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The purpose of this study is to estimate the effectiveness of AR-623 inducing a complete remission in first relapse acute promyocytic leukemia (APL) patients who have not received oral all-trans-retinoic acid (ATRA) within one year.	{ "pile_set_name": "NIH ExPorter" }
The overall goal is to develop a culturally congruent intervention for use in outpatient pharmacological practice by a psychiatrist that will increase retention of Hispanics with major depressive disorder (MDD) in antidepressant therapy. Despite major advances in the treatment of psychiatric disorders, Hispanics continue to underutilize mental health services relative to non-Hispanic Whites and to their own mental health need, as evidenced by lower rates of treatment entry and retention. Hispanic dropout from medication therapy for MDD attains rates two to three times those of non-Hispanic Whites. Mental health utilization in general is lowest among less acculturated, migrant Hispanics, even after controlling for socio- economic factors and psychiatric diagnosis, suggesting that cultural factors are important causes of underutilization. Our pilot work with depressed, culturally traditional Hispanics entering antidepressant therapy suggests that cultural incongruence between patients' and psychiatrists' expectations and concerns about treatment produces ambivalence about taking medication or following physician instructions, which in turn interferes with retention. This association between ambivalence about treatment and dropout is predicted by the "transtheoretical model of change", a schematization of the stages individuals experience in the process of behavior change. However, the validity of this model for Hispanics has not been empirically tested. Motivational Interviewing (MI) is an effective brief psychotherapy that has improved retention among patients with dually diagnosed depression and substance abuse by targeting ambivalence about entering and remaining in treatment. However, MI has not been adapted for Hispanic patients in antidepressant therapy. The specific aims of this study are to: a) culturally adapt and manualize MI for depressed Hispanics in antidepressant therapy; b) explore the efficacy of 4 sessions of this manualized intervention for improving retention during a 12-week open- label trial with a 2nd generation antidepressant and at 6-month follow-up among 65 Hispanics seeking outpatient medication therapy for non-psychotic MDD; c) explore the predictive validity of the "transtheoretical model of change" for treatment retention by examining the relationship between the retention measures and baseline stage of change and stage progression during treatment; d) examine the efficacy of MI for helping patients progress through the stages of change. This study will yield pilot data on the efficacy of a manualized cultural adaptation of MI for enhancing retention of Hispanics in antidepressant therapy and on the predictive validity of the "transtheoretical model of change" in this population. This pilot data will allow us to apply for an R01 to further study this intervention and model in a controlled clinical trial.	{ "pile_set_name": "NIH ExPorter" }
Rotaviruses have been studied extensively in many parts of the world predominately by cross-sectional aproaches. 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 information. 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. Anal swabs and blood specimens were obtained routinely from the youngest age groups, which ranged from 6 months to about 5 years of age during various periods of the study. Surveillance was carried out by a trained medical staff. Children were housed in cottages according to their age. Rectal temperatures were obtained on each child daily; routinely, a physician examined any child with a rectal temperature of 100.6 degrees F or greater. Careful medical records were kept by the nursing staff on all children. Thus, with this background, we plan to investigate the natural history of rotavirus infections in a longitudinal manner employing newly developed techniques such as serotyping of rotaviruses with VP7 specific monoclonal antibodies and determining the epitope-specific serologic response in sequential sera to determine the scope of homotypic and heterotypic responses. 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. In addition, there has been increased emphasis placed on the role of enteric adenoviruses and astroviruses as etiologic agents of gastroenteritis of infants and young children. With the ready availability of assays for detection of both of these groups of agents, we are planning to expand our longitudinal studies to evaluate the role of these viruses in the etiology of pediatric gastroenteritis. Also, the role of these agents will be examined in a cross-sectional manner by examining specimens from infants and young children hospitalized with diarrheal illness at the Children's Hospital National Medical Center, Washington, DC.	{ "pile_set_name": "NIH ExPorter" }
The central objective of this project is to elucidate the neuronal mechanisms of classical conditioning and sensitization. Furthermore, this project will seek to ascertain if postsynaptic neuromodulatory mechanisms elicited by tail nerve shock during classical conditioning interacts in any way with the activity-dependent associative component of classical conditioning. The neuronal circuitry underlying the behavioral plasticity of the Aplysia siphon withdrawal reflex is well defined and relatively simple. Also, the cellular mechanisms present at the sensorimotor synapse of the Aplysia share many of the same properties of CA3-CA1 synapses in the hippocampus. Both synapses have NMDA-dependent LTP and are influenced by neuromodulators, but the complexity of the mammalian system hinders connecting synaptic changes to the actual learning exhibited by the animal. The neural circuitry underlying classical conditioning in Aplysia is well defined and relatively simplistic, therefore synaptic changes are more readily attributed to the behavioral plasticity of the animal. The proposed experiments will increase our knowledge of synaptic plasticity and provide a basis for understanding the synaptic processes underlying human learning and memory. The increased knowledge may contribute to the understanding of human-memory diseases such as Alzheimer's and other related dementias. [unreadable] [unreadable]	{ "pile_set_name": "NIH ExPorter" }
The ultimate goal of treatment for Alzheimer's disease (AD) is to prevent degeneration of brain neurons and synapses. We screened a variety of generic drugs for neuroprotective effect using cell culture and animals models of AD immune pathology. One widely used generic drug with anti-inflammatory properties was found to be highly neuroprotective in cell culture and animal models. Although the same drug showed some clinical benefit, its level of effect was not sufficient to offer a useful therapy. For this reason, we sought to design more potent forms of this generic drug lead. A target discovery program has now identified 4 classes of analogs derived from the parent compound that show >20-fold increase in neuroprotective effect in vitro and improved disease-modifying action against AD pathology in vivo. Importantly, these novel analogs have been designed to eliminate the major dose-limiting toxic side effect of the parent drug. This Phase 2 SBIR proposal seeks to further optimize the analogs for brain penetrating capacity while retaining potency and margins of safety in vivo. Successful completion of Phase 2 will allow selection of a drug, and its backup, for final pre-clinical development. PUBLIC HEALTH RELEVANCE: At the present time there are no effective treatments for Alzheimer's disease. This dementia is a neurodegenerative disorder characterized by loss of memory, declining motor function, and behavioral changes. Death of neurons caused by toxins generated Alzheimer's disease in the brain is thought to be the cause of the dementia. More research is needed to develop effective treatments that will both reduce brain injury and improve intellectual functions in the Alzheimer's disease patient. The proposal submitted here encompasses pre-clinical research on compounds that may help neuron survival and brain function. Exploration of these agents should accelerate the national effort to identify therapies for the prevention of dementia and /or the improvement cognitive function in the Alzheimer's Disease population. .	{ "pile_set_name": "NIH ExPorter" }
This is a phase I study for patients with intrahepatic cancers. Treatment involves a dose-escalating continuous intravenous infusion of Fdurd combined with hepatic radiation. The radiation is given in identical fashion to CRC 1085.	{ "pile_set_name": "NIH ExPorter" }
The Fragile Families and Child Wellbeing Study is a longitudinal birth cohort study of approximately 3700 unmarried parents and 1200 married parents and their children. We seek funding to: 1) perform assessments at age 9 of children's health, language ability, academic achievement, behavior problems and social relationships;2) conduct interviews with children's biological parents, to update information on family resources and parental relationships;3) collect saliva samples for genotyping from primary caregivers and children;4) Interview social parents who live with the child, and;5) collect information from children's teachers on child outcomes and school characteristics. These data will allow researchers to answer the following questions: How do children develop over time, and how do family resources influence children's health and development? How do the resources of unmarried parents evolve over time, relative to those of married parents? How do children's genetic endowments interact with their environments to influence their outcomes? What role do social (non- biological) parents play in the lives of children? How do school environments influence children's social and academic outcomes? The study includes a large number of children and adults who are: poor, members of racial and ethnic minority groups, immigrants and disproportionately affected by welfare, child support, child care, and incarceration policies. The children have diverse and often unstable family structures and child care arrangements. Thus, the sample is well-suited to study how multiple sources of disadvantage influence child health and development. The data are also unique in containing detailed information on mothers, fathers, and their relationships with each other. The information on unmarried fathers, in particular, allows us to follow a group of men that is often under-sampled in national surveys.	{ "pile_set_name": "NIH ExPorter" }
Background. Children up to the age of 5 years affected by HIV are the most vulnerable subgroup of HIV populations globally, especially in low-resource areas. This is because of the strategic, volatile, and vulnerable nature of this highly sensitive period of child development. Mediational intervention for sensitizing caregivers (MISC) has a structured training program to enable caregivers to improve their children's cognitive and social development during everyday casual interactions in the home. In our preliminary NIMH R34 findings, Ugandan HIV children of caregivers receiving MISC training biweekly for a year showed significantly greater gains on the Mullen Early Learning Scales Composite of g fluid intelligence, when compared to children whose caregivers received a standard health/nutrition education intervention (treatment as usual or TAU). The MISC caregivers were also significantly less depressed, and their child mortality that year was significantly lower. Intervention Method. One hundred Ugandan HIV-positive preschool and 200 HIV orphan caregiver/child dyads will be enlisted from Kayunga and Pallisa Districts. These dyads will be randomly assigned by village clusters to either biweekly MISC or health/nutrition education TAU intervention for one year. Child Outcomes are the child development gains on the Mullen, the Early Childhood Vigilance Test (ECVT) of attention, and the Color- Object Association Test (COAT) of memory, the Behavior Rating Inventory of Executive Functioning - Preschool (BRIEF-P), and the caregiver administered version of the Achenbach CBCL. Caregiver Outcomes include an array of emotional wellbeing (EWB) and daily functioning measures validated during the initial qualitative study phase. Study Aim 1 will evaluate if MISC significantly enhances child outcomes when compared to controls for both HIV-positive and orphan children when assessed from baseline to 6, 12, and 18 months. Study Aim 2 will evaluate if MISC significantly enhances caregiver EWB and daily functioning outcomes. To better understand the mechanisms of MISC-enhanced child development, a Secondary Aim is to evaluate the mediating effect of improved caregiver EWB outcomes on corresponding child development gains, and the modifying effects of caregiver HIV illness and functioning on child outcomes. The Overall Impact comes from establishing the feasibility, acceptability, and effectiveness of MISC for HIV orphans and vulnerable children (OVC) and their caregivers in low resource settings;the sustainability of MISC in low resource settings since it is not dependent on published materials or outside resources;the complementary dual impact of significant psychotherapeutic benefit for the caregiver, especially mothers struggling with HIV disease. MISC will also reduce HIV child mortality because in our initial R34 findings, MISC heightened maternal bonding, sensitivity to serious illness, and the prompter seeking of medical care. It also can improve treatment adherence. Finally, our evidence-based MISC caregiver training interventions can be readily implemented globally as a sustainable way to augment OVC cognitive, psychosocial, and medical wellbeing. PUBLIC HEALTH RELEVANCE: Early childhood (up to age 5 yrs) is a period of dramatic change in the cognitive, emotional, social, and behavioral domains;children continuously progress by observing and interacting with the world around them. In the face of economic instability and nutritional, medical and educational deprivation, HIV-affected very young children are the most vulnerable HIV subgroup globally because their families are often the most vulnerable, with little margin for sustaining a favorable developmental milieu for the child. Through strategic caregiver interventions during this sensitive period of child neurodevelopment, our study findings have the potential for positively re-directing the developmental trajectories of tens of millions of HIV-affected children globally.	{ "pile_set_name": "NIH ExPorter" }
GABA-modulin, an endogenous membrane protein, which inhibits non competitively 3H-GABA binding to synaptic plasma membranes, has been isolated and purified to homogeneity using acidic extraction followed by Sephadex column purification and HPLC. GABA-modulin is a peptide of 18,000 MW which inhibits the binding of 3H-GABA and the GABA-induced stimulation of 3H-diazepam binding to synaptic membranes. The role of GABA-modulin in the control of GABA receptor system function is presently being investigated.	{ "pile_set_name": "NIH ExPorter" }
The perifornical hypothalamus (PFH) includes a large population of hypocretin (Hcrt) neurons intermingled with melanin-concentrating hormone (MCH) and other cell types. At present, there are no validated electrophysiological criteria to distinguish Hcrt cells in the PFH during extracellular recording in freely moving animals from other intermixed cell types. Recent electrophysiological studies performed in slices from mouse hypothalamus have shown that Hcrt cells can follow higher frequency electrical intracellular stimulation than adjacent non-Hcrt cells. Our pilot studies in anesthetized and freely moving rats revealed two subpopulations of hypothalamic cells which had different latencies and maximal frequencies of antidromic spikes during LC train stimulation. The goal of the proposed project is to obtain specific parameters of antidromic responses of Hcrt and adjacent cells, which will be labeled juxtacellularly in anesthetized rats with subsequent double immunostaining, to determine characteristics sufficient for identification of Hcrt cells in the freely moving animals. Proposed experiments will answer the following questions: 1) Do Hcrt, MCH and other perifornical cell types, which project to the LC, have specific electrophysiological characteristics? 2) What are the distribution of these Hcrt, MCH, and other cell subpopulations? 3) What are the discharge patterns of identified Hcrt and MCH cell subpopulations across the sleep-wake cycle? Our studies will lay the foundation for future studies and will provide the first data correlating behavior and discharge patterns of Hcrt and MCH cells. A better understanding of the physiological and behavioral role of these cell groups would have implications for development of effective treatments for obesity, depression, narcolepsy and other sleep disorders.	{ "pile_set_name": "NIH ExPorter" }
The Specific Aim of this proposal is to test the feasibility of using an orally available small molecule (FGH10019) to reduce obesity. Obesity contributes to the prevalence of many disease states and accounts for more than 25% of all health care costs in the US according to recent market reports. Prescription pharmaceuticals account for less than 1 percent of the total obesity market because of limited efficacy, poorly understood biology and significant safety issues. We have discovered a diarylthiazole compound that inhibits insulin-mediated adipogenesis by blocking SREBP-1 and 2. Administration of the compounds to obese ob/ob mice led to weight loss, marked reduction of visceral fat, increased insulin sensitivity, and lower blood glucose levels. We have since improved the drug properties of this original compound. Our current lead compound is FGH10019, which has higher potency in blocking SREBP-1 and 2, has improved oral bio-availability in mice, and other improved drug like properties. FGH10019 is effective in reducing weight and improving hyperglycemia in Ob/Ob mice. However, before moving to IND enabling studies, must evaluate this compound in a small animal model that more closely resembles human obesity. In this Phase I study, we will therefore test efficacy of our compound in a high fat diet rat model. To this end, we will carry out the following Tasks: Task 1: Evaluate maximum tolerated dose (MTD) by oral gavage of FGH-10019 to Sprague-Dawley rats. Task 2: Evaluate dose to reduce weight gain and lower triglycerides, LDL and blood glucose. Test of Feasibility: we must observe: 1) MTD at or greater than 100 mg/kg; 2) significant effective dose at 1/10 or 1/5 the MTD and we must observe reduced weight by 8%, due to decrease in fat content, improved hyperglycemia and lowering LDL and TG by more than 25%, without affecting HDL. In Phase II, we will move FGH 10019 toward IND enabling studies and search for improved back-up compounds. PUBLIC HEALTH RELEVANCE: Obesity and related disorders currently account for almost 25% of the total healthcare budget and the problem is getting worse as rates of obesity continue to increase. Diet and exercise are the mainstays of weight loss programs, but in most cases they are not sufficient to address the problem. Prescription drugs are generally of limited use and they can exhibit serious side effects. This is a proposal to develop a truly effective drug tha acts through a novel mechanism for effectively treating obesity and related disorders.	{ "pile_set_name": "NIH ExPorter" }
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 Metal Analysis Core (MAC) has several goals. It aims to provide analytic capability in metal analysis to the ND INBRE partners and faculty in all participating institutions within North Dakota. It also strives to provide technical and professional expertise in metalomics and ionomics to the ND INBRE partners and faculty in all participating institutions of ND. It is also actively developing and establishing an educational network in metalomics and ionomics to serve the post-graduate, graduate and undergraduate students comprising the ND INBRE network. Lastly, it is developing the professional and technical relationships with divisions within the North Dakota Department of Health as metalomics and ionomics applies to the environmental quality of the air, water and soil in ND.	{ "pile_set_name": "NIH ExPorter" }
This project is designed to provide information about the organization of neuronal systems in the mammalian spinal cord that are involved in the neural control of movement, as studied in adult cats in vivo and in the isolated brain stem and spinal cord of neonatal mice studied in vitro. Adult cats are used to examine the organization of specific sets of last-order interneurons in reflex pathways that project directly to motoneurons. Modulation of synaptic potentials produced in motoneurons by input from a variety of afferent systems allows inferences about the convergence onto pathway interneurons of control inputs from primary afferent, supraspinal descending systems, and the spinal central pattern generators (CPGs) that generate rhythmic motoneuron output during fictive locomotion and scratching. Identifiable groups of last-order interneurons receive input from specific low-threshold cutaneous afferents and produce disynaptic EPSPs or IPSPs in specific groups of lumbosacral motoneurons. These disynaptic pathways are powerfully modulated by the spinal central pattern generator (CPG) for locomotion during fictive stepping in decerebrate animals. The specific patterns of CPG modulation in different motoneuron species allow identification of specific sets of segmental interneurons, each with its own patterns of convergence from input control sources. We have found that these patterns of reflex pathway modulation provide important markers for specific states of the locomotor CPG. During FY1997, we compared the control of cutaneous and muscle afferent reflex pathways during fictive locomotion and fictive scratching. To our surprise, we found that oligosynaptic cutaneous pathways from the distal hindpaw, that are markedly facilitated during certain parts of the step cycle in fictive locomotion, are profoundly depressed throughout fictive scratching. In contrast, disynaptic excitation and inhibition produced by group I muscle afferents are powerfully modulated during both fictive locomotion and scratching. In general, disynaptic group I excitation is maximal when the motoneurons are depolarized and active. There was one interesting exception to this pattern. The motoneurons mechanical synergist muscles flexor digitorum longus (FDL) and flexor hallucis longus(FHL) receive mono- and disynaptic group I EPSPs from muscle spindles in either muscle. The disynaptic group I EPSPs in both sets of motoneurons are facilitated during the extension phase of fictive locomotion but during the flexion phase of fictive scratching. The FHL motoneurons are depolarized and active during the extension phase of both movements, while the FDL cells are depolarized and active during the flexion phase. The data suggest that a common set of excitatory last-order interneurons mediates between group I afferents from both muscles and projects to both sets of motoneurons.	{ "pile_set_name": "NIH ExPorter" }
Study will examine how breast-feeding and bottle-feeding a baby affects the nervous system of the mother. Mothers will at one time breast feed then at another time, bottle feed their infant while instruments for non-invasive psychophysiological measures of blood pressure and heart rate. The degree of variability in these measures will be used as an indices of sympathetic and parasympathetic modulaltion of the cardiovascular system.	{ "pile_set_name": "NIH ExPorter" }
Sixteen clinical genetics services in the Washington, DC and Baltimore areas were visited in order to establish potential training sites for genetic counseling practicums. Relationships with counselors interested in teaching and curricula development were also sought.	{ "pile_set_name": "NIH ExPorter" }
Informed consent is an important component of ethical research. Unfortunately, data show that research participants do not always understand key features of the research they participate in. At the same time, consent documents are increasingly becoming longer, more technical, and complex. This project aims to test a simpler, more concise consent form against the standard consent form in a variety of research projects. Both consent forms contain all the elements required by federal regulations, and are approved by an IRB. Participants in these trials are randomized to receive either the standard or the concise consent form and then asked to complete a questionnaire that tests their comprehension of study information and their satisfaction with the process. Our hypothesis is that comprehensionn will be similar in both groups and satisfaction higher in the group that receives the concise form. If the data support our hypothesis, investigators and institutions may be encouraged to write forms that are easier to read and shorter. Two studies with healthy volunteers are complete and published. The data from a third enrolling patient volunteers from around the world is being analyzed and a manuscript will be submitted soon.	{ "pile_set_name": "NIH ExPorter" }
Linear directed movement, such as seen in muscle contraction, cell motility, and intracellular trafficking, is one of the fundamental characteristics of life and is mediated primarily by the myosin, kinesin, and dynein superfamilies of motor proteins. These proteins utilize the free energy of the hydrolysis of ATP to generate molecular movement. At this time, the fundamental structural folds of myosin and kinesin are known. However, many questions remain about the molecular mechanism by which the free energy of hydrolysis of ATP is converted into directed movement. The major goal of this proposal is to understand the structural and functional role of communication pathways in myosin and the minus-end directed motor Kar3. The first objective is to construct and determine the structure of a complex of actin and myosin. A knowledge of the structure of actomyosin is needed, not only to complete the structural picture of motility, but also to understand the structure of the actin filament. The proposed studies will provide insight into the fundamental mechanism of energy transduction in myosin and establish a molecular framework for the development of new small ligands to control the interaction between actin and myosin. The second major objective is to understand the molecular mechanism for Kar3 which belongs to the Kinesin-14 class of motors. Members of this class are minus-end directed, non processive kinesins that operate with a powerstroke mechanism. Kar3 is unusual in that it functions as a heterodimer, forming a complex with either one of the non-motor proteins Vik1 or Cik1. We have recently been shown that Vik1 and Cik1 contain a globular domain that exhibits the same fold as the kinesin motor domain. This domain binds tightly to microtubules but does not hydrolyze ATP. This phenomena demands the existence of intermolecular communication between the motor and non motor domains in order to generate movement. The immediate goal is to understand the nature of the interaction between the Kar3 motor domain and the Vik1 and Cik1 motor homology domains and how this facilitates their biological function. Kar3/Vik1 and Kar3/Cik1 represent an extreme example of asymmetry between motor domains in dimeric kinesins, however, functional asymmetry between kinesin motor domains is observed in other non-processive dimeric kinesins, so that these studies will provide insight into a fundamental phenomena. These investigations will utilize a combination of X-ray crystallography, electron microscopy, kinetic analysis, and functional assays to give a comprehensive view of the structural transitions in these molecular motors.	{ "pile_set_name": "NIH ExPorter" }
We wish to test the hypothesis that the development of B cell lymphomas in humans represents a multistep process which begins in the bone marrow. This idea stemmed from studies of B and T cell malignancies in chickens and mice, and we have obtained support for it in previous studies of humans with pre-B, B and plasma cell malignancies. It contrasts with the prevailing view that B cell lymphomas arise in the lymph node; the nodular lymphomas having as their origin a subpopulation of target cells in germinal centers. In the present studies we will examine the extent of clonal involvement in patients with nodular B cell lymphomas. Cell hybridization techniques will be used to produce two types of monoclonal anti-idiotype (Id) antibodies: One specific for idiotypic determinants expressed by complete immunoglobulin molecules and others specific for V(H) idiotopes expressed on the isolated Mu heavy chains made by the B lymphoma. The former will be used to trace the distribution of the B cells belonging to the neoplastic clone in lymph node, blood and bone marrow tissue samples. The anti-V(H) Id will be used to search for members of the neoplastic clones among the pre-B cell population in the bone marrow. After determining the extent of clonal involvement, neoplastic cells and normal B cells will be isolated from each of the involved tissues and pre-B cells isolated from bone marrow. These subpopulations of cells will then be separately examined by cytogenetic techniques for chromosomal abnormalities, and restriction fragments of the cellular DNA will be analyzed with heavy and light chain gene cDNA probes to determine immunoglobulin gene rearrangements and clonality. Other studies will address the expression of cellular oncogenes in the different subpopulations. In this way we hope to learn more about the origin and the steps involved in the development of B cell lymphomas.	{ "pile_set_name": "NIH ExPorter" }
The specific aims of IDPH in this project to develop and implement an enhanced Pesticide Poisoning Surveillance Program are to: 1. Establish and maintain a scientific advisory committee that includes appropriate state partners, local public health agencies, and other stakeholders to identify relevant Iowa-specific issues and priorities for pesticide poisoning surveillance. 2. Collect and investigate reports of pesticide poisoning. 3. Analyze pesticide poisoning surveillance data. 4. Publish pesticide poisoning surveillance results, interpretations, and conclusions. 5. Conduct an annual evaluation of the accomplishments and impact of the pesticide poisoning surveillance program. The mission of the Iowa Department of Public Health (IDPH) is to promote and protect the health of lowans. IDPH is committed to improving the hearth of all lowans through the delivery of the core functions and essential services of public health. Goal Statement 6-13 and Action Steps 6-13.1 to 6-13.3 in Healthy lowans 2010 (IDPH 2005) establish the reduction of pesticide poisoning in Iowa as a priority for IDPH in its mission to promote and protect the health of lowans. Funding of the enhanced surveillance program will allow IDPH to conduct the following activities: Identify clusters/outbreaks and groups at high risk for pesticide-related illness/injury. Identify high-risk pesticide active ingredients and products associated with pesticide-related illness. Target regulatory, enforcement, consultative, or educational interventions to prevent and control pesticide-related illness/injury. Evaluate the effectiveness of prevention efforts. Data from the National Agricultural Statistics Service of the U.S. Department of Agriculture show that for 1999 to 2002, Iowa was second only to Illinois in pounds of herbicide applied to com, and first among states in pounds of herbicide applied to soybeans. Statistics are not available for the pounds of herbicides, insecticides, and fungicides applied to turf, golf courses, gardens, homes, and fruit and vegetable production However, the pounds of herbicide and pesticide applied to Iowa corn and soybeans alone are sufficient to show that the surveillance of pesticide poisoning should be a priority for Iowa. [unreadable] [unreadable] [unreadable] [unreadable]	{ "pile_set_name": "NIH ExPorter" }
Using phage-displayed random peptide libraries, our goal is to develop tumor-targeting ligands for cancer therapy. The approach is administer the phage library intravenously, surgically sample tumor tissue, and collect phage which has bound to the tumor. Preclinical cytotoxicity testing has been performed and the FDA has issued an IND to proceed with human studies. The rationale for this approach is based on published experiments in animals that resulted in identification of ligands to vascular targets that appear tumor specific and our own preclinical feasibility testing in animal models demonstrating a good safety profile. The benefits of performing this procedure in human cancer patients include the presence of a vast number of possible targets which are otherwise not available in vitro, targets will be in their native configuration, subtraction of the pools of ligands binding to normal tissue, identification of peptide ligands which will be stable in blood, the possibility of identifying unique targets for an individual patient, and the rapidity of the process. Hypotheses: 1) Serial administration of a phage-displayed random peptide library (RPL) is nontoxic to cancer patients. 2) Phage collected from tumor deposits in patients following systemic administration of RPL will containtumor selective peptide ligands. The Specific Aims are 1) Intravenously administer a naive RPL library to cancer patients, surgically harvest phage-bearing tumor nodule, and collect phage from tumor sample. This process will be repeated twice with infusion of phage that bound to tumor. 2) Establish safety of serial systemic administration of naive and enriched RPL collected from patient tumor deposits. 3) Determine peptide sequences of phage collected from harvested tumor specimens. 4) Determine whether candidate clones bind selectively to tumor tissue specimens. Successful completion of this clinical study will result in demonstration of a satisfactory safety profile and short peptides that bind selectively to tumor tissue.	{ "pile_set_name": "NIH ExPorter" }
The molecular mechanisms of resistance to viral entry fusion inhibitors targeting HIVgp41 are not well-understood. Fundamental gaps in knowledge of the energetic and structural interactions which drive binding hamper the long-term goal of development of new drugs with improved resistance profiles. The overall objective of this application is to (1) develop computational structural models to quantify binding for known gp41 fusion inhibitors (both peptides and small molecules), (2) characterize origins of resistance profiles to current inhibitors, and (3) discover new small molecule drug-leads. Based on strong preliminary results, the central hypothesis is that specific interactions within a conserved hydrophobic pocket on gp41, not exploited by the only currently available anti-fusion drug (peptide inhibitor T20), confer improved resistance profiles to next-generation peptide inhibitors and drive binding for small molecule inhibitors. The rationale for the proposed research is that robust computational models allow drug binding to be fully characterized at the atomic level, and this will enable development of HIV drugs with favorable resistance profiles. Thus, the work proposed is directly relevant to the NIH plan for basic and applied research towards discovery and development of novel agents and therapeutic strategies directed against viral factors involved in HIV replication and persistence. The work employs all-atom computer simulations (molecular dynamics and docking), in conjunction with detailed energetic and structural analysis, to test the central hypothesis and accomplish the goals set forth in each specific aim. Aim #1 will determine the molecular basis of resistance to current peptide fusion inhibitors of gp41 to test the hypothesis that binding affinity for T20 is driven primarily by interactions with mutation-prone regions along the binding interface. Aim #2 will characterize the mechanism of action for reported small molecule inhibitors of gp41 which we postulate are due to specific energetic and structural interactions modulated within the conserved pocket. Aim #3 will identify new small organic molecules, which bind specifically to the gp41 pocket, using virtual-high-throughput-screening in conjunction with experimental validation. Active compounds will be characterized structurally using NMR and X-ray crystallography and developed further. The proposal's contributions are significant because results from detailed binding models and computer simulations will allow the molecular basis of recognition to be delineated, which will enable development of improved fusion inhibitors that maintain activity against clinically relevant HIV escape mutations. PUBLIC HEALTH RELEVANCE: Results from the proposed research will be used to uncover the atomic-level structural and energetic determinates which describe binding of membrane fusion inhibitors with the viral entry protein gp41 which mediates HIV infection. The proposal seeks to understand the origins of resistance to gp41 inhibitors, and develop new compounds with improved resistance profiles, thus the finding are expected to be of direct relevance to public health.	{ "pile_set_name": "NIH ExPorter" }
(1) To characterize and purify anti-desmosine antibodies and to develop sensitive immunological assays for monitoring urinary desmosine excretion in papain-treated hamsters and human emphysema subjects. (2) (a) To repeat experiments on enhancement of elastase secretion in cultured mouse periotoneal macrophages exposed to aqueous cigarette smoke solution, using alveolar macrophages (mouse). (b) To further characterize the purified mouse peritoneal macrophage elastase (determine MW, antigenic properties and reactions with endogenous proteinase inhibitors). (3) To examine (a) elastase: alpha 1 AT complexes in sera and lavage fluids of smokers for evidence of incomplete inactivation of the protease; and (b) levels of anti-oxidants (GSH-peroxidase, SOD) in cells and fluids of cigarette smokers with emphysema. (4) To test the effect of alpha 1 AT peptide fragments on alpha 1 AT synthesis in an intact animal model (rat) and in cultured human liver cells (SK-Hep-1), using homologous alpha 1 AT and immunochemical techniques. (5) To screen normal dog lungs for evidence of extracellular leukocyte elastase deposits, using antibody to dog leukocyte elastase and immunoperoxidase staining methods. The results will be correlated with parallel studies on human lung which were completed in the -07 grant period. (6) To repeat and confirm the demonstration of alpha 2 MG synthesis by cultured human alveolar macrophages.	{ "pile_set_name": "NIH ExPorter" }
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. Attention is a critical factor in sensory and cognitive performance. Problems in controlling and directing attention can lead to serious deficits in performance and behavior. More complete information about the neural mechanisms underlying attention will be important for understanding how it facilitates perception and guides behaviors. Our experiments will extend our understanding of the neuronal basis of attention by testing the hypothesis that attentional modulation depends on the same neuronal circuits that mediate sensory response normalization. Response normalization is a well-established mechanism that adjusts sensory signals based on the overall activity in a large group of neurons with similar response properties. We propose that attentional modulations in visual cerebral cortex act through this normalization mechanism, such that attentional modulation cannot occur in conditions when normalization is weak or eliminated. These experiments will provide a critical test of the hypothesis that attention depends on normalization mechanisms. In doing so they have the potential of greatly extending our understanding of the neural mechanisms that underlie attention.	{ "pile_set_name": "NIH ExPorter" }
The main objective of this application is to establish the functional significance of a novel type of aging-related fibroblast- or muscle-specific mtDNA point mutations, which accumulate with extraordinary nucleotide selectivity in the majority of old individuals above a certain age at critical control sites for mtDNA replication. In particular, it is planned to use cellular models of these mutations, constructed by mitochondriamediated transfer of the mutations into mtDNA-less (px) cells, in order 1) to determine in a definitive way whether these mutations have a positive or negative effect on mitochondrial function, by comparing, at the biochemical and bioenergetic level, cell lines carrying a given mutation in 100% or 0% of mtDNA in the same mitochondrial genome and in the same nuclear background; 2) to determine whether these mutations increase or decrease the rate of mtDNA synthesis and affect its initiation at different heavy-strand synthesis origins; 3) to establish whether any identified replicative advantage of the mtDNA harboring these mutations involves preferentially molecules structurally intact and functionally normal or molecules structurally damaged and functionally deteriorated. Furthermore, we will determine whether the muscle-specific mutations arise and/or expand in the differentiated fibers or in undifferentiated muscle precursor cells (satellite cells). It is also planned to investigate the role of the nuclear background and mtDNA haplotype in the observed effects, by analyzing mitochondrial transformants constructed with genetically different px cell lines and different mitochondria donors, to determine the role of the in vivo environment in the appearance and/or expansion of the fibroblast-specific T414G mutation, and to correlate the effects of the mutations in cellular model systems with their effects in ex vivo tissue or cells. The achievements of the above aims will have important implications for understanding the role of these novel mtDNA mutations in aging, as well as their possible involvement in aging-related degenerative diseases.	{ "pile_set_name": "NIH ExPorter" }
Several potential metabolites and transformation products of THP, a urinary excretion product in patients under L-dopa therapy have been prepared and are being evaluated. THP is unstable at pH above 7 and converted into dibenzopryrocolines and dimers of this system. The first dibenzopryrocoline dimer has been recognized and characterized.	{ "pile_set_name": "NIH ExPorter" }
Inhibitory interneurons in the hippocampus regulate pyramidal cells and prevent hyperexcitability that leads to epileptiform activity. Glutamatergic transmission onto interneurons activates these cells to drive these functions. Recently, it has been shown that hippocampal interneurons express the kainate subtype of ionotropic glutamate receptor, and these kainate receptors (KARs) are synaptically activated. Postsynaptic KARs on interneurons contribute to the excitatory postsynaptic potential (EPSP). KARs on interneurons also depress the release of GABA from interneurons onto pyramidal cells, although the mechanisms underlying this effect remain unclear. These two actions suggest that interneuronal KARs play a major role in the control of inhibitory activity and output in the hippocampus, and possibly represent a therapeutic target to limit hyperexcitability during epilepsy. However, at present it is not possible to critically evaluate this possibility, because insufficient information is available about the functions of interneuronal KARs, the mechanisms by which these functions can be regulated, or the mechanisms by which KARs regulate GABA release. This proposal will seek to address these gaps in our understanding of KARs on interneurons. Using whole-cell patch clamp techniques, the activity of KARs on interneurons will be recorded and manipulated using pharmacological tools. Three specific aims will be addressed. (1) Functions for postsynaptic KARs will be identified, by testing four hypotheses: (a) that KARs are calcium-permeable and can initiate calcium-dependent signaling; (b) that KARs are segregated to different afferent pathways than AMPA receptors; (c) that KARs allow interneurons to perform temporal integration at low afferent firing frequencies; and (d) that these three functions are differentially distributed among different interneuronal subclasses. (2) Mechanisms of regulating the KAR-mediated EPSP will be identified, by testing two hypotheses: (a) that the KAR-mediated EPSP is regulated by continuous receptor delivery to, and removal from, the synapse; and (b) that the KAR-mediated EPSP is subject to activity-dependent synaptic plasticity. (3) Mechanisms underlying the presynaptic actions of interneuronal KARs will be identified, by testing two hypotheses: (a) that glutamate can activate presynaptic KARs that directly regulate GABA release; and (b) that the depression induced by KARs is an indirect consequence of interneuronal spiking. These experiments will provide information about the role of interneuronal KARs in the hippocampus, and provide a rational basis for future experiments to assess the possibility of manipulating these KARs to control hyperexcitability.	{ "pile_set_name": "NIH ExPorter" }
The overall goal of this project is to improve the interpretation of magnetic resonance spectroscopy (MRS) data from non-enhancing glioma of various histologic subtypes in an effort to improve the therapeutic planning for patients harboring such tumors. Non-enhancing glioma are most often diagnosed as Grade II, half of which progress to Grade III histology at recurrence. Previous studies by our group have shown MRS to be effective for directing biopsies to the most proliferative and cellular tumor regions that are most likely to contain Grade III foci. Additional studies by our group and others have shown that both the MR spectroscopic and molecular genetic features of purely astrocytic tumors are distinct from those of oligodendroglial tumors. The interesting aspect of such findings stems from the fact that patients with oligodendroglioma (OD) enjoy a more favorable outcome than patients with astrocytoma. The current proposal seeks to evaluate the effectiveness of MRS for post-surgical prediction of the behavior of residual tumor. Particular attention will be paid to tumors with mixed histology (oligoastrocytoma, OA), which may have genetic features and clinical course similar to AS or OD and, therefore, are particularly difficult to treat. The aims of this project will test the following hypotheses: (1) reductions in the creatine+phosphocreatine peak and elevations in the peak corresponding to lactate are observed more often in Grade II OD and Grade astrocytoma (AA) than in Grade II astrocytoma (AS), (2) MRS markers that correlate with prognostic markers for astrocytoma will be different than those that correlate with prognostic markers for oligodendroglioma, and (3) the MRS signature of mixed OA can be used as a surrogate marker for the molecular characteristics of the tumor. We will test these hypotheses by acquiring 3D-MRS data from patients prior to surgical resection and comparing the levels of the aforementioned metabolites within the various histologic subtypes. To more precisely associate metabolic features with the underlying biology, we will also use HRMAS MRS to correlate the MRS parameters with molecular markers of malignant activity (eg MIB-1 and TUNEL) and genetic markers of tumor type (1p/19q chromosomal deletions) and malignant progression (9p and 10q deletions) in tissue biopsies extracted from the tumors of the patients. We will also attempt to classify the OA biopsies based on their molecular, genetic, and MRS features.	{ "pile_set_name": "NIH ExPorter" }
The overall goal of this proposal is to develop the ability to detect and quantitate changes in gene expression in real-time and with a degree of sensitivity suitable to monitor minor changes at a single cell level. This will have considerable value in basic biomedical research, disease mechanism studies and diagnosis, and drug discovery. This revised proposal centers on the use of molecular beacons (MBs) for the analysis of gene expression in living cells. In particular, it focuses on the further development of MBs for studying breast cancer cells and single neurons. During our previous research in studying mRNA expression in single cells, we have encountered several limitations of MBs for cellular analysis. These included susceptibility to degradation by nucleases and single strand binding proteins, incomplete quenching of the fluorophore, and weak fluorescence intensity of conventional fluorophores. The autofluorescence of the cells also proved to limit the sensitivity of MBs for the detection of genes with a low expression. To overcome these limitations, we have proposed many novel solutions including superquenching moieties, bright amplifying fluorescent polymers, and molecular beacons comprised of locked nucleic acid bases. While these advances have not been tested in vivo they have shown promise in solution experiments. In addition to improving MBs as effective molecular probes for intracellular monitoring, we will also integrate advanced imaging methods into our analysis. In our previous results, we have demonstrated the advantages of confocal imaging when applied towards intracellular analysis. In this proposal we plan to demonstrate the advantages of multiple photon imaging and fluorescence time resolved imaging for more sensitive detection of MBs by removing the biological background. Once the MBs are improved and the new techniques are developed we will focus on intracellular applications of MBs for the detection of mRNA using these advancements. Currently, several genes, MnSOD, Cyclin D1 and survivin, have been identified as cancer markers and have been studied in cancer genomics. Using MBs, we will focus on elucidating the effects that different anti-cancer agents have on these genes in different human breast carcinoma cell lines. Given the genetic foundation of learning and memory, we will also use MBs to explore the effects of extracellular stimuli on the gene expression in neurons. The study will focus on EGR1 and Synaptotagmin IV, both of which have been linked to different stages of learning and memory.	{ "pile_set_name": "NIH ExPorter" }
Cytochrome c oxidase is an oligomeric protein complex isolated from the inner mitochondrial membrane of eucaryotic organisms. It is responsible for the terminal step in electron transport from cytochrome c to molecular oxygen. The energy derived from this electron flow is coupled to the conservation of energy in the form of adenosine triphosphate through oxidative phosphorylation. As such, cytochrome c oxidase is an enzyme of immense importance and plays a key role in cell respiration. This project will provide structural information about the three-dimensional arrangement of cytochrome c oxidase through the measurement of distances between protein sites, lipid sites and substrate sites in wild-type and functional structural mutants of cytochrome c oxidase isolated from yeast. The immediate objectives of the proposal are: (1) to chemically modify specific cytochrome c oxidase sites with fluorescent and nonfluorescent chromophores; (2) to reconstitute these labeled cytochrome c oxidase molecules into defined phospholipid vesicles; (3) using the technique of fluorescence resonance energy transfer, measure the spacial relationships between these labeled sites, intrinsic chromophores and covalently attached substrates in isolated and vesicular cytochrome c oxidase; (4) to determine the changes in these spacial relationships in functional mutants of cytochrome c oxidase which contain a single altered polypeptide chain; and (5) correlate these special relationships using reversible crosslinking reagents. The end result of this investigation will be a detailed structural map of the three-dimensional arrangement of the subunits of cytochrome c oxidase with relation to the subunits themselves, to intrinsic heme groups, substrate and to lipid environment. In turn, the study will contribute information about the mechanism of cytochrome c oxidase electron flow as well as mitochondrial membrane biogenesis.	{ "pile_set_name": "NIH ExPorter" }
Project Summary Good control of posture and orientation is vital for animals as they make movements or navigate the environment. Vertebrates rely on the vestibulospinal system to translate gravity sensations from the inner ear into appropriate compensatory trunk (axial) and limb movements to stabilize and orient themselves. Although this system exists in all vertebrates and is crucial for survival, research on it has languished due to the technical difficulties in recording from vestibular and spinal neurons, especially during animal motion. My long-term goal is to define the means by which vestibular and cerebellar pathways influence spinal circuit activity patterns to fine-tune behavioral outputs. The objective of this proposal is to determine how vestibular signals are translated into appropriate compensatory postural adjustments by defining the synaptic circuit by which vestibular neurons govern the activity of spinal motor neurons and interneurons. To surmount the technical difficulties that have limited prior efforts, I propose to use the larval zebrafish. Zebrafish are an excellent system for this line of research because of the accessibility of their brainstem and spinal column, and the strong homologies between zebrafish and mammalian spinal circuits. Thus, circuit mapping between the brainstem and spinal cord can be performed with much greater ease than in mammalian systems, and the results are likely to be applicable across vertebrates. Microcircuit activity can then be translated into behavioral output due to the relative simplicity of the zebrafish body plan, yielding a complete picture of this vital sensorimotor transformation. In Aim 1, a combination of calcium signaling and electrophysiology in vivo will be used to examine differential recruitment of dorsal and ventral musculature while the animal attempts to right itself from side-lying to upright. The requirement for vestibular signals will be tested in mutant animals missing their otoliths (gravity sensors). These experiments will identify how motor pools are activated by vestibular signals to drive self-righting. In Aim 2, vestibular neurons will be stimulated during in vivo recordings from identified spinal motor neurons to test how vestibulospinal drive is distributed to the appropriate pools of motor neurons for self-righting. Finally, Aim 3 will extend this research to spinal interneurons, to identify how descending inputs regulate interneuronal circuits for highly specific modulation of movement. Impairments in vestibulospinal signaling can cause vertigo and falls, a major health hazard in the elderly. Thus, a complete sensory-to-motor analysis of vestibulospinal signaling will advance our understanding of descending control of behavior and potentially identify strategies for improving human postural control.	{ "pile_set_name": "NIH ExPorter" }
Epilepsy is one of the most common neurological disorders, and patients whose seizures are not controlled suffer from many adverse effects. The goal of this study is to investigate the effects of epilepsy on brain structure and function, and to test innovative approaches to treatment when seizures cannot be controlled by currently available approaches. Methods: Patients undergo video-EEG monitoring to determine seizure type and focus localization. Positron emission tomography (PET) and magnetic resonance imaging (MRI) are used to study cerebral metabolism, blood flow, and structure. Antiepileptic drug blood levels are obtained. Recent findings: hippocampal volumes ipsilateral to the epileptogenic zone were significantly smaller in the patients with epilepsy as compared to controls. In addition, mean ipsilateral thalamic, caudate and bilateral lenticular volumes were significantly smaller in the patients with epilepsy as compared to controls. Lateral temporal neocortex shows atrophy as well. These studies revealed that medically intractable temporal lobe epilepsy is associated with volume loss in brain structures outside the presumably involved hippocampus. Volume loss may reflect damage due to the involvement of these structures in recurrent seizure activity. We also found that epilepsy duration had a significant relation to ipsilateral hippocampal formation (HF) volume and ipsilateral/contralateral ratio. Patients with a history of prolonged complex febrile seizures (FS) had smaller ipsilateral HF volume and ipsilateral/contralateral ratio than patients without a history of FS. The effect of age at onset or scan was not significant. Patients with a history of FS did not have earlier age at epilepsy onset or longer duration than those without a history of FS. A history of FS predicted the severity of HF atrophy in our patients. Our study suggests that, after an initial insult, progressive HF damage may occur in patients with persistent seizures. Current Studies: We are conducting a double-blind placebo controlled study of the effect of low-frequency (1/second) transcranial magnetic stimulation (TMS) on the frequency of complex partial seizures. Animal studies suggest that low frequency TMS may induce long-term depression of neuronal excitability. Patients whose seizures are not controlled by currently available anti-epileptic drugs, and have a seizure focus localized by video-EEG monitoring are being enrolled. In the placebo condition, the magnetic coil is directed away from the seizure focus. We have begun a PET study of 5HT-1A receptors in patients with temporal lobe epilepsy. preliminary results suggest reduced binding in both mesial and laterla temporal regions ipsilateral to epileptic foci.	{ "pile_set_name": "NIH ExPorter" }
1. We have replicated high incidence of ventilator associated nosocomial pneumonia (VAP) in sheep, subjected to care similar to that now practiced in patients on mechanical ventilation in a hospital ICU: semirecumbent position with head elevated 30 , and periodic tracheal suctioning. Healthy sheep, anesthetized and paralyzed, were subjected to mechanical pulmonary ventilation in the prone position, with head, neck, and tracheal tube elevated 30 above horizontal, and suctioned periodically. Sheep developed progressive impairment of gas exchange, sepsis, and sometimes death. The trachea and the bronchopulmonary tree were uniformly heavily colonized with pathogens. Early studies, using continuous subglottic suctioning (Hi-Lo evac Mallinckrodt ETT), did not change this poor outcome. However, when sheep were positioned for 72 h prone, with trachea/tracheal tube horizontal and with no suctioning (routinely or otherwise), there was no growth of pathogens in the trachea and the give lobes of the lungs (sampled at 11 sites), normal lung function remained, and lungs appeared normal. We have also shown that the tracheal tube can be oriented horizontally with our custom gantry system, with periodic rotation of sheep from side to side, with equally good results. Importantly, nasogastric feeding was well tolerated (in contrast to conventional practice). We believe this gantry system and routine care based on our studies is readily adaptable to clinical use. 2. We have developed a thin-walled/two stage/lowest dead space/lowest resistance/twin endotracheal tube (UTTS-T-ETT) for use in adult, child, or infant. In the preterm infant, the dead space of this system is lower than that in the healthy, non-intubated newborn. Such a system can permit CPAP with least patient effort, and is likely to reduce greatly the need for mechanical ventilation in patients of all age groups.	{ "pile_set_name": "NIH ExPorter" }
The major impediment to the wide implementation of DNA diagnostic methods is their cost. Technologies to simplify current DNA diagnostic methods and to make them cost competitive with antibody based assays are extremely desirable. Boron Biologicals' new selective silver staining technology could dramatically enhance the price/performance ratio, ease of use and the speed of many currently available DNA based diagnostic methods. Compared to leading alternative techniques, the potential advantages of Boron Biologicals' proprietary technology are: reduced material costs, faster detection procedures, greater safety (eliminating radioisotopes or toxic dyes), greater ease of use for manual systems and greater ease of automation. Preliminary results with model compounds clearly show the possibility of selectively staining DNA probes. Phase I studies will focus on determining feasibility with large probe size DNA fragments in real situations on gels or membranes, and to determine the limit of detection. Methods will also be investigated to improve the yield of modified nucleotides required for the synthesis of probes. Phase II studies will focus on methods of quantitation, demonstration of feasibly with clinic samples and in situ diagnostic methods using selective silver staining. PROPOSED COMMERCIAL APPLICATION: Successful completion of this project will provide a simple, low cost, easy to use, selective DNA detection technology for DNA based diagnostics. DNA probes, if cost effective can replace antibody based diagnostic tools, as well as provide a means for early detection of genetic and infectious diseases. In addition DNA probes also have applications in Forensics, environmental monitoring, etc. to name a few.	{ "pile_set_name": "NIH ExPorter" }
The lipooligosaccharide (LOS) is an important virulence factor in the pathogenesis of gonococcal infection. (2). Our hypothesis is that an understanding of the molecular mechanisms which are responsible for the synthesis and regulation of these macromolecules will assist in the elucidation of there role in pathogenicity. The long term goal of our laboratory is the definition of the biosynthetic pathways which control the expression of Neisserial LOS synthesis. As a prelude to this effort, we have derived from gonococcal strain 1291, a series of 5 different stable LOS mutants. These mutants contain LOS whose oligosaccharides vary progressively by 1 or 2 saccharides from a deep rough mutant to the wild type LOS. In addition, we have prepared a group of monoclonal antibodies which define saccharide epitopes specific for four of these mutant LOSs, hence we have the capability to identify the products of expression of the LOS oligosaccharide synthesis genes from these mutants. During this proposal, we shall define the gene(s) which control the conserved terminal tetrasaccharide residue of gonococcal LOS. This epitope has been chemically characterized and is defined antigenically by the monoclonal antibodies 3Fll and 6B4. This epitope may play a role in gonococcal pathogenesis by molecular mimicry of human antigens since this tetrasaccharide has been demonstrated to be chemically identical to the human l erythrocyte antigen. We have demonstrated that this epitope is present on gonococcal LOS during human infection. During these studies, we will map the LOS oligosaccharide synthesis genes for the epitopes defining each of the LOS mutants and we will determine the relationship between pyocin nucleic acid and the LOS synthesis genes. This will be accomplished by the following specific aims: 1) The gene cluster responsible for the synthesis of the gonococcal LOS oligosaccharide in gonococcal strain 1291 which expresses the terminal tetrasaccharide recognized by monoclonal antibodies 3Fll and 6B4 will be identified by constructing genomic libraries and immunoscreening them with monoclonal antibodies 3Fll and 6B4. The identified clones will be mapped, the genes responsible for the synthesis of the terminal tetrasaccharides identified and their sequence determined. These sequences will be compared known sequences of glycosyltransferases. 2) Complementation studies using the nested deletions from recombinant genomic clones identified in aim 1 and monoclonal antibody epitope analysis will be used to identify the DNA fragments that contain the mutations giving rise to the gonococcal LOS mutants 1291a-e and to order the genes in the LOS synthesis cluster. 3) The relationship between pyocin nucleic acid and the gene cluster responsible for LOS oligosaccharide synthesis will be studied to determine what role, if any, this nucleic acid has in the generation of the gonococcal LOS mutants.	{ "pile_set_name": "NIH ExPorter" }
The goal of this project is to understand how genes are controlled during development. We are studying the Drosophila gene, engrailed (en), which is important for proper segmentation of the embryo and also for formation of the adult. En expression patterns reflect the multiple roles it plays during development. En is expressed in stripes throughout embryonic development, as well as in specific cells in the developing nervous system, head and tail, and the progenitors of the adult tissue (in the imaginal discs). We are interested in how this complex expression pattern is regulated. By making transgenic flies which contain potential regulatory regions driving the expression of the reporter gene beta-galactosidase, we have found that sequences within a 1 kb en intron contain the information to give &n stripes early, but not late in development. This indicated that there are separate programs for controlling &n stripes at different times in development. The ability of the en intron to induce striped expression was dependent upon the type of promoter it was combined with. Promoter specificity was also seen when testing the activity at a homeodomain-binding site in embryos. These data together suggest that the expression pattern of any particular gene is the result of interactions between different elements within a regulatory region. Further studies indicate that the en regulatory region is full of redundancy: temporally overlapping programs for making stripes, more than one control element for striped expression at a particular time, multiple programs for keeping expression off between stripes. These studies are helping us to understand the complexity of controls that go into making an organism.	{ "pile_set_name": "NIH ExPorter" }
This proposal describes a 5-year training program for the development of an independent physician- scientist. The applicant is a junior faculty in Pediatrics and her mentor is Dr. Joshua Hare, Professor of Medicine, and Director of the Interdisciplinary Stem Cell Institute at the University Of Miami Miller School Of Medicine. A leader in the field of stem cell regenerative therapies for cardiovascular disease. Dr. Hare has trained many junior faculty scientists, postdoctoral fellows and graduate students. The curriculum described in this proposal will provide the applicant with the opportunity to acquire a stronger scientific background in stem cell biology as well as neonatal cardiopulmonary physiology. The applicant's research will focus on understanding the role of c-kit in the development of neonatal hypoxia-induced pulmonary hypertension (PH). PH affects approximately 1 in 1000 neonates per year and remains one of the leading causes of mortality in this population, c-kit is a tyrosine kinase receptor which is mainly studied as a stem cell marker; however its role in PH is unknown. Our preliminary data have demonstrated increased c-kit cells in the pulmonary arteries and right ventricles (RV) of neonatal mice with PH. We hypothesize that c-kit cells participate in hypoxia-induced pulmonary vascular remodeling by a) differentiating into myofibroblasts or b) directing the secretion of angiogenic, mitogenic and survival factors which exacerbate pulmonary vascular remodeling. The specific aims are: 1) To characterize the phenotype and temporal-spatial distribution of native and BM-derived c-kit cells in the pulmonary vasculature of neonatal mice with chronic hypoxia- induced PH. 2) To elucidate the mechanisms which regulate the homing and activation of c-kit cells during neonatal chronic hypoxia-induced PH. 3) To determine whether c-kit progenitor cells participate in PH by differentiating into mature pulmonary vascular cells or by secreting mitogenic, angiogenic and survival factors. The University Of Miami School Of Medicine is the ideal environment for this training program as it has the unique combination of state of the art resources and leading scientists of diverse backgrounds. The proposed research training plan will include participation in didactic courses, as well as regular reviewing of the candidate's progress by an expert advisory committee. It is expected that this award will allow the candidate to become an independent investigator with expertise in cardiopulmonary and stem cell biology. RELEVANCE (See instructions): Recently, there has been a flurry of new therapeutic modalities which have vasodilated the pulmonary vasculature, but few have significantly decreased the vascular remodeling that is evident during PH. These studies will expand our fundamental knowledge of the role of stem cells in neonatal pulmonary vascular remodeling and provide a solid foundation for the development of novel therapeutic strategies. (End of Abstract)	{ "pile_set_name": "NIH ExPorter" }
The purpose of the project is to continue the follow-up of a group of patients with bacteriuria of which some are being treated and some are a control group on a placebo and are treated only for acute urinary tract infections. Renal function is being followed by means of serum creatinines every 2 months.	{ "pile_set_name": "NIH ExPorter" }
Much progress has been made in the fight against breast cancer through early detection with screening mammography. Unfortunately, breast cancer can be missed on mammograms due to the difficulty of interpretation and inter-reader variability. For this reason, MedDetect has worked to build a hybrid optical and digital processor for the identification of potentially cancerous lesions on mammograms. The processor is comprised of an optical correlator (OC) combined with a neural network (NN). It is designed to mimic the radiologist - the OC being the eyes rapidly scanning for areas of interest, and the NN being the brain making a recommendation that the case is normal or has suspicious areas. MedDetect's proposed hybrid processor takes advantage of the best of both worlds--the best elements of optical processing and digital computing to create a complete Computer-Aided Diagnosis (CAD) system. Supporting MedDetect in this effort are radiologists Drs. Brenner, Sadowsky, and Levy, University of South Florida algorithm experts and optics experts at Lockheed. If the proposed hypotheses are proven, this innovative technology will be ready for rapid transition into the clinical setting where it can assist radiologists in the early detection of breast cancer. PROPOSED COMMERCIAL APPLICATION: The proposed technology is responsive to a significant market. 25 million mammograms are performed each year and the number is growing. A similar volume of studies exists internationally. The ability to find breast cancer more consistently and perhaps earlier is of great interest to patients, radiologists, and payors. Thus, success with this research should lead to significant business opportunities.	{ "pile_set_name": "NIH ExPorter" }
Project Summary Major depressive disorder (MDD) affects ~4% of the worldwide population, and these numbers are continuously increasing. Moreover, MDD disproportionately affects women, who are twice as likely as men to be diagnosed with the disease. Thus, uncovering the molecular, cellular, and circuit-level mechanisms underlying susceptibility and resilience to depression is critical. Recent work demonstrates that ventral hippocampal (vHPC) projections to nucleus accumbens (NAc) regulate mood and susceptibility to stress. Our group has shown that the transcription factor ?FosB is crucial for hippocampal cell morphology and learning, and several lines of evidence implicate hippocampal ?FosB in depressive-like behaviors (e.g., FosB knockout mice have hippocampal malformations and depressive behaviors, and current antidepressants induce ?FosB in vHPC). However, to date there is no study characterizing the role of hippocampal ?FosB in stress resilience and depression. To this end, we have produced a novel dual-virus CRISPR-Cas9 system allowing knockout of FosB gene expression specifically in vHPC cells projecting to the NAc. In combination with our many well-vetted molecular, physiological, and behavioral tools and models, this will allow us to address the central hypothesis that ?FosB expression in vHPC neurons projecting to NAc mediates cell function and resilience to social stress in male and female mice. To test this hypothesis, we propose two Specific Aims. In Aim I, we will first characterize the epigenetic mechanisms of induction of the FosB gene in hippocampus by chronic stress, determine its role in resilience to stress in both males and females, and uncover its transcriptional targets in vHPC. Indeed, our preliminary data show that inhibition of ?FosB in vHPC induces susceptibility to depression-like symptoms. In Aim II, we will examine the stress induction and cellular effects of ?FosB in vHPC neurons with projections to various brain regions, including NAc, then use our novel dual-virus system to knock down FosB gene expression specifically in vHPC cells projecting to the NAc and observe the behavioral results on stress responses. Here, too, we have strong preliminary data suggesting that ?FosB regulates the excitability of vHPC neurons, and that ?FosB expression in the vHPC- NAc projection cells regulates susceptibility to depression. Together, the proposed studies will elucidate the critical role of ?FosB in stress-induced changes in hippocampal control of limbic circuitry of both males and females, as well as identify novel ?FosB targets that could serve as potential points for sex-specific therapeutic intervention in depression or PTSD.	{ "pile_set_name": "NIH ExPorter" }
Cataract is apparently associated with an irreversible aberration in the lens fiber cell, and perhaps a reversible defect in the metabolism of the lens epithelial cell. Numerous biochemical changes are associated with cateracts, e.g., protein loss and protein degradation in fiber cells, hyperosmolarity of the cortex, and amino acid degradation. There is also degeneration of lens fibers followed by proliferation and migration of the epithelial cells from the equator leading to epithelial cell enlargement and reduction in their nuclear mass. We hypothesize that some dysfunction in the lens fiber cell membrane may underly some of these biochemical and cellular changes. This proposal is designated to elucidate whether specific components of the lens fiber cell are changed during cateract development. To this end we have chosen to study the expression of the gene for the main intrinsic protein (MIP or MP26) of fiber cell membranes in normal bovine, rat or mouse lens and in cataractous lens of the Nakano mouse, and those of rats in which cataractogenesis has been induced by galactose or reversed by removal of galactose from the diet. Parallel studies will also be carried out on human normal and cataractous lenses. We are proposing to use new techniques of molecular biology in these proposed studies on the MP26 gene. Briefly, the main intrinsic protein, MP26, will be isolated to homogeneity, its antisera will be produced in rabbits, and its mRNA will be probed for by an MP26 clone, prepared in this laboratory, in order to follow the expression of the MP26 gene in lens undergoing different experimental conditions. These methods will include the preparation of a lens cDNA library in expression vectors for the immuno-identification of MP26 positive clones or any other desired clones. The MP26 clone will then be amplified and used for the proposed studies on the expression of the MP26 gene. It is expected that these studies will show how a specific fiber cell mRNA is modulated during cataractogenesis.	{ "pile_set_name": "NIH ExPorter" }
A multidisciplinary effort has been initiated to develop (a) an antineoplastic agent effective against slow growing tumors, and (b) a radiosensitizing agent to selectively sensitize hypoxic tumor cells towards treatment with X-irradiation. Butylated hydroxytoluene (BHT) and its analogs represent a class of agents that are free radical inhibitors and have been shown to inhibit enzymatic redox processes and protein biosynthesis in tumor cells. Appropriate chemical modifications of BHT will be made to provide an agent with greater potential for clinical utility than BHT as an antineoplastic agent specifically against slow growing tumors. Gold triphenylphosphine complex of thymidine has been shown to possess antineoplastic activity. A series of gold complexes of various nucleosides will be synthesized in an attempt to obtain an agent which may possess activity against solid tumors. The relative radioresistance of hypoxic cells present in tumors is a critical limitation to the successful radiotherapy of cancer in man. Development of chemical sensitizers has been undertaken to overcome this problem. An extensive structure activity relationship study has been initiated for nitroimidazole analogs to explore the relationship between electron affinity and radiosensitization. A variety of analogs of 2- and 5-nitroimidazole will be synthesized and tested for radiosensitization by in vitro and in vivo techniques.	{ "pile_set_name": "NIH ExPorter" }
Hemophilia A is an inherited bleeding disorder caused by mutations in the coagulation factor VIII (FVIII) gene. Treatment involves repeated i.v. infusions of FVIII concentrates throughout the life of the patient, which creates tremendous discomfort and morbidity, requires sustained compliance, and is extremely expensive. In response to these limitations, we seek to develop a novel technology that uses patients' endothelial colony-forming cells (ECFCs) and mesenchymal progenitor cells (MPCs) to bioengineer a vascular network that is capable of sustained FVIII delivery into the bloodstream. Previously, we demonstrated the feasibility of this approach in a murine proof-of-concept study wherein vascular networks that were genetically engineered to express erythropoietin (EPO) corrected anemia in mice (Lin et al., Blood 2011). Based on this foundational work, we propose a technology whereby ECFCs from hemophiliacs are genetically engineered to serve as autonomous drug delivery vehicles in vivo. The envisioned clinical implementation of our approach would involve: 1) isolating ECFCs and MPCs from patients' peripheral blood; 2) inserting a gene encoding for FVIII into ECFCs; and 3) combining both cell types in a suitable hydrogel and injecting the mixture subcutaneously into the patient. Following implantation, cells will self-assemble into a vascular network. This network will be confined inside a subcutaneous implant, facilitating its removal if necessary, and its microvessels will have the capacity to deliver FVIII into the bloodstream of the patient, correcting the clotting deficiency. To develop this technology and make it applicable to hemophilia A, we propose three Specific Aims. In Aim-1, we will determine efficacy of autologous FVIII-secreting implants in an murine immunocompetent model of hemophilia A. In Aim-2, we will determine feasibility of deriving ECFCs and MPCs from hemophilia A patients' peripheral blood. In Aim-3, we will bioengineer FVIII-secreting implants using patients' cells and determine efficient delivery of the factor in xenograft murine models. Collectively, these studies will establish feasibility of using drug-secreting vascular networks to treat a clotting deficiency. We envision this novel approach has the potential to ultimately liberate hemophilia A patients from frequent injections, stringent compliance, and the exorbitant cost of the factor, improving their overall quality of life.	{ "pile_set_name": "NIH ExPorter" }
Acute coronary thrombosis results in myocardial infarction (Ml) with irreversible loss of functional myocardium unless reperfusion therapy is rapidly instituted. Many patients do not present or are not amenable to prompt reperfusion, and in this large patient population new therapeutic approaches are needed. Initial studies of cell-based therapies post-MI in animal models and in patients have provided encouraging results, but the ideal donor cell population has not been determined. Preliminary data show that transplantation of undifferentiated mouse embryonic stem cells (ESCs) post-MI, resulted in myocardial repair in a mouse Ml model; however, major mechanistic questions and roadblocks remain such as the risk of tumor formation. We hypothesize that cell preparations derived from ESCs that are committed to mesodermal or cardiac lineages can repair the myocardium with minimal risk of tumorigenesis. Secondly, we hypothesize that the optimal benefit from transplanted ESC-derived cells is the result of a short-term reduction in infarct size via paracrine effects and long-term regeneration of myocardium resulting from the proliferation and differentiation of transplanted cells in the absence of significant fusion. The long-term goal of this research is to develop cellular therapies using ESCs to repair the myocardium following Ml by providing essential preclinical studies of efficacy and safety. We propose to: 1) identify and characterize ESC-derived donor cells optimal for myocardial repair with minimal risk of tumorigenesis using cell surface markers and genetic cell-type specific reporters to isolate mesodermal progenitor cells and embryonic ventricular myocytes; 2) develop strategies to condition donor cells for improved survival in the setting of ischemic and oxidative stress typical of the post-MI heart employing in vitro models of ischemia and reperfusion; and 3) determine the effect of transplantation of ESCs and derivatives post-MI evaluating both short-term (infarct size, cell survival, cell proliferation) and long-term (LV structure and function by echocardiography and histology, tumor surveillance with microCT and pathology, cell fate/fusion by co- immunolabeling and Cre/lox donor/recipient techniques, and regenerated myocytes phenotype using cellular electrophysiology) outcomes. Overall, these studies will provide new insights into cell-based therapies in the post-MI setting and bring approaches using human ESCs closer to clinical application. [unreadable] [unreadable] [unreadable]	{ "pile_set_name": "NIH ExPorter" }
Programmed cell death, or apoptosis, is a built-in, signal-induced process by which a cell self-destructs. It is a highly regulated mechanism that is critical for normal development, tissue homeostasis, and the elimination of pathogen-infected cells. In humans, misregulated programmed cell death is associated with tumorigenesis, neurodegenerative diseases, immunodeficiency, and viral pathogenesis. Although many evolutionarily conserved components of the cell death pathway have been identified, the molecular mechanisms involved in cellular regulation of apoptosis are still largely unknown. Since host cell apoptosis can limit virus multiplication, many viruses have evolved diverse strategies to regulate the cell death pathway. The proteins that mediate such viral intervention have provided key insight into the cell death program. The long term objective of this proposal is to define the molecular mechanisms by which apoptosis is regulated through the study of three baculovirus-encoded apoptotic regulators: P35, P49, and IAP. Our approach focuses on the use of baculovirus-infected insect cells as a powerful yet convenient system for molecular analysis of both the induction and suppression of apoptosis. Building on recent advances in the apoptosis field, we use integrated approaches in biochemistry, genetics, and cell biology to determine the molecular mechanism of P49 and IAP anti-apoptotic activity. We focus on P49's novel ability to inhibit an initiator caspase resistant to the pancaspase inhibitor P35 by defining the molecular determinants of caspase selectivity by both irreversible inhibitors. Utilizing the recently discovered capacity of baculoviruses to efficiently deliver apoptotic regulators to cultured Drosophila melanogaster cells, we identify the in vivo targets of P49 and P35 and define the caspase cascade in this model organism. We determine the molecular mechanism of virus IAP anti-apoptotic activity by characterizing the interactions between hybrid IAPs and cellular apoptotic effectors. In concert, we also investigate the functional significance of oligomerization for both viral and cellular IAPs by using novel dominant negative inhibitors. Collectively, these studies are expected to provide new and fundamental information on virus-host interactions and the regulation of programmed cell death in animals. Such knowledge will contribute to the development of therapeutic strategies for apoptosis-associated diseases.	{ "pile_set_name": "NIH ExPorter" }
This year, nearly 1 million older Americans will be diagnosed with cancer. Given improvements in early detection and treatment, most of these individuals will be "cured." However, they may be left with significant morbidity, either from the cancer itself or from its treatment. Furthermore, this population either because of genetic, treatment or lifestyle factors is at increased risk for developing secondary cancers, as well as diabetes, osteoporosis, & cardiovascular disease. Therefore, the older cancer survivor is at even greater risk for developing secondary cancers, as well as diabetes, osteoporosis, & cardiovascular disease. Therefore, the older cancer survivor is at even greater risk of functional decline than the average older adult. However, while cancer may bring a greater risk for functional decline, it may also bring a greater risk for functional decline, it also may bring a "teachable moment." Major lifestyle events, such as a cancer diagnosis, often increase receptivity toward positive lifestyle change. To date, however, little has been done among older cancer survivors to capitalize on this opportunity. Given increased cure rates coupled with the ever growing numbers of elderly, there is significant public health potential for interventions that can reorient the functional decline trajectory of this high risk population. A multi-disciplinary team of investigators in the fields of aging, nutrition, physical activity, behavioral psychology & biostatistics at Duke University Medical Center proposes to test whether a telephone counseling program is effective in improving diet & physical activity behaviors among elderly cancer patients with early stage disease-behavior changes which should ultimately improve their physical function. The specific aims of this study are to determine 1) the efficacy of a diet- physical activity telephone counseling program in improving physical function among elders who are newly diagnosed with early stage breast or prostate cancer in both the short and longer term, 2) differences between experimental & control groups with regard to secondary endpoints (diet quality, physical activity, depression, quality of life, etc.) &3) factors that interact with program efficacy (social support, comorbidity, etc.) We will recruit 420 early stage, breast & prostate cancer patients greater than 65 years throughout North Carolina and randomize them into 1 of 2 arms: 1) EXPERIMENTAL ARM-a group that receives a 6-month telephone counseling program tailored to stage of readiness to improve dietary & physical activity behaviors; & 2) ATTENTION CONTROL ARM- a group that receives telephone counseling in unrelated areas (e.g, cancer screening). Results will increase our knowledge of the use of telephone counseling to deliver multiple risk factor interventions to elderly with early state-cancers-a rapidly growing population that may be highly receptive, where health promotion is greatly needed & where historically little research has been done.	{ "pile_set_name": "NIH ExPorter" }
The Program on Population Processes at the University of Colorado at Boulder, in response to PAR-04-138 for the R21 Developmental Infrastructure for Population Research, requests 5 years of support to build a Population Center on the foundation of a distinguished 30 year old population program. Our Program includes outstanding senior faculty with national and international reputations, new faculty hires, postdoctoral researchers, and graduate students who work on important demographic topics, use cutting-edge methods, publish in the premier demography and disciplinary journals, and obtain federal funding. Importantly, our Program has enjoyed a rich interdisciplinary history, with directors from the departments of anthropology, economics, geography, and currently, for the first time, sociology. We have already accomplished much, but could do far more. With NICHD support, we could (1) expand research in each of our 3 signature themes - migration and population distribution, health, and environment, (2) nourish collaborative and interdisciplinary demographic research, (3) facilitate interaction, among researchers across the U.S. and throughout the world, bridging departments, programs, institutes, and universities, (4) develop junior investigators, (5) raise research quantity and quality through technical support and developmental funds, and (6) increase external funding. To accomplish these goals, we request support for 4 core services - Administration, Information, Statistics and Computing, and Development. The Administrative Core will create new opportunities;produce economies of scale by providing crucial services to all affiliates that would be impossible to provide on an individual basis;and provide secretarial, library, and administrative support for research projects and grants. The Statistics and Computing Core will promote an excellent computing environment for handling large and complex data sets, and in ways that build an unprecedented facility for statistical training and consultation. The Information Core will expand current capabilities significantly, especially in ways that will broaden access to information for affiliates, increase awareness of center activities, and disseminate research findings. The Faculty Development Core will provide crucial seed awards to support new and important demographic research. Together, such support will allow researchers to address larger, more difficult, previously intractable research problems. We are well poised to take full advantage of the resources afforded by a NICHD Center grant and believe that an investment in our program at this time will substantially improve the quality and quantity of our demographic training and research. In fact, we expect that 5 years of NICHD support will position us to compete successfully for an R24 center grant.	{ "pile_set_name": "NIH ExPorter" }
The large majority of genomic loci linked to schizophrenia heritability by genome-wide association harbor regulatory non-coding DNA, including enhancers and repressors, that are not bound to the nearest TSS but instead tethered via chromosomal contacts to genes located elsewhere on the chromosome. Therefore, unsurprisingly, `linear genome' based approaches such as gene expression quantitative trait loci (eQTL) and SNP prioritization algorithms have very limited success in assigning specific target genes to risk loci. Guided by recent Hi-C genome-scale chromosomal contact mapping studies by us and others, we will test in this proposal whether the genetic risk architecture of schizophrenia is associated with cell-type specific vulnerabilities as it pertains to the developmental reorganization of the chromosomal connectome. We predict that neuronal specification into glutamatergic, GABAergic and dopaminergic lineages associated with cell-type specific genome-scale prunings of chromosomal contacts and loss of smaller-scaled chromatin domains. This includes the domain protoype of many of the smaller self-folded `topologically-associated domain' (TAD), with the developmental dissolution of many subTADs nested into larger and megadomain TADs. Furthermore, we predict that differentiating neurons show a disproportionate increase in chromosomal contacts anchored in sequences conferring heritable risk for schizophrenia and related cognitive disorders and traits. We will monitor developmentally regulated shufflings of intranuclear positions for specific GWAS loci and predict mportant differences between the various neural cell types isogenically generated from hiPSCs. If so, then the `functional epistasis', or least co-regulation, of subsets of risk loci sharing the same nuclear sub-territory could be highly dependent on cell type. Last but not least, we predict that targeted mobilization of specific chromatin domains by CRISPR-Genome Organization (CRISPR-GO) approaches can be harnessed for simultaneous targeting of multiple GWAS locis to to specific nuclear compartments such as the nuclear lamina or Cajal body, resulting in multi-layered transcriptome and epigenome changes and cell-type specific phenotypic alterations.	{ "pile_set_name": "NIH ExPorter" }
The ubiquitous, yet poorly understood, phenomenon of intrinsic disorder in proteins has wide-ranging implications for fundamental questions in molecular biology and the design of novel small molecule pharmaceuticals. Intrinsically disordered proteins inherently lack secondary and/or tertiary structure under physiological conditions;they couple folding with binding to their interaction partners with high specificity and, often, extraordinary versatility. The signatures of disorder are found at the heart of transcriptional regulation, protein interaction networks, and a number of diseases. New methods to target and manipulate these protein-protein interactions are urgently required. Understanding the mechanisms in detail, from selection or encounter through intermediates to the bound form, will lead to significant advances in the efficient design of small molecules to control these interactions. Additionally, resolving the principles governing the function of these small, highly specific proteins will further de novo design of peptides and mimics, a growing pharmaceutical aim. This work proposes a combination of NMR spectroscopy, molecular dynamics and molecular biology to gain unique structural insights into coupled folding and binding. Specifically, this work will utilize the complex of the phosphorylated kinase inducible transactivation domain (pKID) of CREB with the KIX domain of CBP as a model system to generate detailed views of the encounter complex and intermediate(s) along an induced folding pathway. Specifically, this proposal aims to (1) define the evolution of secondary structure as distinct from intermolecular interactions in the induced folding pathway, (2) separate the effect of fly-casting from electrostatic steering in docking, and (3) generate detailed models of the binding intermediate(s). For aim 1, the work will utilize relaxation dispersion NMR spectroscopy and the sensitivity of the carbonyl and alpha carbons to secondary structure to quantify the residue specific helical content in the intermediate(s). For aim 2, pKID constructs of varied lengths will be systematically mutated and measured by isothermal calorimetry to separate the effects of charge and construct length on binding thermodynamics. For aim 3, the work will utilize NMR-derived restraints to limit the torsion angles and relative positions in molecular dynamic simulations of the intermediate(s) in the binding pathway. The combination of these studies will significantly advance our understanding of the fundamental mechanisms and guiding principles in the association of intrinsically disordered proteins. The unique structural insights will greatly increase our ability to design small molecules and peptides to bind and disrupt protein-protein interactions. ) PUBLIC HEALTH RELEVANCE: Proteins that inherently lack ordered structure are central to both healthy biological functions and a number of diseases. Disordered proteins often form ordered structure only upon binding an interaction partner forming unique surfaces difficult to target with traditional pharmaceutical design methods. The proposed work will greatly advance our understanding of the mechanisms of disordered proteins as well as our ability to effectively design new pharmaceuticals to target and disrupt protein-protein interactions. )	{ "pile_set_name": "NIH ExPorter" }
Our object is to further develop and strengthen the Harvard Anaesthesia Center established in January 1968 by grant from the National Institute of General Medical Sciences. This research center is based on the facilities of the Harvard teaching hospitals, and on expanded multidisciplinary cooperation. Both research and training have as their aim improved patient care. The research effort emphasizes studies of relevance to Anesthesia and the sciences on which it is based and attempts to make the fullest advantage of interdisciplinary cooperation and advanced technology.	{ "pile_set_name": "NIH ExPorter" }
The a7 nicotinic acetylcholine receptor is being energetically pursued as a drug target for diverse disorders, from Alzheimer's disease to septic shock. We have demonstrated that there are at least three distinct structural motifs which can be used to modify a core agonist structure, such as anabaseine or quinuclidine, to achieve a7 selectivity. For example, selectivity can be achieved through modification of the core agonist with the addition of a large hydrophobic side group such as a benzene ring. The precise chemical structure of the hydrophobic side group determines efficacy and potency, as well as another key feature, the ability to produce stable ion channel desensitization following a transient phase of ion channel activation. The desensitization is due to prolonged binding to the receptor, and the desensitizing properties of specific agents are likely to impact their therapeutic utility for specific indications. We show that drugs which desensitize and do not activate the receptor ion channel can still be effective at treating inflammatory diseases. We will use mammalian cells transfected with a7 alone, or in combination with pro-inflammatory cytokine receptors to test the hypothesis that drugs which induce stable desensitization of the a7 ion channel may still be effective at mediating ion channel independent signal transduction through the intracellular JAK/STAT pathway. We will also test the hypothesis that ion channel activation, in contrast, is essential for the enhancement of LTP, a memory-related process in the hippocampus. We have generated models for how the various a7-agonists dock in the ligand- binding domain of the a7 receptor and have identified amino acids which we hypothesize will have point-to- point interactions with substituents on the hydrophobic side groups of the a7-selective agonists. We will investigate the potential importance of hydrogen bonding and hydrophobic interactions on the binding, gating, and desensitizing properties of the specific receptor/ligand combinations. We will test our hypotheses with site-directed mutations, as well as with novel a7-selective ligands that will be restricted in their ability to form specific point-to-point interactions, for example, agents which are only able to be H-bond donors or acceptors. Wild-type and mutant receptors will be expressed in either Xenopus oocytes or transfected mammalian cells, and we will study ion channel properties by measuring both whole-cell and single-channel currents. We will use the Type 2 positive allosteric modulator PNU-120596 to measure the desensitizing properties of specific ligands and to overcome the intrinsically limited open probability of a7 receptors, making their single-channel currents more amenable to study. We will use tkP3BzPB, a novel highly selective a7 noncompetitive antagonist, to separate ion channel activation dependent and independent forms of signal transduction, and to further manipulate ion channel open probability. Together these studies will provide important advancements leading to the design of a7 agonists with optimized profiles of pharmacological properties for specific indications. PUBLIC HEALTH RELEVANCE: There are many types of nicotine receptors in the brain, and only some of them are related to why people become addicted to nicotine. One type of nicotine receptor that is not the cause of addiction is the alpha7-type receptor, and stimulation of this receptor combats conditions like schizophrenia, Alzheimer's disease, septic shock and other inflammatory diseases. We have identified drugs that will selectively stimulate alpha7 receptors in one of two different ways. One form of stimulation may help alleviate brain diseases;the other may help alleviate diseases like arthritis. We will use our new discoveries about how these drugs work to help make alpha7-stimulating drugs optimally designed to treat specific diseases.	{ "pile_set_name": "NIH ExPorter" }
An inpatient program with selected overnight stays for childhood and adolescent neuropsychiatric disorders is ongoing. The condition currently under study is that of hyperactive children (HAC). Pharmacological compounds under study in these disorders include methylphenidate, amphetamine, piribedil, L-DOPA, tryptophan, Mianserin, clorgyline, and desipramine. Piribedil is safe but clinically ineffective in HAC while L-DOPA is minimally clinically effective. Tryptophan is effective on attention measures. Pharmacokinetic studies with clinical responses are included. Amphetamine half-life in children is about one-third that of adults. Behavior and motor activity responses to d-amphetamine occur during the absorption phase as determined by serial plasma amphetamine following a single dose. Central neurotransmitters and their metabolites are being studied in plasma and urine. Urinary 3-methoxy-4-hydroxyphenylglycol (MHPG) shows a time-related decrease during treatment with d-amphetamine; dopamine metabolites are unchanged. Tyramine and its metabolites are also decreased following d-amphetamine, whereas phenylethylamine is greatly increased following d-amphetamine.	{ "pile_set_name": "NIH ExPorter" }
DESCRIPTION, OVERALL: This Program Project consists of three closely related and integrated proposals focusing on signals which regulate skeletal morphogenesis during embryonic development. Because the same regulatory network is utilized postnatally as the skeleton grows, remodels and repairs itself, these studies will also increase understanding of medically relevant aspects of bone physiology. The projects are highly coordinated, founded in the last 5 years of a highly successful Program Project and a long history of collaborative efforts before that. A unique feature of this team is the exchange of expertise between laboratories whose primary focus has been either developmental biology (Tabin and McMahon) or bone biology (Kronenberg). By continuing the high-resolution histology core and establishing a new core for high-throughput gene expression analysis, the technical capabilities of each individual lab will be extended. All three projects are focused on dissecting the roles of key signaling molecules in skeletal development. Project I will examine the role of PTHrP in chondrogenesis and the role of PTHrP in osteogenesis. Project II will explore the role of Ihh/Shh and Wnt signaling in osteogenesis, and will also develop a fate map of craniofacial skeletogenesis based on a unique code of Fox genes which are suggested to underlie the specification of distinct facial primordia. Project III will study the roles of BMP signaling in chondrogenesis and osteogenesis and will also define the molecular characteristics of the cell types and intercellular signals leading to craniofacial dermal bone formation. These projects are knit together by common themes, complimentary approaches, shared reagents, and direct collaborations. Together these highly related projects will achieve a new level of understanding of the regulation of bone morphogenesis which could not be attained by independent efforts.	{ "pile_set_name": "NIH ExPorter" }
The pathogenic and cytotoxic effects of viruses are largely due to the expression of viral gene products. Therefore, the determinants of these outcomes are the mechanisms underlying the expression of viral genes. The hallmark of herpes simplex virus gene expression is the sequential and coordinately regulated expression of the approximately 80 viral genes. This regulation occurs largely through modulation of RNA polymerase II transcription. Two viral proteins, VP16 and ICP4, function to activate transcription of the five immediate early genes, and the remainder of the HSV genome, respectively. ICP4 appears to be unique in that it doesn't require the cellular coactivators used by many other activators for the activation of viral late genes. However, the cellular requirements for the activation of early genes by ICP4 are greater than those for late genes, suggesting a different mechanism of action. We hypothesize that ICP4 and late promoters, which consist simply of TATA box and Inr element, have evolved to efficiently function with the available polll machinery present late after infection, while ICP4 functions by a different mechanism with the less altered transcriptional machinery present at earlier times post infection to activate early genes. As infection proceeds a number of viral activities result in the altered abundance and activities of cellular proteins. Therefore, it is reasonable to propose that the abundance, subunit composition, and activities of polll trancription factor complexes change as infection proceeds, and that these changes contribute to the regulated cascade of viral gene expression. To address these hypotheses we will: 1) Determine the mechanism of transcription activation of early and late genes by ICP4. 2) Determine how the phosphorylation of Spl during infection affects its ability to activate transcription. 3) Use the combined results from microarray analysis and biochemical fractionation of virus infected cells to determine how the transcription machinery of the cell changes as a consequence of infection and how these changes may affect viral gene expression.	{ "pile_set_name": "NIH ExPorter" }
To identify synergistic drug combinations, we work with Craig Thomas at NCATS on matrix drug screens, which use acoustic dispensing robots to array two drugs in a dose titration series against one another, looking for doses at which the drugs achieve greater cell killing together than individually. We use a chemical library of 2000 drugs that are either approved or in development as cancer therapeutics plus small molecules that serve as tool compounds for important signaling and regulatory pathways, such as NF-kB. In the initial screen using ABC DLBCL lines, we searched for compounds that would synergize or antagonize the toxicity of ibrutinib. Multiple agents targeting the PI(3) kinase pathway strongly synergized with ibrutinib, including drugs targeting the PI(3) kinase catalytic subunit, Akt, or mTORC1. This result is consistent with the notion that ibrutinib primarily targets the pro-survival NF-kB pathway, while having less effect on the PI(3) kinase survival pathway, which is also engaged by BCR signaling. Ibrutinib synergized strongly with PRT-060318, an inhibitor of the SYK tyrosine kinase that is activated proximally in the BCR pathway, and with ABT-199, a BCL2 inhibitor that we have studied extensively in the context of ibrutinib resistance. Another broad class of synergistic compounds included cancer chemotherapeutic agents that elicit a DNA damage response or trigger apoptosis by interfering with microtubules. The reasons behind this synergism is likely due to the ability of NF-kB to antagonize the apoptotic effects of chemotherapy. Indeed, all components of both the CHOP and EPOCH chemotherapy regimens synergized with ibrutinib in killing ABC DLBCL cells, providing impetus to combine ibrutinib with these regimens. Separately, we evaluate small molecules that target essential pathways that are uncovered in our structural or functional genomic efforts in lymphoma. An example of this approach is our study of small molecule IRAK4 inhibitors to target oncogenic MYD88 signaling. In collaboration with Nimbus Therapeutics, we showed that IRAK4 inhibitors reduced IKK activation and the expression of NF-kB target genes, including the cytokines IL-6 and IL-10 that trigger pro-survival JAK/STAT signaling as well as the anti-apoptotic BCL2 family members BCL-XL and A1. The IRAK4 inhibitors synergized strongly with ibrutinib in inhibiting IKK, presumably because both the BCR and MYD88 inputs to IKK activation were blocked. IRAK4 inhibitors also strongly synergized with the BCL2 inhibitor ABT-199, presumably because the downregulation of BCL-XL and A1 by IRAK4 inhibition lowered the apoptotic threshold of these cells. Based on our findings, Genentech acquired the rights to develop the Nimbus IRAK4 inhibitors last year. Based on our identification of chronic active BCR signaling as a key survival pathway in ABC DLBCL, we conducted a phase I/II clinical trial of ibrutinib using gene expression profiling to assign patients to the ABC and GCB subtypes of DLBCL. As predicted by our laboratory investigations, ibrutinib produced a 37% response rate in ABC DLBCL patients but only a 5% response rate in GCB DLBCL, demonstrating that the molecular diagnosis of DLBCL subtypes can inform precision medicine trials. This translated into improved overall survival in ABC relative to GCB DLBCL, including several patients who have remained in complete remission for more than 3-6 years, taking ibrutinib daily without discernable side effects. Based on these promising results, ibrutinib plus chemotherapy is now being evaluated in untreated non-GCB DLBCL patients in a phase 3 randomized trial being conducted by Jannsen. This trial used an immunohistochemical test we developed with our LLMPP colleagues to identify patients with non-GCB DLBCL and is the first phase 3 trial in DLBCL to use molecular profiling for enrollment. This trial has enrolled 800 patients and will read out in 2018. To understand the molecular basis for response to ibrutinib within ABC DLBCL, we resequenced the tumors for recurrent oncogenic mutations. Tumors with CD79B mutations responded more frequently than those with wild type CD79B (55% vs 30%), demonstrating the clinical validity of our observation that these mutations augment BCR signaling. Nonetheless, the majority of responding patients on this trial had wild type CD79B, which is in keeping with our demonstration that chronic active BCR signaling is driven by self-antigen reactivity of the immunoglobulin variable regions. Notably, tumors that had both a CD79B mutation and a MYD88 L265P mutation responded frequently (80%), whereas those with only a MYD88 mutation did not respond. This double mutant genotype occurred in more ABC tumors (11%) than expected by chance based on the prevalence of each mutation individually, providing genetic evidence that the MYD88 and BCR pathways cooperate in these tumors. We showed that ABC cell lines with the double mutant genotype responded to ibrutinib, whereas ABC lines with only MYD88 mutations did not, and that a MYD88 dimerization inhibitor decreased proximal BCR signaling. Together, these observations suggested that the double mutant genotype creates strong addiction to BCR signaling that is hyper-responsive to ibrutinib. Given the frequent responses to ibrutinib in ABC DLBCL tumors with both CD79B and MYD88 L265P mutations, we searched for other lymphoma types that have this double mutant genotype. Recent work has demonstrated that MYD88 L265P mutations are enriched in several types of extranodal lymphoma, including primary breast lymphoma, primary testicular lymphoma, and primary central nervous system lymphoma (PCNSL). Interestingly, the co-occurrence of CD79B and MYD88 L265P mutations in PCNSL is 2-3 times greater than in nodal ABC DLBCL, suggesting that they may be hyper-addicted to BCR signaling due to synergy between the BCR and MYD88 pathways. To explore this hypothesis, we worked with the Lymphoid Malignancies Branch clinical team led by Wyndham Wilson to devise an ibrutinib-based regimen for PCNSL. The basic design of this regimen entailed giving ibrutinib as monotherapy for 2 weeks, followed by the combination of ibrutinib with a set of brain-penetrant chemotherapy agents, given in cycles. By comparing pre-treatment MRI scans with scans immediately after ibrutinib monotherapy, we observed objective responses to ibrutinib in 17/18 treated PCNSL patients, the majority of whom had relapsed/refractory disease. The high rate of response to ibrutinib monotherapy in this trial supports our hypothesis that PCNSL is hyperaddicted to BCR signaling. Although we only had biopsy material available on 4 patients, it was notable that responses were seen in one patient with the double mutant genotype, but also in 2 patients with only a CD79B mutation and 1 with only a MYD88 L265P mutation. Hence, the enrichment in PCNSL for the double mutant genotype was a genetic clue that PCNSL as a whole is typically hyper-addicted to BCR-dependent NF-kB activation. The partial responses to ibrutinib monotherapy in PCNSL were converted into complete responses (CRs) with added chemotherapy in 86% of patients. Notably, 8 of these patients have ongoing CRs with a predicted progression-free survival of 15.5 months, including 5 patients with disease that was refractory to the last therapy. We therefore believe that this chemotherapy platform, which we term TEDDI-R, can serve as the foundation for further improvements in subsequent studies.	{ "pile_set_name": "NIH ExPorter" }
Whereas traditional models for gene regulation posit that recruitment of Pol II to the promoter is both necessary and sufficient for gene expression, we have recently found that release of paused Pol II from the promoter-proximal region is rate-limiting for expression of a large number of genes. Our initial work investigated the prevalence of paused Pol II in Drosophila, employing a combination of global location analysis (using techniques called ChIP-chip and ChIP-seq) as well as in vivo footprinting assays. Surprisingly, these data showed that Pol II pausing is much more widespread than previously appreciated, occurring at thousands of promoters genome-wide. We and others have recently extended these findings to mammalian systems (mouse and human), demonstrating that pausing a prevalent gene regulatory strategy in higher organisms. Moreover, our results reveal that Pol II is pre-loaded in the uninduced state at many genes that respond to environmental or developmental stimuli, suggesting that the presence of Pol II, poised for escape into the gene, facilitates efficient, integrated responses to a dynamically changing environment. Understanding the fundamental properties of paused Pol II, and the factors that govern maintenance vs. release of promoter-proximal Pol II into productive elongation are specific aims of research in the Adelman laboratory. In addition to providing crucial insight into stress-responses, this work is anticipated to elucidate gene expression during the development of cancer and AIDS, since similarly paused Pol II are observed at the mammalian promoters of proto-oncogenes like c-myc, c-fos and junB, as well as at the HIV promoter. As part of our efforts to better define the mechanisms underlying pausing, we have recently developed a novel technique for isolating the short RNA transcripts generated by paused Pol II, and analyzed them through massively-parallel sequencing of individual RNA molecules. This strategy allowed us to pinpoint both the locations of transcription initiation and pausing, at single-nucleotide resolution. Notably, this exciting new technique revealed a role for the DNA sequence within the initially transcribed region in specifying the efficiency of early elongation, providing insights into why polymerase pausing is more prominent at some genes than at others. In probing the molecular mechanisms governing Pol II stalling, the Negative ELongation Factor, or NELF complex, is of particular interest to the laboratory. NELF has been shown to establish paused Pol II at several genes to date, including the junB and HIV promoters. To globally identify targets of NELF, we have performed a microarray analysis on Drosophila cells that were depleted of NELF using RNA interference. We found that many NELF target genes are involved in stimulus-responsive pathways, with a particular enrichment in the innate immune response. To evaluate the physiological relevance of this finding, we have recently performed NELF depletion in the Drosophila fat body (the main immune responsive tissue), followed by microarray analysis of RNA levels to identify NELF target genes. This work confirms that NELF plays a key role in vivo in regulating expression of components of the innate immune system. Follow-up studies in both cells and flies revealed that NELF-mediated Pol II stalling is essential for an optimal immune response to bacterial challenge and indicated that polymerase stalling is necessary for either the binding or activity of the NF-kB transcription factor Relish (Rel). This result is consistent with our earlier suggestion that Pol II pausing helps establish a nucleosome-deprived, accessible chromatin architecture around gene promoters, facilitating future activation. In addition to our work in Drosophila, we are studying the role of polymerase pausing in the mammalian inflammatory response, using primary macrophages derived from mouse. These investigations reveal that many immediate early response genes, like TNF-alpha and junB, possess paused Pol II waiting in their promoter regions in resting, uninduced cells. In contrast, late primary and secondary response genes generally lack stalled Pol II and NELF prior to induction. Overall, we find that there is an interesting relationship between the presence of stalled Pol II and the kinetics of the immune response, in that paused genes exhibit much more transient bursts of transcription activity than do genes regulated through Pol II recruitment. This suggests that there could be a fundamental link between gene regulatory strategy (i.e. the step in the transcription cycle that is rate-limiting for gene expression) and the kinetics or magnitude of gene expression.	{ "pile_set_name": "NIH ExPorter" }
In normal caregiver-infant dyads, caregivers regulate infant attention and state of arousal and affect by closely monitoring the infant and providing appropriate vocal, facial, gestural, kinesthetic and tactile stimulation. Mothers with symptoms of depression fail to provide needed emotional and state regulation for their infants, who exhibit disordered state and behavior in the dyad and who are at risk for later problems of emotion, attachment, and cognition. A substantial body of research shows that caregivers use the vocal channel in particular to modulate infant attention, affect, arousal, and information-processing. Infant-directed (ID) speech possesses exaggerated modulation of the fundamental frequency (Fo) relative to adult-directed (AD) speech, and is known to elicit stronger behavioral and affective responding from infants. Research on the ID speech produced by mothers with symptoms of depression has shown that it tends to have less exaggerated Fo modulation. In a preliminary study involving an associative learning paradigm in which 4-month-olds of nondepressed mothers were taught an association between a voice and a smiling face, ID speech segments from mothers with relatively few symptoms of depression were readily associated with the face, whereas those segments produced by mothers with comparatively more symptoms of depression failed to enter into association with the face. The ID speech of mothers with more symptoms of depression lacked the attention-getting rising Fo contours that are characteristic of normal ID speech. The proposed experiments will seek to replicate this finding, extend the study to mothers with more severe symptoms of depression, and explore in greater detail the acoustic correlates of ID speech produced by these mothers. In addition, associative learning in infants of depressed mothers will be studied to determine if these mothers can associate the ID speech of their mothers, that of other depressed mothers, or that of nondepressed controls with face stimuli. The concurrent and predictive validity of associative learning for performance on the Bayley Scales of Infant Development will also be assessed. The importance of the proposed research lies in its promise to elucidate one mechanism by which maternal depression influences infant associative learning, thereby suggesting strategies to reduce risk for cognitive deficits.	{ "pile_set_name": "NIH ExPorter" }
Project Summary The Washington Department of Health Public Health Laboratories (WAPHL) operates a comprehensive environmental radiation laboratory with advanced radiation emergency response capability, including procedures for measuring radiological contamination in foods. Food safety and security are essential components to commerce in the state and to the United States. Washington is a large agricultural food producer and has several major ports which are active in food importing and exporting. From the ports, food is shipped throughout the Pacific Rim, as well as into other parts of the Unites States and the world. The ultimate objectives of this application are: Maintain expertise in radiological food testing; Build capacity to process a larger number and wider variety of samples quickly and accurately; Develop and strengthen existing collaborative partnerships with FDA and other FERN laboratories, and;Develop protocols and procedures that can be implemented nationally to increase the effectiveness and efficiency of food sample processing for radiological analyses. This application will specifically be used to fund two radiochemists to for procedure development/modification, procedure testing and validation, technology/method transfer to other FERN laboratories, and to coordinate two interlaboratory-comparison emergency response exercises, one in 2012 and the other in 2015, as follow-up to the MENU2010 exercise. Procedure work will emphasize sample preparation and counting for screening and identification by gamma and beta spectrometry aimed at enhancing the speed, throughput and quality of sample preparation.	{ "pile_set_name": "NIH ExPorter" }
In order to study the physiological significance of the coexistence of pancreatic polypeptide and norepinephrine (NE) in peripheral noradrenergic nerves, the effects of pancreatic polypeptides of several species were tested on the isolated rat vas deferens. Neuropeptide Y (NPY) was also studied because of its sequence homology to the pancreatic polypeptides. The contractile responses, which were mediated predominantly by activation of noradrenergic nerves following electrical stimulation, were inhibited by bovine pancreatic polypeptide (BPP), human pancreatic polypeptide (HPP), avian pancreatic polypeptide (APP) and NPY in a dose-dependent manner using a constant flow bath. The decreasing order of the inhibitory responses was as follows: BPP=HPP greater than NPY greater than APP. The inhibitory responses produced by BPP and HPP lasted more than 1 hr and displayed a marked tachyphylaxis. In contrast, the inhibitory effects induced by NPY and APP usually returned to the control level after 20-30 min and had minimal tachyphylaxis. The inhibitory action of NPY was still present during beta-adrenergic blockade. Contractions produced by a single submaximal dose of exogeneous NE in unstimulated preparations were not affected by pretreatment with NPY. The amplitude of contractions was partially reduced 1 min after pretreatment with BPP or HPP; recovery occurred about 15 min after peptide pretreatment in a constant flow bath. These results suggest that an NPY receptor exists presynaptically in the rat vas deferens and that stimulation of the receptor by NPY inhibits the release of NE from noradrenergic nerves. It is concluded that both BPP and HPP act by a presynaptic inhibitory mechanism on noradrenergic nerve terminals and also have a nonspecific inhibitory action on smooth muscle cells.	{ "pile_set_name": "NIH ExPorter" }
Nonablative hematopoietic stem cell transplantation (HCT) with an unrelated donor graft has greatly expanded the number of patients suffering from hematologic diseases and malignancies who can be cured by HCT. However, current dosing methods for HCT regimens lead to substantial interpatient variability in the systemic exposure and commensurate percent donor T-cell chimerism. The parent grants have compelling data that percent donor chimerism is associated with the efficacy and toxicity of nonablative HCT. Our central hypothesis is the pharmacokinetic (PK) and drug-specific pharmacodynamics (PD) of fludarabine and mycophenolate mofetil (MMF), along with relevant metabolites, are biomarkers for donor chimerism. To test this hypothesis, we propose to develop population PK models and limited sampling schedules to characterize an individual patient's systemic exposure and to build popPK-PD models using PD endpoints specific for each drug's activity. We propose three specific aims to be tested in patients receiving fludarabine/total body irradiation conditioning, an unrelated peripheral blood mononuclear cell graft, and postgrafting immunosuppression of MMF and a calcineurin inhibitor. In Aim 1, we will evaluate the PK and PD of plasma fludarabine and intracellular fludarabine triphosphate (FTP), its active metabolite. Using a novel analytical method, we are uniquely poised to measure in-vitro FTP formation by CD4+ and CD8+ cells obtained from patients awaiting HCT and evaluate the cellular mechanisms of FTP formation. We will evaluate fludarabine- specific PD, specifically evaluating the relationship of these PK endpoints with the decline in circulating CD4+ and CD8+ cells. In Aim 2, we will characterize the PK and PD of MMF's active metabolite, mycophenolic acid (MPA), and to confirm our findings that a low MPA AUC increases the risk of low percent donor chimerism and rejection. We also propose to evaluate the genetic mechanisms of MPA clearance and to characterize the PD of MPA and inosine monophosphate dehydrogenase (IMPDH) activity, the enzyme inhibited by MPA. In Aim 3, we propose to evaluate if percent donor T-cell chimerism is associated with the PK and drug-specific PD biomarkers of fludarabine and MMF. We propose to evaluate the PD of donor T-cell chimerism with the PK and drug-specific PD markers evaluated in Aims 1 and 2. Our individual areas of expertise, in combination with our history of collaborations, demonstrate that we are well-poised to test the central hypothesis, thus providing a unique opportunity for these ancillary studies to improve outcomes for patients with hematologic disease or malignancies receiving HCT. The goal of these studies is to identify patient-specific factors related to the balance between the recipient cells and donor cells in patients who receive a hematopoietic stem cell transplant. We will look at two types of patient-specific factors - how the patients'body breaks down and how their bodies immediately responds - to two drugs, fludarabine and mycophenolate. (End of Abstract)	{ "pile_set_name": "NIH ExPorter" }
A novel bioengineered technique to rapidly and permanently repair cut PNS nerves Our long term objective is to performance-optimize to translate for clinical use our novel and innovative technique to produce rapid and permanent repair of acutely and chronically severed mammalian PNS axons to restore the behavioral functions they mediated prior to severance. We apply a well-specified sequence of bioengineered solutions containing polyethylene glycol (PEG) and various anti-oxidizing or oxidizing agents to rapidly and permanently rejoin (PEG-fuse) completely cut-severed ends of rat sciatic axons as a model in vivo system so that PEG-fused axons are physiologically and morphologically intact through the lesion site and their stimulation restores behavioral functions mediated by intact sciatic nerves. Our physiological and morphological measures of axonal continuity are action potential conduction and intraaxonal dye diffusion across the lesion site and EM and immuno-histochemical analyses.. Our measures of behavioral restoration are Foot Fault Asymmetry test and Sciatic Functional Index. We retard Wallerian degeneration by cooling or cyclosporin A and then repair axons chronically severed for up 10 days by PEG-fusion. We performance optimize tissue (biocompatability) responses of acutely or chronically excised sciatic nerve segments that are used as inter- position autografts or allografts to PEG-fuse repair acutely or chronically cut rat sciatic nerves. Nerve severance is a common traumatic injury to PNS axons in humans. Various procedures currently slightly improve the number and specificity of PNS axons that reestablish connections following severance, but not outgrowth rate (~1mm/day) or time (weeks to years) for PNS axons to re-establish those connections. Target tissues may atrophy before re-innervation can occur. Consequently, target tissues are often non-specifically re-innervated and behavioral recovery is often poor. PEG-fusion dramatically improves the speed and efficacy of behavioral recovery following acute cut- or crush-severance of mammalian PNS axons. We can also retard axonal Wallerian degeneration of severed mammalian axons for up to 10 days to increase the time for successful PEG-fusion for up to 10 days post-severance so that nerve injuries do not have to be immediately treated. Our well-specified sequence of bio-engineered solutions and materials needed for PEG-fusion use only FDA-approved chemicals. Hence, our PEG-fusion technique developed on rat sciatic nerves as a model in vivo system should rapidly translate to clinical procedures. The results of our proposed R-01 have high potential for shifting the current emphasis of current research and clinical practice from devising procedures to enhance the results of slow axonal outgrowth to considering rapid repair by our novel PEG-fusion technique.	{ "pile_set_name": "NIH ExPorter" }
Stroke is a major cause of disability including memory impairment. Our laboratory has shown that a novel treatment, anti-Nogo-A immunotherapy results in sensorimotor functional recovery after stroke in rodents. However, the role of anti-Nogo-A immunotherapy in memory recovery after stroke has not, as of yet, been investigated. The purpose of this proposal is to investigate anti-Nogo-A immunotherapy as a therapeutic intervention to improve memory impairment after stroke in aged rodents. Aim 1 will employ the Morris water maze to determine whether anti-Nogo-A immunotherapy after stroke in the aged attenuates memory impairment. The Morris water maze will be used to evaluate spatial reference and working memory. Aim 2 will employ Golgi-Cox analysis to determine whether anti-Nogo-A immunotherapy after stroke induces dendritic plasticity in the hippocampus and entorhinal cortex, two brain structures involved in learning and memory. Neurons in the hippocampus (CA1 pyramidal cells, CAS pyramidal cells, and DG granule cells) and entorhinal cortex (layer II stellate cells, layer III pyramidal cells, and layer V pyramidal cells) of Golgi-Cox processed brains will be examined for structural neuronal plasticity by quantification of dendritic arborization, spine density, and spine morphology. These experiments are relevant to clinical practice by addressing the significant decrease in quality of life caused by memory impairment after stroke. In addition, the translational relevance of these experiments is maximized by modeling the elderly human population, in which stroke is most prevalent, with aged rodents. Furthermore, anti-Nogo-A immunotherapy is a promising therapeutic intervention that may be quickly translated into a clinical trial for stroke. This is evidenced by the Phase 1 clinical trial that has begun in Europe to test anti-Nogo-A as a therapeutic intervention for spinal cord injury. Stroke is a major cause of disability in the United States and in many cases the disability is contributed to by cognitive impairments including memory impairments. This proposal will contribute to the development of treatments for cognitive impairments including memory impairments after stroke. In addition, this proposal will lead to important information regarding the role of neuronal plasticity in brain repair after stroke. [unreadable] [unreadable] [unreadable] [unreadable]	{ "pile_set_name": "NIH ExPorter" }
The control of gene expression is central to the growth and development of all organisms. Disrupting the normal control mechanisms can lead to cell death or transformation. In other cases, subverting the cells transcription apparatus can form the basis for viral infection. Appropriately, to understand the mechanisms of eukaryotic transcription is a major goal in modern biology. This is a proposal to study transcription of RNA polymerase II genes from the model organism Acanthamoeba castellanii. The long-term goal is to establish principles of eukaryotic gene expression at the molecular level. The in vivo structure of an actin gene from Acanthamoeba castellanii will be determined. The results will identify promoter regions that interact with protein in vivo and will be used as a reference point for in vitro studies. An in vitro transcription system will be developed form Acanthamoeba castellanii using the Acanthamoeba actin I and myosin II genes. Promoter elements required for accurate transcription in vitro from the actin gene will be identified and the number and type of factors required for transcription will be determined. The interaction between factors, RNA polymerase II and DNA will be examined by footprinting. The steps of preinitiation complex assembly that lead to initiation will be defined in terms of polymerase and factor binding as well as DNA unwinding or other conformational changes. Purification of transcription factors will be initiated using a combination of conventional and promoter affinity methods.	{ "pile_set_name": "NIH ExPorter" }
HIV transmission via breastfeeding accounts for nearly half of the 350,000 infant HIV infections occurring annually. While HIV is readily transmitted from mother-to-child via breastfeeding, it has been recognized both observationally and experimentally that SIV is not or is only rarely transmitted through breastfeeding to infants of natural hosts of SIV. This lack of SIV transmission via breastfeeding is not attributable to low virus RNA load in the breast milk compartment, as maternal milk virus load in nonpathogenic SIV infection is similar to that of HIV and pathogenic SIV infection. Therefore, the infants of natural hosts of SIV are highly exposed, uninfected population, warranting evaluation of the factors contributing to the impediment of virus transmission in this setting. Elucidation of mechanisms that evolved to protect breastfeeding infants of natural hosts of SIV from postnatal SIV infection will inform immunologic strategies to reduce HIV transmission via breastfeeding. Importantly, we identified robust autologous virus neutralizing antibody response in milk of AGMs. In contrast, an autologous virus neutralization response is not detectable in milk of SIV-infected RMs or HIV-infected women. This effective virus-specific mucosal antibody response in milk of AGMs may impede virus transmission in the infant oral/gastrointestinal tract. Our previous work isolating HIV Envelope-specific monoclonal antibodies from colostrum B cells of HIV-infected women has identified novel HIV Env-specific neutralizing mucosal antibodies produced in the setting of chronic HIV infection. Therefore, identification of SIV Env-specific memory B cell responses in breast milk of nontransmitting natural hosts of SIV will define novel virus-specific mucosal antibody functions that may effectively block of mucosal virus transmission to a highly exposed, uninfected infant population, in contrast to the B cell responses of pathogenic SIV and HIV infection. This proposal is being submitted in response to the NIAID Program Announcement PA-11-217 Mechanistic Studies of HIV-exposed Seronegative Individuals, specifically addressing the topic of mucosal parameters that are distinctly different in the resistant population (SIV-infected AGM mother- infant pairs) compared to the nonresistant population (SIV-infected RMs and HIV-infected humans mother-infant pairs).	{ "pile_set_name": "NIH ExPorter" }
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 goal of this study is to study the cellular and molecular mechanisms of vascular growth in adult hearts. Proliferation of microcirculatory vessels parallels myocyte hypertrophy during normal growth and maturation. Many studies, however, have demonstrated that impaired vascular growth and vascular disease play a major role in pathologic cardiac hypertrophy. This may involve reduced density of coronary vessels and impaired regulation of blood flow. Recent studies have provided more mechanistic information about the role of impaired vascular growth in the evolution of pathologic cardiac hypertrophy. Indeed, it appears that blocking vascular growth during physiological cardiac hypertrophy results in conversion to a pathologic phenotype. Thyroid hormones (THs) are known to stimulate vascular growth and activate the Akt pathway. To this point, however, cardiac vascular growth has not been mechanistically linked to Akt pathway. ____________________ In this study, we will test the hypothesis that TH's promote angiogenesis via an Akt signaling pathway in adult myocardium and this process is accompanied by mobilization of endothelial progenitor cells. To test this hypothesis Aim 1 will extensively characterize the cellular features of T3-induced angiogenesis in adult heart and determine the role of Akt signaling. Aim 2 will use the animals from Aim 1 to investigate the effect of T3 on the mobilization of endothelial progenitor cells. Aim 3 will explore the role of the PI3K-Akt-HIF-1 signaling pathway in TH induced vascular remodeling and vessel integrity during the angiogenic process in vitro.	{ "pile_set_name": "NIH ExPorter" }
Pilot experiments are being performed on the effects of extensive ablations of the rostral limbic cortex on (1) the isolation call and (2) ludic behavior of adolsecent squirrel monkeys. The present phase of the work is concerned with devising a satisfactory surgical procedure.	{ "pile_set_name": "NIH ExPorter" }
The mechanisms of wound promoted tumorigenesis and tumor progression are not fully understood. In particular, the impact of an acute wound such as might occur during biopsy or explorative surgery on an existing tumor has not been addressed. Perhaps the greatest risk is the presence of unsuspected micro deposits of tumor left behind after tumor resection. Animal models to date have described the influence of preexisting wounds on tumor cells, or the influence of wounding on initiated hosts. For example, tumor incidence and tumor volume are higher if melanoma or fibrosarcoma cells are injected into a wound as compared to unwounded tissue, indicating that a preexisting wound microenvironment facilitates the establishment of tumors from a tumor cell inoculate. Likewise, the coinjection of wound fluid and melanoma cells resulted in increased tumor volumes. It has also been shown that full thickness transcutaneous wounding is a sufficient event for tumor expression and growth in both Rous-sarcoma virus infected chickens and v-ras transgenic mice. This demonstrates that wounding can promote tumorigenesis in a host that is already initiated by viral infection or by oncogene expression. In the Rous sarcoma chicken model TGF-beta, a pleiotropic cytokine, has been implicated as a molecular mechanism of wound initiated tumorigenesis. These reports suggest a strong similarity as well as interaction between the wound microenvironment and the tumor microenvironment, and that this interaction can accelerate tumorigenesis and tumor progression in an unfavorable way for the host. Clinically, surgical procedures are typically performed in the proximity of a pre-existing tumor as a necessary component of tumor treatment. While these procedures attempt to eradicate the tumor for the benefit of the patient, local tumor recurrence and implantation of tumor cells along the wound or the needle tract have been described. Although this is often attributed to mechanical tumor spread, the local wound environment itself may similarly influence any residual tumor cells in a negative way. Understanding the mechanisms involved in wound-tumor interactions will help to identify therapeutic targets to prevent a negative impact of such procedures on tumor patients. In order to understand the role of the immune system in wound promoted tumor growth we have begun to study how wounding influences tumor growth in immunocompromised animals. Using athymic BALB/c nu/nu mice we found that wounding does not significantly accelerate tumor growth, indicating that wound promoted tumor growth is mediated by T-cells. We furthermore could show that wound fluid not only increased proliferation rates in vitro, but also accelerated tumor growth in vivo when 4T1 cells were treated with wound fluid generated from BALB/c mice before injection into animals, or when wound fluid was injected in the proximity of the tumor site;wound fluid generated in BALB/c nu/nu animals had no significant effect on tumor cell proliferation or tumor growth. These data indicate that wound promoted tumor growth is relayed by a soluble factor secreted by T-lymphocytes. We currently aim to clarify the role of different T-cell subsets in our model, and started CD8 depletion experiments in collaboration with Lalage Wakefield. If applicable, we will use a similar approach to investigate other T-cell subsets such as CD4 cells by depletion. In parallel, we will harvest tumor tissue at different time points after wounding and analyze the influx of T-cell subsets into the tumor / wound microenvironment using immunohisochemistry and FACS analysis. Since we already demonstrated that wound fluid effects proliferation of tumor cells in vivo and tumor growth in vitro, we will analyze the cytokine expression pattern of wound fluid generated from BALB/c, BALB/c nu/nu mice, mouse plasma and mouse serum using antibody microarrays (Raybiotech);protein expression patterns will be analyzed by 2D-electrophoresis and subsequent mass-spectometry, and protein microarrays will be employed if applicable. Based on these approaches and the current literature, we will establish a shortlist of candidate cytokines or proteins that mediate wound promoted tumor growth. Alternatively, we will start fractionating wound fluid and investigate the effect of these fractions on tumor cells and stromal cells such as fibroblasts or endothelial cells in order to identify new effector molecules. Furthermore TGF-beta has been implicated in wound triggered tumorigenesis in Rous sarcoma virus infected chickens. TGF-beta's has a complex role in tumorigenesis and metastases, and its influence on different cells types depends on the cell type and the signaling context. For example, TGF-beta can stimulate matrix secretion by stromal cells and angiogenesis, and modulates immune function, all of which are altered during tumorigenesis as well as during wound healing. Using a Smad3 knockout model we could show that defect stromal TGF-beta signaling yields smaller tumors in the 4T1 model, and that tumors from Smad3 knockout animals have less CD31 positive vessels. We bred the Smad3 knockout into the BALB/c background to analyze the effect of defective stromal TGF-beta signaling on wound promoted tumor growth. Using this model, we were able to identify several candidate proteins involved in wound promoted tumor growth. In particular, we demonstrated that pretreatment of cells with SDF-1 increased tumor growth while the inhibition of SDF-1 and/or its receptor CXCR4 decreased tumor growth. It was also demonstrated that the inhibition of SDF-1/CXCR4 signaling in vivo reduces the effect of wounds on tumors. We now plan to identify the origin of SDF-1 in wounds/tumors, investigate the role of SDF-1 in wounded promoted tumor growth and to identify the target cell of SDF-1. In addition, we will follow up on the role of T-lymphocytes in wound promoted tumor growth by identifying subsets of T-lymphocytes in wounds and identifying which subset of T-lymphocytes relays wound promoted tumor growth.	{ "pile_set_name": "NIH ExPorter" }
The aim of this research is to examine the mechanism through which social support affects morbidity and mortality from coronary heart disease. Two hypotheses have been proposed to account for this mechanism, both of which operate through effects on risk factors for coronary heart disease, specifically arterial blood pressure and serum lipids. the stress- buffering hypothesis states that social support functions as a social resource for individuals exposed to social stressors; those persons with more support are less vulnerable to the risk-enhancing effects of stressors. The health behavior hypothesis states that persons with more support engage in more positive health behaviors, such as avoiding tobacco; moderate alcohol use; exercise; and, a prudent diet. A major limitation in research to date to evaluate these alternative hypotheses has been the failure to conceptualize and measure social support in a way appropriate to detect its effects in varying social and cultural contexts. This is a major issue in research in sub-cultural communities that are also high risk communities, such as African-Americans. In this project a cross-sectional survey of social stressors, social supports, health behaviors, and arterial blood pressure and serum lipids will be conducted in a black community in the rural South to evaluate the relative strengths of the stress-buffering versus the health behavior hypothesis. Social support will be measured using a culturally appropriate technique which is sensitive both to the distinction between kin and nonkin social support, and the modification of the effects of that support by generational status. Multiple regression analysis and path analysis will be used to evaluate the alternative hypotheses. The research will contribute directly to a better understanding of the mechanism through which social support influences coronary heart disease.	{ "pile_set_name": "NIH ExPorter" }
The current study is designed to define the clinical characteristics of HIV-infected pregnant women, to determine the HIV-specific clinical care offered to them, and to evaluate the potential for participation of these women in perinatal clinical trials. In addition, a 6 month follow-up pediatric data collection form will be completed in order to determine perinatal HIV transmission rates in this population of women and infants.	{ "pile_set_name": "NIH ExPorter" }
?DESCRIPTION (provided by applicant): Protein-protein interactions regulate fundamental cellular events and misregulation of these interactions leads to disease states. Several approaches centered on high-throughput screening and rational design approaches are being evaluated for development of synthetic compounds as protein-protein interaction (PPI) inhibitors. Proteins often utilize small folded domains for recognition of other biomolecules. The basic hypothesis guiding our research is that by mimicking these folded domains we can specifically inhibit chosen PPIs with rationally designed synthetic molecules. Based on this hypothesis, we have developed cell permeable stabilized helices termed hydrogen bond surrogate or HBS helices to target intracellular PPIs. This work has created a foundation for the development of a new class of structure-based therapeutics. As part of these recent studies, we have also developed computational algorithms to predict targets for these helix mimics. The present proposal builds on this success to target therapeutically important interfaces that have not yielded to our past designs. We will build new experimental approaches to overcome hurdles. The three specific aims are to (1) develop a protein surface analysis approach to target recalcitrant helical and non-helical complexes, (2) establish an approach for target-guided assembly of stabilized helices, and (3) assess properties that aid cellular uptake of HBS helices and explore mechanism of entry for stabilized peptide helices. Studies in each Aim will make significant contributions to general approaches to inhibit PPIs and allow advancement of synthetic helices as distinct constructs spanning the molecular size space between small molecules and proteins.	{ "pile_set_name": "NIH ExPorter" }
The overall objective of the proposed research is to continue to use the estrogen induced and -dependent renal carinoma of the hamster as a model to study estrogen carcinogenicity, dependency of these carcinomas on hormones, and the transition of these tumors from hormone dependency to autonomy. Pertinent is the relationship between estrogen carcinogenesis and the steroid receptor system. The presence of a 4 S estrogen binder in the hamster kidney together with its increased quantities in DES treated animals as evidenced by the appearance of an 8 S moiety and the markedly increased levels of progesterone receptor as a result of estrogen treatment during the period of estrogen renal tumorigenesis remains to be defined. Our demonstration that all of the major classes of steroid receptors are present in the hamster carcinoma and its metastases in appreciable quantities provide a unique system to study both in-vivo and in-vitro the harmonal interrelationships involved in tumor response to endocrine therapy as well as the interrelated actions of steroid hormones within a single tissue. Therefore, the multiple steroid hormone receptors found in the hamster renal carcinoma should be a useful model in understanding hormone dependence and responsiveness of such human carcinomas as cancer of the breast and endometrium in which multiple steroid receptors are established clinical findings. Since estrogen carcinogenesis in the hamster is not complicated by other carcinogens or hormonal factors, insight should be gained into the nature of estrogen as a carcinogen. To assess the possible therapeutic application of steroid hormones in the human renal carcinoma, the presence of steroid receptors will be assessed in terms of their frequency of occurrence and concentration.	{ "pile_set_name": "NIH ExPorter" }
Project Summary/Abstract Background. Plexins are transmembrane receptors for the semaphorin axon guidance molecules. Repulsive signals from semaphorin-bound plexins are critical for proper pathfinding and innervation of developing neurons. Plexin signals also play important roles in regulating cell migration, vascular patterning and immune responses. Malfunction of the plexin signaling pathways is implicated in a variety of diseases such as neurological disorders, cancer and autoimmune diseases, and plexins have emerged as new drug targets for these diseases. Essential to the signaling of plexins is their intracellular regions, which contain a R-Ras GTPase activating protein (GAP) domain. The GAP domain contributes to plexin-mediated axon guidance by inactivating R-Ras, which leads to inactivation of integrin and loss of cell adhesion. The plexin GAP domain is normally kept inactive, and its activation requires simultaneous binding of semaphorin and a RhoGTPase (Rac1, RhoD or Rnd1) to the extracellular region and the intracellular RhoGTPase binding domain (RBD) of the receptor, respectively. Objectives. The goal of this research program is to understand the molecular mechanisms of autoinhibition and activation of the plexin GAP domain. Research Design. We use X-ray crystallography in combination with biochemical and cell biological approaches to study the mechanisms. We have solved the crystal structure of the intracellular domain of plexin A3. The structure shows that the GAP domain adopts an inactive conformation, and suggests that the RBD and a N-terminal segment contribute to stabilization of this autoinhibited state. Our analyses of the structures also led to a hypothesis that the plexin intracellular domain can form a specific dimer when plexin is induced to dimerize by semaphorin, and binding of a RhoGTPase to the RBDs of this dimeric plexin allosterically induces a conformational change in the GAP domain which triggers its activation. This proposal is centered around testing this model. In Aim 1 we will perform mutational analyses of the autoinhibition mechanism using a biochemical GAP assay and a cell-based assay. We will also pursue crystal structures of other plexin family members. In Aim 2 we will use the same GAP assay and cell-based assay to test the activation mechanism involving both dimerization and RhoGTPase binding. In Aim 3 we will study the activation mechanism for the GAP domain by determining structures of the plexin intracellular domains in complex with RhoGTPases and R-Ras. These studies together will reveal the molecular basis for the autoinhibition and activation of the plexin GAP domain, and provide new routes to future drug design for diseases associated with plexin malfunction.	{ "pile_set_name": "NIH ExPorter" }
Cholesterol homeostasis is maintained through the coordinated regulation of cholesterol uptake, synthesis, transport, esterification, secretion, and degradation. Recent advances have led to a more complete understanding of the way cholesterol's sorting and transport contribute to the maintenance of cholesterol homeostasis. However, the role of many intracellular cholesterol- binding proteins within various cells in the body, and their effect on cholesterol metabolism/degradation, remains unknown. The overall aim of this application is to elucidate the role of two START domain proteins (StarD4, StarD5) play in cholesterol and bile acid homeostasis in hepatocytes and macrophages using both in vitro and in vivo models. The P.I.s' labs have recently shown that cholesterol transport to the inner mitochondrial membrane represents the rate-determining step controlling bile acid synthesis via the 'acidic' pathway, and that the 'acidic' pathway is able to control nuclear levels of the known regulatory oxysterols, 27-hydroxycholesterol, 25-hydroxycholesterol, and at least one novel sulfated oxysterol. In addition to StarD1, there exists a family of proteins containing a StarD1 homologue domain that potentially is capable of binding and transporting sterols within cells. All proteins in this family have a similar structural lipid-binding domain referred to as the StarD1- related lipid transfer (START) domain. Most recently, they have also shown the following: 1) a coordinated response in the levels of StarD1, 27-hydroxycholesterol, and StarD5; 2) an intracellular redistribution of StarD5 protein from the Golgi to the plasma membrane in response to cholesterol; 3) that cholesterol is a ligand for both StarD4 and StarD5; the later also binds 25- hydroxycholesterol; and, 4) an increase in bile acid synthesis and cholesterol ester formation following StarD4 overexpression. In the studies proposed in this application, in vitro and in vivo models will be used to study the regulation, and physiological function of StarD4, and StarD5 in hepatocytes and in macrophages. Specifically, studies are proposed to: 1) characterize the regulation and subcellular movement of StarD4 and StarD5 in macrophages and hepatocytes (StarD4) in response to cholesterol; 2) characterize the function of StarD4, and StarD5 in macrophages and hepatocytes (StarD4); 3) characterize the physiological role(s) of StarD4 in cholesterol homeostasis in vivo. An understanding of the mechanisms of regulation of cholesterol homeostasis in hepatocytes and macrophages is very relevant to the understanding of the development of arteriosclerosis and heart disease in humans.	{ "pile_set_name": "NIH ExPorter" }
The supplement to the Surgeon General's Report on Mental Health, entitled "Mental Health: Culture, Race, and Ethnicity" (U.S. Public Health Service, 2001) emphasizes the need to develop, adapt, and evaluate assessment and intervention techniques so that they are maximally sensitive and responsive to the needs of particular minority cultural groups. In response to this need, The Community Schools Program at Children's Hospital of Philadelphia (CHOP) proposes to develop a social information processing measure for inner-city African American girls that is scientifically grounded, based in principles of partnership-based research, and informed by advances of video technologies. The new measure, The Assessment of Social Information Processing through Videos (ASIP-V), will draw upon the reformulated social information processing (SIP) theory, which posits that a child's ability to process a series of social cues within the environment will determine whether he/she responds aggressively. With funding from NIMH (K23 Award), the principal investigator has developed expertise in designing social cognitive measures for urban ethnic minority girls. The proposed R21 extends this partnership-based measurement development research, by combining empirically supported literature with consultation from content area experts and community consultants, and focus groups with 3rd and 4th grade girls, playground supervisors, and teachers to design 8 video vignettes to assess inner-city African American girls' SIP. Following this, an open pilot study with forty 3rd and 4th grade girls from a different school will be conducted in order to obtain information necessary to finalize the measure. Then in Year 2, 190 African American inner-city girls will participate in a validation study to determine whether the factor structure of the ASIP-V conforms to the theoretical factor structure outlined in the reformulated SIP theory (Crick & Dodge, 1994), and whether the measure has strong ecological validity and acceptability for inner-city African American girls. Relevance: This study will develop and validate a culturally-relevant.measure that could be extremely useful in future studies examining the social information processing among urban African American girls.	{ "pile_set_name": "NIH ExPorter" }
The principal objective of the proposed research is to clarify the participation and interaction of several metabolically linked chemicals which mediate locally the regulation of blood flow in the coronary circulation. The chemical factors which are the most likely candidates are oxygen, potassium ions, hydrogen ions (or carbon dioxide), and adenosine and its mononucleotides. The model used to investigate the role of these chemicals will be the isolated, Ringer's perfused guinea pig heart. A central component of the experimental design is a two heart perfusion system; one heart will serve as a bioassay organ for the vasoactivity of the effluent of the first heart. The specific goals of this project are to answer several controversial but fundamental questions: 1) Do low levels of O2 and pH, as observed during ischemia, interact to evoke vasodilation? 2) Does the H ion or CO2 per se produce vasodilation and what is the basis for the transient vasoconstriction induced by CO2? 3) Does O2 act directly or indirectly through release of adenyl compounds to effect vasodilation? Such added information will help to advance our knowledge of the mechanism of action and interaction of locally delivered and elaborated chemicals on adjusting coronary blood flow in response to ischemia, occlusion and increased myocardial function. It is believed that the findings elicited from the studies outlined here will contribute, not only to a better understanding of the normal physiological processses controlling blood supply to the heart, but also to establishment of a more comprehensive base of information on which to treat diseases of the coronary circulation rationally.	{ "pile_set_name": "NIH ExPorter" }
The cornerstones of prophylaxis and treatment of infections caused by non-tuberculosis mycobacteria (NTM) are the macrolides, clarithromycin and azithromycin. Although clinically acquired macrolide resistance in mycobacteria is conferred by 23S rRNA gene mutation, intrinsic resistance involves expression of adenine rRNA methylases, or erm genes. Moreover, recent data indicates that other genes can significantly affect the antimycobacterial activity of macrolides. For instance, an rpsA gene allele confers high-level macrolide resistance, and evidence suggests that disruption of trans-translation increases the bactericidal activity of macrolides. In addition, Mycobacteria may have a macrolide efflux system. Therefore, we hypothesize that mycobacteria may have several mechanisms that reduce the antimicrobial effects of macrolides. These mechanisms may include target modification (e.g., rRNA methylation or mutation), target recovery (e.g., trans-translation), and drug transport (e.g., efflux). Consequently, the long-term objective of this work is to characterize the mechanisms that affect the susceptibility of mycobacteria to macrolides. To address this objective, the project is divided into 4 specific aims: (1) to characterize the erm genes of mycobacteria;(2) to correlate the function of the rpsA and smpB genes with resistance to and recovery from the affects of macrolides;(3) to characterize the putative membrane protein genes that affect susceptibility to macrolides;and (4) to define the distribution of genes that affect the activity of macrolides within the Mycobacteriaceae. Mycobacteria are important human pathogens that cause chronic, often intractable disease. Understanding resistance and other processes that reduce drug activity will aid the design and development of new agents with activity against mycobacteria and/or treatment regimens.	{ "pile_set_name": "NIH ExPorter" }
This training program will provide a pathway directing medically trained individuals toward a successful research career in academic otolaryngology. Research experience and training will be available at two levels. At the first level, medical students will perform short (3 month duration) research projects designed to stimulate a general interest in research and a specific interest in otolaryngologic research questions. At the second level, residents selected for the 7-year "Advanced Physician Scholars Program" will be given the opportunity to perform research in depth, in a contiguous two-year period, free of clinical responsibilities. The block research period will generally take place after the surgical internship. It is expected that the research block will provide the individual with a "home laboratory" in which they will continue their investigations with emphasis on increasing their research skills and grant writing experience by competing for small grants for additional funding throughout their subsequent 4 years of clinical training. Attendance at the weekly research meetings and the biweekly combined research seminars between Washington University and the Central Institute for the Deaf, for the involved medical students and all Advanced Scholars, prior to, during, and following the block research time, will additionally provide a "research culture" in which they will continue to be immersed throughout the 7-year program. Such long-term mentoring will provide strong support for successful research career development. These two levels of training provide an opportunity to guide talented and highly motivated individuals toward academic faculty positions, in which they can combine their clinical and research expertise. The specific aim of the program is to train individuals so they can compete successfully for individual NIH-sponsored research awards, such as the Mentored Clinical Scientist Development Award, thereby encouraging a sustained and productive research career.	{ "pile_set_name": "NIH ExPorter" }
In the preceding funding period we have identified recurrent mutations and copy number increases of KIT in 30-40% of melanomas arising on chronically sun-damaged skin (CSD), acral sites. Emerging clinical evidence indicates that these genetic alterations are a strong indicator for clinical responses to already approved KIT inhibitors. By contrast, KIT alterations are absent in melanoma arising on the non-CSD skin, which frequently harbor BRAF mutations, identifying them as biologically distinct. Differences in mutation spectra, chromosomal aberrations, and clinical and histopathological presentation implicate the KIT pathway as an important contributor in acral, mucosal, and CSD melanoma types. Despite these advances, the oncogenic alterations in the majority of these melanomas are currently not known as they infrequently harbor BRAF mutations. Preliminary studies implicate additional genes downstream of KIT as candidate genes, including the scaffolding protein GAB2, the protein phosphatase SHP2, amplification of the two p70S6 kinase genes as additional targets for somatic activation. We hypothesize that together these findings indicate a wider role for KIT pathway activation in these melanoma types, which would have therapeutic and diagnostic implications. We will validate the contribution of genetic alterations in the KIT pathway to the malignant phenotype and response to therapy and aim to discover additional alterations. Our studies will involve comprehensive genomic characterization of archival tissues to identify genetic alterations and determine in which combination they occur (aim 1), functional analyses in vitro models representing the most common of the findings in human tumors to characterize their effect on sensitivity and resistance to targeted agents (aim 2), and tumor samples from patients with KIT alterations who were treated with KIT inhibitors, to determine factors of primary and secondary resistance to treatment in a clinical setting (aim 3).	{ "pile_set_name": "NIH ExPorter" }
In vertebrates lactate dehydrogenase (LDH) exists as a tertrameric enzyme having identical subunits with a molecular weight of 36,000; it is specific for L-lactate. On the other hand, an investigation of LDH's in invertebrates and microorganisms reveals a far greater diversity in specificity, subunit structure, and allosteric properties. With both the primary structure and crystal structure of dogfish M4 and pig H4 LDH known, it becomes meaningful to compare structural homologies which may exist in some of these other LDH's. LDH provides an excellent model for investigating the evolution of enzyme specificity, subunit interaction, and allosteric control and for correlating significant differences in enzyme properties with changes in primary structure. LDH from lobster tail shows unusual sigmoidal kinetics which can be correlated in part to the aggregation state of the enzyme in which the dimer and tetramer are in equilibrium. We have devised a technique for rapidly and selectively isolating peptides from the active site regions of the molecule. The unique nature of lobster tail LDH in comparison to vertebrate LDH's is indeed reflected in a sequence comparison of these active site regions. The complete primary structure is now being elucidated. Again in contrast to vertebrate LDH's, other LDH's exist which are specific for D-lactate. These enzymes are found in mollusks, arachnids, and in several bacteria and are consistently dimeric having a molecular weight of 72,000. We have purified one of these LDH's from horseshoe crab and are presently isolating peptides from the active site region in an effort to correlate the known changes in enzyme specificity with differences in amino acid sequence. A final LDH under investigation has been isolated from Lactobacillus casei. This enzyme is tetrameric although at high pH readily dissociates to a monomer. It is unique in that it is totally dependent on fructose-1, 6-diphosphate for enzymatic activity. Structural studies with this enzyme are in progress. BIBLIOGRAPHIC REFERENCE: Taylor, S.S., Amino Acid Sequence of Dogfish Muscle Lactate Dehydrogenase, J. Biol. Chem. (1977) 252, in press.	{ "pile_set_name": "NIH ExPorter" }
Type-1 diabetes (T1D) is hallmarked by autoimmune attack on the insulin-producing p cells of the pancreatic slets. Infiltration of the islets by autoimmune/inflammatory cells, or insulitis, occurs in a percentage of genetically susceptible individuals, and a fraction of these subjects progress to clinically overt diabetes. Our view of T1D pathogenesis, especially in humans, remains clouded and controversial in many regards. The main reasons for :his unsatisfying picture are the complexity and heterogeneity of T1D, and the fact that it is not usually diagnosed until very late, when most initiating and propagating autoimmune phenomena have already played out. One approach to surmounting the latter problem would be to develop a method to non-invasively image the pancreatic slets in vivo in real time, which has been one of the prime objectives of the collaborative JDC/MGH imaging program. The overall goals of this component of the program, Project 2, has been and remains first, to help evaluate the applicability of novel imaging methodologies (developed within Project 1 and the Core) to mouse models of T1D; second, to exploit successful visualization strategies to answer currently unresolved questions about disease pathogenesis in these models (certain in concert with Project 3); and third, to perform proof-of-principle experiments for translation of successful strategies to human T1D patients (under the auspices of Project 4). During the past funding cycle, Project 2 successfully developed a method to non-invasively image islet inflammation in murine T1D. This technique is based on visualizing, via magnetic resonance imaging (MRI) of magnetic nanoparticles (MNP), the microvascular changes (vessel dilation, vascular leakage) that routinely accompany inflammation, coupled with uptake by neighboring macrophages. A mock clinical trial on recent-onset diabetic mice clearly established the power of the MNP-MRI technique to distinguish between individuals that were or were not responding to anti-CD3 monoclonal antibody therapy. These promising results prompted initiation of a collaborative clinical trial to assess the ability of an analogous MNP, Combidex, to reflect vascular changes in association with human T1D (Project 4). For the next funding cycle, Project 2 proposes to apply the MNP-MRI technique to additional questions concerning the pathophysiology of T1D, but mostly to capitalize on exciting recent advances in fluorescent protein tomography (FTP) made by Project 1 and The Core. At the cutting-edge of whole-body live-rodent imaging, this technique permits non-invasive visualization of proteins and cells of interest in deep tissues (eg the pancreas) of living mice. The Specific Aims are: 1. To develop a system to co-image islet p-cell mass and p-cell activity non-invasively in vivo. A triple- transgenic mouse approach will be taken, exploiting the newly engineered red fluorescent proteins, mRaspberry and dKeima, in conjunction with FTP. Also under this Aim, a series of calibration and validation experiments will put the systems utility to the test. 2. To exploit the p-cell mass/activity co-imaging system to tackle outstanding issues concerning type-1 diabetes in mouse models. Questions to be tackled include: a) How does p-cell mass/activity change during the natural history of T1D in the NOD mouse model? b) How do genetic factors impinge on the mass/activity curves? c) Can the residual level of p-cell mass or activity predict onset of overt diabetes? d) Do different therapeutic regimens influence p-cell mass/activity? 3. To exploit MNP-MRI to guide dissection of the cellular and molecular elements predicting development of clinical diabetes in individual NOD mice. The novel capability of predicting if and approximately when a given mouse will convert from insulitis to diabetes will be used to address the influence of a NK cells b T helper cell subsets and c CD4+25+Foxp3+Treg cells within the infiltrate. Successful achievement of these Aims should provide novel insights into the pathogenesis of T1D, as well as furnish proof-of-principle for human imaging endeavors. Joslin Diabetes Center, Boston, MA PHS 398 (Rev. 04/06) PageM Form Page 2 Project 2: Imaging p-cell attack in mouse models of type-1 diabetes PI: MathiS, Diane PD: Mathis, Diane KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below. Start with Principal Investigator(s). List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Mathis, Diane DMATHIS JDC PI OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Kulkarni, Rohit JDC Consultant Weir, Gordon JDC Consultant Human Embryonic Stem Cells ^ No CH Yes If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list: http://StemcellS.nih.gov/reqistrv/indeX.aSP. Usecontinuation pages as needed. If a specific line cannotbe referencedat this time, includea statement that one from the Registry will be used. Cell Line PHS 398 (Rev. 04/06) Page^O Form Page 2-continued Number the following pages consecutively throughout the application. Do not use suffixes such as 4a, 4b. Project 2: Imaging p-cell attack in mouse models of type-1 diabetes PI: Mathis, Diane PD: Mathis, Diane The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. PROJECT 2 TABLE OF CONTENTS Page Numbers Cover Page 93 Description,	{ "pile_set_name": "NIH ExPorter" }
The leukemic cell is characterized by maturational arrest at a stage of hematopoietic development where the ability to proliferate is maintained. Human leukenic cells cultured in vitro can be stimulated to develop into a non-proliferating cell. Thus, the leukemias can be viewed, in part, as a disorder of differentiation as well as proliferation. One of the most potent agents stimulating differentiation of leukemic cells is 120tetradecanoyl phorbol 13-acetate (TPA), a phorbol ester. By substituting for diacylglycerol, TPA activates the CA2+- phospholipiddependent kinase, protein kinase C. The ability of diacyglycerol derivatives to mimic TPA stimulated cellular responses indicates that protein kinase C activation may mediate a portion of the cellular effects exerted by TPA. However, diacylglycerol derivatives are unable to stimulate differentiation of the human leukemic cell line U937. In our laboratory, a non- protein kinase C, TPA stimulated kinase activity has been identified. Protein kinase C and the TPA stimulated kinase activity can be distinguished by differences in substrate specificity. The inability of diacylglycerol derivatives to stimulate differentiation or to activate this kinase suggests that activation of the TPA stimulated kinase may be important in mediating TPA stimulated differentiation. The goal of this proposal is to characterize the nonprotein kinase C, TPA stimulated kinase and to determine if activation of this kinase is crucial in mediating the signal stimulating differentiation of the leukemic cell. Using column chromatography, this kinase will be characterized and compared to other non-protein kinase C phosphotransferase activities stimulated by TPA. Using 32P-labeled intact cells and two dimensional electrophoretic analysis, endogenous substrates phosphorylated by this kinase will be defined. The effects of other agents inducing U937 differentiation (lymphokines, dibutryl cAMP, etc.) will be evaluated for effects on this kinase activity. A better understanding of the mechanism controlling differentiation of human leukemic cells would aid in developing therapeutic modalities specifically designed to stimulate the in vivo differentiation of leukemic cells. Such a therapy, in addition to providing an alternative form of treatment, would theoretically avoid many of the toxic side effects inherent to the currently used antineoplastic therapy. The proposed research could also provide insight into the mechanisms mediating the many diverse effects exerted by phorbol esters in other biological systems.	{ "pile_set_name": "NIH ExPorter" }
Description: (Adapted from applicant's description) The overall objective of this project is to investigate the adult consequences of childhood learning disability (LD), math disability (MD), and particularly reading disability (RD). Developmental course and consequences are examined from two complementary perspectives: 1) from an epidemiologic perspective, these investigators will determine the outcome of RD and MD; and 2) from a fine-grained, analytic perspective, they will investigate the neurobiologic and cognitive mechanisms serving reading in adults with a childhood history of RD. They propose to study a precious resource, the cohort of 445 children recruited initially in 1983 and prospectively followed from kindergarten. In one Project, this large and virtually intact sample (n=401) will be studied at adulthood (ages 23-29 years). The sample, the measures and the longitudinal framework allow them to address two central themes: 1. Nature and determinants of outcome at adulthood; 2. Neurolinguistic and neurobiologic mechanisms in adults with childhood histories of reading disability. In the first, the investigators inquire of a large sample of adults who have been prospectively assessed for RD and MD: What is the nature and determinants of outcome of childhood RD and MD at adulthood? In the second, the investigators address mechanisms in adults with childhood history of RD, asking the question: What are the underlying cognitive and neurobiologic factors contributing to the development of fluency and automaticity in skilled readers and the relative success or failure to develop these components of skilled reading in groups of disabled readers? Although initiated in 1983, this study incorporates many of the recommendations made in this RFA. The Connecticut Longitudinal Study is unique in its ability to address some of the most pressing questions in learning disability research and practice. Innovative in using cutting edge neuroscience together with a prospectively defined population of a range of readers, some with persistent, others with compensated reading problems, this study weds the power of an epidemiologic survey, longitudinal follow-up with the power of modern imaging technology to provide a new dimension to studies of skilled reading, specifically automaticity and fluency.	{ "pile_set_name": "NIH ExPorter" }
Summary: The goal of this project is to apply and develop molecular biological methods and animal models that can ensure that vaccines and cell substrates are free from viral, and particularly retroviral, contamination and to assess the oncogenic and infectivity risks associated with residual cell-substrate DNA. Reverse transcriptase (RT) assays measure the presence of retroviruses. The conventional RT assay was insenstive and not quantitative. This changed with the advent of PCR-based RT PBRT) assays, which are a million-fold more sensitive than conventional RT assays. We set up the three PBRT assays at CBER in order to compare their sensitivities, specificities, and reproducibilities. Modifications were made to the assays to eliminate one problem with the assays, viz., their high background signals. We also modified the assay such that RT activities of cellular DNA polymerases were substrantially reduced. Recently, we have adapted the PBRT assay for use with the real time quantitative system, the TaqMan system, for use with the Perkin-Elmer 7700 system. This modified assay, the TM-PERT assay, is linear over at least 6 orders magnitude and is as sensitive as the original assays. As part of our investigation into the chick RT activity, we began a study to determine whether pseudotypes can form between a retrovirus core and an envelope glycoprotein (Env) from paramyxoviruses or orthomyxoviruses. If so, then this may provide a means by which the chick RT particle could enter human cells. As a model system, we investigated measles, mumps and influenza virus Envs with HIV core particles. Our results show that pseudotypes can form in vitro, and thus there is a theoretical possibility of such pseudotypes forming in vivo. The consequences of such pseudotype formation remains unknown. We have demonstrated that mixed pseudotypes [i.e., pseudotypes containing envelope glycoproteins (Envs) from two different viruses] can form between measles virus and HIV-1 Envs. This raises the possibility that such virions can form in vivo. This may have implications for HIV pathogenesis, since HIV particles containing an Env of measles virus would be able to infect non-CD4 cells; such infected cells would have unknown consequences on HIV pathogenesis. We have developed Q-PCR assays to detect the genomes of primate polyomaviruses (SV40, JCV, BKV). Assays for these viruses will be a first step in our program to establish quantitative assays for the detection of adventitious agents. With these Q-PCR assays, we are quantifying the levels of the human polyomaviruses BKV and JCV DNA in human blood with the aim of determining whether the primate polyomavirus SV40 is present in human blood. We have shown that antibodies to SV40 can neutralise BKV and antibodies to BKV can neutralise SV40. We are developing a reporter-gene neutralisation assay for SV40, BKV, and JCV in order to test whether exposure to one polyomavirus can affect the probability of being infected with another polyomavirus. We have initiated studies to develop quantitative assays to assess the biological activity of residual cell-substrate DNA. For many years, DNA resulting from cell substrates has been considered to pose a risk to vaccine recipients receiving products manufactured in neoplastic cells. This is one of the main reasons for these cells not being used for vaccine production to date. The risk is perceived to be either from an oncogenic potential and an infectious potential. We are developing quantitaive assays to assess both types of risks. In collaboration with NCI and CDER, we are exploring animal models to asssess DNA oncogenicity. We have developed an in vitro system to quantify the infectivity of retroviral DNA.	{ "pile_set_name": "NIH ExPorter" }
cDNA from hepatitis A virus (HAV) has been cloned into pBR322. Six cDNA clones which together span the entire genome were isolated and ligated together to form a single clone thought to represent full length HAV cDNA. Transfection of both tissue culture cells (in vitro) and marmosets (in vivo) with these plasmids failed to generate HAV. Fine structure mapping of the HAV cDNA indicated that about 40 base pairs had been deleted during the ligation process. The deletion was repaired, but transfection of marmosets and tissue culture cells still failed to generate HAV. An additional modified construct, differing by two nucleotides thought to be important for infectivity, also failed to generate HAV in marmosets. The entire full length construct was sequenced; comparison with the sequence of the parent clones used to create the construct did not reveal any differences. The cDNA was placed into an RNA transcription vector and plus strand RNA was made in vitro from the cDNA. Transfection of marmosets with the RNA failed to generate HAV. Preparation of plus strand HAV RNA, synthesized in vitro from minus strand HAV RNA and poliovirus RNA polymerase, is in progress.	{ "pile_set_name": "NIH ExPorter" }
Understanding the physiologic relationship between genomics and proteomics is a common theme and focus for investigation. The use of cell models and organ systems remains a fundamental approach to achieving such an understanding. Enamel is a complex bioceramic tissue that is a reflection of its unique molecular and cellular formation. From their ectodermal origins, ameloblasts exist for the sole purpose of producing a mineralized shell. The shell is almost entirely devoid of protein and is deposited over the "bone-like" dentine. We have recently discovered TIP39kDa, a protein that favors the secretory surface of ameloblasts (Tomes' processes). Antibodies to TIP39kDa react to secretory vesicles within Tomes' processes, and also the immature enamel matrix. TIP39kDa expression is not unique to ameloblasts, but preliminary data suggest that it may be unique to cells with a significant secretory function. This project will pursue an experimental strategy to identify a definitive function for TIP39kDa, and also explain the nature of the multiple messenger RNA isoforms observed for TIP39kDa. The first aim will test the hypothesis that TIP39kDa is involved with the cells secretory activity by down-regulating TIP39kDa expression within cell and organ cultures, and look at the subsequent levels of a secreted protein (amelogenin). For this I will employ both an antisense RNA and a ribozyme approach. The second aim will be to use the TIP39kDa gene promoter in an appropriate reporter system and produce transgenic animals to define organs for which TIP39kDa plays a role. The third aim will identify the subcellular localization of TIP39kDa. For this I will employ available TIP39kDa antibodies and green fluorescent protein tags. Regular database searches will be performed to assist in the identification of functional domains. The fourth aim will be to screen a yeast two-hybrid mouse embryonic library to look for TIP39kDa protein partners. This data will be used to help identify a physiological role for TIP39kDa. The fifth and final specific aim will seek to answer the question of how multiple messenger RNA isoforms of TIP39kDa are generated as evident from previously published data. These first four specific aims will assign a subcellular location and function to a TIP39kDa that, in the case of ameloblasts, has a preference for Tomes' processes, secretory vacuoles and the immature enamel matrix. The fifth specific aim will answer a very basic question related to how TIP39kDa is processed post-transcriptionally, and the potential significance of this. [unreadable] [unreadable] [unreadable] [unreadable]	{ "pile_set_name": "NIH ExPorter" }
There are a number of different chemomechanical coupling systems. The most ubiquitous in the actin-myosin-ATP system. Another, also common in eucaryotic cells, is the tubulin-dynin system. In bacteria it appears that energy supplied to the membrane is used via a coupling protein (the mot gene product) which converts it into the rotation of the flagellar filament. The mechanism of this process is not understood. We think that it is fundamental for understanding and affecting bacterial cell function. Our proposal is aimed at elucidating this process and determining how general it is among bacteria and what role it plays in infection. We plan to isolate and study this protein. However, since we have not assay procedures that can specifically detect this protein we have to use less conventional approaches. We plan to use genetic techniques to amplifY the mot genes and to identify the gene products. The products will be isolated and characterized. We will try to determine how it functions, and we will see if analagous proteins would be found in nonflagellated bacteria. Finally, we will set up systems to evaluate the role of motility and chemotaxis in the infectious process.	{ "pile_set_name": "NIH ExPorter" }
This proposal focuses on creation of a sequencing core for the Undiagnosed Disease Program (UDP) as well as comparison of the utility of genome-wide sequencing (GWS; also known as whole genome sequencing) versus Whole Exome Sequencing (WES) for the identification of causal variants. Illumina and the Medical College of Wisconsin (MCW) have worked together to advance genomic sequencing into clinical medicine; this proposal is joint between these entities. All of the necessary components for the UDP sequencing core are functional at MCW and Illumina and required capacity and turnaround are met. Both groups have championed GWS as opposed to WES for genetic discovery leading to the second focus; comparison of GWS and WES for diagnostic success. MCW uses both WES and GWS; along with obvious advantages in detecting non protein coding variants, we find significantly better coverage of actionable genes with GWS, and a higher diagnostic success rate. We thus propose to conduct GWS for all participants enrolled in the UDP creating the opportunity to compare utility of WES versus GWS. With an integrated team and using innovative lab and bioinformatics techniques we propose to test the hypothesis that GWS will produce at least 25% more diagnoses than WES. Aim 1 will generate clinical grade GWS for all UDP cases sequenced and perform read mapping and variant calling. Sharing of the data generated and the methods developed will enable the UDP network to directly compare diagnostic use of WES and GWS. Aim 2 will undertake clinical grade tertiary analysis of the data using our clinically validated analysis platform; we will also provide clinical interpretation and report generation for all cases requested. These will be produced using our existing clinical methodology and tools. Aim 2 will also support dissemination of the methodology and offer tertiary analysis and clinical interpretation to all UDN sites. Aim 3 will confirm the NextGen sequencing results using Sanger and, through gathering of this data, determine whether this step will be necessary in the future. We envision that all of the laboratory operations, methodologies, and tools developed will be made available and will be suited for cloning in additional currently non network hospitals and large clinics. Relevance: This application is highly relevant in that it seeks to establish MCW as the sequencing core for the UDP. In addition to meeting this goal, the application seeks to extend the UDN benefit by determining whether application of GWS as compared to WES provides a diagnostic advantage.	{ "pile_set_name": "NIH ExPorter" }
The proposed research is concerned with the biochemical dissection of morphogenesis. Four major systems are under investigation. (a) The retino-tectal projection in the chick embryo. Using in vitro assays for cell recognition between retinal cells and tectal surfaces, cell surface moieities are selectively destroyed with lytic enzymes and the effects on recognition are evaluated. One morphogenetically-active compound has been identified, so far, as the ganglioside GM2. (b) The role surface glycosyltransferases in chick embryo morphogenesis will be tested by specific perturbation of the enzyme classes. Preliminary data suggest that such interference leads to predictable classes of morphogenetic defects. (c) In order to gain further understanding of the biochemistry of the glycosyltransferases, the liver conjugating enzymes are being investigated. These glucuronyltransferases are advantageous for study because they are easily assayed, they are inducible in the chick embryo, with induction, they appear first in the internal cell membranes and, later, on the cell surface and, finally, genetic defects for some of these enzymes are available in the rat and human. (d) Neural crest cells from wild-type and crest-defective mouse mutations will be grown on substrates of hyaluronic acid and chondroitin sulfate in order to test and compare their abilities to migrate on, and spontaneously glycosylate, these substrates. Preliminary experiments with patch and dominant spotting mutants show marked differences in both these parameters.	{ "pile_set_name": "NIH ExPorter" }
Although intrauterine growth retardation complicates 4-5% of all pregnancies, and the fetus is particularly susceptible to acute stress, very little is known about the physiologic mechanisms involved in the fetus' inability to tolerate stress, or how the mechanisms might be modified. The broad objective of these studies is to investigate the response of the fetus, which is growth retarded due to uteroplacental insufficiency, to acute stress, and to determine if these responses may be modified. Fetal growth retardation is produced by a gradual embolization of the uteroplacental vascular bed with non-radioactive microspheres. The development of autonomic regulation is studied. The effects of acute hypoxemia, or combined hypoxemia and acidemia on the cardiovascular and metabolic responses is investigated. Beta-adrenergic agents will be used to determine if the responses may be modified in a beneficial manner. BIBLIOGRAPHIC REFERENCES: Pickart, L.R., Creasy, R.K. and Thaler, M.M.: Polycythemia and Hyperfibrinogenemia as Factors in Experimental Intrauterine Growth Retardation. Am. J. Obstet. Gynec., 124(3):268-271, 1976. Char, U.C. and Creasy, R.K.: Glucose and Oxygen Metabolism in Normally Oxygenated and Spontaneously Hypoxemic Fetal Lambs. Am. J. Obstet. Gynec., 127(5): 499-504, 1977.	{ "pile_set_name": "NIH ExPorter" }
DESCRIPTION Traumatic brain injury (TBI) is a major source of disability among military and civilian populations. Unfortunately, the pathophysiology of the motor, cognitive, and affective dysfunction that can result from TBI remains incompletely understood. In right handed people with typical brain organization, the left hemisphere is dominant for the praxis systems that mediate how (postural and spatial-temporal movement parameters) we perform skilled purposeful movements, and the right hemisphere mediates action-intention systems that determine when we start, continue, stop, and inhibit our actions. Each hemisphere also controls movements of the limbs on the opposite side of the body. The corpus callosum is the major white matter structure that transmits information between the two cerebral hemispheres, and it is particularly susceptible to damage from TBI (Rubens et al, 1977), including mild TBI (Inglese et al, 2005; Wilde et al, 2008). Damage to the corpus callosum can impair the interhemispheric communication necessary for bimanual coordination, isolate the left hemisphere praxis systems from the right hemisphere (which controls the left hand), and isolate the right hemisphere action-intention systems from the left hemisphere (which controls the right hand). The purpose of this study is to test the hypothesis that specific and dissociable types of motor dysfunction result from traumatic injury to the left hemisphere, the right hemisphere, and the corpus callosum. For this proposed study, we will recruit approximately 16 experimental participants with moderate to severe TBI from closed head trauma, 16 with mild TBI from closed head trauma, and 16 matched controls who have not sustained TBI. This sample size was determined based on feasibility of recruitment within the two year time period of the CDA1 program. Data from this study will then be used for a power analysis to determine the sample size necessary for continuation of this project for future research including application to the CDA2 program. The AIMS of this study are to utilize behavioral-motor tasks to 1) test the hypothesis that people with TBI will have motor impairments resultant from dysfunction of the left hemisphere praxis systems (impaired production of gestures and deft fine finger movements) and right hemisphere action intention systems (motor impersistence and impairments of reaction times and motor response inhibition), and that in some people with a history of TBI, there will be a double dissociation of these types of impairments thus demonstrating that TBI can lead to selective impairments of these two systems; 2) test the hypothesis that people with TBI will have motor impairments attributable to callosal disconnection: apraxic deficits greater in the left hand than the right, action-intention deficits greater in the right hand than the left, and impaired bimanual in-phase and antiphase movements; 3) correlate the presence of motor impairments due to damage to praxis systems, action-intention systems, and callosal disconnection with impairments in daily functional ability as measured by the Disability of Arm Shoulder and Hand (DASH) scale. A characterization of distinct types of motor dysfunction that result from TBI is essential to understand the pathophysiology of TBI and the nature of the motor disability, to monitor recovery, and to guide individualized rehabilitation strategies that will be a focus of future research. PUBLIC HEALTH RELEVANCE: The Department of Defense reports 233,425 cases of TBI among service members of the US military from 2000-2011, with at least 94% of these cases caused by closed head injuries (Defense and Veterans Brain Injury Center website, 2012). Motor impairments are common after TBI, and their presence correlates with both functional disability and performance on other neuropsychological measures (Clifton et al, 1993). The nature of these motor impairments is not fully understood. This study aims to demonstrate the pathophysiological basis for distinct types of motor impairments that result from closed head trauma and to correlate these different types of motor impairments with functional disability. This research will add to our knowledge of the disability and prognosis after TBI and has the potential to lead to individualized rehabilitative interventions through future research.	{ "pile_set_name": "NIH ExPorter" }
The Bioinformatics Shared Resource (BSR) is a new Shared Resource that performs statistical and computational analysis of highly multivariate biological data arising from cDNA or oligonucleotide microarrays, serial analysis of gene expression (SAGE), tissue microarrays (TMA) or proteomics. Studies using these high-throughput technologies are becoming increasingly important in cancer research as investigators broaden their search for potential therapeutic targets and try to elucidate the pathways and mechanisms relevant to diagnosis and prognosis. As a new component of the CCSG at The University of Texas M. D. Anderson Cancer Center (UTMDACC), the BSR works cooperatively with the biologists and technicians of the institution's Genomics Core Facility to provide bioinformatics services to cancer researchers throughout the institution. Through this cooperative effort, the BSR assists researchers in the application of state-of-the-art methodology for the development, conduct, and analysis of studies using high-throughput technologies. Programs of the CCSG are supported through the seven primary services performed by the faculty and analysts of the BSR: (1) Assist researchers in developing effective experimental designs; (2) Develop and implement curatorial tools for the acquisition, structural preservation, storage, and retrieval of data from high-throughput technologies; (3) Develop new statistical methods for data analysis; (4) investigate analytical methods published in the literature; (5) Apply state-of-the-art methodologies in the rigorous analysis of experimental data; (6) Link experimental results to public databases that annotate gene descriptions and functions; and (7) Provide education and knowledge transfer to biologists and statisticians through weekly bioinformatics seminars, research publications, and public a Web site. The BSR operates under the guidance of directors of the core laboratories and facilities to provide high-throughput technologies in the areas of pathology, molecular pathology, and cancer genetics. The BSR has 55 users from 16 programs; 64% of the users have peer-reviewed funding.	{ "pile_set_name": "NIH ExPorter" }
Prostate cancer is the second leading cause of cancer death in men in the United States. Localized prostate cancer can be cured by androgen ablation, but when the disease escapes the confines of the gland, the prospects for cure decrease drastically and the disease becomes castrate resistant. Bone is the primary site of castrate-resistant disease progression, which is associated with a poor prognosis. The fibroblast growth factor (FGF)/FGF receptor (FGFR) complex, a signaling axis involving multiple FGF ligands and receptors, mediates tumor-stromal interactions and is one ofthe most commonly altered signaling pathways during prostate cancer progression. Expression of FGFR1, multiple FGF ligands, and FGFR adaptor, FRS2a has been observed in prostate cancer epithelial cells. Our recent studies have defined a mouse model of prostate cancer highly dependent on FGF signaling, and have implicated FGFQ in the osteoblastic progression of human prostate cancer cells in bone. The results of our preliminary studies support a notion that during bone metastasis, the prostate cancer cells that aberrantly express both FGF and FGFRs creates a new compartment in bone as source and recipient of additional FGF-mediated signaling, thus subverting homeostasis. The implication ofthe FGF axis in prostate cancer progression suggests that FGFR blockade represents a new therapeutic opportunity for men with castrate-resistant prostate cancer. Recently, TKI258, a receptor tyrosine kinase inhibitor (TKl) with strong activity against FGFR1-3 (IC50 < 40 nM), has become available and is being used as an experimental new drug for solid tumors. The main goal of this proposed project is to establish the feasibility of using TKI258 to modulate FGF signaling in men with castrate-resistant prostate cancer and to correlate FGF signaling modulation with clinical disease progression. We will assess the effect of TKI258 on human prostate cancer xenografts growing in the prostate and bone of castrated immunodeficient male mice (Aim 1), and also on mouse models of prostate cancer (Aim 2) to identify markers of response to TK1258 therapy directly related to FGF signaling. We will then perform a proof-of-principle clinical study with TKI258 in men with castrate-resistant prostate cancer and bone marrow infiltration (Aim 3). The study will create an annotated tissue resource and will permit validation of FGF signaling responsive markers emerging from Aims 1 and 2. This will be the first clinical study to assess the effect of TKI258 in prostate cancer. RELEVANCE (See instructions): Prostate cancer is the second leading cause of cancer death in men in the United States. In the proposed study we will perform a proof-of-principle clinical study with a receptor tyrosine kinase inhibitor, TK1258 in men with castrate-resistant prostate cancer and bone marrow infiltration. The results of this study will be the foundation for development of further therapies of prostate cancer patients based on targeting the fibroblast growth factor signaling pathway.	{ "pile_set_name": "NIH ExPorter" }
The lipogenic enzyme ATP-citrate lyase (ATP-CL) is subject to dietary regulation by sucrose. The level of hepatic ATP-CL is increased several fold in the sucrose refed rat over the level found in the liver of starved rats. This induction of ATP-CL by dietary sucrose has been shown to be due to an increased amount of ATP-CL. This increased amount of ATP-CL has been shown to be due to an increase in the rate of synthesis of ATP-CL rather than to a decrease in the rate of degradation of ATP-CL. Dietary sucrose either stimulates the release (or synthesis) of a substance or is converted to a substance which is the molecular inducer of ATP-CL. The identity of this molecular inducer, the step of protein synthesis affected by the molecular inducer, and the mechanism of induction of ATP-CL are not known. The following research proposal is designed to determine the step of protein synthesis which is acted on by the molecular inducer to yield increased synthesis of ATP-CL. A homogenous preparation of rat liver ATP-CL will first be prepared in order to make an antibody specific against rat liver ATP-CL. This anti ATP-CL will be used to identify nascent and completed ATP-CL and to precipitate polysomes containing nascent ATP-CL. The determination of the puromycin released nascent ATP-CL, in vitro completed ATP-CL and polysomes containing nascent ATP-CL during starvation and sucrose refeeding will determine at which step, if any, of translation (i.e. chain initiation, chain elongation and chain termination) sucrose exerts its molecular effect. The amount of ATP-CL mRNA will be measured by determining the amount of mRNA in the polysomes containing nascent ATP-CL specifically percipitated by anti ATP-CL and by measuring the effectiveness of mRNA isolated from the different dietary states to produce ATP-CL (measured by precipitation with anti ATP-CL). If sucrose is shown to increase the amount of ATP-CL mRNA, then the rates of synthesis and degradation of ATP-CL will be determined by following the appearance and disappearance of (C14) uridine in the mRNA fraction of polysomes containing nascent ATP-CL precipitated with anti ATP-CL.	{ "pile_set_name": "NIH ExPorter" }
The primary goal of the Prevention Research Program is to provide prodromal participants with a structured, high quality clinical care setting within which the proposed Center research can take place. The objectives are 1) to recruit patients with prodromal symptoms and demographically comparable healthy controls; 2) to conduct diagnostic and screening evaluations of potential participants to determine study eligibility and to coordinate their participation in the following TRCBS research projects: Long-Term Memory Processes, Development of Automaticity, Attention and Dual-Task Interference, Associative Learning and Emotional Regulation, Social Cognition, and Stress and Emotional Reactivity; 3) to sustain subjects' participation in the TRCBS research protocols for a period of at least 12 months by providing extensive case management, psychological, and (when appropriate) psychiatric services; 4) to conduct repeated standardized assessments of diagnosis, clinical symptoms and functional status and to ascertain cases who convert to schizophrenia and other psychotic disorders within 1 year. To achieve the first objective, we will engage in community outreach activities and partner with community mental health sites to generate referrals of patients with prodromal symptoms. Staff will attend disposition planning meetings at local mental health sites to ascertain cases and give talks to educate local community mental health programs, high schools, colleges, and relatives' groups about the schizophrenia prodrome and our services. To achieve the second objective, we will conduct diagnostic and screening interviews of established reliability and predictive validity and continue to develop efficient screening instruments to diagnose the prodrome. To achieve the third objective, we will provide prodromal patients with case management, social and life skills training, family education, ongoing monitoring of symptoms and functional outcome, psychiatric evaluation and, when clinically indicated, psychiatric treatment. By offering ongoing evaluation and case management to participants, we hope to detect conversion to psychosis earlier than would otherwise be typical. Intervention early after onset of psychosis is associated with better treatment response and long-term prognosis. To achieve the fourth objective, we will repeat diagnostic and symptom assessments at 1-, 3-, 6-, and 12-month follow-ups and assessments of functional status at 6- and 12-months. Each TRCBS research project will evaluate a set ofneurocognitive and/or emotional processes in order to determine whether baseline functioning in these systems, and deterioration over time, are associated with onset of psychosis. This information could improve theoretical specification of the mechanisms underlying psychosis onset and improve the sensitivity and specificity of prediction of schizophrenia, so that future primary prevention efforts can be targeted to the individuals who need it most.	{ "pile_set_name": "NIH ExPorter" }
Project Summary/Abstract Veterans and other Americans who survive stroke often face disabling motor impairments that impede performance of activities of daily living and limit free-living activity. Prominent among these are diminished locomotor function and impaired balance that not only foster a sedentary lifestyle and physical deconditioning, but also increase the risk injuries due to falls. Recent research from our group at the Baltimore VAMC and others around the world has demonstrated how motor learning based interventions can modify brain activity and improve motor functions in persons with stroke. Now there is a major research opportunity to advance the effectiveness of these interventions by applying new robotics technologies to improve neuromotor control of essential functions such as gait and balance. One critical area for performance of walking and standing balance is the control of the ankles, as they are a major conduit of mechanical power in gait and also modulate torques affecting the motion of the whole body center of mass when balancing. Thus the current proposal is designed to investigate two approaches for using an impedance controlled ankle robot to improve gait and balance function among stroke survivors with chronic lower extremity hemiparesis. One approach uses the ankle robot in a seated visuomotor training program that focuses on improving paretic ankle motor control that may transfer to gait and balance functions. The other approach follows the dominant rehabilitation paradigm of task-specific training by integrating use of the ankle robot during treadmill exercise training to assess effects on the same outcomes. The effectiveness of both robotics approaches will be compared to that of a treadmill exercise program without robotics. The study tests the hypothesis that, in persons with chronic lower extremity hemiparesis, 6 weeks of seated ankle robot training will improve paretic ankle motor control with major improvements in standing balance and moderate improvements in gait, whereas the same amount of training on the treadmill with the ankle robot will improve gait function more than balance. Both robot-trained groups will outperform the treadmill only group on balance, while the treadmill + robot group will make the greatest gains in gait and the seated robot group will make some improvement in gait but will show greater gains in ankle motor control and balance. Aims: In a 6-week intervention (18 sessions) with persons with chronic lower extremity hemiparesis 1) Compare effects of seated visuomotor ankle robot training vs. treadmill + robot training on paretic ankle impairments and motor control;2) Compare effects of seated-robot vs. treadmill + robot training on functional mobility and balance outcomes;and 3) Compare the effectiveness of both robotics approaches to a standard treadmill exercise protocol of the same duration. This proposal will establish the initial comparative efficacy of two motor learning based approaches using a modular impedance controlled ankle robot and contrast motor control and functional gait and balance outcomes among them. As a pilot study we will establish initial deficit profiles for users that respond to each intervention across the 6-week period. This will begin to fill the knowledge gap on how to apply robotics technologies in the elderly stroke population. Such findings have the potential to change the paradigm of stroke care based upon advances in our understandings of activity-dependent brain plasticity and the critical need for improving mobility in the rehabilitation of Veterans and others with disabilities. Our results will provide the requisite information to design a larger Merit Award trial that refines or combines these interventions to reduce risk factors for disabilities and limited social participation in the growing stroke population. PUBLIC HEALTH RELEVANCE: Relevance of the Proposed Work to the VA Health Care Mission The VA patient care mission seeks to improve the general health status and quality of life of Veterans after disabling diseases such as stroke. Using a novel ankle robotics device, this study tests two new approaches aimed at improving gait and balance function after a stroke. In one approach subjects use the robot to play ankle videogames and the other uses the robot while walking on a treadmill. The goal is to re-learn motor control of the weaker ankle to improve gait and balance. These approaches may eventually help in other neurologic disabilities, including neurologic injuries among returning Veterans from recent wars. Generally, such uses of robotics may help to reshape rehabilitation models within the VA by augmenting the effectiveness and scope of therapy by allowing longer training sessions, facilitating more intensive sessions, and enabling therapists to work with larger numbers of VA patients at a given time.	{ "pile_set_name": "NIH ExPorter" }
Physical activity clearly favors bone formation although the optimal exercise regimen for preventing loss at the sites of interest, namely thoracolumbar spine, proximal femur and distal radius, has not been determined. The purpose of this study is to evaluate the potential effect of low and moderate intensity stationary cycling on bone mass in the elderly population.	{ "pile_set_name": "NIH ExPorter" }
The Breast Cancer Detection and Demonstration Project (BCDDP) screening program began in 1973 in 29 centers in 27 widely dispersed geographic areas of the United States. Initial screening was complete on over 280,000 women over a 2-year period. From the original 280,000 participants in the screening phase of the BCDDP, approximately 64,000 were selected for 4 years of long-term follow-up (LTF) beginning in 1978, to assess the biology and natural history of breast disease, and to test hypotheses relating to detection, etiology, and survival. Those selected for LTF included all breast cancer cases found during the screening phase, all benign breast cancer cases, all those recommended for biopsy, and a sample of "normals." The LTF database will facilitate the exploration of important questions regarding the etiology and natural history of breast cancer. The size of the subcohorts and breadth of data available on them make this population unique. The large number of cases of both breast cancer and benign breast disease with histologic information available should allow particularly useful analyses of several risk factors in relation to these conditions. The first 5 years of LTF was completed in all centers in September 1986, and a further continued follow-up has begun. This project is being conducted jointly by the Cancer Prevention Studies Branch of the Division of Cancer Prevention and Control and the Environmental Epidemiology Branch of the Division of Cancer Etiology.	{ "pile_set_name": "NIH ExPorter" }
Summer vacation represents an important time away from the school setting for a majority of children attending public school in the US. This break is characterized by large amounts of free time and involvement in a wide variety of formal and informal activities, time spent with friends and family, and travel. Yet for many children, particularly those from low-income households, summer vacation represents a ?window of vulnerability? in which dramatic declines in both health and academics occur. During the summer months (typically 3 months) children gain a larger amount of body weight compared to the amount of weight gained over the school year, and weight gain during summer reverses weight losses achieved during school. For academics, it is well established that children from low-income households experience greater declines in reading and math during the summer than their middle-to-upper income peers. Numerous programs that address these issues currently exist (e.g., summer school, summer weight loss or fitness programs). However, these programs are designed and delivered at the expense of the promotion or prevention of the other ? academic focus without health or health focus without academics. Our long-term goal is to develop summer programming for widespread dissemination that addresses both lifestyle behaviors (i.e., physical activity and nutrition) and academic performance. The objective of the proposed study is to establish the efficacy of an innovative ?Healthy Summer Learners? (HSL) program for low-income, minority children. The rationale for the proposed research is that no summer programs incorporate curriculum that addresses both unhealthy weight gains and academic achievement simultaneously. This application seeks to fill this void by testing a prototype learning and health- oriented summer program focused on promoting physical activity and nutrition, along with content that provides quality learning experiences to develop reading and math skills. To accomplish this objective, we will evaluate over 2 summers the effectiveness of a 10-week Healthy Summer Learners program delivered within a Boys & Girls Club summer camp using a randomized design in a sample of rising 4th graders. The aims of the study are Aim 1: Evaluate the impact of Healthy Summer Learners on children?s weight gain and academic performance from beginning (end of Spring school year) to the end of summer (beginning of Fall school year), and Aim 2: Evaluate the feasibility and acceptability of Healthy Summer Learners to children, parents, and program staff. This work is significant and represents an important step towards addressing important public health goals ? obesity and learning ? through a comprehensive program delivered during a timeframe ? summer vacation ? in which substantial and long-lasting negative effects occur. This application is innovative because of the dual approach of targeting both health and learning in a single, comprehensive program.	{ "pile_set_name": "NIH ExPorter" }
PROJECT SUMMARY Inducing a robust state of tolerance that is stably maintained despite adverse perturbations is essential for the ability of tolerance mechanisms to maintain the acceptance of the transplanted organ for the life of a recipient. Successfully induced tolerant states in the clinic can be persistent, however in a subset of patients, long-term surviving allografts can be lost. This loss has been correlated with pre-transplant donor-specific antibodies, and in some cases, occurred after episodes of infection. These observations underscore the need to identify individuals who have achieved robust tolerance from those who achieved a less robust/metastable tolerance, where infections may trigger alloreactivity and allograft rejection, and where interventions or closer monitoring may be necessary. While many factors are likely to contribute to a fully robust state of tolerance, in the current funding period, we focused our investigations on the mechanisms that enforce donor-reactive T cell tolerance induced to fully mismatched heart allografts with anti-CD154 in combination with donor spleen cells in mice. We determined that multiple redundant induced T cell tolerance mechanisms maintained transplant tolerance in this model, namely constrained donor-reactive T cell numbers and T cell receptor avidities to levels approximating those in nave mice; induced cell-intrinsic hypo-responsiveness and expression of negative co-inhibitory molecules by the persisting donor-specific T conventional cells; and increased percentages of FoxP3+ cells within the donor-specific CD4+ T cells. We also demonstrated that pro- inflammatory cytokines produced during infection overrode some of the T cell tolerance mechanisms, resulting in allograft rejection, but that tolerance returned spontaneously when the infection was cleared. Finally, we showed that the restored tolerance after infection was eroded in quality and more susceptible to reversal. That established tolerance mechanisms remain responsive to environmental cues highlights the need for tolerance to be robust at initiation as well as resilient, in order for it to successfully maintain graft acceptance for the life of the recipient. Recipient sensitization and the ensuing immunological memory is currently considered the most important barrier to achieving tolerance in the clinic. Clinical studies have identified previous transplant rejection and pregnancy as important sensitizing events. This proposal builds on our current findings and focuses on how prior sensitization after graft rejection (Aim 1) or pregnancy (Aim 2) prevents the establishment of specific T cell tolerance mechanisms. The insights gained from these studies will provide granularity into how T cell tolerance mechanisms cooperate to mediate robust transplant tolerance, guide the identification of biomarkers for robust tolerance, and ultimately, lead to rationally designed therapies that are tailored to achieving robust tolerance in sensitized recipients or for restoring robust tolerance after erosion by infection.	{ "pile_set_name": "NIH ExPorter" }
The overall goal of the proposed experiments is to determine the molecular mechanisms involved in the regulation of cardiac muscle contraction by troponin and to determine its role in familial hypertrophic cardiomyopathy (FHC). SPECIFIC AIM 1. Cardiac TnT (CTnT) Isoforms and the Regulation of Contraction. CTnT isoforms are generated mainly by alternative splicing of two exons encoding the N-terminal variable region and are expressed in the human heart as four isoforms (HCTnT1 through HCTnT4). The expression of these isoforms at the protein level has previously been found to differ in the normal and failing adult and fetal human heart. Based on recent results from this laboratory we hypothesize that HCTnT N-terminal isoforms modulate Ca2+-sensitivity and the relaxation properties of cardiac muscle. In order to determine the fundamental role of HCTnT isoforms in the regulation of cardiac muscle contraction and to test this hypothesis, we will substitute the various CTnT isoforms into reconstituted skinned muscle and thin filament systems and study their individual properties. SPECIFIC AIM 2. Functional Consequences of Tn FHC Mutations. Our working hypothesis is that mutations in Tn subunits lead to changes in the interactions between the CTn subunits and or changes in their interaction with other thin filament proteins, which in turn lead to changes in the Ca2+-affinity of CTnC and or changes in muscle mechanics eventually resulting in FHC. We propose to address the following issues: 1) to determine the in vitro properties of different Arg mutations in HCTnl (Arg 21, Arg 145 and Arg 204). Arg FHC mutations in HCTnT and HCTnl have been reported to be mutated to six different amino acids (Cys, Gly, Trp, Leu, Pro, and Gin); 2) to determine the in vitro properties of different Arg mutations in HCTnT (Arg 92, Arg 94, Arg 130 and Arg 278); 3) to characterize the first FHC mutant found in HCTnC; and 4) to determine the effect of different ratios of TnT, Tnl and TnC FHC mutants on the Ca 2+- sensitivity and level of actomyosin ATPase activity. [unreadable] All of the Specific Aims listed above focus on the effect of cardiac Tn, containing CTn subunits with FHC mutations, on the regulation of cardiac muscle contraction. Knowledge on these mutations will provide insights into the mechanisms by which these mutations cause cardiac disease as well as provide information about the importance of various regions in these CTn subunits. [unreadable] [unreadable]	{ "pile_set_name": "NIH ExPorter" }
The hearing impaired often struggle in complex acoustic environments (e.g., cocktail parties) where good localization abilities are particularly important. Many hearing-impaired people use only one assistive hearing device (e.g., hearing aid or cochlear implant), effectively limiting them to monaural (single ear) listening. Understanding the limitations monaural listening imposes on sound localization is of particular importance when evaluating the differences (e.g., costs, risks, and benefits) between monolateral and bilateral assistive devices. We hypothesize that listeners can discern the location of sound sources based on information in the monaural spectral profile. This hypothesis is tested in a series of psychophysical experiments that integrate the techniques of monaural studies of spectral profile analysis and binaural studies of spatial hearing. The overall goal of this research is to understand how the variability in speech affects the monaural localization abilities of normal-hearing listeners. The proposed work begins with the simple stimuli typically used in studies of spectral profile analysis and systematically builds towards speech-like stimuli. Although primarily a study of monaural localization, understanding how the variability in speech influences our perceptions is also important for understanding studies of binaural spatial hearing, speech intelligibility, and cochlear implant processing. The studies outlined here will provide important baseline data for evaluating (1) the degree to which monolateral assistive devices preserve information for monaural localization and (2) the benefits of bilateral assistive devices. In addition, determining the factors that lead to good monaural localization will aid in the design of better assistive devices (both monolateral and bilateral). Relevance The findings of this study will aid in the design of monolateral assistive hearing devices that preserve information useful for localization thereby reducing the difficulties that hearing-impaired individuals have in the prototypical cocktail party. The findings will provide important baseline data so that the benefits of assistive devices can be more accurately assessed. Additionally, comparisons between the results of this proposed work and previous studies have the potential to highlight shortcomings of current assistive devices and provide a guide for the design of the next generation of technology. [unreadable] [unreadable] [unreadable] [unreadable]	{ "pile_set_name": "NIH ExPorter" }

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