes are due to intrinsic or extrinsic factors, examine effects of agents that induce individual P-450s, and determine configurations of the hydroxy-MNAN metabolites. (2) We will develop systems for culturing adult rat esophagus, while still maintaining MNAN metabolism. We will study MNAN metabolism in cultures of newborn and adult rat forestomach, hamster forestomach and hamster esophagus, and by freshly incubated adult and fetal human esophagus, and study effects of inducers and other agents on these cultures. (3) We will examine MNAN metabolism by rat liver and esophageal microsomes and reconstituted cytochrome P-450 (\"P-450\") systems to determine which P-450s metabolize MNAN. Gel electrophoresis and specific antibodies will identify the individual P-450s in adult and developed esophagus and forestomach. (4) We will examine MNAN alkylation of DNA to give 06- and N7 alkylguanine and 04-alkylthymine, using 3H-MNAN or radioimmunoassay. In vivo persistence of alkylated bases in esophageal DNA will be measured, and DNA alkylation by 2- and 4-oxo-MNAN. (5) We will determine whether MNAN is taken up more extensively than dimethylnitrosamine by rat esophagus. (6) We will compare carcinogenicity of MNAN injected into newborn, 3-day-old and adult rats and hamsters, test for carcinogenicity of 2- and 4-oxo-MNAN in adult rats, and determine whether inducers can affect MNAN carcinogenicity. These studies are designed to investigate (a) why MNAN is a specific carcinogen for the rat esophagus, (b) what intrinsic or extrinsic factors control the striking changes in MNAN metabolism from newborn to adult rats and hamsters, and (c) whether human esophagus could be susceptible to nitrosamine carcinogenesis."}
{"text": "We continue to study the streptococcal infections associated with acute glomerulonephritis (AGN) and acute rheumatic fever (ARF) in Trinidad by identifying the streptococcal strains isolated and/or the type specific antibodies which develop. We also assess patient-response to these infections by titrating antistreptolysin O and antihyaluronidase in serum samples and compare these responses in patients with ARF or AGN and in normal children. We also shall study the response to streptococcal DNAse B in representative patients and school-children. We are examining local immunological responses in patients with ARF by assaying: 1) saliva, for immunoglobulins, ASO, AH and anti group A carbohydrate and 2) joint fluid, for immunoglobulins and complement components. We have completed observations on the course of serum B1C globulin and fibrin degradation products in patients with AGN. These were made in view of the possibility that activation of the coagulation system with subsequent activation of plasmin may be responsible for the decreased C3 found in these patients. We found no evidence for this occurrence. We also are studying the effects of both \"nephritogenic\" and \"rheumatogenic\" streptococcal strains in peritoneal chambers on rats, according to the studies of Lindberg and Vosti."}
{"text": "Studies on DNA-DNA homology between bacteria of food and human clinical origin will be performed to determine the human pathogenic potential of refrigerated food spoilage bacteria in the genera Pseudo- monas and Achromobacter. The bacteriostatic properties of the fluorescent pigment produced by certain refrigerated food spoilage psuedomonads will be studied with respect to inhibition of Achromobacters on fish and meat. The fluorescent pigment from Pseudomonas septica will be purified, and its mechanism of bacterial inhibition (DNA, RNA, or protein synthesis) determined. The use of gas chromatography and mass spectroscopy will be applied to the genus Pseudomonas in an attempt to further characterize the identify refrigerated food spoilage bacteria of this genus. The mechanism of temperature restriction of an obligately psychrophilic phage-host system involving an intense fish spoilage strain of Pseudomonas putrefaciens is to be studied."}
{"text": "This facility, operating since 1990, provides cost-effective and accurate chemical analyses for molecules relevant to cancer research of Cancer Center (CC) investigators. It is also a base for consultation related to the quantitation of analytes and strategies in analytical chemistry. Services and expertise in advanced methods are available including state-of-the-art techniques, particularly liquid chromatography mass spectrometry (LCMS) and multiplexed immunoassays. In addition, routine analyses and determinations of clinical markers are offered to investigators. A host of new assays have been implemented in the current cycle due to needs of CC members. No other laboratories are available to provide many of these services in the State of Hawai'i, particularly regarding type, speed, cost, and combinations of assays, many of which are custom designed by this core to fulfill the specific requirements of CC projects. The high quality of services is continually monitored by taking part in internal and external quality assurance programs including the participation in round robin tests organized by the National Institute of Standards and Technology. The availability of this core provides state-of-the-art services, improves feedback to the investigators, stimulates inter-disciplinary collaborations, and provides cost effective service to CC investigators. The current use at full capacity is anticipated to continue for the foreseeable future. Newly funded projects requiring services of the resource will outnumber projects terminating in the coming years. Equipment obtained through the CCSG support grant in the current cycle has greatly enhanced the potential of this resource. This facility has shown remarkable growth: its productivity increased by 84% since the last cycle and has grown accordingly in personnel and revenue. The great value of this core is underscored by a total of 79 cancer related peer reviewed publications during FY06-11 that involved services by the ABSR (58% inter-programmatic, 13% intra-programmatic)."}
{"text": "Hip fracture and resultant surgical repair entail a significant loss of muscle mass and function. Both injury and surgery, in and of themselves, initiate a cascade of events which lead to the loss of muscle mass. The general premise of this project is that this initial loss of muscle protein during surgery and the accompanying convalescence predispose a generally compromised patient to poor functional or morbidity outcomes. Further, we propose that muscle protein is lost due to a significant alteration in the muscle protein metabolism. The general goal of this project is to investigate interventions designed to ameliorate the negative muscle balance between protein synthesis and protein breakdown. We hypothesize the enhancement of muscle anabolism is required for these patients to recover muscle strength and function. We first propose to restore muscle protein synthesis throughout hospitalization with the administration of an essential am/no acid formula. We further propose that the amelioration of hypercortisolemia after surgery will result in a decreased muscle protein breakdown. The amelioration of hypercortisolemia after surgery will be accomplished with a common antifungal agent, ketoconazole, in order to investigate the resultant affects on muscle protein breakdown. We will measure lean body mass and muscle volume, as well as functional outcomes before surgery and discharge from acute hospitalization to determine the efficancy of these interventions. We will utilize stable isotope methodology to quantify muscle protein metabolism after acute hospitalization. We will also amplify these results by investigating cellular markers of protein breakdown to corroborate our metabolic endpoints. [unreadable] [unreadable] [unreadable]"}
{"text": "Our long-term goal is to determine how cells establish and then maintain progressive motility in response to nondirectional external signals during wound repair. In wound repair, fibroblasts and endothelial cells repopulate the immature matrix to form both the supporting matrix and vasculature required to regenerate the tissue structures. The initial migration is driven by signals that arise from within the wound bed. However, once within the wound bed, the cells must distribute often m the absence of a gradient of signals. A central question is how cells establish the asymmetry required forprogressive motility. Soluble growth factors induce the migration of both the fibroblasts and endothelial cells. This motility requires asymmetry of biophysical cell processes within the cell. At the front, the cells must extend lamellipodla and form new adhesions, while rear de-adhesion and retraction is required. Between these two cell regions, contractility occurs to bring the cell body forward. Key molecular switches have been identified which regulate each biophysicalprocess. What remains unknown is how these signaling pathways initiate the biophysical processes in their correct spatial orientation. While an external signaling gradient would be an attractive explanation, the literature suggests that for eukaryotic cells, the key regulators are intracellular. This would be particularlz true for chemokinetic agents, such as EGFR and VEGFR ligands, which can induce progressive cell motility even in the absence of an external gradient. We hypothesize that cellular asymmetry needed for productive motility is accomplished by subeytoplasmie restriction of the activation of key biochemical switches. We propose to test the following molecular mechanisms: I. That PLCgamma activity is limited to the leading lamellipod by cdc42. We will use imaging and molecular perturbations to determine where and how PLCgamma/-1, required for front-direct actin reorganization, interacts with cdc42, the small GTPase that orchestrates the motility vector. These studies are based on preliminary data. II. That m-calpain drives de-adhesion in the cell body and trailing edge secondary to restricted availability of phospho-inositides. We will determine how m-calpain activity is restricted to the cell body and tail region, based on preliminary findings of phospho-inositides being required for m-calpain activation by growth factors. III. That contractile forces are asymmetric within the cell during EGF-induced motility. Biophysical deconstruction will identify quantitative and qualitative differences in contractility across a motile cell. These investigations wil! define molecular bases of the spatial restriction of signals and responses. This will enable the design of'smart scaffolds for cell and tissue engineering directing the synthesis of both the matrix and the vascular bed that is reauired to sunnort tissue function."}
{"text": "This research study is looking at the interactions of the combination of ritonavir and saquinavir and efavirenz."}
{"text": "Epithelial Na channels (ENaC) are expressed in the distal nephron where they serve as the final sites of Na reabsorption, thereby playing a key role in the regulation of extracellular fluid volume and blood pressure (BP). ENaC gain-of-function mutations are associated with hypertension, whereas loss-of-function mutations are associated with hypotension. ENaCs are comprised of three structurally related subunits termed ?, ? and ?. These subunits are assembled into a trimer within the endoplasmic reticulum (ER) and, following ER exit, the subunits undergo post-translational processing. In particular, cleavage at defined sites of the extracellular domain of the ? subunit by the proteases furin and prostasin has a key role in activating ENaC in heterologous systems, presumably by releasing an inhibitory domain within the subunit's ectodomain. Experiments are proposed to first define the functional role of ? ENaC in vivo by generating mice with distal nephron-specific ? ENaC disruption. These will be followed by studies assessing the functional role of ? ENaC subunit proteolysis in vivo by generating mice that express ? ENaC subunits in the distal nephron that lack key defined proteolytic cleavage sites for furin or prostasin. The specific aims are: 1. Generation and characterization of mice lacking ? ENaC selectively in ENaC-expressing cells in the kidney. The proposed studies are directed at generating a mouse model that has disrupted ? ENaC expression in the nephron. To accomplish this, two mice will be used: 1) a mouse expressing Cre recombinase either in principal cells of the collecting duct or in the entire nephron;and 2) a mouse containing loxP-flanked exons critical to ? ENaC function. Breeding of these animals will result in distal nephron-knockout of ? ENaC. These ? ENaC knockout animals will allow us to define the role of ? ENaC in the regulation of blood pressure, renal electrolyte excretion, and collecting duct ion transport. 2. Generation and characterization of mice with renal-specific reconstitution of wild-type or mutant ? ENaC. These studies are designed to define the role of ? ENaC proteolysis in the in vivo regulation of ENaC activity. Prostasin-dependent cleavage of the ? subunit, in concert with furin cleavage, fully activates the channel. The proposed studies are directed at generating a mouse model that expresses a ? subunit with mutations in the furin or prostasin cleavage sites selectively within the distal nephron. To accomplish this, native renal ? ENaC will be disrupted and ? ENaC expression reconstituted using;1) a ? subunit with a mutation in the furin cleavage site;2) a ? subunit with a mutation within the prostasin cleavage site;or 3) a wild type ? subunit (as a control). The introduced ? subunits will bear epitope tags to facilitate detection and proteolytic processing. These animals will allow us to (i) define the extent of proteolytic processing of the ? subunit at the prostasin or furin cleavage sites under basal conditions and in the setting of volume depletion;and (ii) determine the role of ? subunit proteolytic processing at the prostasin or furin cleavage sites in the expression of functional channels. PUBLIC HEALTH RELEVANCE: The proposed studies are designed to determine the physiologic role of cleavage of the epithelial sodium channel in the kidney. The studies will define the importance of two different cleavage sites within the sodium channel in regulation of kidney sodium and potassium excretion and maintenance of normal blood pressure. This work has the potential to uncover mechanisms involved in the development and/or maintenance of hypertension and disorders of potassium excretion."}
{"text": "Mechanical ventilation (MV) is a clinical tool to sustain pulmonary gas exchange in patients that are incapable of maintaining adequate alveolar ventilation. Although MV is often a life-saving intervention, prolonged MV promotes problems in weaning patients from the ventilator. While several factors can contribute to difficult weaning, weak inspiratory muscles are a major factor. In this regard, MV results in diaphragmatic inactivity that promotes the rapid onset of inspiratory muscle weakness due to diaphragmatic atrophy and contractile dysfunction (known as ventilator-induced diaphragm dysfunction (VIDD)). Although MV-induced diaphragmatic atrophy occurs due to both increased protein breakdown and decreased protein synthesis, the mechanism(s) that regulate these processes are poorly understood and thus, no clinical therapy exists. Hence, our long-term goal is to identify biological targets that will assist in the development of a therapeutic strategy to preven VIDD and therefore, protect against weaning difficulties. Although numerous signaling events can contribute to VIDD, we predict that the forkhead boxO (FoxO) family of transcription factors are important because they regulate the expression of genes involved in both the ubiquitin-proteasome and the autophagy pathway of proteolysis. Moreover, gene targets of FoxO may also contribute to the depression of protein synthesis. HYPOTHESIS: We will test the hypothesis that activation of FoxO-dependent transcription is required for the decreased protein synthesis, increased expression of proteasome/autophagy genes, and fiber atrophy that occurs in the diaphragm during MV. APPROACH: Our hypothesis will be tested in a well-established rat model of MV. Cause and effect will be determined by using an adeno-associated virus vector for gene delivery to express a dominant negative FoxO to prevent activation of FoxO target genes in the diaphragm during prolonged MV. SPECIFIC AIMS: Aim 1 will establish if FoxO-dependent transcription is essential for the rapid decrease in diaphragmatic protein synthesis that is observed during prolonged MV. Aim 2 will determine if FoxO-dependent transcription is required for the MV-induced increase in the expression of proteasome and autophagy genes along with diaphragmatic atrophy. SIGNIFICANCE: Collectively, this work can provide the foundation for new therapeutic strategies in the prevention of VIDD, a major contributor to the inability to wean patients from the ventilator."}
{"text": "Individuals with apolipoprotein (Apo) E deficiency develop hypercholesterolemia and atherosclerosis. Similarly, ApoE-deficient (ApoE-/-) mice elevate plasma ApoB48-carrying lipoproteins and develop atherosclerosis in a manner that resembles the human disease. The primary cause of atherosclerosis in ApoE-deficient patients and mouse models is the deposition of ApoE-deficient, ApoB48-carrying (E-/B48) lipoproteins in the arterial wall. The deposited lipoproteins recruit monocytes into the arterial intima and transform them into macrophages, which participate in the pathogenesis of atherosclerosis mainly through two mechanisms: 1) releasing inflammatory mediators, and 2) forming foam cells. An uncontrolled macrophage uptake of E-/B48 lipoproteins could be a mechanism underlying foam cell formation in ApoE-/- mice. Our laboratory recently demonstrated that E-/B48 lipoproteins reduce cellular cholesterol efflux from macrophages and down-regulates lysosomal hydrolase expression. In addition, the degradation of E-/B48 lipoproteins by macrophages declined over time. These novel findings indicate that both reduced cholesterol efflux and decreased degradation of E-/B48 lipoproteins could contribute to foam cell formation. Our preliminary studies also revealed that incubation of macrophages with E-/B48 lipoproteins enhanced eukaryotic translation initiationfactor 21 (eIF-2a) phosphorylation, which is linked to one of the unfolded protein response (UPR) signaling pathways. Thus, interaction of E-/B48 lipoproteins with macrophages may activate UPR signaling pathways, which in turn regulate gene expression and induce atherogenic events, such as triggering foam cell formation. In the proposed studies, we will test the hypothesis that activation of UPR signaling pathways is a mechanism by which E-/B48 lipoproteins regulate gene expression, induce foam cell formation and promote atherosclerosis. This project includes four specific aims: 1) to determine whether E-/B48 lipoprotein-induced changes in gene expression result from altered transcription or translation or both in mouse macrophages;2) to determine whether activation of UPR signaling pathways is a mechanism underlying E-/B48 lipoprotein-induced gene expression changes;3) to determine whether activation of UPR pathways is a mechanism underlying E-/B48 lipoprotein-induced foam cell formation;and 4) to determine the effect of inhibiting eIF-2a phosphorylation on atherosclerosis in ApoE-/- mice. If our hypothesis is correct, inactivation of UPR signaling pathways will attenuate E-/B48 lipoprotein-induced gene expression, and suppress foam cell formation and atherosclerosis development. PUBLIC HEALTH RELEVANCE: Myocardial infarction and stroke are the leading caused of death in the United Sates. Atherosclerosis is the primary cause of myocardial infarction and stroke. Formation of lipid-laden foam cells in the vessel wall is the early stage of atherosclerosis. The goal of this proposal is to determine the involvement of endoplasmic reticulum stress in foam cell formation. Endoplasmic reticulum stress is a cellular event that reduces the level of some proteins but increases the level of other proteins in cells. Our preliminary studies indicate that lipoproteins obtained from mice deficient in apolipoprotein E can cause endoplasmic reticulum stress and induce foam cell formation. The experiments designed in this proposal will study whether endoplasmic reticulum stress is the cause of foam cell formation. Finding from this work should contribute to understanding of the molecular mechanism of foam cell formation, and provide strategies for treatment or prevention of atherosclerosis by inhibition of endoplasmic reticulum stress."}
{"text": "This project undertakes an analysis of the mechanisms of transformation and gene regulation by human adenoviruses. Deletion mutants of adenovirus type 2 (Ad 2), isolated from laboratory stocks, or constructed in vitro, will be used to identify regions of the viral genome involved in transformation or the regulation of gene expression. The phenotypes of mutants with apparent defects in transformation or regulation will be carefully studied to determine how the deleted regions of the genome are involved in these processes."}
{"text": "This application requests continuation of funding for a very successful and well-established post-doctoral training program (dating back to 1991) in Major Psychoses and Translational Clinical Neuroscience. It seeks to integrate training in translational clinical neuroscience with an emphasis on the study of schizophrenia and other major psychoses using a lifespan trajectory perspective. That is, trainees may study illnesses during their characteristic age of onset, but they may also examine them from the perspectives of neurodevelopmental and/or aging processes. This program in integrative translational clinical neuroscience will place strong emphasis on all three aspects: integrative, translational/clinical, and neuroscience. The ultimate goal is to train a group of young investigators who will combine a high level of sophistication about the complexities of clinical phenomena with a firm grounding in some specific area of neuroscience, such as neuroimaging, cognitive neuroscience, and genetics/genomics/molecular biology. The program supports five trainee slots/year. Trainees remain in the program for a minimum of two years, and many elect to continue their training for a total of three years because of the complexities inherent in doing sophisticated integrative translational clinical neuroscience. The minimum experience required for entry into the program is a doctoral degree (M.D., Ph.D.), but many of our trainees have also completed residency training and have M.D./Ph.D.s. Relevance An integration of basic and clinical science will, we hope, help identify the mechanisms and causes of the major psychoses and to suggest improved methods for treating or preventing them. The disorders studied through this program (schizophrenia, depression, and bipolar illness) are three of the top ten contributors to the global burden of disease, according to a recent study by the World Health Organization. Thus it is crucial that we train a young generation of investigators who will advance knowledge about these diseases over the coming decades. PUBLIC HEALTH RELEVANCE: The disorders studied through this program (schizophrenia, depression, and bipolar illness) are three of the top ten contributors to the global burden of disease, according to a recent study by the World Health Organization. Thus it is crucial that we train a young generation of investigators who will advance knowledge about these diseases over the coming decades. 20"}
{"text": "The main objectives of this project are: 1) to measure the effect of polycyclic hydrocarbons on the microsomal enzymes of macrophages and lymphocytes; 2) to develop a dependable, convenient in vitro assay for aryl-hydrocarbon hydroxylase (AHH) in human peripheral blood monocytes; and 3) to find whether interaction of polycyclic hydrocarbons with macrophages facilitates the induction and/or expression of contact hypersensitivity to these compounds."}
{"text": "The absence of a suitably HLA-matched donor precludes hematopoietic cell transplantation (HCT) in many patients, as the risks of graft-versus-host disease (GVHD) and rejection are formidable when extensive HLA barriers are transgressed. HLA-mismatched HCT without rejection or severe GVHD would make this treatment available to patients lacking a matched donor, and could maximize graft-versus-tumor (GVT) effects. The toxicity of allogeneic HCT can be diminished using non-myeloablative conditioning. The major goal of this Program Project is to develop non-myeloablative approaches to HLA-mismatched HCT, while minimizing GVHD and exploiting the potent GVT effect of anti-MHC GVH alloreactivity. Two major strategies will be explored in animal models, and a third will be characterized mechanistically. Nonmyeloablative allogeneic HCT that includes T cell depletion of the recipient and donor inoculum can lead to mixed hematopoietic chimerism across extensive MHC barriers without GVHD. When donor lymphocyte infusions (DLI) are administered to established murine mixed chimeras, the GVH alloresponse associated with subsequent DLI leads to powerful GVT effects without GVHD. Recipient-derived professional antigenpresenting cells (APC) in mixed chimeras maximize GVH alloresponses and thereby maximize GVT, while GVHD, a disease of epithelial target tissues, is avoided. We have taken these observations from mice to a unique large animal (pig) model and into clinical trials for the treatment of leukemias and lymphomas. Two of the Projects (Project 1 in mice, Project 3 in large animals) focus on the administration of delayed DLI to mixed allogeneic chimeras prepared with non-myeloablative conditioning that includes T cell-depleting mAbs, in order to achieve maximal GVL effects without GVHD. The goals of expanding donor hematopoietic stem cells (HSC) ex vivo and in vivo and improving immune recovery in Project 4 will ultimately be applicable to the clinical application of this approach, since large numbers of purified (T cell-depleted) donor HSC may be required to achieve engraftment across extensive HLA barriers. Project 4 includes an initial clinical evaluation of the ability of PTH to expand the numbers of mobilized HSC in patients failing conventional mobilization treatment. Large animal studies to evaluate the use of PTH to enhance allogeneic HSC engraftment (Project 3) will provide a critical bridge between the pre-clinical and clinical applications of these strategies (Project 4). Like Project 1, Project 2 involves confinement of the GVH alloresponse to the lymphohematpoietic system, via a novel strategy involving IFN-gamma. A mechanistic understanding of this phenomenon will lead to the development of novel strategies for separating GVHD and GVL clinically. Core A will provide multicolor (up to 11-color) flow cytometry and high speed cell sorting services to support all of these projects. Core B (Administrative) will be responsible for administering the grant, and Core C will provide statistical and database support for all of the Projects."}
{"text": "This proposal seeks continued support for the development and continuation of an interdisciplinary Center for Research in Oral Biology. The central goal or purpose of the Center is to assist in the national effort to reduce the toll of oral disease and to promote the general level of oral health. The Center is achieving this goal by developing and supporting meritorious and relevant research projects, by creating an intellectual and physical environment conducive to optimal research productivity, by providing opportunities for significant interaction between investigators with diverse backgrounds, by producing both basic and applied knowledge related to the oral region, and by encouraging the incorporation of relevant research findings into dental education, dental practice, and the surrounding community. The types of projects supported have been the subject of intensive discussion within the Center and by the Scientific Review Committee, and within the committee appointed by the Vice President for Health Affairs to study the future pattern of the Center. The emphasis in the past has been on high quality basic research programs. This emphasis continues, but we are increasing our percent effort directed toward application of basic research findings to problems of patient diagnosis and care and to measures which will lead to improvement of the oral and dental status of the population."}
{"text": "The broad objective of the Opportunities Fund Management Core (OFMC) will be to facilitate early stage support for innovative, exploratory, and R&D projects, particularly those which have the potential to lead to practical products in the fields of radiation biodosimetry and prediction and prevention of acute and delayed injuries. The OFMC will support 1) Pilot projects (basic and translational research) and 2) Advanced Development projects that lead to Investigational New Drug (IND)-enabling studies and Medical Countermeasure (MCM) development. The OFMC will generate an SOP (Standard Operating Procedure) for the operation of the Core, which after Steering Committee approval, will be the template for its operation. The OFMC will coordinate the process of soliciting proposals, as well as coordinating the review of the proposals by an external panel of experts convened via teleconference for the purpose. The OFMC will tally the scores given by the review panel at the teleconference, and present the rank ordered list to the Steering Committee for funding decisions. Innovative and translational research will be strongly emphasized in the review process. The OFMC will also be responsible for coordinating all necessary documentation of financial, IRB, and IACUC requirements with sub-recipient institutions, as well as financial oversight and reporting. The OFMC will also coordinate annual progress reports, and collect progress reports with renewal requests to be considered by the Steering Committee. The OFMC will also help to facilitate Presentation opportunities for the pilot project recipients. For instance, pilot awardees may be given the opportunity to prepare a poster for the annual CMCRC meeting, and will be encouraged to present at national meetings, such as the Radiation Research Society Annual Meeting. !"}
{"text": "Abstract Bioluminescence imaging (BLI) systems are installed in thousands of facilities and labs, and with their straightforward and low cost workflow for longitudinal studies, are the most commonly used preclinical modality for assessing tumor models in rodents. There currently is no high throughput and low cost system enabling BLI images to be combined with anatomical images of soft tissue to confirm tumor volume, context, or vascularity. In our Phase I work, we demonstrated the feasibility of mapping data from our whole body ultrasound (US) system to 2D BLI images. The additional US data dramatically reduced inter-user quantification variability of the BLI signal (>80%). Furthermore, we showed that our clinically translatable microvessel imaging technology, acoustic angiography, can be mapped to the BLI data. Those tumor microvessels were analyzed using patented vessel analysis algorithms, yielding quantifiable vascular morphology metrics, previously shown to be reliable predictors of tumor malignancy and response to therapy in humans. Thus our commercialized hybrid modality US+BLI device, the Alpheidae Platform, will allow angiogenic tumors and anti-angiogenic therapies to be studied in ways current in vivo imaging tools do not allow. In Phase II, we propose to bring the Alpheidae Platform to market, leveraging whole body tissue and vascular US imaging to improve cancer research with BLI. The team includes experts in optical imaging system design, photoacoustic system design, and US imaging system design. Specifically, in Phase II, we will address the following aims: (Aim 1) Hardware R&D for multi-animal tri-modality imaging. Six animals will be scanned sequentially by our robotic system in each of the three modes. Throughput for six animals will be <15 min. (Aim 2) Software and algorithm R&D to enable automated targeted US and PA acquisitions based on BLI images, and leveraging algorithms for improved spatial resolution in both US and PA datasets. (Aim 3) Validation studies within in vivo tumor drug response study. Device performance will be assessed by comparison to standard BLI in a murine breast cancer model. Its capacity to reliably predict eventual drug response within one week of starting therapy will be the criteria for success. Once Phase II is completed we will have created a novel high throughput and portable tool enabling tumor tissue and microvessel images to be mapped to BLI data. Importantly from a commercial perspective, the Alpheidae Platform can be sold for a fraction of what competitive systems cost. From a translational research perspective, the device includes a clinically translatable US microvessel imaging approach for tumor assessment, and thus forms a direct link between preclinical findings in mice and actionable clinical cancer assessment protocols."}
{"text": "Receptors for transplantation antigens may be visualized directly on unsensitized cells with the use of an anti-idiotypic antisera. It should be possible to eliminate these cells specifically by treatment of the unsensitized population with sera directed at the specific binding site. Sera have been raised to cells cytotoxic for transplantation alloantigen in strain combinations selected so that only the variable portion of the antigen specific receptor could be recognized. Bona fide anti-idiotypic sera have not been raised. Refinements in the approach under way include 1) use of cytotoxic populations as immunogen with a demonstrably high proportion of receptor-bearing cells, 2) use of parent strains differing only by a point mutation to restrict the heterogeneity of the immune response of one against the other, and 3) use of continuously cultured cytotoxic cell lines to increase the homogeneity of the receptors used as immunogen."}
{"text": "Occupational exposure to arylamines used in the manufacturing of industrial dyes was the first known cause of human bladder cancer. These carcinogenic arylamines, specifically, 4-aminobiphenyl (4-ABP), beta-naphthylamine, and benzidine, are also contained in tobacco smoke, and are the most likely causative agents responsible for the raised rates of bladder cancer in smokers. Oxidation of arylamines is recognized as a critical first step in turning these chemical species into their carcinogenic metabolites capable of causing DNA damage to urothelial cells. Hair dyes represent another substantial source of arylamines in humans, and we recently showed that sustained use of permanent hair dyes in women is a risk factor for bladder cancer, especially among those deficient in arylamine detoxifying enzymes. Subsequently, 4-ABP was detected in bottles of commercial hair dyes bought in a US store. Then, using hemoglobin adducts of 4-ABP as a biomarker of exposure, we showed that other, presumably diffuse, sources of 4-ABP exposure may be related to bladder cancer in nonsmokers. Thus, the latest evidence indicates arylamine exposure as the primary cause of bladder cancer in the United States. Ten years ago, we reported that a reason for the 3-fold increased risk of bladder cancer in US white versus Chinese men despite their comparable smoking habits may be the higher prevalence in the former population of individuals with deficient arylamine-detoxifying enzymes, which are under genetic control. We capitalized on this finding and launched a population-based case-control study involving both a Los Angeles and a Shanghai, China component to explore genetic factors that play a major role in determining bladder cancer risk in arylamine-exposed individuals. This already completed database consists of roughly 1300 cases of incident bladder cancer (750 cases in Los Angeles, 550 cases in Shanghai) and about an equal number of control subjects. Our initial goals were to investigate the roles of selected polymorphic, arylarnine-metabolizing genotypes/phenotypes in bladder cancer, and in the Los Angeles component, to use hemoglobin adducts as biomarkers of exposure to arylamines in examining nonsmoking-related bladder cancer. In this application, we aim for a more comprehensive understanding of the arylamine-bladder cancer etiologic link through the following extensions on our Los Angeles/Shanghai database: (1) Completion of arylamine hemoglobin adduct measurements on Shanghai subjects in order to compare the respective effects of these adducts on risk between Los Angles and Shanghai study subjects;(2) Addition of genotypes involved in arylamine metabolism, in cellular response to oxidative stress and in DNA repair;and (3) Development of a Bayesian hierarchical statistical model to allow for an efficient, pathway-driven examination of multiple gene-arylamine interactions in bladder cancer. The ultimate goal of this research is to assess individual bladder cancer risk for the purpose of preventive interventions."}
{"text": "Recent literature in mental health services has identified a serious gap between what is known about mental disorders and their treatment from university-based clinical research and what services are actually provided to consumers in typical community practice settings. This IP-RISP \"Biosocial Factors in Rehabilitation for Schizophrenia\" will build a partnership between university-based clinical services researchers and practitioners and consumers from a psychosocial rehabilitation service agency. The goal of this IP-RISP is to implement a research and practice agenda that will translate recent findings on psychobiological deficits in schizophrenia and their remediation into community based rehabilitation practice. We will address questions relevant to: (i) improving the effectiveness of community-based psychosocial rehabilitation interventions for functional disability in schizophrenia, and (ii) adapting and infusing new knowledge and new interventions into psychosocial rehabilitation practice settings in the community. This IP-RISP will address four practice-research aims and two infrastructure aims. The four practice-research aims are: 1) To introduce knowledge on functional outcomes and psychobiological factors to practitioners and consumers of psychosocial rehabilitation services at Portals;2) To partner with agency clinicians and consumers in adapting and infusing (transporting) psychosocial intervention strategies for the remediation of psychobiological deficits into existing rehabilitation services;3) To partner with agency clinicians and consumers in implementing controlled pilot research on whether targeted psychobiological service elements increase the effectiveness of psychosocial rehabilitation services in the community by improving consumer outcomes;4). To facilitate the sustainability of the new service configurations by incorporating consumer, practitioner, and administrator perspectives. The two infrastructure aims are: 5). To develop a shared university/agency infrastructure consisting of a database and shared clinical-research personnel specifically: a). To increase the clinical utility of an existing database used to measure psychosocial outcomes in a community psychosocial rehabilitation setting, and b). To train and support clinical-research personnel who will participate in both clinical and research agendas at the community service setting. 6). To build a platform for ongoing psychobiological data collection consisting of an agency-based laboratory for assessing and measuring relevant psychobiological variables such as neurocognition, social cognition, and psychophysiological responsivity. The aims will be met during four activity phases consisting of: a) partnering among agency staff, consumers, and researchers;b) introducing and adapting new service elements into rehabilitation practice;c) implementing pilot research on the effectiveness of the new intervention elements;and d) assessing the implementation experience."}
{"text": "PRODUCTION OF TWO THOUSAND CAPSULES OF 4 MG AZA-TDCPOP: 03/04/19 THROUGH 03/03/20. The batch of product was produced by the contractor and sent to clinical repository. The shelf-life was also evaluated. The drug product will be used in the NCI clinical trials."}
{"text": "Program Summary. Amyotrophic lateral sclerosis (ALS) affects an estimated 30,000 individuals in the United States and 450,000 worldwide. The disease is characterized by loss of motor neurons leading to loss of muscle control and muscle wasting. Patients typically progress from diagnosis to death in 3-5 years. While there are two approved therapies to treat ALS, their effects are limited. The lack of available treatments for ALS patients remains a significant unmet medical need. Unlike numerous other rare diseases, ALS is not monogenic, thus it is unlikely that a single therapy will be able to address the entire patient population. Despite recent advances in sequencing technology, 90% of ALS cases are sporadic making determination of a genetic cause more difficult. Nonetheless, many of the genes originally found in familial cases also appear in the sporadic population including C9orf72, and Optineurin. One of the genes that has recently been shown to be associated with ALS in a dominant fashion is tank binding kinase 1 (TBK1). Using patient derived cells, it has been shown that the mutations lead to a haploinsufficiency and loss of function. Furthermore, studies on TBK1 have shown that it is a key regulator of autophagy and mitophagy and interacts with Optineurin, p62, and Ubiquilin, all of which have also been shown to be associated with ALS. To develop therapies for TBK1 patients, we propose to develop a cellular system with TBK1 haploinsufficiency that can be used to develop an assay to assess the efficacy of potential drug candidates to increase TBK1 function from the remaining allele. We propose to use CRISPR-Cas9 to disrupt a single allele of the TBK1 gene in human induced pluripotent stem cells (hiPSCs) as a phenotypic model of TBK1 patients. Once we have generated a single allele knockout of TBK1, we will assess the phenotype in hiPSC-derived motor neurons in comparison to parental cells. Disease relevant phenotypes will be examined including levels of activated TBK1, autophagy, and mitophagy. Our drug discovery efforts are focused on increasing the levels of activated TBK1 and thus rescuing any deficiency found in the downstream pathways of autophagy and mitophagy. We have identified a molecular target that modulates TBK1 activity and have preliminary data showing that pharmacological modulation of this target leads to an increase in mitophagy in human microglial cells. We are continuing to pursue this target and anticipate that we will have a significant number of compounds to assay once the TBK1 knockout cells are available. The demonstration of activity in a cellular model of ALS is crucial to support our ultimate goal of developing a therapeutic for this patient population. In addition, many ALS associated genes, including TBK1, have also been shown to be associated with Frontotemporal Temporal Dementia (FTD), and autophagy and mitophagy defects are associated with several neurodegenerative diseases, thus this work may have implications for drug discovery beyond ALS."}
{"text": "Description (from applicant): One of the long standing unanswered questions in one of the most frequently studied models of carcinogenesis, chemical hepatocarcinogenesis, is what is the cell of origin of hepatocellular carcinomas (HCC)? The classic cellular pathway, championed by Emmanuel Farber, is that HCC develop from altered hepatocytes progressively through foci and preneoplastic nodules. However, studies on oval cells, which appear early during hepatocarcinogenesis, indicate that HCC may arise from \"bipolar\" liver progenitor cells located within terminal ducts or from pluripotent progenitor cells located adjacent to the ducts. In addition, recent studies on female mice and rats receiving bone marrow transplants from male donors indicated two possible origins of liver stem cells: from bipolar progenitor cells in the terminal ducts or from periductal stem cells. It is further postulated that these periductal progenitor cells may arise from circulating bone marrow stem cells either by differentiation into liver cells or that bone marrow cells may fuse with liver cells. In Specific Aim 1, the applicant proposes to develop a transgenic Fischer rat line expressing EGFP which can be used as a donor for bone marrow transplantation studies. In Specific Aim 2, three models of carcinogenesis: a. Continuous DEN, which features foci of altered hepatocytes preceding HCC with little or no oval cells will be used to determine if HCC arise from hepatocytes; b. Solt-Farber model, which features prominent proliferation of ductal oval cells to determine if HCC arise from bipolar ductal precursor cells; c. Choline-deficiency-ethionine, a model which features periductular proliferation of oval cells will be used to determine if preneoplastic lesions or HCC arise from periportal oval cells. In each of these protocols, DDPIV- (canalicular enzyme) female rats will receive a bone marrow transplant from DDPIV+, EGFP+, male donors. The origin of the populations of cells responding by proliferation will be identified by the presence of donor or recipient markers, and the types of cells identified through labeling with both liver lineage and littoral cell markers, when appropriate. In Specific Aim 3, the possibility of fusion of donor bone marrow stem cells with recipient liver cells will be tested and the possibility of fusion in etiology, progression or metastasis of HCC determined. Knowledge of the cellular origin of cancer is critical for understanding how cancer evolves and for developing prevention strategies."}
{"text": "ABSTRACT Pattern recognition receptors sense pathogen-associated molecular patterns and mediate the earliest host innate immune response to infection. The cytosolic nucleotide oligomerization domain (NOD) like receptors (NLRs) are a highly conserved group of cytosolic proteins that play a central role in the immune response to diverse microorganisms, environmental insults and cellular danger signals. We have recently demonstrated a role for Nlrp6 in the control of enteric virus infection. Nlrp6 controls enteric virus infection in the intestine by interacting with a RNA sensor, Dhx15. Here we propose to define the mechanisms of NLRP6-mediated anti-viral pathways, to identify whether other helicases may also interact with NLRP6 (Aim 1), whether other relevant RNA viruses are recognized through this pathway, and whether mutations may underlie certain human cases of vaccine failures (Aim 2). A fuller understanding of the triggers and physiologic function and signaling pathways for NLRs will provide key insights to immune mechanisms involved in host defense and immune-mediated diseases."}
{"text": "The acetylcholine receptor of Torpedo californica is an oligomeric glycoprotein which is embedded in the membrane of the multinucleated electroplaque cells of the electric organ. The arrangement of the subunits of the receptor may be an important indication of their function. I propose to study this arrangement by chemical crosslinking in order to map the nearest neighbor proteins of each subunit and by surface labeling, enzymatic proteolysis, and chemical fragmentation in order to determine the exposure of each subunit at the cytoplasmic and external surfaces of the membrane. A primary culture of the electroplaque precursor cells will be isolated for the purpose of studying the biogenesis of the acetylcholine receptor. Both receptor synthesis and degradation will be investigated. The synthesis of the receptor may be regulated at the transcriptional, posttranscriptional or translational levels. The control mechanisms will be studied by isolating mRNA and using this mRNA as a template for in vitro translation."}
{"text": "This proposal outlines an experimental strategy aimed at understanding age-dependent dysregulation of stem cell function in the Drosophila model. The adult Drosophila intestinal epithelium is maintained by resident stem cells which contribute to all gut epithelial cell types. These intestinal stem cells (ISCs) divide in response o tissue damage and aging, but appear to lose control of their proliferative limit. Specifically, agig leads to ISC hyperproliferation, which is in itself similar to pathological conditions we find in mammalian models of cancer. Thus, understanding the mechanisms for age-associated stem cell dysfunction may illuminate on the mechanisms of cancer in higher organisms, such as humans. We will begin filling gaps in our current understanding of stem cell aging by addressing two relevant problems in the field. In the first aim, we will attempt to understand the dynamics of gut aging, specifically focusing on the contribution of cell autonomous and cell non-autonomous aging cues on ISC aging and hypeproliferation. We will utilize well established methods in Drosophila system which will allow us to genetically manipulate the ISCs and their lineage in the gut. We will also use antibodies that reveal the rate of gut cell aging. Our second aim will expand on the new techniques in metabolite profiling to characterize the endogenous small molecule content of the aging environment in flies. We will then use computational methods and stringent statistical analysis, which efficiently identifies the most probable longevity candidates and will test their role on lifespan and stem cell aging. While our metabolite profiling may offer structural information of small molecule regulators of the stem cell aging, the complementary gene expression studies may illuminate on upstream genetic circuitries which can be functionally analyzed in cell-specific manner in the fly gut."}
{"text": "This proposal addresses the need for definitive evidence on the role of Foxp3+/T regulatory cells in the acceptance of physiologically relevant full MHC incompatible organ grafts in various settings of operational tolerance. In the initial year of funding (ARRA), spontaneous acceptance of MHC-mismatched mouse renal allografts was shown to depend exquisitely on Foxp3 cells using diphtheria toxin (DT) to transiently deplete circulating and intragraft Foxp3 cells in B6.Foxp3DTR mice bearing life sustaining DBA/2 renal allografts. The accepted grafts had distinctive nodular perivascular infiltrates of Foxp3 cells and dendritic cells (DC). Deletion of foxp3 cells caused dissolution of the nodular aggregates and widespread infiltration of the cortex, tubules and blood vessels by T cells, typical of acute rejection, and manifested by a rise in BUN over 7 days. Similar nodular aggregates of Foxp3 cells occur in grafts of patients who have accepted MHC mismatched kidneys on mixed chimerism tolerance induction protocols. Specific aim 1 will extend these observations to other forms of induced tolerance, mixed chimerism and neonatal tolerance, some of which are expected to be Foxp3 dependent;those with deletional mechanisms are expected to be Foxp3 independent. We will seek distinctive pathologic features in the graft, indicating Treg activity, such as the co-expression of latency associated protein (LAP) and Foxp3 or DC expression of indoleamine 2,3-dioxygenase (IDO), that might be used to distinguish foxp3 dependent from foxp3 independent forms of graft acceptance. The donor reactivity and regulatory potential of the cells in grafts and in lymphoid organs will be tested by ELISPOT and compared between the different forms of acceptance and acute rejection. The dependence of systemic tolerance on the intragraft infiltrates will be tested by graft nephrectomy in recipients with accepted kidney and skin grafts that have a remaining native kidney. Specific aim 2 will seek to identify the molecular and cellular basis for the dramatic strain differences in susceptibility to spontaneous graft acceptance, comparing BALB/c (rejected) and DBA/2 (accepted) kidneys (both H-2d) in B6 recipients. We will test for strain differences in DC function, cytokine/chemokine/complement mediators, minor histocompatibility antigens and ability to generate Foxp3 cells. Follow-up experiments seek to alter identified factors to promote acceptance of BALB/c kidneys. Specific aim 3 visualize interaction of Foxp3+ cells with other T cells, recipient and donor dendritic cells and tubular cells in accepting and rejecting renal grafts using a custom real time confocal, multiphoton endomicroscope. This system permits observations of the same graft over time, live video recording, delineation of 3-4 different cell types and computer assisted measurement of cell motility and interactions. These studies are designed to reveal the significance and in vivo function of Foxp3+ Treg in murine renal allograft acceptance and provide insights potentially applicable in clinical renal transplantation. PUBLIC HEALTH RELEVANCE: The mouse kidney transplantation system offers an optimal combination of fidelity to human pathological processes, a rich spectrum of outcomes without drugs, and the ability to manipulate and observe the components of the immune response for mechanistic studies. These studies will provide insights into Treg physiology in renal allografts that will clarify their therapeutic relevance and reveal features that distinguish pathologic from beneficial intragraft infiltrates, of considerable importance in clinical transplantation and tolerance induction studies."}
{"text": "The primary goal of this study is the exploitation of the guinea pig inclusion conjunctivitis animal model for studies referable to human chlamydial infections. Thus, attempts will be made to determine the pathogenic events involved in the transmission of chlamydiae from the eye to the lung and gastrointestinal tract and to assess the clinical and pathologic significance of chlamydial infection in these sites. In addition, efforts will be made to study the mechanism of resistance or immunity to infection with Chlamydia trachomatis resulting from previous exposure and to determine the potential role of previous exposure in heightening the disease response from hypersensitivity."}
{"text": "The overall goal of this competing renewal application (Years 16-20) is to train postdoctoral physician fellows for successful research careers in academic infectious diseases. Support is again requested for 3 fellows/year for 5 years. During the current funding cycle, 6 fellows were appointed to our Research Training Program (2/year), including 2 from underrepresented minorities. Two fellows have completed the Training Program and are in academic or public health positions, and the 4 other appointees remain in training (2 in their 3rd year and 1 each in their 2nd and 1st year of training). Collectively, these trainees have published 20 papers (16 first authored) in peer-reviewed journals. Each year, we have carefully assessed the recruitment efforts and performance of the trainees and mentors in our Program and have implemented important changes to strengthen it. We will offer training in the 3 areas of greatest mentoring strength in infectious diseases at the University of Pittsburgh: HIV/AIDS, Molecular Epidemiology and Microbiology, and Sexually Transmitted Diseases. There is a critical need for well-trained physician investigators in each of these areas. The mentoring faculty, consisting of 6 MD and 9 PhD scientists from the Schools of Medicine and Public Health, have exemplary records of research accomplishment, funding and training of postdoctoral fellows. Physicians who have completed the first clinical year of infectious disease fellowship will be eligible for the Research Training Program. Appointees are selected by the Program Executive Committee, consisting of the PI and senior mentoring faculty. Appointees develop a three-year research Training Plan with their mentor for approval by the Executive Committee. The performance of the trainees is carefully reviewed by the mentor (weekly), the PI (quarterly), and the Executive Committee (biannually). The latter occurs at Fellows Research in Progress Meeting (July and January), during which all trainees present their work and are challenged to defend their research findings and plans. Issues related to responsible conduct of research are discussed throughout the Training Program. The goal of the Program is for each trainee to develop expertise in an area of infectious disease research that will serve as the foundation for a successful investigative career. Our Program will increase the capacity for research that is highly relevant to the NIAID mission."}
{"text": "[unreadable] [unreadable] This conference grant is for the 7th biennial workshop on \"Molecular Aspects of Myeloid Stem Cell Development and Leukemia.\" This 7th workshop in a series held since 1995 is scheduled for May 13-16, 2007 in Annapolis, MD. The workshop hosts ~50 speakers, with a total of ~100 participants. Leukemia is a disease of hematopoietic stem cells that undergo a block in differentiation and a deregulated expansion. The workshop focuses on genes located at translocation breakpoints in leukemia such as AML1, TEL, RARalpha, and MLL, and on genes aberrantly expressed due to epigenetic alterations or function abnormally due to mutations. Mutations or abnormal levels of these factors are causally related to leukemogenesis. Murine gene-targeting studies and sequence analyses of patient samples show the critical importance of these same genes for normal hematopoiesis. The functions of these factors are combinatorial in hematopoietic development, and the complexity of interactions within overlapping networks makes it difficult for single investigators to gain wide scientific and clinical perspective to understand their roles in normal hematopoiesis and leukemogenesis. The goal is to bring together investigators with expertise in complementary aspects of stem cell biology, normal myelopoiesis and leukemia. Basic and clinician-scientists from the USA, England, Europe and Japan come together to discuss their latest findings on stem cells and hematopoietic regulators, e.g. transcription factors, including those who work on leukemia mechanisms and therapeutics, signal transducers and cell cycle components dysregulated through genetic mutations or epigenetic modifications. The aim is exchange of information through informal presentations and discussions. Young investigators (graduate students, postdoctoral fellows) comprise ~50% of participants and have the opportunity to present their work as posters or oral talks. Participants achieve greater understanding of the critical factors that regulate lineage specification and malignant hematopoiesis, and their relevance in the clinic in terms of leukemia. The workshop uniquely brings together basic scientist, clinician-investigators, and junior investigators in a manner that truly promotes communication and open exchange of ideas, unpublished results, cross-fertilization and collaborative research between scientists working on normal and leukemic stem cells and myeloid development, along with translational leukemia research and leukemia therapeutics. [unreadable] [unreadable] [unreadable]"}
{"text": "The work carried out this year represents a continuation and extension of earlier studies on the role of carbohydrates in biological systems. Two separate areas are covered in this report. (a) The previously described findings on the hormonal regulation of sialylation in rat thyroid cells have been examined in human thyroglobulin isolated from patients with Graves disease and from patients exhibiting endemic goiter. In contrast to normal controls or material from endemic goiters, the thyroglubin recovered from patients with Graves disease was characterized by severe hypersialylation and aberrant localization of the sialic acid residues within the carbohydrate core. (b) Techniques designed to isolate and characterize individual nuclear protein fractions have revealed the presence of enzymatically degradable glycogen in the pore- lamina fraction of the nucleus and the presence of one or more complex glycoproteins within the nuclear matrix."}
{"text": "Adult dental caries, including both coronal and root caries, is an infectious disease that afflicts the majority of Americans aged 55 and older and is the most common chronic disease at midlife. In addition, adult caries exacts a significant economic toll, and this economic toll continues to grow. Despite the high prevalence and bacterial pathogenesis of caries, no FDA-approved anti-microbial treatment for caries is available to the American dental professional. The Prevention of Adult Caries Study (PACS) is a randomized, double-blind, placebo-controlled Phase III clinical trial conducted under FDA Investigational New Drug license #45,466. This study is designed to evaluate the efficacy of a topical, temporary, 10% w/v chlorhexidine dental coating in reducing caries increment in at-risk adult dental patients. This study will follow 1000 patients over a 13-month study period at four centers with vastly different populations. The primary endpoint for this study will be caries increment. The centers participating in this proposed research are: Center for Health Research, Kaiser Permanente Northwest, in Portland, Oregon;Tufts University Dental Clinic in central Boston, Massachusetts;Dental Service of Massachusetts Clinical Center in Southborough, Massachusetts, and;the dental clinic of the Tuba City Regional Health Care Corporation in Tuba City, Arizona. Kaiser Permanente's Center for Health Research in Portland, Oregon will act as the data-coordinating center, and Tufts University will act as the Administrative Center. This application from the Kaiser Permanente Portland Clinical Center is part of the coordinated set of applications in support of the PACS study. Please refer to the Administrative Center Application for a complete description of the study Protocol and Manual of Procedures."}
{"text": "Telomeres are the natural ends of linear chromosomes and crucial for genome stability, cellular viability and chromosome integrity. Telomeres shorten during each cell division, representing a cellular clock and limiting the replicative lifespan. To overcome this limit, cancer cells have to activate telomere maintenance mechanisms, which counteract telomere shortening and endow the cells with immortality. Ninety percent of cancers do so by activating telomerase, a reverse transcriptase complex that adds telomeric repeats to chromosome ends. The remaining 10% of cancers take advantage of a recombination-based mechanism for telomere length maintenance, called ALT (Alternative Lengthening of Telomeres). While telomerase activation is the more frequently used mechanism and widely investigated, it is becoming clear that ALT pathways can be activated upon telomerase inhibition, emphasizing that both telomere maintenance mechanisms have to be understood before telomere elongation can be successfully targeted as cancer therapy. ALT-dependent telomere lengthening is based on recombination between long and short telomeres, but the mechanisms are not currently understood. ALT has long been considered the result of defects in cellular recombination pathways, but despite intense efforts no deficiencies in recombination regulators have been identified in ALT-cells. Recent data suggest the hypothesis that ALT relies on aberrant recombination pathways needs to be reexamined and is likely incorrect. We discovered that ALT is likely a consequence of poor histone placement at repetitive regions, such as telomeres. We can induce ALT dependent telomeric recombination by suppression of isoforms of the histone chaperone Asf1. Upon Asf1 down regulation all characteristics of ALT emerge in primary and transformed cells, which include telomere sister chromatid exchange, telomere length heterogeneity, the formation of single stranded telomeric C-circles and the colocalization of PML, RPA and TTAGGG repeats in ALT associated PML bodies. The three intellectually connected but independent aims of this proposal are designed to investigate our novel concept for the ALT mechanism, which suggests that improper nucleosome placement at telomeres leads to single stranded loops at telomeres, which then readily recombine with each other. In AIM1 we will investigate telomere structure in cells with suppressed Asf1, as well as nucleosome placement and DNA damage signaling at telomeres and throughout the nucleus in Asf1 suppressed cells. We will also address whether Asf1 dependent ALT activation is capable of long-term telomere maintenance. AIM2 is designed to investigate why Asf1 suppression has telomere-specific effects and whether it leads to changes in telomeric chromatin in primary and transformed cells, therefore defining an ALT specific epigenetic signature. In AIM3 we will investigate the Asf1 status of ALT tumor cells, whether ALT can be suppressed by Asf1 expression, and whether ALT is an epigenetic state that can be induced by constitutive Asf1 suppression."}
{"text": "Ongoing comparative analysis of the protein compositions of the pigment epithelial (PE) cell plasma membrane from normal and dystrophic (RCS) rats will be extended in greater detail. The goal of this research has been to determine if the RCS mutation results in retinal degeneration by affecting the production of a PE plasma membrane protein that is involved in the recognition and phagocytosis of shed rod outer segment (ROS) membranes. Two dimensional (2D) electrophoretic analysis of the protein composition of plasma membrane enriched fractions from normal and dystrophic rat PE suggests that a 183,000 MW glycoprotein is underglycosylated in the RCS PE. In order to elucidate the chemical nature of this apparent decreased glycosylation, the carbohydrate compositions of the normal and RCS 183,000 MW proteins will be analyzed by sequential degradation with exo- and endoglycosidases, gel filtration chromatography and high performance liquid chromatography (HPLC). Cultured rat PE cells will be assayed for enzymes commonly found in the plasma membrane of other absorptive epithelia. Electroblots of 2D maps of PE cell membranes will be stained with antibodies specific for enzymes that test positive in the preliminary assays. Alternatively, subunits for some of the enzymes will be identified by use of specific radioactive affinity labels or histochemical stains. In this manner, specific functions will be identified for proteins previously characterized by molecular weight only in 2D electrophoretic analysis of PE plasma membrane proteins. This information will be valuable for narrowing down the number of spots in the complex 2D map that may be involved in recognition/phagocytosis of ROS membranes by the PE. Another important component of the PE cell plasma membrane, the receptor for serum retinol binding protein (RBP), will be detergent solubilized from swine PE. An assay for detergent solubilized receptor will be devised, and subsequently employed in purification of the receptor by lectin affinity chromatography, gel filtration chromatography, and HPLC. Antibodies will be prepared to the purified receptor. The anti-RBP receptor antibody will be used to determine if distribution of the receptor in the plasma membrane is altered as a secondary effect of retinal degeneration in the RCS rat. The long term goal of this research project is to elucidate, on a molecular level, the cause of inherited retinal degeneration by determining how alterations in the proteins of the PE plasma membrane are involved in both primary and secondary effects of inherited retinal degeneration."}
{"text": "The recent release of the Surgeon General's Call to Action to Promote Sexual Health and Responsible Sexual Behavior evidences the continued interest and concern regarding sexual behavior and its outcomes, particularly among adolescents. Research and great deal of resources on the prevention of negative sexual outcomes such as unintended pregnancy and sexually transmitted infections (STI), with only mixed results. The Theory of Planned Behavior (TPB) has been used to study a wide range of health behaviors, including safer sex behaviors such as condom use. Comprised of various sociocognitive elements (attitudes, norms, and perceived behavioral control), the TPB postulates that these elements predict intentions to perform a behavior, and that these intentions, in turn, predict the actual behavior. Despite the proliferation of the TPB in studies of sexual behavior, there remain several compelling issues worthy of study. This research aims to explore four different issues relating to the TPB and adolescent sexual behavior: 1) The predictive value of past behavior has received some attention in the literature on the TPB, but the issue of whether or not past behavior should be included in the model remains unresolved; 2) In addition, a growing body of research on attitudes toward sexuality suggests that having a \"sex-positive\" attitude, one that endorses sexuality and sexual behaviors as positive aspects of one's life and personality, increases one's ability to advocate for themselves in the sexual arena, such as for the need for safer sex. This suggests a new course for preventive interventions for teens, and calls for further empirical consideration; 3) Third, although the TPB has many strengths, it fails to consider the influence of macrocontextual factors on behavior. Gender differences in social power, for example, may hinder the ability of an adolescent girl to use safer sex if her male partner is unwilling, regardless of her personal perceptions of behavioral control ; 4) Last, as advocated in the Surgeon General's report, it is necessary to consider physical, sexual, and mental health as interrelated rather than discrete domains, and to fully explore the connections among them. In order to accomplish these objectives, the TPB will be applied to data from the National Longitudinal Study of Adolescent Health (Add Health). Add Health's size, sample diversity, breadth of focus, and longitudinal design each enable researchers to tackle a wide range of issues, including those mentioned above. This proposed work strives to contribute to existing knowledge by exploring emerging areas of interest in the field of adolescent sexuality while still remaining grounded in a well-established theory of health behavior prediction."}
{"text": "Our long term goal is to develop immune therapies for disease caused by Human Papillomavirus (HPV). While the field of cancer immunotherapy has shown proof of principle in humans, namely, that established malignancies can be recognized and eradicated by a tumor-specific adaptive T cell response, reproducibly achieving this effect has been difficult. One reason may be that therapeutic vaccines tested to date have not been immunogenic enough to eliminate established disease. Another reason may be immunologic suppression directly mediated by developing and progressing tumors, which are operative in the lesion microenvironment. This project will test the hypothesis that HPV-specific adaptive T cell responses can be elicited by therapeutic vaccination in subjects with high grade cervical dysplasia (CIN3), and that the application of a topical TLR7 agonist, imiquimod, directly on the lesion, will enhance access of CD8+ T cells to the lesions. CIN3 lesions are relatively accessible, and some of them do regress within our 15-week study window. The disease provides rational, non-self antigenic targets for therapeutic vaccination. This project will test a heterologous DNA prime, recombinant vaccinia-based boost regimen to enhance the immune response against HPV16 E6 and E7, which are expressed in a functionally obligate manner in cervical cancer and its precursor lesion, high grade cervical dysplasia (CIN3). This project also provides the opportunity to analyze target lesions directly to determine if we can identify evidence of mechanisms that would mitigate the function of immune effector responses, namely, impairment of homing and function of immune cell subsets. In fact, we found that CD8+ T cells accumulate at CIN3 lesions, but in the case of persistent disease, fail to access lesional epithelium. While patterns of immune cell infiltration have been identified to predict clinical outcome in the clinical setting of invasive disease, to our knowledge, they have not been identified early in disease, before development of an overtly invasive phenotype. This work is an opportunity to study immune responses that are localized to incipient neoplasia, and immune homeostatic mechanisms which could be locally uncoupled to enhance both access and function of effector immune cells to eliminate disease."}
{"text": "Lead poisoning, particularly in children, remains a major health problem in the United States today. Nearly 19% of the children in a recent series of screening programs from 1969-1971 had blood levels of lead of 60 micron g/100 ml or higher, amounts that are generally accepted by physicians as being severe enough to require treatment. Lead intoxication is known to affect the erthropoietic system, producing anemia. One possible mechanism for the production of anemia is inhibition of heme biosynthesis in erythropoietic tissue. A dialyzable factor, identified as a polyglutamated folic acid derivative (pteroylpolyglutamate), has been discovered that protects against lead inhibition of uroporphyrigogen I synthetase, an enzymatic step of the heme biosynthesis in erythropoietic tissue. A dialyzable factor, identified as a polyglutamated folic acid derivative (pteroylpolyglutamate), has been discovered that protects against lead inhibition of uroporphyrinogen I synthetase, an enzymatic step of the heme biosynthetic pathway. This pteroylpolyglutamate has also been found to be an activator of the enzyme which indicates that such a pteridine derivative functions as a coenzyme for this enzymatic reaction. The objectives of the proposed research are to elucidate the role that pteroylpolyglutamates serve in the regulation of inhibition of uroporphyrinogen synthetase activity by lead and other inhibitors, to learn how drugs, hormones and pteridine derivatives regulate this enzyme and the synthesis of heme, to extend these studies to extrahepatic tissues, such as the brain, kidney, bone marrow and endocrine system, and to the neonate. The results of these investigations will provide valuable information on the mechanism of the conversion of porphobilinogen to uroporphyrinogen and the synthesis of heme, and its regulation by lead and other inhibitors. This information will contribute to our knowledge of lead and drug toxicity and its treatment."}
{"text": "Abstract. In the past two years, elevated HEF1 expression have been shown to induce glioblastoma invasion and lung cancer and melanoma metastasis in both animal models and in humans: our preliminary studies have now suggested an equally important role for HEF1 in breast cancer progression. Our studies of HEF1 since we first described this protein in 1996 have elucidated a complex function. HEF1 acts to assemble signaling complexes at focal adhesions in interphase cells, at the centrosome in pre-mitotic cells, and at the cilium in cells emerging from quiescence. Taken in sum, these data suggest that deregulation of HEF1 may represent a point of cellular vulnerability based on coordinate dysregulation of these discrete signaling complexes. If so, restriction of HEF1 action may offer a useful therapeutic approach. The goal of this proposal is to better understand the mechanisms by which the HEF1 scaffolding protein controls both cell cycle progression and cell attachment signaling. Our work in past cycles of RO1 CA63366 defined interactions of HEF1 with the oncoproteins Src and FAK as critical for HEF1 action in promoting cell migration and invasion. Subsequently, we defined HEF1 interactions with Aurora-A, Ajuba, and HDAC6 as essential for regulation of mitotic progression and ciliary disassembly. These two sets of signaling relationships have been considered in isolation. However, accumulating evidence suggests roles for Src in mitosis, and for Ajuba and HDAC6 in interphase cells. We propose that HEF1 is a core component of a signaling module that functions both in mitosis and interphase cells, and timed migration of HEF1 and its partners between intracellular compartments allows coordinated regulation of cell division and cell migration/invasion. We propose deregulated HEF1 expression most commonly marks later rather than earlier stages of cancer development because prior lesions inactivating mitotic checkpoints allow cells to tolerate HEF1 scaffolding defects. This proposal has 3 aims. In Aim 1, we will assess the factors regulating HEF1 movement from focal adhesions to centrosomes, allowing HEF1 activation of AurA at mitotic entry. We will determine whether Src and FAK contribute directly to this process. In Aim 2, we will define the role of Aurora A, Ajuba, and HDAC6 in allowing HEF1 to return to focal adhesions at cytokinesis, and the importance of interactions between HEF1, Ajuba, and HDAC6 in interphase for HEF1-dependent cell migration and invasion. In Aim 3, we will use murine knockout and transgenic models to study HEF1- dependent breast cancer development, and to analyze the relationship between deregulated HEF1 expression, genomic instability, and metastasis."}
{"text": "Multiple sclerosis (MS) is a chronic autoimmune disease characterized by the degradation of CNS myelin. The etiology of the disease is still unknown;both genetic and environmental factors have been implicated. Oligodendrocyte apoptosis may represent an initiating event in MS, with improper removal of apoptotic cell bodies and released myelin fueling the immune response. Low density lipoprotein receptor-related protein-1 (LRP-1) is a cellular receptor, which functions in endocytosis and cell signaling. We have demonstrated that LRP-1 is a key endocytic receptor for myelin in vitro. LRP-1 also inhibits the inflammatory response by controlling the NF?B pathway. We, therefore, hypothesize that LRP-1 could be directly involved in regulation of the development and/or progression of MS. We will test this hypothesis by a combination of in vivo and in vitro experiments. As complete LRP-1 deficiency is lethal, the role of LRP-1 in a mouse EAE model will be determined using mice with conditional LRP-1 deficiency in the myeloid lineage. Our preliminary results suggest that LRP-1 deficiency in this model leads to a significant increase in EAE symptoms. Effector functions of LRP-1- deficient macrophages will be characterized. Finally, we propose to identify LRP-1 ligands in myelin preparations, using a proteomics discovery approach. This research grant proposal should provide the basis for understanding the function of LRP-1 in MS and for potential development of novel therapeutics for MS. PUBLIC HEALTH RELEVANCE: Multiple sclerosis (MS) is a chronic disease characterized by the immune system attack on myelin sheath, which is necessary for proper brain function. This proposal is focused on a cell protein, LRP- 1, which we show is important in the clearance of degraded myelin, and controls of the immune response. We hypothesize that LRP-1 regulates the sequence of events leading to MS development. We aim to identify the mechanisms by which LRP-1 functions in MS, with potential of discovering ways leading towards development of novel treatments for MS patients."}
{"text": "Inactivation of p53 is the most common genetic alteration detected in human cancers. Underscoring the importance of p53 in preventing tumorigenesis, mice in which p53 has been disrupted invariably develop lymphomas and other cancers. The overall goal of this proposal is to expand our understanding of the p53 tumor suppressor pathway, including upstream effectors and down-stream mediators, and the mechanisms by which it prevents pediatric cancers. Individuals diagnosed with Li-Fraumeni Syndrome (LFS) generally carry a germline p53 mutation and are remarkably predisposed to cancer at an early age. The mutations are highly penetrant and are associated with brain, breast, bone and adrenal cancers. We have recently identified a group of unrelated pediatric patients with adrenocortical carcinoma (ACC) in which 35 or 36 patients have a identical germline p53 mutation (R337H). Analysis of intragenic polymorphisms eliminate a founder effect. Tumors deleted the wild-type allele and express high levels of mutant p53 in the nucleus, indicating that p53 is functionally inactive in vivo. However, these patients are not from cancer-prone LFS families and over-expression of 337H in cell culture-based assays fail to detect a functional defect. Our hypothesis is that 337H is functionally inactive under physiologic conditions and that this mutation contributes to th4e development of ACC through a loss of p53-mediated cell cycle arrest and/or apoptotic activities. To test this hypothesis, we propose to address the following specific questions: 1) Does the 337 H mutation disrupt p53 biological activity under physiological conditions?; 2) Does the 337H mutant have altered biochemical and biophysical properties?; 3) Does the 337H mutation contribute to tumorigenesis? These studies should establish a new class of p53 mutation, a low penetrant allele, that would previously go undetected but nevertheless, significantly contributes to the development of human cancers."}
{"text": "This goal of this proposal is to uncover the molecular mechanisms for sensing and regulating intracellular iron in the model eukaryote S. cerevisiae. To maintain optimal intracellular iron levels, iron transport and storage is tightly regulated in al eukaryotic cells ranging from yeast to humans. However, there are significant gaps in our understanding of iron regulation mechanisms at the cellular and molecular level. We will address these gaps by teasing out the molecular details of iron regulation in yeast and defining the roles of each component in the iron signaling pathway. In yeast, the monothiol glutaredoxins Grx3 and Grx4, the BolA- like protein Fra2, and the aminopeptidase P-like protein Fra1 function together in an iron-responsive signaling pathway that controls nucleocytoplasmic shuttling of the iron-responsive transcription factor Aft1. Under iron replete conditions, this pathway induces dimerization of Aft1 (and presumably its paralog Aft2), favoring their localization to the cytosol. We have demonstrated that Fra2 forms [2Fe-2S]2+-bridged heterodimers with Grx3 or Grx4 and characterized the Fe-S coordination chemistry of these complexes. In addition, we have strong evidence that [2Fe-2S] Fra2-Grx3 transfers a [2Fe-2S] cluster to Aft2, facilitating Aft2 dimerization. Aft1/2 dimerization, in turn, is proposed to inhibit activation of the iron regulon. Despite our significant progress in defining the molecular interactions between several components in this signaling pathway, some key aspects of the iron sensing and regulation mechanism remain unresolved and will be addressed in this proposal. We will uncover the mechanistic details of Fe-S transfer from Fra2-Grx3/4 to Aft1 and Aft2 and determine the impact of Fra1 on this process by using mutagenesis, biochemical analysis, and biophysical spectroscopy to examine the kinetics and efficiency of cluster transfer to Aft1 and Aft2 and identify residues in Grx3/Grx4/Fra1/Fra2/Aft1/Aft2 that are critical for both donor-target recognition and Fe-S transfer (Aim 1). We will test whether the Fra-Grx complex transfers an Fe-S cluster to Aft1/2 and induces dimerization in vivo by determining how mutations in Fra2, Grx3/4, or Fra1 affect protein-protein interactions within the iron signaling pathway, Fe binding to Aft1/2, and Aft1/2 subcellular localization and dimerization in vivo (Aim 2). Finally, we will elucidate the mechanism by which Fra-Grx mediated Aft1/2 dimerization inhibits activation of the iron regulon by testing if Fra-Grx-mediated dimerization of Aft1/2 disrupts movement of Aft1/2 to the nucleus, binding of Aft1/2 to its DNA targets, or recruitment of transcriptional co-activators using both in vivo and in vitro protein-protein and protein-DNA interaction assays (Aim 3). Since several key proteins in this pathway are conserved in humans and essential for viability, exploiting the yeast system to define their functional and physical interactions will provide a fundamental understanding of their roles in human iron metabolism. PUBLIC HEALTH RELEVANCE: Both iron deficiency and iron overload are significant human health issues: iron deficiency is the most common and widespread nutritional disorder in the world, while iron overload disorders are some of the most common genetic disorders in the U.S. This proposal is designed to define the sensing and control mechanisms for maintaining optimal intracellular iron levels using bakers' yeast as a model system."}
{"text": "Project Summary/Abstract Persistent HIV health disparities impacts African Americans and are a public health priority. Despite declining HIV rates nationally, African American communities continue to experience disproportionate HIV burden and increasingly account for the largest share of HIV-related morbidity and mortality in the United States. These concerning disparities necessitate research to inform robust responses that target priority subgroups within this population. High rates of incarceration in many African American communities contribute to high HIV risk environments, where the risks persist after release from the criminal justice system. Formerly incarcerated African American youth (FIAAY, ages 18-24 years) in particular are at heightened risk in terms of sexual behavior, illicit drug use, and challenged rates of HIV testing behavior after release. A prominent barrier to HIV testing and timely diagnosis in this and other vulnerable populations is HIV-related stigma. Thus, more research needs to not only examine stigma-related factors that reduce HIV testing behavior, but also create a basis for interventions that can reduce stigma and increase HIV testing and HIV preventive behaviors in priority populations, such as FIAAY. Towards these goals, my proposed study focusing on FIAAY in Louisville Kentucky will allow me to: 1) develop a risk profile of HIV-negative or HIV status unknown FIAAY at greater likelihood of having stigmatizing beliefs related to HIV infection and longitudinally examine the likelihood of HIV testing based on this profile and 2) delineate mediating and moderating mechanisms by which stigma reduces HIV testing in this population. These actions will help generate knowledge for the development of a multilevel intervention targeting individual and structural factors to reduce HIV-related stigma and increase HIV testing and prevention among this population of vulnerable youth. This Career Development Award will provide the necessary knowledge and skills to conduct this and future research with criminal justice-involved populations. This entails enhancing my skills in advanced statistical methods such as survival analysis and structural equation modeling. In addition it will foster content expertise in decision science and the criminal justice system, which will help inform intervention development. Study methods will include longitudinal survey data collection (baseline, 12-months, 24-months) from 274 FIAAY in Louisville, KY. This research will inform the development of stigma-related HIV-risk profiles for FIAAY who are less likely to test and delineate mediators and moderators of the stigma-HIV testing relationship. If a relationship between stigma and testing is not detected, other psychosocial factors will be examined to determine mediators and moderators of HIV testing. This study has important downstream benefits for HIV prevention and treatment in this highly vulnerable population."}
{"text": "Solving the structure with a view to understanding the function of membrane proteins remains one of the grand challenges in all of Biology. Given that a third of the human genome codes for membrane proteins, many of which serve signal transducing, structural and transport roles and are targets of disease causative and treatment agents, the health consequences of meeting the challenge are great. A multifaceted approach will be taken to advance our understanding of membrane protein function by producing structure grade crystals for use in macromolecular crystallography. Emphasis is placed on crystallization in lipidic mesophases by what is referred to as the 'in meso' or cubic phase method. This is proving to be a generally useful approach for membrane protein structure determination. With this method crystallization takes place in a membrane environment that is likely to be favored by the target membrane protein. We have built a unique, state-of-the-art robot that performs in meso crystallization in high-throughput fashion and that requires miniscule amounts of protein, lipid and precipitant. It will be used in the current application to produce 3-D crystals for the high-resolution structure determination of a select group of membrane proteins. The target group covers a broad range of membrane protein types including eukaryote and prokaryote, integral and peripheral, monomeric and multimeric, as well as protein-protein and protein-peptide complexes. Some of the target proteins will be produced in-house, some will be provided by individual collaborators, while others will be supplied through the NIH Structural Proteomics Initiative. [unreadable] [unreadable] In line with the NIH Structural Biology Road Map, and in parallel with the proposed structure study, effort will be devoted to improving crystallogenesis and to broadening the range of membrane protein targets that yield to structure determination. This will be achieved by implementing a synthesis program whereby lipids with desirable physico-chemical characteristics are produced for use in crystallization trials. The molecular mechanism of crystal nucleation and growth will be studied too with a view to more rational and successful crystallogenesis. Success in obtaining crystals, and ultimately the atomic structure of membrane proteins, will enhance our understanding of some of the most fundamental processes underlying cellular function that are integral to human health. [unreadable] [unreadable] [unreadable] [unreadable]"}
{"text": "Dietary restriction (DR) is the only experimental manipulation known to retard aging in mammals; however, the mechanism responsible for the life prolonging action of DR is unknown at the present time. One attractive explanation is that the effect of DR arises at least partially through changes in gene expression. Using the heat shock gene hsp70 as a model system to elucidate the mechanism by which aging and DR alter gene transcription, we have found that the changes in hsp70 transcription that occur with age and DR are correlated to changes in a specific transcription factor: HSF (heat shock transcription factor). In higher eukaryotes, HSF is found in non-stressed cells in an inactive (i.e., non- DNA binding) monomeric form in the cytoplasm. An increase in temperature, or other stress, results in the activation of HSF to an oligomer that binds the heat shock element (HSE) in the promoter of the hsp70 gene. We have shown that age and DR alter the activation of HSF. The objective of the research described in this proposal is to elucidate the molecular mechanism whereby aging and DR alter the activation of HSF. Specific Aims: 1. To determine if the changes in HSF activation are due to an alteration in HSF expression. The protein and mRNA levels of HSF will be measured as a function of age and DR. 2. To determine if the changes in HSF activation arise from alterations in the oligomerization of HSF. The oligomerization, phosphorylation, and translocation of HSF will be measured in cell extracts obtained from rats of various ages fed ad libitum and a DR diet. 3. To determine if the changes in HSF activation can be correlated to alterations in the physiochemical properties of HSF. The hydrodynamic properties of HSF, the affinity of HSF for the HSE, and the interaction of HSF with the hsp70 promoter will be studied as a function of age and DR. 4. To determine if the changes in the activation of HSF with age and DR arise from changes in the levels/activities of factors that inhibit the activation of HSF. A novel system has been designed for screening an expression cDNA library from the liver of old rats for the presence of cDNAs coding for inhibitors of HSF activation. 5. To demonstrate that HSF is responsible for the changes in the induction of hsp7o transcription. The ability of recombinant HSF to reverse the changes in the in vitro transcription of an hsp70-promoter/reporter template will be studied, and transgenic mice will be used to determine if the overexpression of HSF can reverse the age-related decline in hsp 70 transcription."}
{"text": "PROJECT SUMMARY/ABSTRACT The C9ORF72 GGGGCC repeat expansion associated with familial and sporadic amyotrophic lateral sclerosis (c9ALS), frontotemporal lobar degeneration with TDP-43 inclusions (c9FTD) and combined presentations of ALS with frontotemporal dementia (c9FTD/ALS) is the most common cause of familial ALS, ALS-FTD and FTD identified to date, and is reported in about 6% of sporadic ALS and FTD. The causes of phenotypic variability in c9ALS are not established. Wide ranging clinical expression of c9ALS is a challenge to elucidating the pathogenesis of the repeat expansion, as genotype-phenotype correlation calls for molecular genetic data and correlative clinical data in manifesting carriers of the repeat expansion and asymptomatic mutation carriers. Similarly, studies of disease pathogenesis using patient-derived biospecimens, including skin fibroblasts and assessment of postmortem neuropathology, require detailed clinical phenotypic data in order to associate biospecimen-derived data with clinical expression of disease. Studies in this P01 to elucidate the molecular pathogenesis of c9ALS will require ascertainment of patients with c9ALS, their extended family members who carry the C9ORF72 repeat expansion, and appropriate comparison groups with collection of clinical data, biospecimens and post mortem tissue. In order to identify symptomatic and asymptomatic C9ORF72 repeat expansion carriers for collection of longitudinal clinical data and biospecimens for Project 1, to provide clinical data and biospecimens of c9ALS and ALS patients with normal C9ORF72 repeat length for in vitro studies of disease mechanisms in Project 2, and to provide clinical data and post mortem tissue for investigation of the neuropathology of c9ALS in Project 3 the goals of Core B are: to identify for Project 1 at least 50 probands with c9ALS, and at least 10 ALS patients with normal C9ORF72 repeat length (c9(-)ALS) (Aim 1); through genealogic studies of c9ALS probands and c9(-)ALS patients ascertained in Aim 1 identify for Project 1 the following 3 categories of first, second and third degree blood relatives without symptoms of ALS or dementia: 1) at least 50 c9ALS relatives carrying the C9ORF72 repeat expansion; 2) at least 5 c9ALS blood relatives who do not carry the repeat expansion; and 3) at least 5 blood relatives of c9(-)ALS patients (Aim 2); collect as called for in Project 2 single time point biospecimens including skin tissue from 1) at least 10 patients with c9ALS and 2) at least 10 c9(-)ALS patients ascertained through Aim 1 (Aim 3); and, offer all study subjects participation in our deeded autopsy program, and for those who die during the course of the project, conduct autopsies to harvest brain, spinal cord and other tissues in support of Projects 1 and 3."}
{"text": "We will investigate the factors regulating the subcellular distribution of the high conductance, calcium- and voltage-gated BK channel, a critical ion channel that regulates neuronal firing output in health and disease. Despite its powerful role in modulating excitability, experimental evidence indicates that it is sparsely distributed at the plasma membrane, a phenomenon that is regulated via interactions with the brain-specific accessory subunit, ?4. Conventional methods to study membrane protein localization have relied heavily upon overexpression of tagged proteins, a method that can significantly alter protein distribution by changing the stoichiometry of the target with its regulatory factors. To accurately determine how BK channels are distributed across the cell, it is important to be able to determine the location of individual molecules at endogenous expression levels to preserve critical concentration-dependent interactions with regulatory partners. We have developed a novel protein/dye tag with high-fluorescence emission that enables single-molecule detection, for both high- and low-abundance proteins. To preserve normal channel expression levels, we will generate a transgenic mouse where this tag has been inserted into the endogenous BK channel gene. The localization of this channel in primary neurons derived from these animals will be evaluated, and its accessory subunit and activity-regulated surface distribution will be determined."}
{"text": "The relationship between aging and carcinogenesis was explored by transforming cultured cells derived from various sources with oncogenes. To simulate the in vivo state, we prepared primary culture cells from lung and skin tissues. Young and old rats were mainly used as donors. For in vitro studies, fibroblasts from human fetal lung and rat whole embryos were passaged to appropriate population doublings. Cells were transfected with the calcium phosphate precipitated oncogene DNA, then subjected to a focus assay. The number of foci, which indicate the number of transformed cells, were examined to determine if older cells respond differently to the transfected oncogenes than younger cells. Our results indicate that cellular aging alters the intrinsic susceptibility to oncogene induced neoplastic changes at the cellular level."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. As part of its dissemination strategy, BioCAT takes part in any facility wide workshops that are organized by the APS and other institutions that covers techniques supported by BioCAT. The main purpose of the National School on Neutron and X-ray Scattering is to educate graduate students on the utilization of major neutron and x-ray facilities. Lectures, presented by researchers from academia, industry, and national laboratories, will include basic tutorials on the principles of scattering theory and the characteristics of the sources, as well as seminars on the application of scattering methods to a variety of scientific subjects. As part of this activity Prof. Irving gave a lecture on non-crystalline diffraction in the formal sessions. As part of the school students conducted six short experiments at Argonne's Advanced Photon Source and Oak Ridge's Spallation Neutron Source and High Flux Isotope Reactor facilities to provide hands-on experience for using neutron and synchrotron sources. As part of this activity BioCAT hosted 14 graduate students to provide training in small angle x-ray scattering techniques. The admission process to the school is highly selective and so we view this as a highly effective way of reaching the next generation of researchers."}
{"text": "More than 200,000 patients are treated every year for peripheral nerve injury. Approximately 90% of patients never recover full movement of an injured limb. Slow axon regeneration and/or misdirected axon targeting are most often blamed for these poor functional outcomes. Increased neuronal activity, such as through exercise or electrical stimulation, enhances axon regeneration following peripheral nerve injury. We have shown that brain derived neurotrophic factor (BDNF) expression in the neurons whose axons are regenerating is required for this enhancement, but also that signaling through androgen receptors is required in both males and females. The source of the ligands for these receptors, androgens, is not known, especially in females. We hypothesize that their availability could be altered by the effects of neuronal activity on steroid converting enzymes. By measuring the expression of these enzymes in both neuronal and non-neuronal cells in response to increased neuronal activity, we anticipate that we can identify the sources of androgen that promote enhanced axon regeneration. The targets of androgenic activity required to promote axon regeneration also are poorly known. By evaluating the effects of increased neuronal activity on axon regeneration in mice in which the androgen receptor gene is knocked out in a cell-type specific manner, we will be able to identify the cellular requirements for androgen receptor-mediated enhancement of axon regeneration. In this proposed study, we will use a combination of newly available technologies to investigate these roles. Clinically, androgen therapy is already in use. Knowledge of androgen production and signaling is needed to tailor this potential therapy for peripheral nerve regeneration and could be applied to other neurodegenerative diseases."}
{"text": "Schizophrenia is currently thought to be a complex genetic disorder, with altered neuronal development, perhaps very early in life, important in determining vulnerability. Neurogenesis and neurodevelopment occur in the adult olfactory epithelium, and probes of the olfactory system may provide insights into the neuro-biological basis of altered neurodevelopment in this disorder. Indeed, studies of patients with schizophrenia have demonstrated robust behavioral, structural, and functional impairments of the olfactory system. Our work, along with others', has indicated that olfactory brain regions are affected by both neurodegenerative and genetically-mediated neurodevelopmental processes. This project represents the only systematic effort to examine the underpinnings of these chemosensory impairments from a life-span perspective. To date, our efforts have established that: 1)pervasive and stable olfactory deficits exist across the lifespan in schizophrenia;2)no significant medication effects are seen;3)similar psychophysical, structural, and functional deficits exist in first-degree relatives of patients;and 4)underlying facial structures such as nasal and palate volumes are abnormal. Over early fetal life, cerebral morphogenesis proceeds in embryological intimacy with craniofacial morphogenesis. As such, quantitative analysis of craniofacial and cerebral dysmorphology along with a detailed psychophysical assessment of the olfactory system may provide important information concerning the developmental origins of schizophrenia. In this application, we propose an in-depth assessment of the structural and functional abnormalities of the olfactory system from an early neurodevelopmental perspective. We will study 40 schizophrenia patients, 40 otherwise healthy first-degree family members, 40 unrelated healthy controls, and 40 high-risk subjects who present with early symptoms of psychosis. New methods will be utilized to examine the neurodevelopmental contributions to chemosensory dysfunction in patients including nasal/palate volume, structural MRI, and quantitative examination of facial morphology. Predictive utility of these impairments will be probed by examining subjects at increased risk for the development of psychosis. Our working model is that olfactory impairment reflects developmentally disturbed processes of neurogenesis and synapse formation and that these measures may represent specific markers of embryological dysmorphogenesis underlying schizophrenia. Schizophrenia is currently thought to be a complex genetic disorder, with early developmental abnormalities in brain structure and function, being important in determining vulnerability to illness. This study will examine changes in smell abilities in schizophrenia along with detailed measurements of the nasal and oral cavities and mapping of facial and brain structure and topography. We believe that robust olfactory impairment seen in patients reflects a disturbance of an early developmental process and that the analysis of these functions and structures may provide important information concerning the developmental origins of schizophrenia."}
{"text": "Clinical-grade T cells rendered specific for CD19 have demonstrated anti-tumor activity. We are now proposing a translational study to investigate the temporal-spatial biodistribution and microenvironment associated with adoptively transferred CD19-specific T cells as achieved using positron emission tomography (PET). To target aggressive B-cell malignancies, we have initiated two clinical trials to infuse autologous and allogeneic T cells that have been genetically modified to express a CD19-specific chimeric antigen receptor (CAR) which recognizes CD19 on the cell surface, independent of MHC. This new R01 grant application establishes an inter-disciplinary (chemistry, imaging, biostatistics, bioinformatics, nuclear medicine, gene therapy, and immunology) and multi-institution (MDACC and TMH) team, partnering with industry (CellSight Technologies, Inc.) to investigate a platform for imaging infused CAR+ T cells by PET. This will be accomplished by coexpressing a mutant of herpes simplex virus-1 thymidine kinase (sr39tk) with the CD19-specific CAR in T cells using the Sleeping Beauty (SB) transposon/transposase system which we have adapted for clinical translation. We will synchronously electro-transfer two DNA plasmids expressing the SB transposons (i) CAR and (ii) sr39tk, using a new method we dub ?double transposition?. Aim #1 seeks to determine if non-viral gene transfer will produce T cells that co-express CD19-specific CAR and sr39tk under control of constitutive and conditional promoters. The sr39tk reporter gene will be fused to hygromycin phosphotransferase (Hy) and thus CAR+sr39tk+ T cells will be selectively propagated in presence of cytocidal concentration of hygromycin B on &#947;- irradiated artificial antigen presenting cells that co-express CD19 along with desired T-cell co-stimulatory molecules. Aim #2 seeks to undertake longitudinal &#956;PET imaging of infused human CAR+sr39tk+ T cells with the reporter probe [18F]FHBG in immunocompromised mice to assess biodistribution and sensitivity of detection. T-cell activation status will be imaged by comparing (i) conditional expression of sr39tk under control of NFAT promoter with (ii) the new PET probe [18F]F-AraG developed at CellSight. T-cell hypoxia will be assessed by introducing a molecular sensor for oxygen to test whether sr39tk can report low oxygen tension. Aim #3 seeks to translate these pre-clinical data to a new clinical study infusing CAR+sr39tk+ T cells in patients undergoing gene therapy with CD19-specific T cells. This trial will be a companion study to our existing trial (IND# 14193) infusing CAR+ T cells after autologous hematopoietic stem-cell transplantation for research participants with advanced B-lymphoid malignancies. The PET probe [18F]FHBG, marketed by CellSight Technologies, will be manufactured for clinical imaging at TMH, per IND #61880. In aggregate, these studies will test the central hypothesis that CD19-specific CAR+sr39tk+ T cells can be imaged in humans using PET. These studies will establish principles and practices for translating PET-based imaging of CAR+ T cells and provide the first human imaging data on the biodistribution of genetically modified T cells."}
{"text": "Research Project 1 Summary HIV infected individuals have heightened cancer risk. Particularly for those caused by human oncogenic viruses such as EBV, HPV and KSHV. AIDS Malignancies are a sanitary problem for Latin American Countries as Argentina, even in the post HAART era. There is scant information regarding cancer among HIV-infected individuals in Argentina. Moreover, translational research in AIDS malignancies is an area traditionally neglected where most Argentinean clinicians and oncologists rarely collaborate. The recent convergence of efforts of Dr. Mesri laboratory at University of Miami Miller School of Medicine's Sylvester Comprehensive Cancer Center (UMMSM/SCCC), Dr.Omar Coso at IFIBYNE ? UBA ? CONICET and Dr. Pedro Cahn from Fundacin Husped the most prestigious ONG devoted to AIDS studies, diagnosis and prevention have converged in this consortium proposal under the U54 format, including other Senior Prestigious Researchers and Junior Scientists in every Consortium Component. Project 1 aims to ?Define oncogenic signaling networks in AIDS-associated viral cancers as targets of chemoprevention and treatment.? The proposal will make use a variety of strategies at the molecular, cellular level and even through animal models to identify molecules (mainly proteins and miRNAs) implicated in the onset of viral cancers and investigate its relationship with HIV infection, using different approaches and targeting different molecules. Putative Molecules identified as responsible for the onset of cancer by laboratory approaches will be studied in Human Samples with the aid of Project 2. Relationship with the OGB Core will allow enhancement of the research capabilities a significant step further by using unbiased genomic analysis, as well as IHC techniques, to obtain high throughput data that will validate data obtained in the laboratory and prompt new questions into the bench making possible to address research questions that were unimaginable only a few years back. Mentoring is of paramount importance to this project. Dr Coso will lead the studies in KS related molecular signaling working as a PI to Project 1. On the other hand and combining with his role in the CE Core, he will supervise Dr Ana Raimondi, exploring the possibility of targeting mTORC1 in HPV models. Dr. Coso will also team with his former trainee Dr Julian Naipauer, who has developed a new cell and animal model of KSHV oncogenesis, conducting experiments in the laboratory and mentoring for the future development of his scientific career as a viral oncologist upon return to Argentina."}
{"text": "The Scientific Review Committees (SRC) provide thorough scientific reviews of all protocols to be activated at the Moffitt Cancer Center. Support of the SRC process is the primary focus of the staff within the Protocol Review and Monitoring (OPRM). Specific goals of the PRMS are: ? Appropriate and thorough scientific and statistical review of Moffitt protocols ? Efficient processing and accurate tracking of Moffitt clinical research protocols, and ? Monitoring of scientific progress for ongoing clinical research trials. OPRM support staff process the documents necessary for the review committees and facilitate timely communication of SRC actions between the committee and investigators. All correspondence is saved electronically and noted within the research database (Oncore?). Support staff members also assist investigators in the development of protocol-specific data and safety monitoring plans. The OPRM office maintains an accurate protocol-tracking database for use by the regulatory staff, principal investigators, study teams, and committee members to monitor protocol progress through the review processes, including SRC and IRB review, budgeting, and contracting as appropriate, and an operational review process to allow ancillary departments to review and sign off on protocol feasibility. Other OPRM staff members assist Moffitt investigators with efficient, accurate review and processing of research protocols and submission to the IRB for human subject approval and work with monitors in the data and safety monitoring process. Moffitt has committees that perform scientific review, each meeting monthly. Two committees review clinical research proposals, while the third committee reviews tissue and data studies. Meeting dates are staggered to allow for SRC meetings approximately every 10 days. The three SRCs provide the initial scientific peer review of the protocol and monitor these protocols throughout the term of the study for scientific progress (e.g., meeting accrual goals) and for changes that might impact the objectives of the study. The PRMS requests CCSG Support of $247,671, which is 28% of its operational budget."}
{"text": "The Cancer and Stem Cell Biology (CSCB) Program Area was established in 2010, in response to increasing evidence that the concepts and tools of stem cell biology can be used to gain new insights into cancer biology, and to take advantage of the tremendous expansion and productivity of the stem cell research community at UCLA. The primary goal of the CSCB Program Area is to link basic and translational investigators interested in the unique biological processes shared by malignancy and stem cells. The fundamental biology that characterizes these two fields can be viewed as the control of the opposing processes of self-renewal and differentiation. The balance of these processes is, in turn, determined by cell intrinsic and micro-environmental cues. These common mechanisms are studied by CSCB investigators using normal stem cells and their malignant counterparts from two experimental platforms: Hematopoiesis and Epithelium. Similarly, clinical research projects in CSCB focus predominantly on hematologic and genitourinary malignancies. Scientific interactions between the two platforms are driven by three main integrating themes that are relevant to the study of malignancy and stem cell biology irrespective of the specific tissue/cell source: 1) the regulation of growth and differentiation in tissue-specific stem cells during malignant transformation and normal development; 2) the role of microenvironment in tumor formation and stem cell regulation; 3) the use of pluripotent stem cells as a model to study basic mechanisms of self-renewal and transformation. The CSCB Program Area is comprised of 31 members, representing three schools and twelve departments; six members are"}
{"text": "We propose to carry out the detailed structure analysis of selected nucleotides, nucleotide analogs and modified nucleotides of transfer RNA to obtain further support for the established rules of the stereochemistry of nucleic acid constituents as well as gain additional insights into the conformational perturbations on nucleotides wrought by base and sugar modifications. The strain energy incurred by a nucleotide when it assumes the unfavorable conformations will be studied by obtaining precise information on bond distances and bond angles of cyclic nucleotides. Structural studies on metal salts of nucleotides typically contain a large number of water molecules. Hence, these studies will be aimed at providing information on the preferred binding sites and coordination schemes of the metals with the nucleotide base, sugar and phosphate groups as well as the role of water molecules on nucleotide conformations. Our continued studies of the nucleotide antibiotics is beginning to disclose some of the relevant structural features for antibiotic action. Whenever possible we focus our attention on studies of oligonucleotide structures. These studies which are being performed in conjunction with theoretical potential energy calculations are designed to uncover the broad as well as the intricate rules that govern the structure of nucleic acids. BIBLIOGRAPHIC REFERENCES: Nygjerd, G., McAlister, J., Sundaralingam, M. and Matsuura, S., \"The Crystal and Molecular Structure of the Fluorescent Base Yt (1H-4, 6-Dimethyl-Imidazo (1,2-a) Purine-9-One) in tRNA.\" Acta Cryst., B31, 413 (1975). Prusiner, P. and Sundaralingam, M., \"Molecular and Crystal Structure of the Modified Nucleoside 2' -O-Methyladenosine. A Novel 2'-exo-3'-exo (2T3) Sugar Pucker.\" Acta Cryst., in press (1975)."}
{"text": "ABSTRACT Chronic neuroinflammation of the olfactory epithelium, as occurs in chronic rhinosinusitis (CRS), results in the loss of the sense of smell, due in part to cytokine-mediated neuronal death and inhibition of olfactory regeneration. Within the scope of the parent grant, the exploration of the effect of chronic neuroinflammation on the olfactory system extends to the axonal projections of the olfactory epithelium to the olfactory bulb (OB). In the brain, including the OB, neuro-immune interactions are increasingly believed to play an important role in the pathogenesis of Alzheimer's disease (AD) and its related dementias. Neuroinflammation is a prominent feature of AD that is highly correlated with disease escalation. The precise etiologic relationship between inflammation and AD histopathology is uncertain, but recent evidence increasingly suggests that systemic inflammatory disease is a risk factor and exacerbator of ADRD. The OB, which is an early site of AD histopathologic change, is connected and in close proximity to the olfactory epithelium. Directly exposed to the outside environment, the delicate olfactory neuroepithelium is subject to microbial exposures and local immune responses. In chronic rhinosinusitis, the olfactory epithelium becomes infiltrated with inflammatory cells producing a variety of pro-inflammatory mediators. A case-control study has linked dementia with a history of chronic rhinosinusitis, suggesting that longstanding nasal inflammation may propagate effects to the brain. The major goal of this project is to use a mouse genetic model of chronic olfactory epithelial inflammation to investigate whether neuroinflammation spreads to the OB over time, either driving deposition of amyloid plaques and neurofibrillary tangles, or exacerbating disease histopathology in mouse AD models. We further plan to investigate the role of the MAP kinase JNK in AD pathophysiology, which is an inflammatory signaling mediator that we have been studying as a therapeutic target for olfactory neuroprotection in CRS. Using genetically-modified mouse strains and pharmacologic agents, we will explore the roles of specific cytokines implicated in ADRD in mediating olfactory inflammation-induced AD-like changes in the OB, and whether these can be modulated by inhibiting JNK activity. The experiments described in this proposal will afford new insights into neuroinflammatory mechanisms that may trigger early stages of ADRD."}
{"text": "Project Summary In the past decades, extensive research has aimed to identify risk factors, causative agent, treatments and preventive strategies for Alzheimer's disease (AD), the most common type of dementia. These efforts have predominantly focused on proximate behavioral and biological factors, but the upstream social determinants of cognitive decline and AD and related dementias (ADRD) are not well-understood. The overall objective of this proposed project is to ascertain how marriage, an important but overlooked social risk/protective factor, is linked to trajectories of cognitive decline and risk of developing dementia in late life. Marriage has long been identified as the most important type of social relationships that holds the greatest significance for health during adulthood. However, surprisingly, scientists know little about whether and how marriage influences cognitive decline and progression to ADRD. Rapid social changes in marriage, family formation, and family dissolution mean that an increasing number of older Americans are entering late life with complex marital histories, which reshapes key aspects of their lives, and may in turn affect cognitive function over the life course. We hypothesize that the complex life-course traits of marital relationships such as current marital status, histories of marital dissolutions, and marital strain, will influence trajectories of cognitive decline and dementia risk over one?s life course via multifaceted economic, psychosocial, behavioral, and biomedical pathways. This project breaks new ground with an innovative interdisciplinary life-course research design that conjoins data from three NIH-sponsored, national, longitudinal datasets: (1) Health and Retirement Study (HRS), (2) National Health and Aging Trends Study (NHATS), and (3) National Social Life, Health and Aging Project (NSHAP) to examine a full life course picture of marital relationships including marital status, marital biography and marital quality linked to cognitive trajectories and dementia. We will apply advanced statistical methods to systematically examine economic, psychosocial, behavioral and biomedical mechanisms through which marriage influences cognitive trajectories and dementia risk over the life course, and further assess potential gender differences in the processes. The interdisciplinary research team is characterized by a complementary set of talents and experience in population health, cognitive aging, marriage and social relationships, neurology, neuropsychology, bio-demography, epidemiology and quantitative methodologies that uniquely position them to carry out this innovative project."}
{"text": "While HIV/AIDS can be managed with antiretroviral drugs, these agents do not clear the virus and require life-long administration. Recently, we discovered a completely new class of compounds that interfere with the HIV-1 virulence factor called Nef. This viral protein is critical to HIV-1 replication in vivo, immune escape of HIV-infected cells, and AIDS progression. During the past year, we successfully completed Phase I of our STTR project aimed at development of Nef antagonists suitable for clinical testing. Working with the Fox Chase Chemical Diversity Center, we evaluated 50 analogs of our original diphenylpyrazolodiazene Nef inhibitor, some of which display tighter Nef binding while retaining potent antiretroviral activity and improved ADME properties. Several analogs prevent Nef-mediated downregulation of MHC-I on HIV-infected CD4+ T-cells, resulting in activation of autologous anti-HIV CD8+ CTLs. These data suggest that our Nef antagonists may restore recognition of HIV-infected cells by the patient's own immune system as a path to functional cure. In this Phase II application, we will expand our Nef drug development efforts with the following Specific Aims: Aim 1: Perform lead optimization medicinal chemistry. Based on SAR developed during Phase I, we propose to synthesize 100-150 new Nef inhibitor analogs to find suitable compounds for in vivo testing in Aim 3 using HIV-infected humanized mice. Our approach will employ structure-based design while considering analog ADMET and PK properties in parallel. All analogs will also be tested for Nef binding affinity, effects on Nef-mediated enhancement of HIV replication and reversal of CD4 and MHC-I downregulation by Nef in HIV-infected patient cells. Aim 2: Ensure suitable drug properties via in vitro and in vivo ADMET evaluation. Up to 15 compounds per year that meet Nef-binding and functional criteria (Aim 1) will be evaluated using in vitro ADMET assays including microsomal stability, CYP 3A4 inhibition, solubility and plasma protein binding. Three to six (IV) and 2-3 (PO) compounds will be evaluated in mouse PK studies, with the 2-3 most promising compounds advancing to in vitro non-GLP safety assays. These data are essential for choosing the best compounds for in vivo testing in humanized mouse models of HIV/AIDS (Aim 3). Aim 3: Test the hypothesis that Nef antagonists can suppress HIV replication and T-cell loss in a humanized mouse model of AIDS. Humanized mice infected with Nef-deleted HIV-1 exhibit dramatically lower viral loads and substantially less T cell depletion than those infected with wild-type virus. The most promising compounds identified in the first two Aims will therefore be administered to humanized mice to monitor effects on HIV replication, CD4+ T cell loss and immune system function. Successful completion of these goals will provide a comprehensive package to support advanced development, safety testing in large animals and advance the project further toward an IND submission for Phase I safety testing in normal volunteers."}
{"text": "Patients with B-cell malignancies [non Hodgkin lymphoma (B-NHL), B-chronic lymphocytic leukemia (B-CLL) and acute lymphoblastic leukemia (B-ALL)] respond to T cells redirected with chimeric antigen receptors (CARs) specific for CD20 or CD19 antigens, and encoding costimulatory endodomains. Targeting these antigens, however, does not distinguish between normal and malignant B cells, so that this approach, when effective, causes profound B-cell aplasia. It is therefore important to identify targets expressed more selectively by B-cell malignancies. B-NHL and B-CLL cells express monoclonal immunoglobulins (Igs) that contain either ?- or ?-light chains. As a proof of principle, we hav implemented a phase I clinical trial in which patients with relapsed/refractory ?+ B-cell malignancies are infused with autologous T-cell products engineered to express a CAR that targets the ?-light of human Igs, and also contains the CD28 costimulatory endodomain (CAR.?.CD28TM.CD28). This CAR would target normal and malignant ?+ cells, but spares the subset of normal B cells that express the ?-light chain. This study is currently ongoing, and it enrolled 10 patients. Two patients achieved complete response and 4 patients experienced disease stabilization. Although promising, this study also shows some limitations such as the suboptimal in vivo expansion/persistence of these cells. We discovered a specific role of the CD8? stalk that when incorporated in CARs expressing either CD28 or 4-1BB signaling domains dramatically enhance their antitumor effects. Our central hypothesis is that the inclusion of the CD8??stalk in the CAR.? (CAR.?.CD8?.CD28), rather than CD28 transmembrane domain and signaling domains, will enhance the expansion and persistence of CAR-T cells in vivo and promote higher rate of clinical responses. In the proposed phase I study we will address the mechanistic role of the CD8? stalk in CAR signaling, and then conduct a phase I clinical study in which each patient will receive two T-cell products expressing either the current CAR.?.CD28TM.CD28 or the new CAR.?.CD8?.CD28 allowing us to clearly evaluate the expansion/persistence of each T-cell subset within the same patient. On completion of this first-in-man study we will know whether this novel CAR can substitute the CD19-specific CAR currently used for mature B-cell malignancies. We will also develop preclinically the specific CAR that targets the ?-chain in order to implement a strategy that covers the great majority of patients with B-cell mature malignancies."}
{"text": "Human granulocytic anaplasmosis (HGA) is an emerging tick-borne disease caused by Anaplasma phagocytophilum, an obligate intracellular bacterium of neutrophils. A. phagocytophilum infection impairs neutrophil function by transcriptional reprogramming, where the reprogrammed neutrophil promotes inflammatory recruitment of new neutrophils, tissue injury, ineffective regulation of inflammation, and poor antimicrobial responses. We studied altered neutrophil function with A. phagocytophilum infection and focused on how the nuclear effector protein AnkA, when delivered into the host cell where it binds to promoters of genes regulated with infection, induces epigenetic chromatin remodeling and transcriptional reprogramming. The granulocyte transcriptome with A. phagocytophilum infection shows a number of differentially transcribed genes that promote infection [3-6]. Given the meager genomic resources of A. phagocytophilum, it is difficult to explain the extent of host transcriptional change and functional reprogramming by individual translocated effector proteins. This implies that the bacterium exerts influence over global gene transcription, including chromatin and histone remodeling, perhaps by targeting conserved mechanisms of transcriptional regulation such as in cellular differentiation and neoplasia. AnkA has properties that suggest function as a matrix attachment region-binding protein that could regulate access of chromosomal territories to transcriptional modifiers, a new paradigm in bacteria-host interactions. We hypothesize that AnkA binds to promoters of some transcriptionally regulated genes and modifies or recruits modifiers of epigenetic chromatin marks or transcription factors. In addition, we hypothesize that A. phagocytophilum reprograms the global neutrophil transcriptome by altering the epigenome through AnkAs action on nuclear matrix, chromatin, and transcriptional apparatus recruitment. We propose the following aims: 1. To identify AnkA binding sites in the CYBB promoter and to define AnkA domains or motifs involved in CYBB promoter binding and transcriptional activity. 2. To determine whether AnkA affects host gene transcription through direct action at the CYBB promoter or through recruitment of chromatin remodeling or transcription factors. 3. To determine whether AnkA functions as a matrix attachment region-binding protein that tethers DNA to nuclear matrix, regulates DNA loopscape, and permits docking of other chromatin modifiers in global transcriptional regulation. The effects that bacteria have over cellular transcription are increasingly recognized. Testing these hypotheses will provide evidence of a potentially powerful mechanism for prokaryotic control over eukaryotes. The long- term goals are to develop a mechanistic understanding of how bacteria with intimate host cell associations circumvent host functions. This information could allow rational preventions and therapies for HGA, but could also span biology and medicine, since such molecules could be engineered as epigenetic tools or therapies. PUBLIC HEALTH RELEVANCE: Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by an intracellular bacterium, Anaplasma phagocytophilum that must live within neutrophils and similar cells. Interestingly, neutrophils are the major early host defense cells, and A. phagocytophilum alters their function to benefit survival of the bacteria. The altered function occurs mostly because the mRNA-producing machinery of the cell is altered by infection with this bacterium. We learned that one protein made by the bacterium, AnkA, is transported to the neutrophil's nucleus where mRNA is made, and its presence there changes how mRNA is made. In fact, the magnitude of changes in mRNA made by the cell are very difficult to explain based on AnkA altering mRNA made from individual genes. We propose to study the exact way that AnkA changes mRNA production from single genes, and to study whether it also affects the structure and function of chromosomes in a way that mRNA production is drastically altered. This information could provide evidence of an entirely new way for bacteria to control host cells, could define some aspects of normal cell function, and might provide new tools, even new drugs, for studying cells and their function in health and disease."}
{"text": "Huntington's Disease (HD) is an autosomal dominant neurodegenerative disorder characterized by chorea, dementia and personality disorder affecting between 5 to 10 persons per 100,000 in the U.S. Although the mean age of onset of HD is about 40 years of age, manifestations of the disease may occur as early as 2 and later than 80 years of age. There is no effective treatment for HD which is progressive and follows an inexorable course leading first to total debilitation and then death some 15 to 20 years after onset. Typically the HD patient requires several years of full time nursing care and the disease can represent an extraordinary psychological and financial burden for both their family and society as a whole. The Huntington's Disease Center Without Walls is a basic research center that was established in 1980 with clearly stated goals: the discovery of the nature of the genetic defect causing HD, the elucidation of the mechanisms whereby this defect acts to produce the clinical and pathologic features of the disorder, and the development of effective approaches to its treatment. The Center is multidisciplinary, encompassing in a single cooperative program a broad range of research approaches including genetics, epidemiology, biochemistry, and neuroanatomy. In the past grant cycle we accomplished the goal of identifying the HD mutation. The disorder is caused by an expanded, unstable CAG trinucleotide repeat in a gene encoding a large novel protein named huntington. In this renewal period we intend to study the dynamic behavior of the HD CAG repeat, its clinical correlates, factors that modify these parameters, the origin of repeat instability, the connection between CDA expansion and impairment of energy metabolism, the consequences of CAG expansion on neural development and anatomy, on structure, expression, localization and interactions of huntingtin protein and on metabolism of cultured neuronal cells. The HD Center comprises a team of highly interactive investigators with a unique combination of talents and considerable experience and dedication to HD. We are confident that the next 5 years will see tremendous strides toward achieving our goals of understanding the nature and consequences of the genetic defect, and of being able to provide an effective treatment for this devastating disorder."}
{"text": "Salmonella are one of the most important causes of gastrointestinal infections. The overall goal of our research is to understand how the innate immune response helps to protect against salmonella infections. The first aspect of innate immunity that we will study is PMNs. We previously reported that human epithelial cells make the potent chemotactic peptide IL-8 when invaded by salmonella, which implies that PMNs are important for the host defense against this pathogen. We then showed that PMNs are crucial for defense against salmonella that have virulence plasmids, but not those without virulence plasmids. This proposal focuses on the role of PMNs in protecting the intestine from salmonella infections, using a model of oral infection in mice. The role of PMNs will be established by making mice neutropenic with a monoclonal antibody (RB6-8C5) to the Ly-6G antigen that is specific for myelocytes. We will look for evidence that one or both chemokines are made in vivo in infected mice and in vitro using both cultured murine intestinal epithelial cells and colon organ culture. The second component of the innate immune response that we will investigate is lipopolysaccharide binding protein (LBP). We have confirmed the report by Jack et al (Nature, 389,742,1997) that LBP-deficient mice are more susceptible to salmonella infections, but the mechanism of action of LBP has not been elucidated in infection. We used mice that had the wild-type Nramp1 allele so are genetically resistant to salmonella infections. LBP deficient mice had lower levels of 2 ELR+ CXC chemokines. We will now study the role of CXC chemokines in host resistance to Salmonella by blocking the CXC receptor with antibody. The third goal is to determine whether complement mediated phagocytosis is critical for early resistance to salmonella infections. We will study the course of infection in mice that are deficient in C3 and determine whether neutropenia makes them more susceptible to infection. We will compare isogenic strains of salmonella that express either the group B or 0 LPS antigens in C3 deficient and neutropenic mice, in order to determine how the structure of the LPS affects virulence in mice."}
{"text": "The value of this study of normal and abnormal embryology as an experimental approach to determine factors leading to regeneration of flexor synovial tendons has been demonstrated in the past granting period. The failure of synovial differentiation in paralyzed chick embryos leads to the choice of early motion as an inducer of regeneration to be studied in adult chick tendons in this grant. Suturing techniques to allow early motion as well as the effects of early motion will be evaluated in animal flexor digital tendons by light and electron microscopy, microvascular studies and biomechanical testing. Both an Instron testing machine and an arthrograph will be utilized for the mechanical studies. As well as the above applied studies, basic investigations will be continued in chick embryos and tendon organ culture. In addition, collagense activity in injured tendons and organ culture will be investigated as an additional modifiable factor in tendon healing. This enzyme activity will be determined by quantitative assay and localized in tissue by immuno assay."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Sub-project 4 description Obesity is associated with an increased risk of cardiovascular death. One potential contributing factor in obesity-associated cardiovascular deaths may be related to the pro-thrombotic and pro-inflammatory states induced by increases in adipose mass, both of which are critical components of the pathogenesis of the clinic manifestations of atherosclerosis. Platelets play a central role in arterial thrombosis, are activated in inflammatory states, and are directly influenced by specific adipokines, and therefore have the potential to serve as an essential mediator of the cardiovascular consequences of obesity. alpha-Granules are essential to normal platelet activity. Many inflammatory factors and cytokines are stored in [unreadable]-Granules. Activated, but not resting platelets are able to alter the chemotactic properties of endothelial cells by inducing the secretion of monocyte chemoattractant protein (MCP-1). Similarly, transforming growth factor-beta (TGF-beta) is released from activated platelet alpha-granules and has been shown to augment the release of type-1 plasminogen activator inhibitor (PAI-1) from adipose tissue. Therefore, platelet secretion may play an important role in the pro-inflammatory and pro-thrombotic consequences of diet-induced obesity. The cGMP/PKG pathway plays a critical role in platelet secretion. In addition, we have recently identified a role for Src family kinses, especially the Lyn kinase, in platelet secretion. Therefore, we will manipulate these signaling pathways to identify a role of platelet secretion in obesity-associated pro-inflammatory and pro-thrombotic states."}
{"text": "Membrane traffic is required for a broad range of essential cellular functions, such as controlling the accessibility of cell surface receptors, the translocation of glucose transporters in response to insulin, antigen presentation, neuronal transmission and the establishment and maintenance of epithelial cell polarity. Therefore, the regulation of membrane traffic is directly relevant to a broad range of human diseases including cancer, diabetes and neural degeneration. Rab GTPases are key regulators of membrane traffic. By recruiting and activating a functionally diverse set of effectors, a single Rab GTPase can coordinate the various sub- reactions within a given stage of membrane traffic, including vesicle budding, delivery, tethering and fusion. Furthermore, our results indicate that adjacent stages of transport can also be coupled through coordinated rab regulation. We recently defined a rab guanine nucleotide exchange factor (GEF) cascade in which one rab, in its GTP-bound state, recruits the GEF that activates the next rab along the exocytic pathway. We also have preliminary evidence for a rab GTPase activating protein (GAP) cascade operating in a counter current fashion. Here the downstream rab recruits the GAP that inactivates the upstream rab. The net effect is rab conversion in which a given patch of membrane starts out labeled with one rab, but over time becomes labeled with another rab. Since each rab recruits and activates a distinct set of effectors, this leads to a functional maturation of the membrane. We will explore these two cascade mechanisms in further detail and test the physiological consequences of uncoupling adjacent stages of membrane traffic. We will test the role of a Sec4p effector in SNARE assembly and explore the roles of several new putative Sec4p effectors. Through these studies we will begin to define the exocytic pathway as a fully coordinated system, rather than as a collection of isolated sub-reactions. Membrane traffic is the mechanism by which material is transferred between different compartments within the cell and the regulation of membrane traffic is directly relevant to a broad range of human diseases including cancer, diabetes and neural degeneration. This study addresses the molecular mechanisms by which different stages of membrane traffic are coordinately regulated."}
{"text": "Aging and stress responses are tightly linked~ indeed, most interventions that extend lifespan appear to do so at least in part through potentiation of stress responses. Nevertheless, the genetic and mechanistic basis of this central relationship remains poorly understood. Here we will test the hypothesis that microRNAs (miRNAs) coordinate stress-responsive pathways in response to lifespan-prolonging interventions. We propose the following: To identify critical new miRNAs that link stress responses to aging in C. elegans, we profiled expression of small RNAs during development, aging and in stressed conditions (heat shock, starvation, hypoxia and oxidative stress). Since miRNAs that are differentially regulated during stress are likely to be mechanistic regulators of the stress response, we propose to characterize ten of the most differentially expressed miRNAs and determine their position in the gene-regulatory architecture of stress responses. We will also integrate our previous profiling and functional analyses of aging-associated miRNAs with these results to identify miRNAs that are associated with both stress and aging and test whether these genes provide mechanistic links between these conditions. To investigate network-level roles of microRNAs in regulation of stress and aging, we propose to elucidate the underlying regulatory network of genes and miRNAs involved in aging and stress. In order to identify critical new sets of miRNAs and pathways that link these processes, we have integrated transcription-factor binding site information, with miRNA target predictions to build a preliminary interaction network of the known regulatory relationships between transcription factors, aging- associated miRNAs, and miRNA biogenesis genes. We also propose to determine targets of key miRNAs biochemically via CLIP-seq and RNA-seq in the presence and absence of the miRNA. These data will allow us to improve the known miRNA-mRNA regulatory interaction network. Then, using this network, we will find miRNAs that comprise feedback loops and highly connected interaction nodes. We will test whether these highly connected miRNAs play critical roles in aging, stress responses, or in integrating the two. To identify miRNA mediators of lifespan extension due to dietary restriction. Our preliminary data point to miR-71 and miR-228 as key network nodes connected to pha-4 and skn-1, transcription factors critical for the response to dietary restriction and other stressors. We will characterize the roles of thes miRNAs and use our gene- regulatory network to identify other such candidate miRNAs. We also propose to identify miRNAs differentially expressed in dietary restriction via deep sequencing and include these in our network analysis above. We are uniquely well situated to carry out this work, as the Slack lab combines extensive experience in miRNAs and aging biology with leading expertise in genome-wide small-RNA characterization. MiRNA analogues and antagonists are pharmacologically tractable~ thus identifying critical aging and stress responsive miRNAs in C. elegans may lead directly to lifespan and healthspan-prolonging interventions in humans."}
{"text": "Cyanide is an archetypal chemical threat. This powerful natural toxin is encountered in many different settings and potential exposures range from industrial accidents to major terrorist attacks. Cyanide has devastating consequences. Higher quantities lead rapidly to seizures, cardiovascular collapse and death, while lower doses are associated with substantial long-term morbidity including debilitating central nervous system injury. Currently available antidotes simply do not match the complexity or scale of the potential mass casualty threat scenarios, and there is an urgent need to develop novel countermeasures. The major aims of this CounterACT Center of Excellence proposal are 1) to identify novel classes of cyanide countermeasure using unbiased approaches in a validated zebrafish model system, 2) to explore in vivo the structure activity relationships (SAR) of these novel countermeasure classes and using medicinal chemistry generate a series of optimized lead compounds and 3) to validate these optimized leads in established murine and rabbit models of cyanide toxicity. Together these individual interdisciplinary aims are focused around the overarching goal of this collaborative U54 application: The identification of at least 3 novel cyanide countermeasures ready to move directly to formal preclinical testing and Phase I clinical studies at the completion of the current application. Successful generation of three validated countermeasures using this pipeline will not only lay the foundation for parallel work in preclinical development (through the CounterACT Preclinical Development Facility or CPDF as outlined below) and for subsequent clinical development of these compounds (in a logical extension of the current proposal), but will also form a generalizable approach for the accelerated development of countermeasures to any existing or emerging chemical threat."}
{"text": "The purpose of this study is to follow children who are currently enrolled in or were previously enrolled in pediatric ACTG treatment or drug research studies to look for any late consequences of therapy in growth, neurologic and neuropsychologic function, long-term survival and quality of life."}
{"text": "Project Summary/ Abstract: Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is the most common pediatric neurodegenerative disease caused by a mutation in the CLN3 gene. Children with JNCL experience progressive visual, cognitive, and motor deterioration with a decreased life expectancy (late teens-early 20s). There is currently no cure for JNCL, making research to identify novel drug targets crucial to extend the survival and quality of life of afflicted patients. Previous studies have shown that astrocyte activation precedes and predicts regions of neuronal loss in JNCL, suggesting a defect in supportive glial functions. Glutamate levels are elevated in the JNCL brain and neuronal loss is thought to occur, in part, via glutamate excitotoxicity. Currently, little is known about aberrant glutamate-glutamine cycling in astrocytes and no reports investigating neuron-astrocyte cross-talk or regulation in JNCL exist. Our prior studies revealed a significant decrease in glutamine synthetase coupled with altered expression of the glutamate transporters in CLN3?ex7/8 astrocytes both in vitro and in vivo. This proposal will utilize mice harboring a 1.02 kb mutation spanning exons 7 and 8 of CLN3 (CLN3?ex7/8) to study glutamate regulatory pathways both in vivo and in vitro. My preliminary data indicates that primary CLN3?ex7/8 astrocytes display decreased glutamate transporter expression and activity when exposed to stimuli present in the JNCL brain. By extension, this disruption in glutamate homeostasis in the context of CLN3 mutation threatens to disrupt neuron-astrocyte signaling pathways, which if chronically perturbed can induce neuron excitotoxicty. Indeed, my preliminary studies have demonstrated reduced Ca2+ signaling in CLN3?ex7/8 astrocytes concomitant with heightened Ca2+ transients in CLN3?ex7/8 neurons, suggesting that disruptions in glutamate cycling interrupt critical homeostatic cell signaling networks. Concurrent with impaired Ca2+ signaling, preliminary electrophysiology conducted on hippocampus slice cultures from 30-day-old mice show that CLN3?ex7/8 mice have higher population spike amplitude and field excitatory post-synaptic potentials. This data indicates that the CLN3 mutation causes an increase in neuron activation that would potentate elevated glutamate release. Collectively, these findings formulated the hypothesis that the CLN3 mutation perturbs glutamate cycling pathways, which potentially creates a toxic cellular milieu, culminating in neuron death. The Specific Aims of this proposal are: 1) To identify the mechanisms responsible for astrocyte glutamate dysregulation and altered astrocyte-neuronal signaling networks in the context of CLN3 mutation; and 2) To evaluate whether the S100B synthesis inhibitor ONO-2506 can limit neurotoxicity in CLN3?ex7/8 mice by enhancing glutamate transporter expression and glutamate uptake. Establishing key regulatory mechanisms of glutamate and astrocyte-neuron cross-talk in JNCL may unveil novel therapeutic targets to extend the quality-of-life for children suffering from this devastating neurodegenerative disease."}
{"text": "With a few important exceptions, all the neurons in the mammalian nervous system fall into two major classes: excitatory neurons, using glutamate as a neurotransmitter, and inhibitory neurons, using GABA (and in some regions also glycine) as a neurotransmitter. GABAergic neurons play the critically important roles of counterbalancing excitation and of channeling the flow of sensory and motor information in time and in space; loss of cortical GABAergic neurons or impairment of their function is a major factor underlying epilepsy and degenerative motor diseases. Their critical roles notwithstanding, GABAergic neurons remain poorly understood; in most regions they are interspersed among the more numerous excitatory neurons, and studying them has been seriously hampered to date by the lack of a means to identify them reliably in living tissue. The need addressed by the proposed study is for a novel method to identify and visualize GABAergic neurons in living preparations. We have recently made the first step towards addressing this need, by generating several lines of transgenic mice in which expression of green fluorescent protein is driven in a cell-specific manner in putative GABAergic neurons. These lines are potentially an extremely powerful research tool for studying GABAergic neurons in living preparations; however before they can be used for such studies, the molecular genetics of the transgene needs to be characterized, the fidelity of GFP expression in GABA neurons needs to be validated, and normal GABAergic function should be verified. Once these aims are accomplished, the mice will be made available to the scientific community through the NIH-sponsored Mutant Mouse Regional Resource Centers. The Exploratory/Developmental R21 Grant mechanism of support is appropriate, because the proposed project will demonstrate the validity and feasibility of a new tool, the GAD67-GFP transgenic mouse, which could have a major impact on the field of neuroscience by allowing investigators to address crucially important questions regarding GABAergic neurons, questions which were so far difficult or impossible to address."}
{"text": "AS-101 (Ossirene) induces the production of several hematopoietic growth factors in vitro, and protects mice from hematologic toxicity of external beam radiation or chemotherapy in vivo. The BRMP previously demonstrated similar effects of IL-1 before chemo/radiotherapy in animals, while a BRMP clinical trial demonstrated acceleration of platelet recovery from high-dose carboplatin when IL-1 followed chemotherapy. This protocol tests the ability of AS-101, by two doses and several schedules, to produce similar effects with high-dose carboplatin in pts. The objectives of this study are: 1) determine the toxicity of Ossirene and high dose carboplatin, 2) determine whether the sequence and/or schedule of drug is important, 3) determine which dose of Ossirene is superior in limiting bone marrow suppression from carboplatin. Pts. received carboplatin 800 mg/m2 and Ossirene, 3 or 6 mg/m2 pre or post carboplatin. Nine pts. were entered on this study. There were 2 minor responses. Pts. experienced significant hematologic toxicity, however toxicity did not prevent treatment."}
{"text": "A variety of endogenous and environmental DNA damaging agents pose a constant threat to DNA. Ultraviolet (UV) light from the sun is a common source of DNA damage in organisms, including humans. Exposure to UV light causes lesions in DNA that can impede DNA polymerases resulting in stalled replication forks. Failure to re-start stalled replication forks can have serious effects on cells, including interruptions in the cell cycle and chromosome breakage. Thus, UV-induced DNA lesions can destabilize the genome, increasing the chance of mutations. Such genome instability can lead to cancer. One way cells minimize the deleterious effects of damage that blocks DNA replication is through use of a specialized class of DNA polymerases that can replicate across the lesions in a process called translesion synthesis. DNA polymerase 7 (C. elegans POLH-1) is especially important for translesion synthesis past UV-induced lesions. Polymerase 7 and other translesion polymerases have high error rates when replicating undamaged template. Thus, their access to DNA replication forks must be tightly regulated to prevent mutagenesis. How translesion polymerases are removed from replication forks after TLS, to minimize their engagement with undamaged DNA, is not currently known. Recent work from our laboratory has elucidated a possible mechanism for how this might occur. According to our model, when POLH1-1 is recruited to replication forks, it is SUMOylated by GEI-17 E3 SUMO ligase, and stabilized long enough to perform translesion synthesis. The mechanism by which GEI-17 senses DNA damage to SUMOylate POLH-1 is not known. After translesion synthesis, POLH-1 is ubiquitinated by CDT-2 ubiquitin ligase, targeting it for degradation. Thus, we hypothesize that CDT-2-mediated degradation of POLH-1 limits its access to undamaged template, controlling POLH-1's mutagenic capacity. The experiments described in this proposal will address two outstanding questions related to this hypothesis. In Aim 1, I will use genetic and biochemical approaches to clarify the mechanism for how GEI-17 is recruited to sites of DNA damage to SUMOylate POLH-1. In Aim 2, I will use high throughput DNA deep sequencing to examine whether CDT-2 controls mutagenesis. PUBLIC HEALTH RELEVANCE: The variant form of the genetic disorder Xeroderma pigmentosum (XP-V) is caused by loss of polymerase 7 function. XP-V patients are hypersensitive to sunlight and have an increased predisposition for skin cancer. This pathology suggests that human DNA polymerase 7 limits the carcinogenic potential of UV light. A more complete understanding of polymerase 7 function, coming from my studies of POLH-1, could contribute to the eventual development of advanced cancer treatment therapies. PUBLIC HEALTH RELEVANCE: The experiments in this proposal will explore the regulation of DNA polymerase POLH-1 in Caenorhabditis elegans. Homologs of POLH-1 (DNA polymerase 7 in humans) minimizes mutations caused by UV light, thus limiting the development of cancer. Patients with a defect in the function of polymerase 7 are highly sensitive to UV light and have a predisposition for cancer. Thus, a detailed understanding of the molecular activities of polymerase 7 and its regulation may contribute to the creation of new cancer treatments."}
{"text": "Graft versus host disease (GVHD) is a debilitating and often fatal immunological condition induced by lymphoid cells grafted either experimentally or naturally into an immunoincompetent foreign host. Since children receiving bone marrow transplants or blood transfusions during the period of brain development are at risk of therapeutically-induced GVHD, we have studied the effects of GVHD on neonatal cerebellum, a well-defined model of developing human brain. The disease causes a decrease in cell accumulation (DNA synthesis and content and the numbers of cells in specific populations) and function (RNA content, synthesis and translational capacity; the number of cells migrating; and in the growth of Purkinje cell dendrites). The lack of inflammatory response or lymphocytic infiltration into the cerebellum suggests that a blood-borne factor is responsible. Amazingly, we found that many of the deleterious effects of GVHD on brain development could be reversed after the disease was stopped with alloantiserum immunotherapy. Here, we propose to study further GVHD-induced brain growth retardation and its amelioration with regard to: the timing of cell cycling and migration (autoradiography); cerebellar coordination of motor function (swimming behavior); the prenatal induction of GVHD (effects on prenatally formed cells and severity of postnatal events); the amount and distribution of the various forms of mRNA and the activity of each (cell-free protein synthesizing systems and 2-d gel electrophoresis; the significance of newly synthesized proteins during the disease). To investigate possible mechanisms involved in GVHD-induced brain growth retardation, we will address these questions; 1) Does cerebellar damage occur nonspecifically, i.e., regardless of histocompatibility antigen specificities? 2) Does GVHD serum inhibit growth in dividing cells in organ culture? 3) Is the blood brain barrier intact in GVHD? 4) How does GVHD effect the adrenal glands and their production of corticosterone? The general questions we expect to answer are: 1) In what manner does an immune process cause readily reversible alterations in neuronal cell functions which may be correlated with motor behavior? 2) Can immune processes regulate histogenesis and protein synthesis in non-neuronal rapidly dividing cell populations."}
{"text": "The purpose is to determine the effectiveness and tolerability of ucb L059 given in a twice daily regimen to existing AED treatment and to also determine the pharmacokinetic parameters (the measuring of the drug in the blood and urine) of both ucb L059 and its breakdown product ucb L057 in the age group of 6 to 12 years."}
{"text": "The menisci are C-shaped fibrocartilage disks that occupy the periphery of the knee joint and serve a number of significant mechanical functions including load distribution across articular cartilage, and joint stabilization. In the face of such mechanical demands, it is not surprising that the meniscus can become damaged. Unfortunately, surgical options for the treatment of damaged menisci are limited, primarily due to the poor vascularity and healing capacity of the central two thirds of the meniscus. Allograft implantation is complicated by the threat of transmission of infectious diseases, the difficulty of matching meniscal shape and material properties, and problems with maintaining cell viability. While meniscectomy (the complete or partial removal of the affected tissue) which is the most common orthopedic procedure performed in the United States can lead to the early onset of osteoarthritis. Providing a meniscal substitute engineered to function much in the way of the native tissue could delay or even avoid the onset of osteoarthritis. Meniscal substitutes, whether cell-seeded degradable scaffolds or synthetic implants, should ideally help to distribute loads across adjacent articular cartilage much in the way of the native tissue, thereby protecting the underlying articular cartilage. However, the properties of a substitute that are required to ensure that this functional capability is met are unclear. This is caused in part by the absence of a robust, comprehensive and physiologically relevant model within which the effect of substitute design variables can be parametrically studied. This lack of such a test, or series of tests, makes the design and evaluation of candidate scaffolds impossible, retards the regulatory pathway to commercialization, and leads to a situation where scaffolds are implanted in humans with little information about their ability to perform mechanically in the highly-loaded environment of the knee joint. Our goal is to define the relationship between meniscal substitute material and structural properties and joint contact mechanics under multiple physiological activities across a range of patient populations. The main element of the framework required to achieve this goal is a statistically-based, rapidly computable interpolator that will be built using results from experimentally-validated knee specific computational models. We will gather information about factors that most markedly affect the functional performance of the native meniscus and use this information as a template to identify the design space into which substitutes should fall if they are to function appropriately. Our goal is not to design a meniscal substitute; rather, we will demonstrate that our approach can establish a workable design space for use by those developing solutions for meniscal repair. Our efforts will culminate in the development of a new paradigm for the development and screening of any complicated tissue substitute, whether a non-degradable implant or a cell-seeded degradable scaffold prior to initiating more time consuming and costly animal and clinical trials. PUBLIC HEALTH RELEVANCE: Our goal is to define the relationship between meniscal material and structural properties and joint contact mechanics under multiple physiological activities across a range of patient populations. The main element of the framework required to achieve this goal is a statistically-based, rapidly computable interpolator that will be built using results from experimentally-validated computational models of the intact and meniscal substituted knee. Our approach will serve as a tool for rapid evaluation and design of functional substitutes and provide a new paradigm for the development and screening of any complicated tissue substitute, whether a non- degradable implant or a cell-seeded degradable scaffold."}
{"text": "PROJECT SUMMARY Caspase-2 (Casp2) has been described in the literature as an excellent target for Alzheimer's disease (AD) and has been implicated as therapeutically relevant in Huntington's disease (HD). Several non-apoptotic roles of Casp2 that may be relevant to neurotherapeutic discovery include its regulation of autophagy, oxidative stress, endoplasmic reticulum stress, and its effect on dendritic spines. Our own collaborative work provides evidence that ?tau314 (a non-fibillar, N-terminal tau fragment) levels are correlated with AD in humans. In rTg4510 mice, which express the hP301L tau associated with frontotemporal dementia (FTD), we have shown that Casp2 cleaves tau to form ?tau314 (H2N?YKPVD314), a heretofore-unreported tau fragment, and have linked this soluble tau fragment to reversible cognitive dysfunction. Given these exciting and promising results, we have initiated a comprehensive program to identify potent and selective Casp2 inhibitors. This platform includes standard structure-based design of covalent inhibitors based on the YKPVD314 cleavage site in tau and high-throughput screening (HTS). Herein we propose to employ fragment-based screening (FBS) to expand and complement the scope of this program, increasing the likelihood we will discover small-molecule inhibitors of Casp2. The single specific aim of this exploratory project is to identify and characterize fragments (low molecular weight and soluble compounds) that bind to Casp2. A commercial in-house fragment library will be screened against Casp2 using differential scanning fluorimetry (DSF). Active ligands will be characterized by surface plasmon resonance (SPR) and X-ray crystallography. Confirmed actives will be validated using SAR (structure-activity relationship) by commerce. Our working hypothesis is that the collection of well- characterized scaffolds we discover will constitute excellent starting points for future work focusing on the optimization and development of potent and selective small-molecule inhibitors of Casp2. The potential impact of this project on human health is considerable. There is an unmet medical need for therapeutic agents that can halt or reverse the cognitive decline associated with AD. This work will have a positive impact on the field of therapeutics to treat the cognitive decline observed in several diseases of the central nervous system, particularly AD, HD, and FTD. The eventual development of a selective inhibitor of Casp2 would address this unmet medical need and would represent a significant advancement in the field of AD."}
{"text": "The goal of this project is to increase the ability of virus vectors to deliver genes to the postnatal nervous system and to use these vectors to alter cell fate in the nervous system. Two strategies will be undertaken to expand the range and longevity of gene delivery to neural cells in the mouse nervous system. In the first scheme, retrovirus vectors will be generated which can confer stable gene expression on astrocytic cells grafted into the brain. The phosphoglycerate kinase promoter will be used to regulate expression of brain-derived neurotrophic factor (BDNF) for degenerating dopaminergic neurons in newborn weaver mice. These vectors will also be used to genetically modify astrocytic lines capable of migrating in the brain. In addition, one of the astrocytic lines will be converted to a packaging cell line which can be used to deliver retrovirus vectors to endogenous neural cells in vivo. Retrovirus vectors will also be generated to aid in functional analysis of the NF2 gene product in Schwann cells and tumor cells. Further we will try to develop an animal model for neurofibroma formation by retrovirus-mediated delivery of antisense RNA to repress expression of neurofibromin or by homologous recombination to knock-out the normal NF1 allele in Schwann cells from heterozygous NF1 knock-out mice. The behavior of genetically altered Schwann cells will be evaluated in a peripheral nerve injury-repair model. In the second gene delivery scheme we will try to improve herpes vectors for delivery of genes to neurons. A \"piggy back\" system of herpes simplex virus vectors will be developed using a combination of amplicon and recombinant virus vectors to combine complementary features of both vector types and to make them mutually dependent on each other. This system will be designed to allow limited replication of the vectors in glia-derived cells using glia-specific promoters to regulate expression of critical genes. Amplicon vectors will be used to deliver NGF and Oct2 to neurons at the time of infection in order to encourage entrance of the virus into latency, at the same time making preparation of vector stocks feasible by using a tet operon system to control expression of these genes in culture. The recombinant virus vector will be deleted for genes contributing to neurovirulence including ICP4 (or ICP27), ribonucleotide reductase, gamma 34.5 and UL41. The herpes vector system will be evaluated for gene delivery, stability of transgene expression and pathogenicity to neural cells in the brain. These new gene delivery systems will provide a basis for therapeutic intervention in animal models of Parkinson's disease and neurofibromatosis."}
{"text": "Studies to define the host immune response to human tumor has concentrated in part on characterizing the T cells within the tumor bed. The picture that has immerged is complex with respect to the functional responsiveness of these cells. Tumor infiltrating lymphocytes (TIL) contain responsive T cells that display normal effector functions. It is also clear that a significant portion of TIL from a variety of tumors are defective in their proliferative response. We have demonstrated that TIL derived from human RCC and B cell lymphomas have a reduced proliferative response that is reflected by a decrease in the IL2Ralpha surface expression and mRNA levels. While TIL can produce comparable levels of IFNgamma when compared to PBL they also display a diminished capacity to produce IL2. Our hypothesis is that while a segment of TIL are responsive the majority display a state of unresponsiveness that is rather limited in scope. Other functional parameters are thought to be intact even though most TIL have a diminished ability to proliferate and produce IL2. Aim 1 will determine which subsets of TIL are most affected. Cell sorting experiments \"will determine if the proliferative defect is unequally distributed among T cell subsets (CD4+ and CD8+ cells) and if the defect relates to the state of activation of T cells in the tumor bed. Aim 2 will define the extent of the unresponsiveness by determining if other functional parameters are intact in the affected populations. Purified TIL subsets will be used to document whether TIL are defective in their ability to produce IL2. Whether the defect is limited to IL2 will he determined by testing TIL for the production of other cytokines such as IL4, TNFalpha and GM-CSF. Experiments will also determine if TIL are as competent as PBL to produce mRNA for perforin and serine esterases. Additional work will define the role various stimulation routes play in the unresponsiveness. An analysis of multiple functions induced by IL2R and TCR stimulation will help define the extent to which each of these receptors are affected. Experiments will also determine if triggering TIL via CD28 is normal since this pathway appears to be distinct from that induced by TCR. Aim 3 will determine if the reduced IL2Ralpha surface expression and IL2 production of TIL is related to alterations in transcription. RT/PCR will be used to quantitate mRNA levels of TIL and PBL. Included will be studies that determine if the reduction in mRNA levels is due to changes in the kinetics of mRNA accumulation, rate of transcription or mRNA stability. The goal of the proposed work is to begin to understand how broad the defect is, and to identify the subsets affected as well as which receptor pathways are altered in the unresponsive TIL. It is important to characterize quantitatively and qualitatively the T cell unresponsiveness within the tumor. An understanding of this anergy may provide ways to reverse or bypass this defect allowing for T cell activation and the development of a potent host immune response to tumors. Understanding T cell anergy in disease states such as cancer will be important for the development of new treatment strategies."}
{"text": "The objective of this study is to evaluate the effectiveness of therapeutic stockings to reduce the incidence of foot ulcers in high-risk diabetics, and to evaluate self-reported functional status among treatment groups. We expect that subjects treated with therapeutic stockings will have fewer foot ulcers and that their self-reported functional status will be higher than patients receiving standard care. Persons with diabetes with previous foot ulceration have a yearly recurrence rate of more than 30%, even at centers with specialized foot clinics. Therapeutic stockings are an inexpensive and practical mechanism to reduce pressure and shear forces on the foot and thereby reduce the likelihood of ulceration. However, there is very little clinical evidence to support the use of \"therapeutic stockings\". We will randomize 400 persons with diabetes and a history of foot ulceration to receive standard therapy (ST) consisting of education, regular foot care and protective shoes and insoles;or standard therapy with the addition of therapeutic stockings. The stockings used in this study are designed to reduce pressure and friction at the stocking-foot interface. The NeuroQol instruments will be administered to evaluate self-reported functional status. We will patients at centers in Temple, Texas, Georgetown, Texas, and two centers in Manchester, UK. Patients will be evaluated at least every three months for regular foot care after screening and provision of protective shoes and insoles. Patients will record daily use of therapeutic stockings, insoles and shoes in a log book. Patients will use a step counter to record daily activity. Therapeutic stockings are an inexpensive and practical tool to protect the feet of high risk patients from pressure and shear forces. This simple measure may significantly reduce the risk of foot ulcers in high risk patients."}
{"text": "A continuation of the CCOP grant will enable Hematology-Oncology Associates of Central New York, P.C. (HOACNY) to continue its active participation in a wide variety of ongoing clinical cancer research studies, including treatment and prevention trials. The goal of the program participation is to continue translating experience in cooperative group clinical research into expert cancer prevention, cancer control, diagnosis, and treatment (curative, palliative, supportive) for the greater community of central New York (Syracuse and surrounding rural counties). The Syracuse Hematology-Oncology CCOP aims to continue pursuing multi-disciplinary and multi-specialty participation in and awareness of clinical treatment and prevention protocols. The CCOP physicians and staff will continue in efforts to reach the professional and lay publics with current practice in the prevention and management of cancer and to encourage participation in research which has long term benefits for the entire community. The CCOP plans to increase recruitment of minorities to research trials and to maintain its high level of female participation in clinical trials. More specifically, the goal of the Syracuse CCOP physicians and staff is to maintain current levels of participation in clinical treatment trials while increasing the number of registrants on prevention and control trials. In the process of recruitment, the CCOP staff will be able to continue providing health education and screening related to cancer prevention and control."}
{"text": "We have continued during the past year to study those metabolic properties of the red cells of different animals which are associated with their viability in vivo and in vitro and which play a part in the transport of oxygen. We have also continued investigations on metabolic changes which take place during the maturation of the red cell. Our previous studies on the red cells of the alpaca, which presented interesting problems because of the unique adaptations of this animal to high altitude, have been extended to another cameloid, the dromedary. A program was begun this year to see what role the red cell of the diving animal might play in the special requirements of these animals for transport and conservation of oxygen. Metabolic intermediate analyses and glycolytic rate determinations have been performed on red cells from the bottlenose dolphin, the California sea lion and the gray seal. Work which has been in progress in this Laboratory for several years to try to explain the peculiar metabolic properties of pig red cells has continued with in-vivo experiments on C14-glucose utilization by the circulating red cells of the domestic pig. A project has been started this year to learn about the energy metabolism of fish red cells and analyses have been made of the glycolytic intermediates and soluble nucleotides of red cells from mackerel, dogfish and salmon. Work is also continuing on a comparison of the metabolic properties of mature circulating erythrocytes with reticulocytes obtained from rats and rabbits after treatment of the animals with phenylhydrazine and after severe bleeding."}
{"text": "This 2nd revision of an application for a competing renewal of an Independent Scientist Award seeks funding to support development of the candidate's research on the treatment of late-life bipolar disorders and his mentorship of young investigators. This research program addresses the need for evidence that can help individualize the management of these underserved patients by examining proposed clinical and neurobiological predictors of inter-individual differences in benefit from first line pharmacotherapies. Having initiated the multi-center GERI-BD study of acute treatment of manic bipolar elders, and its Coordinating Center, the candidate aims to use this platform to explore the utility of selected measures of frontostriato-limbic impairment as predictors of resistance to anti-manic treatment. Three Pilot Studies will focus on affect-laden executive tasks, neuroimaging of white matter integrity, and neurotransmitter-related genetic variation. Conducting these Pilot Studies will enable the candidate to expand his knowledge base in cognitive and affective neuroscience, neuroimaging and pharmacogenetics, and related analytic issues, through interaction with expert advisor/collaborators. This career enhancement is intended to foster development of his research to address the needs of elderly patients who do poorly with optimal standard treatments, and to study the management of other phases of bipolar illness in late life. It is also intended to support continuation and expansion of his mentorship activity. As the population ages, greater numbers of elders present with bipolar disorder. They suffer from severe symptoms, disability, and high mortality. These studies can provide findings that use relatively non- invasive and cost effective methods, derived from neuroscience and neurobiology, to develop safe and effective treatment approaches."}
{"text": "Chronic kidney disease-mineral and bone disorder (CKD-MBD) results in complex skeletal and metabolic phenotypes. Because of this, patients with advanced kidney disease have an increased risk of fractures. In patients with age-related bone loss, bone mineral density (BMD) estimates are a helpful predictor of fracture development. In CKD, however, the picture is unclear. While it may be of use, the presence of other metabolic derangements indicate that bone quality likely plays a greater role in the pathogenesis of CKD-related fractures than in those associated with age-related bone loss. Current treatment in patients with CKD-MBD is focused on suppressing elevations in parathyroid hormone. This is accomplished by calcitriol supplementation. While the effect of calcitriol in osteoporotic patients has been studied, its impact on bone quality and fracture risk n CKD patients is currently unknown. Also, recent analyses suggest that raloxifene, a selective estrogen receptor modulator, might be a useful intervention for patients with late stage kidney disease. This is supported by beneficial renal outcomes in patients on raloxifene as well as evidence demonstrating an additional non-cellular mechanism by which to improve bone quality. So, we hypothesized that CKD leads to alterations in bone quality that can be corrected by raloxifene and its combination with calcitriol. This will be tested through the use of a slowly progressive model of CKD-MBD, the Cy/+ rat. This study will compare the quality of skeletal tissue (independently of mass) in normal and Cy/+ rats. This will be accomplished by examining bones from 30-week-old rats for changes in bone quality. Specifically, outcomes will include tissue-level mechanical properties, mineralization, collagen composition (cross-linking, morphology, and mechanics), and bone matrix hydration (MRI). We expect rats with CKD to display lower bone strength, lower mineralization, lower collagen stiffness, lower matrix-bound water, and higher non-enzymatic cross-linking of collagen. The second major goal is to examine the effects of raloxifene on bone abnormalities present in Cy/+ rats. 25-week-old animals will be treated for 5 weeks (equivalent to one bone remodeling cycle). Primary outcomes will be determined by skeletal analyses (histology, microCT, bone density, mechanical testing, and bone quality measures), though the biochemical, renal, and vascular components of CKD-MBD will be assessed as well. Gene expression analyses will be performed to begin to identify signaling pathways involved in changes in biomechanical bone quality. We predict that raloxifene will improve the mechanical properties of bone by improving bone quality, while calcitriol will improve mechanical properties by increasing bone mass. Combination therapy should exceed all other treatments by positively impacting both quality and mass. An understanding of the detrimental impact of CKD on bone quality is a crucial step in preventing fractures in these patients. This study provides an important step in achieving this goal by examining these changes and their potential corrections in a rat model with the spontaneous and progressive development of chronic kidney disease."}
{"text": "The broad objective is to facilitate the immunologic control of cancer by providing information on tumor associated antigens. Specific aims are: 1. To elucidate the structures and antigenic roles of the carbohydrate and protein portions of CEA. 2. To isolate CEA-like materials from the tissues of both malignant and nonmalignant diseases in which elevated CEA levels have been reported in the serum. 3. To examine the CEA-like materials isolated to see if they are chemically identical to the material isolated from colonic adenocarcinoma. If they are different, attempts will be made to identify the chemical nature of the differences. 4. To assist other research groups in research on CEA and other tumor antigens through provisions of reagents and in training personnel in radioimmmune assay techniques. 5. To search for new tumor antigens which may be susceptible to isolation and purification for structural studies. 6. To develop and apply techniques other than radioimmune assay which may be helpful in establishing the role of the antigen in the evolution of the malignancy and may point the way to its application in the control of cancer. 7. To explore and develop techniques, such as mass spectrometry, which will enable structural information to be obtained on very small amounts of complicated materials. 8. To apply these and other techniques to structural studies of new tumor antigens as they become available."}
{"text": "The broad long-term objective of this research is to find and describe the genes responsible for 2 highly incapacitating hereditary disorders, namely, Diastrophic Dysplasia (DD) and progressive Myoclonus Epilepsy (PME). Nothing is presently known about the pathogenesis of these disorders. When these genes have been described the pathogenesis will become clarified and the way paved for treatment and prevention. It is particularly appropriate for these disorders to be studied in Finland, because they are rare elsewhere but common in Finland. Approximately 150 patients with DD (100 living) and 160 with PME (110 living) have been diagnosed in finland. The distribution of the genes is uneven considerable enrichment in small sub-isolates. This feature makes it possible to use homozygosity mapping in addition to conventional linkage. The first specific aim of this project is to generally map the genes causing DD and PME, with no available data about the nature of the genes of their products, the first will be to search for linkage with random RFLPs. Over 1500 RFLPs that span over 95% of the human genome are available; over 300 polymorphic probes are already in use in the laboratory of the PI. by choosing probes located less than 20 cM apart and distributed as evenly as possible along the chromes, region after region of the human genome will be excluded until positive linkage is found. Blood samples have already been collected and permanent lymphoblastoid cell lines established from 32 DD patients and 75 of their family members representing 14 pedigrees. These samples are sufficient to exclude or establish linkage. For PME the corresponding figures are 20 patients, 30 family members, representing 12 pedigrees. The collection of further samples for PME is underway and will be completed in 1989. Homozygosity mapping will be done using patient DNA from unrelated affected individuals. The second specific aim is to find markers as close as possible to the genes. This will be done by saturating the region with probes. Once closely linked, preferably flanking, markers have been identified, a method based on segregation analysis for carrier detection, and presymptomatic and prenatal diagnosis will be devised. The results of this research should increase the understanding of the biology of DD and PME and provide methods to diagnose and prevent these disorders."}
{"text": "Human vision seems to be a continuous process, uninterrupted by the eye blinks and saccades that usually occur every few seconds. We propose to explore this phenomenon in the following experiments: (1) Measure the time course of blinks that occur (a) spontaneously, without voluntary control or attention, (b) reflexly, in response to a puff of air or looming visual stimulus, and (c) voluntarily, on signal from the experimenter. (2) Evaluate the time course of any neural suppression that is found to accompany each blink. (3) Measure the extent and time course of the optical attenuation produced by lid closure. (4) Simulate the same attenuation in the open eye by external changes in the illumination of the visual field, and compare the visual effects of attenuation by lid closure with those of the equivalent reduction of external illumination. (5) Test the statement by Davson (1972) that, --\"A movement of the eyes is generally accompanied by a blink and it is thought that this aids the eyes in changing their fixation point.\" (6) Test the extent to which the eyes rotate during blinks and the related question of whether blinks result in an apparent displacement of the visual field."}
{"text": "Pediatric cardiac arrest is a common and devastating condition which remains poorly understood. Mortality rates are extremely high and brain injury is the most common cause of death. The majority of research regarding cardiac arrest over the past 50 years has focused on improving rates of return of spontaneous circulation (ROSC), with significant progress leading to increased survival rates. However, without interventions to minimize organ injury, there is an increase in long-term health issues associated with our improved resuscitation practices. This has been termed the post-cardiac arrest syndrome, consisting predominantly of long-term neurological deficits. Indeed, several interventions that have been useful in improving ROSC, have not shown benefit in improving long term outcome. There have been numerous pre- clinical translational studies of cardiac arrest in adult animals demonstrating various pharmacological interventions to improve neuronal survival following global cerebral ischemia. However, to date, there are very few studies in pediatric cardiac arrest, as models are scarce. In the current proposal, we describe the first pediatric cardiac arrest model utilizing mice to study the effects of cardiac arrest on neuronal survival and test new therapies. Epidemiologic studies in adults have suggested that females have better outcomes after CA when compared to males. Numerous experimental studies in adult animal models have recapitulated this clinical data, showing that female animals exhibit significantly less brain injury following cerebral ischemia than males. The sex difference observed in experimental adult animals can be nearly completely explained by the high levels of estrogen in female animals, as removal of endogenous sex steroids (ovariectomy) increases female brain injury to male levels and estrogen replacement returns female injury to intact animal levels. Not surprisingly, we did not observe a gender difference in ischemic injury in pediatric mice, consistent with pre- pubertal state of low estrogen in both male and female animals. Interestingly, when estrogen is exogenously administered we observe a remarkable sex-difference in response to estrogen neuroprotection. We observed that a single intravenous estrogen dose administered at a clinically relevant time point following CA/CPR (30 min) provides protection to the female brain, while having no effect in the male brain. Therefore, the aims of the current proposal are designed to further characterize our novel pediatric cardiac arrest model and begin to elucidate the molecular mechanisms of sexually dimorphic estrogen neuroprotection observed at this developmental stage. Therefore, we will 1) establish the role of estrogen in determining neuronal injury following pediatric cardiac arrest and 2) determine the relative contribution of estrogen receptors alpha and beta (ER? and ER?) in estrogen neuroprotection. 3) Finally, determine the molecular mechanism of estrogen neuroprotection in pediatric cardiac arrest."}
{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mm-cpn is an Archaeal type II chaperonin of ~1 MDa. Its structural organization is simpler than TRIC, the mammalian type II chaperonin, because, while also a 16mer, it possesses only a single subunit isoform. We propose to determine its structures in different chemical states at near atomic resolution."}
{"text": "Cancer research relies on technologies that can interrogate the entire genome, detecting deletions, copy number variants and point mutations responsible for disease. It is becoming increasingly clear that epigenetic lesions in DNA and histone modification are at least as important, and probably more important, than genetic lesions as cells progress towards malignancy, and acquire various attributes, such as drug-resistance. Cancer research at Cold Spring Harbor Laboratory has been at the forefront of this \"cancer genomics\" approach, as well as research in epigenetic mechanisms that contribute to these and other diseases. Examples of research topics are summarized in the application, ranging from the role of methylation in breast cancer and chemoresistant AML to replication-induced senescence. Further, we plan to examine epigenomic profiles in model animals and plants, which undergo developmentally programmed changes in epigenetic profiles, and which use RNA interference to guide DNA methylation and histone modification patterns. To do this, we are requesting funds to purchase a Genome Sequencer FLX instrument from Roche Applied Science, which can reliably determine the sequence of the epigenome, even when cytosines are converted to uracil, because longer read lengths permit unambiguous mapping back to the reference genome. PUBLIC HEALTH RELEVANCE: As we learn more about how information is stored in our genome and controls biological processes it has become apparent that in addition to our basic DNA structure, modifications to it, such as the addition of methyl groups in specific places is of crucial importance. Changes in methylated DNA regions can change gene expression in normal development as well as lead to cancer if and possibly other diseases if not properly controlled. We are requesting equipment that will allow us to examine, in great detail and at very large scale, the changes to methylation in the genome so that we can better understand how this process occurs in normal biological processes as well as which genes it regulates in cancer."}
{"text": "In the developing nervous system, axons are guided to their targets by extracellular guidance cues. While much progress has been made in the characterization of these cues, the downstream signaling pathways are poorly understood. The goal of this project is to determine the role of the cytoplasmic proteins encoded by mig-10, a gene that is required for pathfinding of the SMDD axon. Preliminary evidence suggests that mig-10 might act to modulate signaling downstream of slit, a repulsive guidance cue. The expression pattern and locus of function will be determined for each of two MIG-10 isoforms. The hypothesis that mig-10 may modulate slit signaling will be tested by ectopically expressing both isoforms in the AVM, a neuron that is known to be sensitive to axonal repulsion by slit. Genes that interact with mig-10 will be identified through a candidate gene approach and a genetic screen. The products of the mig-10 gene are similar to a family of mammalian proteins that includes GRB-7, GRB-10, and GRB-14. These proteins are overexpressed in several types of cancers, but their function is poorly understood. Information from this project will be beneficial in understanding disorders of the nervous system as well as the molecular basis of cancer."}
{"text": "Retinoids are known to be important in a variety of processes during development. Previous studies have shown that both retinoid and FGF signaling are required for anteroposterior patterning. The specific aims are: 1) to determine if ets domain transcription factors interact with RAR and FGF signaling pathways, 2) to show the necessity for RAR regulation of Xcad expression in early anteroposterior development and 3) to show RAR and Wnt signaling act synergistically to pattern the trunk and posterior. We will employ MO-mediated gene knockdown and microinjection of specific reagents to determine the function of RAR isoforms, Wnt signaling as well as interactions with ets domain transcription factors. Whole mount in situ hybridization, real time PCR, microinjections, and embryological manipulations will be employed to discern the effects of loss-of-function in certain genes and their affects on the function of RAR, Wnt and FGF signaling. The long-term goal of the proposed research is to understand the role of retinoid signaling during vertebrate axial patterning. The results should be widely applicable to the study of other vertebrate model systems."}
{"text": "The long-term goals of the Duke/North Carolina Central University (NCCU) \"Building Interdisciplinary Research Careers in Women's Health\" (BIRCHW) program are (1) to contribute to improvement in women's health by providing training and experience in research methodology to new investigators interested in addressing important questions, (2) to foster innovative approaches to these questions by encouraging investigators to utilize the wide range of intellectual capital available through interdisciplinary training and collaboration, (3) to build the capacity of Duke and NCCU to provide leadership in women's health research, and (4) to serve as a model for broad-based interdisciplinary research training and collaboration for both universities. The program will reach these objectives by achieving the following specific aims: (1) providing didactic and practical training in the conduct of research in women's health for junior faculty across a broad spectrum of disciplines, focusing on 4 broad areas: Clinical Trials and Outcomes, Decision Making, Health Disparities, and Basic and Translational Research; (2) building upon existing interdisciplinary faculty relationships to foster productive and innovative collaborations; (3) creating new interdisciplinary research relationships between Duke and NCCU faculty; (4) providing additional mechanisms for the ongoing exchange of ideas, methods, and insights among the community of researchers at Duke interested in women's health; (5) providing a mechanism for recruitment and career development of junior faculty committed to the long-term goals of the program; and (6) enhancing overall research productivity of faculty by encouraging and facilitating interaction between clinician and non-clinician researchers. Trainees in the program will have a minimum of 2 years of mentored research training, including weekly seminars on research methods and career development topics, experience in peer review and grant writing, and completion of at least one project working in conjunction with a senior faculty research mentor. Trainees will be expected to complete and submit at least one application for independent research training by the end of their time in the program."}
{"text": "Mutations in LMNA, the gene for lamin A/C, have been shown to cause several forms of muscular dystrophy, a cardiomyopathy, familial partial lipodystrophy, one form of Charcot Marie Tooth neuropathy, mandibuloacral dysplasia, and Hutchinson Gilford progeria syndrome (HGPS). HGPS patients have many aging-like phenotypes, including a wizened appearance, alopecia, osteoporosis, and coronary heart disease. HGPS is a merciless disease; death occurs on average at age 13, nearly always from atherosclerosis in the coronary and cerebral arteries. A recent NIH workshop on HGPS concluded that a thorough understanding of vascular disease in HGPS syndrome represents a key research priority. In most cases, HGPS is caused by a de novo single nucleotide substitution in exon 11. This mutation generates a cryptic splice site, resulting in an in-frame deletion of 50 amino acids near the carboxyl terminus of the lamin A. Interestingly, the deletion prevents the endoproteolytic processing of prelamin A. The objective of this grant application is to create mouse models of HGPS for the purpose of exploring mechanisms underlying the strikingly increased susceptibility to atherosclerosis in HGPS. The Principal Investigator (PI), Dr. Stephen G. Young, is well-positioned to pursue this objective. For the past 10 years, the PI has worked both on the posttranslational processing enzymes required for the biogenesis of lamin A and on the genetic underpinnings of atherosclerosis. For both projects, the PI has relied heavily on studies with gene targeted mice. Recently, the PI showed that the targeted inactivation of Zmpste24 prevents the proper endoproteolytic processing of lamin A, leading to the accumulation of prelamin A. Of note, Zmpste24-/- mice have a host of phenotypes similar to those in HGPS. The PI has also created mice lacking other enzymes involved in the posttranslational processing of the lamins (protein farnesyltransferase, Rcel, and Icmt). The first aim of this application is to create, with gene-targeting approaches, authentic mouse models of HGPS. The second specific aim is to compare, at both cellular and whole-animal levels, phenotypes in HGPS mouse models and the Zmpste24-deficient mice. The third specific aim is to understand mechanisms underlying the strikingly high susceptibility to atherosclerosis in HGPS. The PI's laboratory has extensive experience in analyzing atherogenesis in mice and is thrilled with the prospect of exploring mechanisms of atherosclerosis in HGPS."}
{"text": "The primary objective of this research project is to study the modulating effects of green tea polyphenols on reducing hepatocarcinogen-induced oxidative damages in high-risk human populations. Oxidative damage induced by reactive oxygen species in vivo plays important roles in human hepatocarcinogenesis primarily caused by chronic infection of hepatitis B/C viruses and exposure to dietary aflatoxins. The level of 8-hydroxy-2'- deoxyguanosine, a biomarker for oxidative DNA damage, increases in hepatitis B virus surface antigen positive and aflatoxin-exposed humans and in aflatoxin- treated animals. Dietary antioxidants are important components of cancer modulating agents, which have been proven to effectively target carcinogen biomarkers, including oxidative damages, in high-risk human populations. Among various identified dietary associated antioxidants, green tea and its polyphenols have been shown to be safe and highly effective in inhibition of a variety of carcinogen-induced oxidative damages, mutagenesis, and tumorigenesis in in vitro bioassays and in vivo animal models. The general hypothesis underlying this proposal is that green tea polyphenols have a protective effect against oxidative stress or damage induced by aflatoxin and hepatitis B/C viruses through the mechanisms of modulating aflatoxin metabolism and oxidated DNA damage. The specific aims include: (1) to determine antioxidative role of green tea polyphenols in inhibition of the level of 8-hydroxy-2'-deoxyguanosine in urine samples collected from an intervention study of 120 participants who are double positive for hepatitis B virus surface antigen and aflatoxin-albumin adducts, and (2) to determine the modulating effect of green tea polyphenols on excretion of carcinogen detoxifying product, aflatoxin Bl-mercapturic acid in urine samples collected from the study participants. The results of this proposed study will help to understand the mechanisms of antioxidative role of tea polyphenols in modulating human hepatocarcinogenesis caused by hepatitis B/C viruses and aflatoxins."}
{"text": "Skeletal muscle insulin resistance, particularly that associated with central obesity, is a serious public health problem that is largely due to exercise deficiency. Exercise/muscle contraction, like insulin, stimulates muscle glucose transport by translocation of GLUT4 glucose transporters from intracellular sites to the cell surface. As this acute effect of exercise wears off, it is replaced by an increase in insulin sensitivity. Exercise also induces an adaptive increase in GLUT4 that results in increased insulin responsiveness. These effects are among the most important health benefits of exercise. While there has been much progress in the elucidation of insulin action, little is known regarding how contractions stimulate glucose transport. The initial signals appear to be the decrease in high-energy phosphates and the increases in cytosolic Ca2+ during contractile activity. These signals activate AMPK and CAMKII, both of which appear to mediate the increase in glucose transport. Thus the initial and final steps in the stimulation of glucose transport by contractions have been identified. However, the steps connecting activation of CAMKII and AMPK to GLUT4 translocation are unknown. Our goals are to elucidate the steps in the pathway by which contractions stimulate glucose transport and to investigate the mechanisms responsible for the increase in insulin sensitivity after exercise. Relative to the first goal, one of our aims is to determine if the contraction-mediated and the second, i.e. not wortmannin-inhibitable, insulin signaling pathway overlap or merge. Relative to this aim we will address the questions: Are small G-proteins of the Rho family activated by contractions? Does stimulation of glucose transport involve activation of tyrosine kinases? and, Is phosphatidic acid involved in the stimulation of glucose transport by muscle contractions? The second aim relating to the first goal is to determine whether activation of Rab4 is involved in the stimulation of glucose transport by contractions. It is our working hypothesis that this is the step at which the contraction-stimulated pathway and wortmannin-inhibitable insulin-signaling pathway merge. Relative to this aim we will address the following questions: Is phosphorylation of AS160 a step in the pathway by which contractions stimulate glucose transport? Is Rab4 activated in response to muscle contractions? Does inhibition of Rab4 activation prevents stimulation of glucose transport by contractions? and, Is activation of atypical protein kinases C-zeta/lambda involved in the stimulation of glucose transport? Relative to our second goal, one of our aims is to evaluate the hypothesis that during recovery from stimulation GLUT4 undergoing endocytosis cycle into a storage pool that is accessible to a weak insulin stimulus. Another is to determine whether the p38 MAPK is involved in mediating the exercise-induced increase in insulin sensitivity. A third is to answer the question, is activation of PKC-zeta/lambda involved in the exercise-induced increase in insulin sensitivity?"}
{"text": "ABSTRACT Despite the availability of the human papillomavirus (HPV) vaccine that can prevent over 34,800 HPV-related cancers in the US every year, only 51% of girls and boys were up-to-date by 2018. Rural populations are the most impacted by HPV-related cancers. Best practices like the Announcement Approach training and systems communication have proven effective in increasing HPV vaccination, but rural providers struggle to access and implement such best practices. These data prompt the question: ?How can academic centers support HPV vaccination in rural primary care practices?? Although never tested for HPV vaccination, the ECHO (Extension for Community Healthcare Outcomes) Model is a promising implementation strategy (practice facilitation) that allows ?experts? at academic centers to connect with primary care providers to discuss best practices in care and complex cases managed within local practices. The objective of this R01 is to test two ECHO-delivered HPV vaccination communication interventions in rural primary care clinics. The first will provide Announcement Approach training (HPV ECHO); the second will provide this approach plus systems strategies to communicate with parents who initially decline vaccination (HPV ECHO+). The rationale for the project is that ECHO is a robust, highly-accessible platform to deliver best practices to rural providers and address the context-specific communication needs of parents. Our long-term goal is to improve HPV vaccination rates in rural clinics and reduce the health inequity rural populations experience in cancer outcomes. Aim 1 is to evaluate the impact of HPV ECHO and HPV ECHO+ on HPV vaccination among adolescents. We will conduct a 3-arm cluster randomized trial with 36 primary care clinics in rural Pennsylvania. Clinics will be randomized to: HPV ECHO, HPV ECHO+, or control. Our primary outcome will be change in HPV vaccine initiation (?1 doses) among adolescents, ages 11-14, at 12-month follow-up. Aim 2 is to evaluate the impact of HPV ECHO and HPV ECHO+ on implementation outcomes. Guided by implementation science frameworks, we will conduct a mixed-methods evaluation to compare interventions on acceptability, adoption, cost, penetration, and sustainability. Aim 3 is to evaluate the impact of interventions? vaccine information on secondary acceptance of HPV vaccination at the clinic level. We will also follow a subset of 200 vaccine-declining parents for up to 12 months to assess exposure to and impact of vaccine information from study arms versus naturally-occurring sources (e.g., social media) on secondary acceptance. Our expected outcome is to demonstrate the effectiveness of a highly efficient and scalable implementation strategy, ECHO, to support HPV vaccination in rural clinics. This study is innovative in leveraging existing infrastructure at academic centers to deliver best practices for HPV vaccination where they are needed most and in developing a greater understanding of the influences on decision making among vaccine-declining parents. We expect the project to have a significant impact on HPV vaccine uptake as we address the communication needs of both rural providers and parents."}
{"text": "[unreadable] Cystic Fibrosis is a fatal lung disease caused by misfolding and premature proteasomal degradation of the mutant Cl- channel CFTRAF508. CFTRAF508 is synthesized in the endoplasmic reticulum (ER), but its folding arrests at an unknown intermediate step and it is selected for degradation prior to passage to the plasma membrane (PM). Loss of CFTR function leads to defects in the hydration of mucosal layers that line glands and airways and chronic infections that death occurs due to lung failure. Hope for the treatment of CF comes from observations that folding defects in CFTRAF508 are rescued by compounds that either alter the cellular folding environment. This competitive renewal application for 2RO1 GM56981 seeks to aid in the development of therapeutics to treat Cystic Fibrosis by elucidating defective steps in the CFTRAF508 folding pathway and identifying ER quality control (ERQC) factors that select CFTRAF508 for degradation. The three major objectives of the proposal are as follows 1. We seek to define the mechanism by which mutations in CFTR cause it to misfold and be degraded the ubiquitin proteasome system. 2. CFTR is a polytopic protein that exposes surfaces in the ER lumen, ER membrane, and cytosol. 'Thus, we will identify the components of the ERQC machinery that sense the folded state of different sub-domains in CFTR. Then, we will determine how the action of the ER localized and cytosolic QC factors is coordinated. 3. We propose to apply the knowledge obtained in our basic studies to develop approaches to block the selection of CFTRAF508 for premature degradation and promote its proper folding. Overall, our studies will provide basic information on protein QC that will aid in the development of therapeutics to treat CF. [unreadable] [unreadable] [unreadable]"}
{"text": "Malignant gliomas are associated with high morbidity and mortality. Standard therapies such as surgery, radiation therapy and chemotherapy do not offer effective options and local recurrence is the norm. However, being locally malignant nonmetastatic tumors, they may be amenable to control using effective tumor-targeted local therapies. TNF- related apoptosis inducing ligand (TRAIL/Apo2L) induces apoptosis in gliomas in vitro and prolongs survival in xenograft rodent models. It is nontoxic to normal organs in animal models including primates. However, the efficacy of local intracranial delivery of TRAIL, its toxicity to brain-adjacent-to-tumor and selectivity towards gliomas in humans remain to be fully explored. In this study, we will test the efficacy and toxicity of TRAIL in a living human glioma & brain slice model and in xenograft models. Our preliminary data suggests that soluble TRAIL is active against gliomas in vitro and in vivo and that its activity is modulated by the Akt pathway. We hypothesize that: a) soluble untagged TRAIL will be effective against human glioma and nontoxic to normal brain 2) Inhibition of Akt can sensitize human gliomas to TRAIL and increase survival in a glioma model 3) resistance to TRAIL is mediated by differing mechanisms in normal and tumor cells permitting selective targeting of resistance mechanisms in tumors. To test these hypotheses, we propose the following specific aims 1) determine the efficacy and toxicity of soluble untagged TRAIL expressed by an adenovirus in a mouse intracranial xenograft and an ex-vivo living human glioma slice model. 2) determine the in vivo effect and relevance of Akt inhibition to TRAIL-induced apoptosis in gliomas. 3) determine the mechanisms of TRAIL resistance in normal brain and resistant glioma cells in human living tumor/ brain slice model and the effects of modulating these mechanisms on TRAIL sensitivity. The combined use of human living brain slices and intracranial xenograft models are aimed at providing direct and highly relevant data on the efficacy of TRAIL against human gliomas and its toxicity against the brain. The results of this study could also potentially provide a basis for clinical trials using TRAIL or analogues against gliomas and allow rational selection of agents to modulate the tumor resistance to TRAIL. [unreadable] [unreadable]"}
{"text": "This application attempts to clarify the mechanistic basis for the association between excessive adiposity and risk factors for prostate cancer-specific morbidity and mortality in men with clinically localized prostate cancer. The overall hypothesis is that excess adiposity alters prostatic exposure to nutritional, hormonal, and immunologic factors influenced by weight status and implicated in prostate cancer progression, and that inter- individual variation in gland-level exposure to these factors mediates the effects of adiposity on the pathologic presentation and course of the disease. To test this hypothesis, we will: Aim 1) conduct a prospective 2-year cohort study of incident cases of clinically localized prostate cancer to measure the association of adiposity with pathologic tumor features at diagnosis and 2-year risk of biochemical (PSA) failure after radical prostatectomy;Aim 2) measure fatty acids, insulin-like growth factor (IGF) axis activity, modulators of inflammation, and sex steroid hormone profiles in prostate tissue and periprostatic fat collected at surgery, and use mediation model analysis to determine which physiologic variables account for the effects adiposity on tumor characteristics and risk of biochemical failure;Aim 3) and evaluate the effects of post-treatment changes in adiposity on 2-year risk of biochemical failure. The cohort will consist of 540 men enrolled in the urology clinics of four Chicago-area medical centers who are awaiting prostatectomy for clinically localized prostate cancer. Adiposity will be quantified by anthropometry and dual energy x-ray absorptiometry at diagnosis and one year after surgery. Pathologic tumor characteristics will be determined by a single pathologist, and biochemical outcomes will be ascertained using a standard clinical protocol. Potential mediators to be studied include fatty acids, the IGF-1 receptor signaling pathway, adipose tissue-derived cytokines, eicosanoids products of arachidonic acid, and metabolites of estrogen and testosterone. Lifestyle factors that associate with more rapid progression of clinically early-stage prostate cancer are of increasing concern to health care providers and public health professionals. This research, to be completed over 5 years, will identify some of the biological reasons why obesity associates with more \"aggressive\" prostate cancer at diagnosis and decreases a man's chances for cure with surgery. It will also shed light on ways to prevent prostate cancer recurrence that may involve new or existing medicines or lifestyle changes. PUBLIC HEALTH RELEVANCE: Lifestyle factors that associate with more rapid progression of clinically early-stage prostate cancer are of increasing concern to health care providers and public health professionals. This research, to be completed over 5 years, will identify some of the biological reasons why obesity associates with more \"aggressive\" prostate cancer at diagnosis and decreases a man's chances for cure with surgery. It will also shed light on ways to prevent prostate cancer recurrence that may involve new or existing medicines or lifestyle changes."}
{"text": "Our long term goals are to understand the principles which underlie the information processing capabilities of neurons in the central nervous system. The goal of this proposal is to examine mechanisms of information processing in one region of the auditory brainstem, the dorsal cochlear nucleus (DCN). In the most superficial layer of this nucleus, a system of fine, unmyelinated parallel fibers (axons of granule cells) make numerous synapses on the principal projection neurons of the nucleus (pyramidal cells) and on small interneurons (cartwheel and stellate cells). The parallel fibers provide the substrate for unique and extensive interactions occurring orthogonal to the tonotopic axis of the DCN. Four specific hypotheses about the mechanisms of information processing by neurons in the mammalian DCN will be investigated. First, we will investigate the hypothesis that the activation of a specific subset of excitatory amino acid receptors, the N-methyl-D-aspartate (NMDA) receptors, can contribute to nonlinear facilitation of parallel fiber and auditory nerve inputs to pyramidal cells. Second, we will investigate the hypotheses that postsynaptic calcium entry associated with parallel fiber synaptic activity, either through NMDA receptors or voltage-sensitive calcium channels, can result in the long-term modification of synaptic or voltage-dependent conductances in postsynaptic cells. Third, we will test the hypothesis that the voltage- dependent discharge patterns of dorsal cochlear nucleus pyramidal cells are in part produced by a transient potassium conductance. Fourth, we will test the hypothesis that cartwheel cells are inhibitory to pyramidal cells. The experiments to test these hypotheses use intracellular, whole-cell tight-seal, field potential recordings, and optical recordings of calcium activity from an in vitro brain slice preparation, and whole-cell tight-seal voltage-clamp recordings from acutely isolated neurons. The results of these studies will provide important information about the function of the parallel fiber system in the cochlear nucleus, and will help to generate new theories about the role that this system plays in processing incoming acoustic information. These experiments will also set the stage for subsequent investigations of changes in central physiology that may occur as a function of peripheral acoustic trauma and aging."}
{"text": "Many theories have been proposed for the statistical mechanics of protein folding, typically derived from theories for less structured systems, such as random heteropolymers, diffusion-nucleation, random energies, or spin glasses. While these have certainly captured some important features of the physical chemistry of real proteins, such as cooperativity of folding and rapid folding from the random coil state, their derivation requires making some broad assumptions about the average behavior of polypeptides. In fact, the proteins of biological relevance consists of apparently very rare, moderately long amino acid sequences that permit the chain to fold rapidly- to a unique complicated native conformation, which depends greatly on the sequence. This suggests that theories focussing on average properties of long-chain heteropolymers may be over-generalizing and neglecting the important features of rare sequences folding to rare conformations. On the other hand, neither nature nor computer has sufficient time to exhaustively explore all conformations and all sequences for even small proteins. The idea here is to simply an otherwise realistic representation of polypeptides by reducing chain length, number of conformation states per residue, and choices of amino acid types until all sequences and all conformations can be exhaustively enumerated. By varying these parameters in the computationally feasible range, general conclusions can be detected and extrapolated to parameter values corresponding to real proteins. Since this model is so different from most theories, it is able to test their assumptions and conclusions about protein folding, such as the nature of the energy landscape and order parameters to describe the progress toward the native state. Questions to be addressed include: is there a general way to describe the folding of all proteins, or do some proceed by a recognizable pathway while others have innumerable routes? Can this model reproduce and explain the currently available experimental results on folding mechanisms and intermediates for certain particular proteins."}