text
stringlengths 1
32.7k
| meta
dict |
|---|---|
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. Background: In the present study, we report the effects of cooling ejaculated and epididymal rhesus monkey (Macaca mulatta) sperm with and without the presence of a cryoprotective agent, glycerol. Methods: Water transport data during freezing of ejaculated and epididymal sperm cell suspensions were obtained at a cooling rate of 20 [unreadable]C/min in the absence of any cryoprotective agents and in the presence of 0.7 M of glycerol, as well. Using previously published values, the macaque sperm cell was modeled as a cylinder of length 73.83 [unreadable]m with a radius of 0.40 [unreadable]m and an osmotically inactive cell volume, Vb, of 0.772Vo, where Vo is the isotonic cell volume. This translated to a surface area, SA to initial water volume, WV ratio of ~ 22 [unreadable]m-1. Results/Discussion: By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best-fit membrane permeability parameters (reference membrane permeability to water at 0 [unreadable]C, Lpg or Lpg[cpa] and the activation energy, ELp or ELp[cpa]) were found to range from: Lpg or Lpg[cpa] = 0.0020[unreadable]0.0029 [unreadable]m/min-atm;ELp or ELp[cpa]) = 10.6[unreadable]18.3 kcal/mole. By incorporating these membrane permeability parameters in a recently developed equation (optimal cooling rate, Bopt [unreadable] 1009:5 x exp(-0.0546 x ELp) x (Lpg) x (SA/WV);where the units of Bopt are [unreadable]C/min, ELp or ELp[cpa] are kcal/mole, Lpg or Lpg[cpa] are [unreadable]m/min-atm and SA/WV are [unreadable]m-1), we determined the optimal rates of freezing macaque sperm to be ~ 23[unreadable]C/min (ejaculated sperm in the absence of CPAs), ~29 [unreadable]C/min (ejaculated sperm in the presence of glycerol), ~24 [unreadable]C/min (epididymal sperm in the absence of CPAs) and ~ 24 [unreadable]C/min (epididymal sperm in the presence of glycerol). In conclusion, the subzero water transport response and consequently the subzero water transport parameters are not significantly different between the ejaculated and epididymal spermatozoa under corresponding cooling conditions.
|
{
"pile_set_name": "NIH ExPorter"
}
|
DESCRIPTION OF OVERALL CENTER (provided by applicant): This application is submitted in response to RFA DK-04-014, Silvio O. Conte Digestive Diseases Research Core Center, requesting continued funding of the Texas Gulf Coast Digestive Diseases Center (called DDC for simplicity). This Center serves basic and clinical scientists at institutions within the Texas Medical Center (Baylor College of Medicine, The University of Texas Health Science Center at Houston) and at the University of Texas Medical Branch in Galveston. Our qualifying Funded Research Base is $22,182,225 million (direct costs) and includes 44 separate awards from NIDDK totaling $9,016,286 million (direct costs). Reflecting the goals of these projects, this is a Center for Gastrointestinal Development, Infection and Injury. This Center was developed from a core group of investigators who began interacting formally in 1996, to explore the need and interest in establishing a DDC. These interactions were initiated by a Pediatric Training Program in Gastroenterology at Baylor whose faculty represent key investigators in our DDC. Institutional resource commitments in space, funds and personnel support this effort, including new positions in basic and clinical departments for multidiscliplinary, independent, faculty to establish new research programs. This Center facilitates on-going Digestive Diseases research, promotes translational research between basic and clinical areas, develops new projects, nurtures new investigators, and provides GI educational activities. Support is requested for an Administrative Core, four Basic Science Cores (Morphology, Gene Expression and Proteomics, Gastrointestinal Immunology, Integrative Biology) and one Clinical Core (Study Design and Clinical Research Support). Pilot/Feasibility and Enrichment Programs, including a Career Development Initiative, to support innovative ideas and new investigators in Digestive Disease research and foster collaboration are a key part of the DDC, and have been extremely successful The Center draws together a multidisciplinary group of investigators, including basic scientists with proven track records of success, and well-coordinated clinical programs dealing with pediatric and adult GI patients. Center leaders are senior scientists-administrators experienced in directing interactive, multidisciplinary programs. A large, multi-ethnic population of infants and adults with Digestive Diseases emphasizes the need and the opportunities for this Center. Various groups involved in GI research and education in the Gulf Coast Area of Texas have shown the willingness to enthusiastically work with the Center. This NIDDK-funded DDRCC will promote and coordinate Digestive Disease activities in the area.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Traumatic brain injury (TBI) is becoming an increasing concern for the Veterans Administration, as the number of returning soldiers who have acquired a TBI in combat continues to rise. Still, little is known about the relationship between brain injury severity, resultant neuropathology, and subsequent neurobehavioral disability. At present, we still lack a clear biomarker of TBI that is both sensitive to the history and/or presence of injury, and one that scales with severity of injury. Therefore, the overall objective of the proposed research is to fill this need by validating an in vivo, non-invasive biomarker for TBI diagnosis. Through the use of diffusion tensor imaging (DTI), we seek to investigate the integrity of specific white matter tracts in the thalamus and brainstem, in the hopes of identifying a potential consistent biomarker of TBI. To accomplish our objectives, we will recruit a total of 72 veterans with a history of mild TBI due to blast-related injury, 24 non-injured controls and 24 extra-cranial injured controls. Patients will be matched for post traumatic stress disorder symptom severity, time in theater and combat exposure. Patients will also complete a 3 Tesla MRI with high resolution DTI of the thalamus and brain stem, and structural imaging to quantify atrophy and lesions. In addition, in order to evaluate the functional integrity of the brain stem-thalamic-prefrontal circuit, tests of oculomotor function (fixation, visually guided saccades, anti-saccades) will be administered. Cognition, post-concussive symptoms, mood, anxiety and post traumatic stress disorder symptoms will be evaluated using a battery of neuropsychological tests sensitive to mild TBI. The data will be analyzed to determine whether (a) thalamus projection fibers and (b) brain stem fibers are affected by blast-related TBI and determine whether damage to these fibers results in neurophysiologic and neuropsychological impairment. The primary outcome will determine whether this measure is clinically relevant in returning OIF/OEF veterans, and whether or not these methods are valid in assessing and diagnosing mild TBI due to secondary blast injury.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The horse family, Equidae, is being used as a model system to explore the evolution of highly reiterated DNA sequences (buoyant density satellite DNAs) within mammalian genomes. Of particular interest is the relationship between the DNA sequences found in heterochromatin of species with high diploid number, e.g. Equus przewalskii, 2N equals 66, and the heterochromatic DNA of species with lower diploid chromosome number and altered patterns of heterochromatin distribution, e.g. Equus zebra hartmannae, 2N equals 32. Buoyant density satellite DNAs are to be purified and localized by in situ hybridization. Restriction enzyme analysis and filter hybridization will provide information concerning nucleotide sequence homologies.
|
{
"pile_set_name": "NIH ExPorter"
}
|
A successful drug development program requires a complete understanding of the clinical pharmacology of the agents being evaluated. The Clinical Pharmacology Section (CPS) has as its primary interest the use of pharmacokinetic and pharmacodynamic concepts in the development of novel anticancer agents. The CPS is directly responsible for the pharmacokinetic/pharmacodynamic analysis of numerous Phase I and II clinical trials conducted within the NCI. In addition, the CPS provides direct pharmacokinetic support for many studies performed elsewhere in the extramural community. Within the section, we utilize compartmental and noncompartmental approaches to define the disposition of agents. Also, we are often required to characterize the plasma protein binding properties and metabolism of new agents through in vitro techniques. Several of our clinical trials have used adaptive control with a feedback mechanism to target particular plasma concentrations (e.g., suramin, CAI). The drugs with which the CPS has had its greatest experience include: suramin, phenylacetate, phenylbutyrate, TNP-470, PMEA, AZT, PSC 833, CAI, DAB486IL2, IgG-RFB4-SMPT-dgA CD22, IgG-HD37- SMPT-dgA CD19, ormaplatin, UCN-01, flavopiridol, thalidomide, 9AC, intraperitoneal cisplatin, intraperitoneal carboplatin, and paclitaxel. Currently, we are characterizing the pharmacokinetics of multiple day dosing of PSC 833 when administered with vinblastine and paclitaxel. This is an attempt to characterize any interactions between PSC 833 and either of these agents both in the clinical pharmacokinetic data, as well as through in vitro metabolism studies. Recently, we have completed a Phase I trial of COL-3, a matrix metalloproteinase inhibitor. - pharmacokinetics, pharmacology, drug development, - Human Subjects & Human Tissues, Fluids, Cells, etc.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The proposed research is motivated by problems arising in the statistical design and analysis of cancer clinical trials. In each trial, outcome-adaptive decisions are made repeatedly as the trial progresses and the data from patients treated previously in the trial become available. Possible decisions include choosing a patient's dose or treatment, dropping a treatment, stopping the trial, adaptive randomization, and selecting an optimal treatment or multi-course treatment strategy. Because outcome-adaptive methods use more available information on a more timely basis, they are more scientifically valid and more ethical. At each interim analysis, the underlying model must be re-fit and decision criteria re-computed, which requires either updating the posterior under a Bayesian model, or computing a test statistic under a frequentist model. We evaluate average design behavior by computer simulation, which requires the interim decision criteria to be computed many times. Consequently, the proposed methods are computationally intensive. We also will apply more formal criteria, including Bayesian A-, D-, or T-optimality and decision theoretic methods. Depending on the application, the trial may involve various types of multiplicities, including multivariate outcomes, multiple disease subtypes, multiple treatments, or multiple courses of treatment per patient. We will explore and apply Bayesian regression models, hierarchical models, and latent variable models to accommodate these multiplicities and account for heterogeneity and association. Proposed research projects include developing models, methods and designs for (1) Dose-finding in clinical trials where the doses of two agents are varied, with the goal to find several acceptable dose pairs; (2) Dose finding in clinical trials where patient outcome is a multivariate ordinal variable representing several qualitatively different toxicities having different clinical importance; (3) Clinical trials in diseases with multiple subtypes; (4) Evaluating treatment effects on a disease presenting in multiple body sites in each patient; (5) Carrying out both dose-finding and treatment selection with multiple treatments; (6) Accounting for patient heterogeneity in Bayesian models when clinical outcome is multinomial; and (7) Frequentist selection and testing designs based on a bivariate outcome including response and toxicity. In order to promulgate the methods to the statistical and medical communities, user-friendly computer software for implementation will be made freely available.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Human diseases transmitted by mosquitoes have serious negative impacts on populations in tropical countries and also affect populations in temperate regions (for example, the recent emergence of West Nile virus in North America). Anopheles gambiae is a major vector of malaria parasites in sub-Saharan Africa. Better understanding of the biology of A. gambiae may lead to improved strategies for disrupting malaria transmission and for controlling other mosquito vectors of human diseases. The recent release of the DNA sequence of the A. gambiae genome has been a major achievement that should make possible rapid progress in molecular studies of the function of gene products in this mosquito. This proposal for an Investigator-Initiated Small Research Grant is to initiate pilot studies on the biochemistry of two groups of proteins in An. gambiae: serpins and laccases. Serpins are serine protease inhibitors, which have potential to regulate insect immune responses. However, their functions in mosquitoes have not yet been defined. Laccases are copper oxidases that can oxidize diphenols as well as other types of substrates. Laccases may function in insect cuticle sclerotization or immunity, but they have been the subject of little previous biochemical study. The aims of this project are to obtain full-length cDNA clones corresponding to the five laccase-like genes and a selected group of the 14 serpin genes from An. gambiae and to use these cDNAs as probes to study expression of the genes and for production of recombinant proteins for initiation of biochemical studies. These aims are intended to produce preliminary results to support future more extensive proposals on An. gambiae serpins and laccases. This project will be a start toward development of a new research area for the PI (mosquito biochemistry) that will build from his experience in biochemistry of other groups of insects.
|
{
"pile_set_name": "NIH ExPorter"
}
|
In earlier studies conducted by the applicants, the "Estimated Survival Probability" (ESP) index was developed and proposed as a technique for grading the severity of hospitalized trauma on the basis of ICDA codes. This index was developed using data from the 1973 Hospital Discharge Survey (HDS). In this proposed study, data from three insight into the nature and strength of the associations between outcome of hospitalized trauma and such variables as age, sex, bed size of hospital, number of conditions mentioned (both trauma and non-trauma), number and type of surgical codes and other factors, with length of stay and discharge status (alive or dead) used as outcome variables. Modern methods of contingency table analysis will be used to test the appropriate hypotheses concerning these associations. It is felt that the improved ESP index will be a useful tool to investigators involved in retrospective studies of hospitalized trauma, and that the insight gained from this study into factors associated with outcome of trauma can be used in the planning of emergency medical services.
|
{
"pile_set_name": "NIH ExPorter"
}
|
PROJECT SUMIV1ARY (See instructions): In skeletal muscle, the interaction between two proteins, the dihydropyridine receptor (DHPR) in the plasma membrane/transverse-tubules and the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR), is essential for linking elect R1cal excitation to contraction (excitation-contraction coupling, EC coupling). In particular, it is thought that conformational changes of the DHPR (containing Cav1.1 as its principal subunit) in response to depolarization cause RyR1 to open and release calcium from the SR, and that this signaling depends on physical links between the two proteins. Significantly, mutations of the DHPR or RyR1 in humans can result in the inherited disorder of malignant hyperthermia susceptibility (MHS), whereby volatile anesthetics cause dysregulation of calcium release that can lead to a fatal rise in body temperature unless there is rapid intervention. With the long-term objective of understanding the interactions between the DHPR and RyR1, and the mechanisms of MH, the specific aims of Project 3 are: Aim 1.1: To use whole cell patch clamping to measure L-type Ca2+ current and charge movement (which a R1se directly from the DHPR), together with Ca release from the SR, to determine how MHS mutations (RyR1R2435H and -T4826I; Cav1.1-R174W) in mouse and human myotubes affect bi-directional signaling. Aim 1.2: To determine the effect of dantrolene on bi-directional signaling in MHS myotubes. Aim 1.3: In collaboration with Core D, to determine if MHS mutations alter the frequency or disposition of DHPR tetrads in myotubes. Aim 2: To measure membrane currents and myoplasmic Ca2+ transients in dissociated FDB fibers from adult (3-6 mo) and aged (12-18 mo) male and female MHS mice (Cav1.1-R174W and RyR1-R163C,-R2435H, and -T4826I) to determine the heterogeneity that results from differences in gender, age and locus. Aim 3: To measure membrane currents and Ca^* transients in FDB fibers from 3-6 month old male Het RyR1T4826I MHS mice that have been crossed with mice over-expressing SERCA1 (enhanced SR Ca2+ filling) or dnTPRC6 (reduced SOCE), or which were administered 4-OH-BDE49 (reduced RyR1 leak) or salicylamine (YKA scavenger) to determine if modification of one of the 4 key elements associated with MHS can mitigate or abrogate alterations in RyR-DHPR bi-directional signaling. Aim 4.1: To determine whether Ca^* currents, charge movements or voltage-gated Ca^* transients are differentially affected by volatile anesthetics in WT or MHS mutant (RyR1-R1630, -R2435H, -T4826I; Cav1.1-R174W) FDBs. Aim 4.2: To determine whether effects of volatile anesthetics on Ca2+ channel function are prevented by treatment with dantrolene. Aim 5: To use expression in myotubes of proteins harboring MHS mutations newly discovered by Core C in order to determine their effects on bi-directional signaling and hypersensitivity to volatile anesthetics.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Cystic Fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Relative to wild type CFTR, the most common mutation deltaF508-CFTR, present in >85% of CF patients is expressed at low levels in the plasma membrane and has reduced in vivo activity. Thus to correct the deltaF508-CFTR mutation both the number of plasma membrane channels and the activity of individual channels must be increased. Our data suggest that the reduced in vivo activity of deltaF508 - CFTR is due to a reduced level of phosphorylation. In this study we will test two hypotheses, 1) that differences in CFTR activity can be caused by differences in channel phosphorylation, and 2) that differences in the activity of wild type and deltaF508-CFTR are due to differences in phosphorylation. By activating CFTR under various experimental conditions and by examining CFTR with serine to alanine mutations at phosphorylation sites, we will generate CFTR with different phosphorylation states. Then by comparing the channel kinetics of different phosphorylation states, we will generate structure function relationships. Our studies will provide. biochemical evidence for the hypotheses that PKC-dependent phosphorylation is required for PKA-dependent activation and that protein phosphatases show selectivity with respect sites of dephosphorylation. These studies will assist in the development of drugs to treat CF patients with deltaF508-CFTR. In specific aim I, two-dimensional peptide mapping, site-directed mutagenesis and mass spectroscopy will be used to identify sites of PKC- and PKA-dependent CFTR phosphorylation and establish experimental conditions where CFTR phosphorylation differs. In specific aim II, channel kinetic parameters will be determined for CFTR that has been phosphorylated on different sites. Alterations in mean open and closed times, Po and the number of active channels will be correlated with phosphorylation at specific sites. In specific aim III, we will compare phosphorylation and channel kinetics of wild type and deltaF508-CFTR to determine if differences in channel activity are correlated with differences in phosphorylation.
|
{
"pile_set_name": "NIH ExPorter"
}
|
This investigation will assess the neurological development and audiological outcome of term, healthy newborns who developed severe non-hemolytic hyperbilirubinemia.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Secretory processes for electrolytes and proteins is a continuing objective of this work. The salivary glands of adult and immature, and the microperfused main duct of SM control of neural (autonomic nervous system) factors. The investigation of growth regulation in early postnatal rats by dietary, chemical and surgical manipulations are expected in delineate changes in glandular physiology and biochemistry associated with the time of weaning. The postnatal development of electrolyte and protein secretory ability of SM and PA glands as well as the concurrent development of their neural elements (especially autonomic receptors) will be investigated by electrophysiological, histochemical (catecholamine fluorescence) and biochemical (endogenous norepinephrine and choline acetyltransferase activity) methods; histologic changes at the light and EM levels that accompany secretory and neural development will also be assessed. In adult and immature rat, the functional status of glands modified in size by dietary or surgical manipulations will be evaluated by their ability to secrete electrolytes and protein in response to autonomic stimulation. The concourrent changes in the nerve activity or number of and affinity of autonomic receptor binding sites or levels of cyclic nucleotides will be also determined in "atrophied" and enlarged glands of adult or immature rats. In the immature, manipulations leading to enlargement or atrophy of glands is also expected to lead to premature or retarded maturation of neural elements and secretory activity. Secretion of amylase and calcium by SM and PA will be an important aspect of this work since calcium is known to exert an important role in many biological processes including stimulus-secretion coupling and ion transport. Further clarification of autonomic control of salivary secretion is important for understanding of pathophysiology of cystic fibrosis. In addition, delineation of ductal secretory and reabsorptive processes of ions and their autonomic regulation is essential to an understanding of the steps by which saliva is formed and secreted.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Vitamin A is essential for normal immune function and has shown promise for treatment of different infectious diseases in children. The purpose of the project is to determine whether periodic, oral vitamin A supplementation reduces morbidity and mortality for HIV-infected children in sub-Saharan Africa. The goals of the project are being met through a randomized, double-masked, controlled clinical trial of oral vitamin A supplementation, 60 mg retinol equivalent, every three months for HIV-infected children from 12 to 36 months of age. The project is collaboration between Makerere University and the Johns Hopkins University at the Mulago Hospitals in Kampala, Uganda. The project has involved HIV screening of 23,439 women, identification of 3,751 HIV-infected women, and HIV screening of 1241 infants. The project has enrolled 300 HIV-infected children and is in-progress, and children are scheduled to reach 36 months of age by January 2002. Interim analysis shows that groups appear to be similar at baseline (with different characteristics measured at different ages). The application notes a trend in possible mortality benefit to the children receiving vitamin A. The study is being reviewed annually by a data and safety monitoring committee. In this competing continuation application, it is proposed to complete the clinical trial, laboratory studies, and data analysis over the next 3 years. If vitamin A supplementation is shown to reduce morbidity and mortality for HIV-infected children, this would be an appropriate, low-cost (8 cents for capsules per year), safe, and widely applicable intervention to increase survival for HIV-infected children in developing countries.
|
{
"pile_set_name": "NIH ExPorter"
}
|
All-trans Retinoic acid (RA) and its analogs have emerged as dermatological agents and as potential cancer chemopreventive/chemotherapeutic agents. As a class, these compounds share teratogenic potential and the tendency to cause vitamin A toxicities. However, the O-acyl glucuronide metabolite of RA has been suggested to be a less toxic, active material. The synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) shows some unique activity as a breast cancer chemopreventive with further reduced toxicity and teratogenicity. Our own studies with 4-HPR-O-glucuronide (4-HPROG) have shown it to be an even less toxic, more effective preventive and therapeutic for DMBA-induced rat mammary tumors relative to 4-HPR. However, these glucuronides as well as the retinamide 4-HPR are unstable to hydrolysis back to the parent retinoid making it unclear what the active forms of the molecules are. We have been synthesizing stable C-linked analogs of 4- HPR and 4-HPROG for evaluation as breast cancer chemopreventive/chemotherapeutic agents. A number of these analogs show increased potency preventing DMBA-induced tumors and further reduced toxicity relative to 4-HPR. Like 4-HPR, these analogs bind weakly to the known nuclear retinoid receptors and, unlike RA, cause apoptosis in tumor cells in a manner that is not inhibited by retinoid receptor antagonists, leaving their mechanism of action incompletely understood. The specific aims of our proposed continued research are: 1) to scale up production of our preferred analog (4-HBR, 4-HBRCG, or 4-HBRCglucose) based on a current chemotherapy/chemoprevention experiment underway, 2) to complete chemoprevention evaluation of the chosen analog in the DMBA-induced rat mammary tumor model, 3) to begin study of the chosen analog in a second (MMTV/ErbB2 mouse) mammary tumor model, 4) to conduct predictive teratology studies of the chosen analog(s), and 5) to explore mechanism of action issues with the chosen analog(s) focusing on our observations that 4-HPR and our analogs appear to induce ER stress. We will focus here on establishing the necessity of these phenomena for the actions of these compounds as well as determining molecular players upstream of these processes which trigger these events. PUBLIC HEALTH RELEVANCE: Despite recent advances, breast cancer-associated morbidity and mortality remains a serious problem in the USA. The analogs of 4-HPR we are developing, show real promise as more effective and less toxic preventive and therapeutic agents for breast cancer. A clearer understanding of how these promising molecules function would further enhance our ability to develop and exploit them.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Age associated losses of strength occur slowly over the adult lifespan. Most research has focused on what occurs in the elderly. However, the primary processes start at a much earlier age. In young and middle-aged workers, the early losses contribute to work related injuries and for some occupations, job performance. The causes of age related strength losses are multifactoral. Research has focused on age-associated loss of muscle that has been referred to as sarcopenia, While sarcopenia has been mainly studied in the elderly where the greatest changes in muscle mass and strength manifest, by age 50 the average individual has lost 10% of their maximal strength. While sarcopenia is clearly a major factor in strength loss, there are other contributors to the changes in maximal function. To understand sarcopenia these other factors need to be studied. Our goal is to understand the time course of strength loss, factors that contribute to the loss, the degree to which the exercise response differs between old and young individuals, and the forms of motivators and alternative exercise programs that might impact on the losses. We have used several different approaches to address these issues. First are descriptive studies using the Baltimore Longitudinal Study of Aging (BLSA). These studies focus on describing the characteristic losses that occur in muscle strength mass, and physical functioning that occur with aging by examining the entire adult lifespan and their impact on function and longevity. We have previously demonstrated that declining muscle strength and rate of change of muscle strength are independent contributors to mortality in men when considering age, physical activity and muscle mass. We have further shown that muscle power, and speed of movement are further independent sarcopenic factors that contribute to longevity. The observations suggest that central nervous system processes are contributing to the importance of sarcopenia on longevity. Second, working with collaborators at the University of Maryland, we are examining genetic contributions related to muscle hypertrophy and strength. We have identified several genes that contribute to the inherited aspects of how much muscle and strength we have. As an example, we reported that IGF-II genotype is related to muscle strength but not muscle mass. This is consistent with the mortality data, where muscle mass and muscle strength have common and different effects on outcome. Also, we found that IL6 and CNTF genotype had some influence on body composition which impacts on sarcopenia. Likewise, longer androgen receptor repeat in exon 1 in men is associated with higher testosterone blood levels and with greater levels of fat free mass. Third are intervention studies to alter the time course of strength and muscle mass changes. In previous work we demonstrated that the exercise response to resistive training is very similar in young and old subjects. However, while the response to strength training may be similar by age, there are clear differences in muscle responsiveness as represented by gene expression, and body compostion change differences. We have been interested in alternative strategies for exercise intervention. With Dr. Laura Talbot, we have examined two alternative exercise strategies using subjects with osteoarthritis of the knee. First was neuromuscular electrostimulation (NMES), a passive activation of the knee extensor muscles using an electric stimulator. We tested a protocol that used NMES at much lower force levels, to make the stimulation more acceptable (i.e. with less or no pain), and to minimize the likelihood of injury in individuals with osteoarthritis of the knee. The NMES group used a portable electrical muscle stimulator to incrementally increase the intensity of isometric contraction over 12 weeks up to 20-40% of their maximal voluntary strength. We found an increase in muscle strength in response to this passive activity, and a decline in knee pain immediately following the treatment (though not a sustained effect). The second approach was to use home based pedometer driven motivational program resulted in improved walking, increased knee extensor strength, and modest functional improvements. We are currently in the planning stages of a study to examine whether the use of electromyostimulation can positively impact the course of recovery from traumatic war-related below the knee amputation. During recovery and rehabilitation, quadriceps muscle strength declines following traumatic amputation. NMES may offer a passive form of exercise that can maintain strength during this time period.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The long term goal of this research is to better understand the mechanisms of intracellular pH (pHi) regulation in adult ventricular muscle. In this project the applicant will focus on two modulators of cardiac pHi control systems, vasoactive peptides (angiotensin II, endothelin 1) and chronic (4-5 days) acid-base disturbances (metabolic acidosis and alkalosis). These peptides are potent positive inotropic agents which exert a wide range of effects on the heart and are currently thought to exacerbate myocardial damage during ischemia/reperfusion. Their cardiac effects may be mediated, in part, by changes in pHi. Similarly, the effect of prolonged acidosis and alkalosis on cardiac pHi control systems may represent an important adaptive response to chronic myocardial ischemia. The applicants will test the hypothesis that both modulators alter the kinetic properties and pHi dependence of Na/H exchange, Na-HCO3 cotransport, and Na- independent Cl-HCO3 exchange. Experiments are also designed to identify the sarcolemmal receptor which mediates the pHi action of the peptides. The applicant will also investigate the intracellular signaling role of Cai, calmodulin, Ca/calmodulin-dependent protein kinase II, and protein kinase C in mediating the pHi effects of ATII and ET1. The experiments will be performed at 37oC on ventricular myocytes, enzymatically isolated from normal adult rabbits, guinea pigs, and humans (only rabbits will be used in the chronic acid-base studies). Intracellular activities of H+ and Na+ will be measured with the fluorescent indicators (SNARF-1, BCECF) and SBFI, respectively. In some experiments, measurement of pHi and Nai will be made during internal dialysis of cells with a suction pipette. A rapid solution switcher will be used for rapid (less than 3 msec) external application of the peptides.
|
{
"pile_set_name": "NIH ExPorter"
}
|
This is a proposal for continuing support of the Center for Demography of Health and Aging at the University of Wisconsin-Madison. The overall goal of the Center for Demography of Health and Aging is to sustain and improve a major research and training program in the demography of health and aging. The institutional architecture of CDHA is designed to create links between social demography and biomedical and epidemiological research on health and aging. Major themes of ongoing and developmental research activities within CDHA include (1) Early and Mid-Life Determinants of Late-Life Health and Well-Being; (2) Race, Ethnicity and Socioeconomic Status; (3) Demography of Aging; (4) Trends in Chronic Disease and Disability, and Burden of Illness; and (5) Health, Work and Retirement. During its first decade, CDHA has grown from 39 to 63 faculty members; has successfully turned pilot projects into grants; has grown from about $3.3 million to $74 million in NIA support; has created unique data resources and a secure data enclave; and has substantially expanded training activities. CDHA coexists with the Center for Demography and Ecology and thus shares and extends a well-developed research infrastructure in administration, computing, print library, data library, and GIS facilities. CDHA's administrative core (A) will provide leadership and administrative support for the Center. The program development core (B) will support faculty, staff, and research assistants engaged in innovative pilot research projects that are likely to lead to major NIA support. The external innovative network core (C) will continue a popular, networked current awareness service for research in the demography of aging and support regular workshops, conferences, and visits. The external research resources core (D) will support user-friendly provision of large-scale public data resources in the demography of health and aging. The statistical data enclave core (E) will continue to develop facilities for the analysis of sensitive data under secure conditions.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Notwithstanding considerable advances, the establishment of the complete primary structure of a protein remains to a large extent a slow, manual procedure requiring skilled operators. It is thus an expensive undertaking and human operators are singularly ill-suited to the sort of tedious repetitive functions which constitute the procedure. It is therefore proposed to design, build and develop a machine capable of taking a sample of a pure protein, breaking it up into peptides, separating the peptides, and repeating the purification of each fragment until purity is achieved as judged from the stoichiometry of its amino acid composition, determining the sequence of each peptide by some form of repetitive Edman degradation from the NH2-terminal end, and finally collate and classify the total evidence to yield the complete amino acid sequence together with the experimental evidence employed for arriving at that sequence. Ideally, the system will be entirely self-contained and all on-line decisions will be taken by the controlling computer. Such a system would have extremely wide and fundamental biological and biomedical applications, as the cost per residue will be drastically reduced, the time required for sequence determination will be measured in days rather than months, and the overall size of pure protein sample needed is likely to be at least an order of magnitude smaller than for manual procedures. For several months, the members of the Departments of Biological Sciences, Chemical Engineering, Computer Sciences, Electrical Engineering, and the Design and Development Center of the University listed below as investigators, reduced all chemical procedures envisaged to their logical outline and various mechanical, electronic and control options were considered. It became the informed opinion of the group that the machine proposed above can indeed be built and made as foolproof as necessary to provide reliable operation.
|
{
"pile_set_name": "NIH ExPorter"
}
|
CORE C: Cell and Molecular Biology - Ying Jin, Ph.D. - PI This Core has existed for 10 years within the Department of Neurobiology and Anatomy to facilitate the molecular analysis of cells and tissues in the SCI site and within transplants. Most importantly, the core prepares cells for grafting experiments in the individual projects. All projects will use the Cell and Molecular Core. The facility isolates, expands and stores primary fibroblasts, neural stem cells and cell lines as required. We genetically modify cells by viral transduction or transfection and provide quality control for all cells and reagents used in the core. The staff of the Cell and Molecular Core provides assistance for experimental design and data analysis. The core has developed a computerized ordering system in which individual investigators & staff request control, labeled or genetically modified cells for transplantation. Our records show that over the past 5 years the core has provided over 3 x 1 0 ^ cells, including unmodified and modified fibroblasts, neuronal and glial restricted precursors (NRP and GRP), neuroepithelial cells (NEP) and cells from immortalized cell lines. The core maintains viruses and transduced cell stocks in long-term storage and serves as a database and repository for cDNA clones, vectors, viruses and cells. Efficient and cost-effective ordering of tissue culture and molecular biology supplies as well as large scale testing of new sera and improved growth media, enzymes, kits or equipment needed for molecular analysis (thermocyclers, Q-PCR) are key core functions. We are also continuously developing new improved plasmids and viruses and have recently added and adeno-associated virus vector (AAV) as an option to our program. This will allow us to specifically transfect graft neural stem cells with growth promoting and therapeutic factors to be delivered at or near the site of injury.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Title: Context-specific functions of CDK8 Project Summary: The long-term goal of this project is to elucidate the function and regulation of the CDK8 module, a key component of the transcription cofactor Mediator complex, in the versatile model organism Drosophila melanogaster. The four subunits of the CDK8 module ? CDK8, CycC, MED12, and MED13 ? are either mutated or amplified in cardiovascular diseases and a number of human cancers, such as melanoma and colorectal cancers. Elucidating the function and regulation of the CDK8 module in different biological contexts is essential to understanding the pathological consequences of CDK8 module misregulation, which is important for the design of clinical strategies to treat these diseases. Studies in the previous funding cycle of this project have demonstrated that CDK8-CycC serves as a critical regulatory node linking nutrient intake to fat metabolism and developmental timing in Drosophila development. These studies have shown that CDK8-CycC is a direct inhibitor of SREBP (sterol response element binding protein)-dependent gene expression, and that CDK8-CycC positively regulates ecdysone receptor-activated gene expression. The overall objective of this project is to unravel the function and regulation of CDK8 in different developmental contexts. Studies in the previous funding cycle illustrate that Drosophila is an ideal and powerful experimental system to achieve this long-term goal. Two new aims are proposed in this funding cycle. Aim 1 will determine the role of CDK8 in regulating the expression of telomeric retrotransposons and telomere biology in Drosophila. Our RNA-seq analyses revealed a specific upregulation of telomeric retrotransposon expression and a significant increase in telomere length in cdk8 and cycC mutants. We have also discovered similar deregulation of telomeric retrotransposon expression and telomere length in med7 and Scalloped (Sd) mutants. The transcription factor Sd functions downstream of the conserved Hippo pathway. Thus we propose to examine the unexplored functions of the CDK8 module and Sd in regulating the expression of telomeric retrotransposons and telomere length in Drosophila. Aim 2 of the proposal will identify and validate upstream regulators and downstream effectors of CDK8 in Drosophila. We have established novel and robust wing phenotypes caused by specific alterations of CDK8 activities, allowing us to perform a dominant modifier genetic screen to identify factors interacting with CDK8 in vivo. We have identified 26 genomic loci whose haploinsufficiency modifies these CDK8-specific phenotypes; further genetic analysis led us to identify genetic interactions between CDK8 and the components of the epidermal growth factor receptor (EGFR) and Dpp/TGF? signaling pathways, as well as several specific genes. In parallel, we have performed immunoprecipitation coupled to mass spectrometry (IP- MS) analyses to identify proteins that can interact with CDK8. Combination of these biochemical and genetic screens puts us in a unique position to systematically identify and validate upstream regulators and down- stream effectors of CDK8 in vivo, which will impact our understanding of the context-specific functions of CDK8.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Cardiovascular disease remains the leading cause of mortality of both women and women in the United States, so the health benefits gained from a better understanding of the mechanisms of cardioprotection could be enormous. Considerable epidemiological data indicates that moderate alcohol consumption decreases both the incidence of cardiovascular disease and the mortality associated with myocardial infarction. The cardioprotection associated with moderate alcohol consumption is analogous in many ways to late phase pre-conditioning indicating that they may share common, but yet undefined, molecular mechanisms. The cardiovascular protective effects of alcohol can be categorized into two major responses, (1) effects on systemic circulatory components (blood vessels and blood components), (2) effects on myocardium. This proposal will focus on delineating the mechanisms of the cardio-protective effects of alcohol on the vasculature. Preliminary data show that (1) moderate alcohol enhances vascular function, a form of cardioprotection; (2) moderate alcohol results in induction of nitric oxide synthases (NOS) in the vasculature; (3) induction of NOS involves two isoforms, iNOS and eNOS; (4) bioavailability of .NO may also be increased by induction of superoxide dismutases (SOD); (5) increased bioavailability of .NO may be responsibl4e for the cardiovascular protection associated with moderate alcohol consumption. These data have led to the hypothesis that increased nitric oxide (NO) bioavailability is a critical event in the vascular and cardioprotective events associated with chronic moderate alcohol. This hypothesis will be tested by completion of the following Specific Aims: (1) the vascular protective events associated with chronic moderate ethanol are due to the increased bioavailability of .NO resulting from induction of nitric oxide synthases and increased production of .NO and (2) the vascular protective events associated with chronic moderate ethanol are due to the increased bioavailability of .NO resulting from increased superoxide dismutase and a consequent decrease in superoxide (O2). The information gained from the accomplishment of these specific aims will provide insights into the mechanism which lead to increased .NO-dependent mechanisms in alcohol-induced cardiovascular protection.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The primary purpose of this study is to investigate the efficacy of RS-79070, a ganciclovir prodrug, as induction therapy in subjects with newly diagnosed peripheral cytomegalovirus retinitis. It will assess the safety profile of RS-79070 and the pharmacokinetics of ganciclovir following administration of RS-79070 in the target population.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Our ongoing in vivo and in vitro studies of functionally important cellular populations and of the role of individual proteins in effector functions of T-lymphocytes have obvious immunopharmacological implications. Our studies identify the cell types and key proteins that should be targeted for efficient immunomodulation when there is a clinical need to inhibit or to enhance the intensity of the effector phase of the immune response. Our ongoing studies are concerned with finding most efficient inhibitors of enzymes we have implicated in lymphocyte functions. We found that it is possible to influence cellular responses of intact cells using peptides which mimic inhibitory domains of known protein kinases and have demonstrated that such peptides are able to block T-cell responses. The problem of peptide proteolysis due to ecto-peptidase activities is expected to be dealt with by the use of hydrophobic tail-modified peptides to improve intracellular intake. A novel set of molecular targets for immunomodulation is being explored in studies of ecto-protein kinases, ecto-phosphatases, and ecto-ATPase. The data suggest that after adequate characterization of these ecto-enzymes and the development of specific peptide inhibitors, it will be possible to take advantage of the extracellular location of this set of ecto- phosphorylation/dephosphorylation enzymes.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Mice transgenic for an intact bovine growth hormone (bGH) develop a diffuse form of glomerulosclerosis which mimics the renal lesions that occur in human diabetes. In contrast, mice transgenic for a mutated form of GH, G119K mice, (mutated in the third helix) do not develop renal lesions and are resistant to steptozotocin-induced diabetic nephropathy. These observations led us to postulate that the third helix of the GH molecule contains a domain that is critical for the development of glomerulosclerosis. When we crossed bGH mice with mice transgenic for the G119K mutation, the resulting offspring had glomerular lesions which paralleled the ratio between the intact and mutant bGH. This data confirmed the direct role of GH in the development of the glomerular lesion. Further evidence in support of this hypothesis was obtained in mice, in which we placed a transgene coding for IGF-1 BP1. These mice have essentially no free IGF-1 in the circulation. As a result of the absence of feedback regulation the circulating GH levels are quite elevated. The homozygous mice developed a severe form of glomerulosclerosis resembling that found in the GH mice. These observations confirm that IGF-1 may not be a significant mediator of glomerulosclerosis in GH induced disease. Finally the lack of IGF-1 appeared to impair nephrogenesis, since we found a 10 to 20 % reduction in nephron number in the IGF BP1 transgenic mice, in their littermates, as well as in mice born of transgenic mothers. These data suggest that while IGF-1 plays a role in the development of the kidney, it does not seem to be involved in glomerulosclerosis.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Cognitive therapy has been shown to be at least as effective as, if not more effective than, pharmacotherapy in the treatment of non-bipolar outpatient depressives and, perhaps, more effective in terms of preventing subsequent relapse. To date, no studies have examined the efficacy of combining cognitive therapy with pharmacotherapy. Similarly, no studies have attempted to extend cognitive therapy into a variety of subtypes of depressions, e.g. bipolar, inpatient (and/or psychotic) depressions. Further, no adequately controlled study has yet compared the relative efficacy of group cognitive therapy with individual cognitive therapy, either with or without medications. This project consists of a coordinated series of studies examining the comparative efficacies of cognitive therapy, pharmacotherapy, and combined cognitive-pharmacotherapy in the treatment of depression. Five specific studies are proposed including three with outpatient samples (2 non-bipolar, 1 bipolar), and two with inpatients (1 non-bipolar, 1 bipolar). Treatment efficacy will be evaluated in terms of response to treatment, prevention of relapse, and minimization of dropout rates. The use of a common core battery and a common treatment protocol will facilitate comparisons across populations and settings. Finally, a series of cross-lagged panel analyses and multivariate analyses will be utilized to explore: 1) a variety of hypotheses regarding the mechanisms of change associated with each treatment modality (e.g., the relationships between biochemical indicies, cognitive processes, and levels of syndrome depression), and 2) potential predictors of both response to treatment and maintenance of treatment gains.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Viruses are among the best-known and studied pathogens and infect virtually every living organism from bacteria to humans. As viruses are parasites of their hosts, the life cycle of any virus is inextricably tied to that of the host cell Despite this dependence, all viruses share a number of essential tasks that they must accomplish for survival. Most viruses must find and recognize a cell in which they can replicate, release their genome, generate new viral components and assemble them into precursors that mature into stable, progeny virions that are primed to be transmitted and infect a new host. These tasks are accomplished by viruses in different ways as a result of adaptation to different cellular environments. Each task involves interactions between components within the context of the whole virion and hence requires the visualization of the entire structure at which the techniques of cryo-transmission electron microscopy (cryoEM), cryo-electron tomography (cryoET), and three-dimensional (3D) image reconstruction ('cryo-reconstruction') excel. We will exploit these powerful tools to study a diverse set of viruses, including those that infect humans and other mammals, protozoa, insects, bacteria, and fungi. Numerous projects funded by the current grant have illustrated the structural response of different viruses to the common tasks of the viral life cycle. This proposal involves ongoing as well as new studies that focus on structural investigations of viruses and virus complexes and dynamic events that lie beyond the current realm of crystallographic technology. The large number and extent of our studies are made possible through several fruitful collaborations, which provide important correlative information such as from biochemical, genetic, and X-ray crystallographic experiments. Icosahedral and non-icosahedral and enveloped and non-enveloped viruses will be studied. These include representatives of several different virus families, all of which make excellent model systems for studying form and function: Iridoviridae, Papillomaviridae, Partitiviridae, Parvoviridae, Podoviridae, Siphoviridae, Tetraviridae, Togaviridae, and Totiviridae (and also some yet unclassified viruses). Specific examples include: three totiviruses and one partitivirus that infect protozoa, which in turn cause human disease (Tricomoniasis, Leishmaniasis, Giardiasis, and Cryptosporidiosis); several adeno-associated viruses being developed as gene delivery vectors; Sindbis virus, a BSL-2 select agent; recombinant, virus-like particles of several human papillomavirus serotypes, which together cause >90% of cervical cancers; Sf6 bacteriophage, which infects Shigella flexneri that causes bacillary dysentery in humans; and many other insect and fungal viruses, including one that targets a fungus that infects plants and also causes human allergies. Lastly, the use of bacteriophage P22 capsids and polyheads as nano-particles for drug delivery will be investigated.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Project Summary The Administrative Core will manage and oversee all program components of the Oklahoma Center for Microbial Pathogenesis and Immunity (OCMP&I) Phase-1 COBRE. The Administrative Core will coordinate communications among investigators, organize meetings, and be responsible for the overall financial management of the budget. In addition, the Administrative Core will organize and support mentors, internal and external advisory committees, and coordinate all meetings and collaborative activities of the OCMP&I COBRE. The specific aims of the Administrative Core are. Specific Aim 1: To Provide Administrative Management of Oklahoma CMP&I COBRE Activities, Specific Aim 2: To Provide Mentoring and Career Guidance for Junior Investigators to Achieve Independence, Specific Aim 3: To Develop a Vibrant Pipeline of New Junior Investigators, Specific Aim 4: To Establish and Utilize External Advisory and Internal Advisory Committees, Specific Aim 5: To Establish Milestones and Monitor the Progression of Junior Investigators to Independence, Specific Aim 6: To Administer a Pilot Project Program, Specific Aim 7: To Foster and Augment Interactions with Other IDeA- Supported Programs, Specific Aim 8: To Host an Annual OCMP&I Symposium and a Grant- Writing Workshop. Accomplishment of these aims will ensure this Phase-1 COBRE has well- managed, programmatic growth and success during the establishment of the OCMP&I.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Measurements of fluorescence polarization and lifetime of fluorescein and bidpy-labeled proteins.
|
{
"pile_set_name": "NIH ExPorter"
}
|
In this application we propose to continue our studies aimed on understanding the molecular basis of the pathogenic process in transmissible spongiform encephalopathies, a group of fatal neurodegenerative diseases such as Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle. It is believed that the infectious pathogen responsible for these diseases is a misfolded, proteinase-resistant form of the prion protein, PrPSc, which is derived from a precursor PrPC by a poorly understood mechanism. While the structure of PrPC is well characterized, one of the most enduring mysteries in the field is the molecular structure of infectious PrPSc conformers. In this project, we propose to undertake a major effort to bridge the gap in understanding the structural properties of abnormally folded prion protein aggregates, with specific aims focused on: (i) determining the conformational structure of proteinase K- resistant form of the recombinant Syrian hamster prion protein, PrPres, obtained by brain PrPSc- templated protein misfolding cyclic amplification (PMCA) reaction;(ii) determining medium- resolution structure (site- and region-specific structural constraints) for brain-derived hamster PrPSc;(iii) elucidating the nature of structural differences between PrPSc corresponding to the hyper and drowsy strains of TSE. This study will employ a number of complementary biophysical methods including site-directed spin labeling, solid-state NMR spectroscopy, hydrogen/deuterium exchange, and hydroxyl radical-mediated footprinting coupled with mass spectrometry.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Project highlights: using miniaturized to 1536-well plate format with 8 different media, collections of MMV box, NPC and NCGC MIPE libraries were screened. The positive compounds are being tested for further compound selections. During this period, the NCGC has fostered and maintained over 180 active collaborations with both NIH and extramural investigators, facilitating drug discovery efforts across the entire spectrum of human disease. These efforts have led to over 100 high-throughput screens and nearly 60 medicinal chemistry campaigns, providing our collaborators and the general research community a wealth of publications and promising small molecule leads. In addition, the NCGC has undertaken a number of informatic challenges to make better use of existing drug and disease target information and provide the general public with easily accessible resources, further catalyzing the development of new therapies for human disease.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The proposed study is concerned with an evaluation of the antiarrhythmic activity of a series of compounds synthesized in our Medicinal Chemistry Laboratories. The drugs in question are characterized by being quaternary ammonium compounds. Each of the agents will be assessed for its actions against digitalis-induced arrhythmias as well as rhythm disorders associated with experimentally-induced myocardial ischemia and/or infarction. All agents will be examined for their ability to protect the heart against the development of ventricular fibrillation by studying the drugs in animals subjected to periods of myocardial ischemia followed by abrupt reperfusion and by studying the influence of the drugs upon the electrical ventricular fibrillation threshold. The detailed cardiovascular pharmacology of these agents will be explored. Studies will be carried out on the electrophysiologic effects of the quarternary-ammonium drugs in the ischemic and normal canine myocardium in an effort to determine their possible mechanism of action and to compare their actions to the more commonly employed antiarrhythmic drugs. An attempt will be made to better understand the relationships between chemical structure and antiarrhythmic and/or antifibrillatory activity. BIBLIOGRAPHIC REFERENCES: Lucchesi, B.R. and Lomas, T.E.: The cardiac effects of the oral hypoglycemic agent glyburide. In Micronase (Glyburide) Pharmacological and Clinical Evaluation. Ed. by Harold Rifkin. Excerpta Medica, 1975. Princeton, N.J., pp. 91-104. Lucchesi, B.R., Burmeister, W.E., Lomas, T.E. and Abrams, G.D.: Ischemic changes in the canine heart as affected by the dimethyl quaternary analog of propranolol, UM-272 (SC-27761). J. Pharmacol. Exp. Ther. (in press, 1976).
|
{
"pile_set_name": "NIH ExPorter"
}
|
SAFE, SILENCING-RESISTANT, NON-ONCOGENIC GLOBIN EXPRESSION CASSETTES. A proof of principle that gene therapy for the hemoglobinopathies is feasible has been obtained in mice. The major aim of this project is to move on from this important milestone to address a major remaining issue relating to the potential for oncogenesis by lentiviral-mediated insertional mutagenesis. We propose to design new gene therapy cassettes that will have a minimal impact on the flanking sequences thereby reducing the risk of oncogenic insertional activation events. SPECIFIC AIM 1 is to assess the effectiveness of insulators in blocking activation of genes in the flanking sequences and to test newly discovered insulators. The only known method to protect the flanking sequences near integration sites of enhancers in transgenes is the use of insulators and enhancer-blockers such as DNAsel hyper-sensitive site 4 of the chicken p-globin gene cluster (cHS4). While cHS4 activities are well demonstrated in artificial constructs, whether this element is effective for all promoters is not known. We propose to answer this question and to test other regulatory elements which might have complementary activities. SPECIFIC AIM 2 is to assess the role of the LCR in silencing, its potential to cause insertional mutagenesis in human hematopoietic cells, and to develop novel LCRs with a shorter range of action. We have obtained evidence that paradoxically transgene silencing can be caused by the presence of the LCR. We propose here to determine the generality of this novel mechanism of transgene silencing. SPECIFIC AIM 3 is to determine if replicators incorporated in transgenes can prevent silencing at large number of sites and to measure their effects on the flanking sequences. We have obtained evidence that silencing can be released by inclusion of a replicator in the expression cassette. We propose here to determine if this effect is general. Little is known about the effects of replicators on transgene expression and on the flanking sequences. We propose to fill some of the gaps in our knowledge of these elements. Relevance: Spectacular advances in the field of gene therapy have been made in the last few years but a major possible side-effect remains the risk of treatment-induced leukemias and lymphomas. We propose here to develop therapeutic expression cassettes which will cause less side-effects. hESC line WA01 and WA09 will be used in this project. [unreadable] [unreadable] [unreadable]
|
{
"pile_set_name": "NIH ExPorter"
}
|
The objective is to gain knowledge of the genetics and physiology of poliovirus. Two specific areas will receive primary attention: viral RNA synthesis and viral genetics. The study of RNA metabolism will focus on the recent evidence that the host-factor needed to allow initiation of minus strand synthesis by the viral polymerase may be terminal uridyl transferase (TUT). The association of host-factor with TUT will be examined critically, primarily through the generation of monoclonal antibodies. The possibility will be examined that viral RNA synthesis involves first the formation of a hairpin, and then its cleavage leaving VPg at the 5'-end. Viral genetic studies will concentrate on the use of an infectious polio cDNA clone to generate and study defined mutants. Five good mutants have already been generated using three insertion or deletion methods and more mutants will be sought using the same methods. Complementation and recombination among the mutants will be studied. Inertypic recombinants will be examined to determine what sequences are involved in recombination. The phenotypes of the mutants will be examined to link individual proteins to specific functions and to understand the range of functions involved in poliovirus growth. Because poliovirus is a prototype of all picornaviruses, these studies will be applicable to many viral systems.
|
{
"pile_set_name": "NIH ExPorter"
}
|
[unreadable] We propose to develop an x-ray microtomography system specifically designed for nondestructive 3D imaging of orthopedic implant coatings to investigate fracture mechanics for the purpose of improving the implant technology. The proposed x-ray microtomography system offers unique advantages over visible-light and electron microscope based methods for nondestructive 3D imaging of orthopedic implants in the study of fracture mechanics and development of advanced coating materials. Specifically it would allow 80nm-resolution studies of subsurface cracks in situ in an unperturbed state without the need of cutting the sample to risk altering the crack. It would also allow the cracks to be seen in or through any material regardless of optical transparency (such as simulated body fluid) and would enable the same crack to be imaged repeatedly to monitor its progress. The hard x-ray microscope will have much wider usage for fracture mechanics studies of other types of implant coatings, such as commercial hydroxyapatite, and nondestructive imaging of internal structures in any solid materials. [unreadable] [unreadable]
|
{
"pile_set_name": "NIH ExPorter"
}
|
Recent research on attention in adults has distinguished three separate components of attention: alertness, selectivity, and processing capacity. Studies have shown that these components may be isolated along a time dimension and that they operate independently on incoming visual information. Current studies of age trends in visual information processing have revealed striking differences in attention and memory between children and adults. The present proposal outlines a program of research to investigate more precise@y the exact nature of observed age differences in attention. Using tachistoscopic procedures with age groups from five years of age through adulthood, studies are designed to examine whether age differences in attention reflect differences in alertness, selectivity, or processing capacity. It is hoped that the research outlined here will provide a more fine-grained analysis of developmental trends in visual information-processing skills.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Adolescents are at considerable risk for becoming infected with HIV, and most HIV-infected adolescents acquire HIV via risky sexual behaviors. Alcohol consumption, substance use, childhood delinquency, criminality, and mental disorders have been found in many studies to be significant risk factors for sexual behaviors that increase risk of HIV infection among adolescents. However, developmental trajectories of HIV sexual risk behaviors and their relationship with other risk factors have not been well established because long-term prospective studies are relatively limited. Disentangling the complex associations of social and personal factors with HIV sexual risk behaviors requires systematic examination of patterns of HIV sexual risk behaviors over time and identification of factors that influence the change or continuity of these risky sexual behaviors. Drawing upon key concepts from problem-behavior theory (Jessor &Jessor 1977) and age-graded life- course theory (Sampson &Laub 1993), the proposed study seeks to provide a better understanding of the developmental trajectories of HIV sexual risk behaviors among adolescents as they transition from adolescence to young adulthood. Based on the National Longitudinal Survey of Youth, 1997 (NLSY97) covering years of 1997 to 2005, the proposed study will identify distinctive trajectory patterns of HIV sexual risk behaviors over a long observation period (e.g., approximately ages 15 to 25) and associated risk and protective factors at individual, family, school, peer, and community levels. We are particularly interested in how drug use, mental health problems, and criminality influence HIV sexual risk behaviors, as well as how trajectory patterns and risk and protective factors differ by gender or ethnicity/race. The specific aims of the proposed study are (1) to identify risk and protective factors (individual, family, school, peer, and community) associated with initiation and trajectories of HIV sexual risk behaviors;(2) to identify distinctive patterns of initiation and trajectories of HIV sexual risk behaviors;and (3) to describe gender and ethnic differences in the factors, processes and patterns resulting from the above aims. The NLSY97 data provide an excellent opportunity to investigate developmental trajectories of HIV sexual risk behaviors and their associations with drug use, mental health status, delinquency, and criminality among adolescents. Comprehensive data have been collected from a large nationally representative sample of youth since 1997 with multiple waves of assessments on sexual activity, drug use, mental health, delinquency, and criminality. Results of the proposed study will have implications for developing improved HIV prevention strategies for youths. PUBLIC HEALTH RELEVANCE: HIV is a significant public health problem. The proposed study seeks to provide a better understanding of the developmental trajectories of HIV sexual risk behaviors among adolescents who are observed over a long- period of time. Using longitudinal data from the National Longitudinal Survey of Youth, 1997 (NLSY97) covering 1997 to 2005, the proposed study will identify distinctive trajectory patterns of HIV sexual risk behaviors as these youths transition from adolescence to young adulthood and will examine how trajectories of risky sexual behavioral are influenced by drug use, mental health problems, and criminality. Results of the proposed study will have implications for developing improved HIV prevention strategies.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The Office of Alternative Medicine at NIH recommends that all healthcare professionals and students be exposed to alternative theories and techniques of primary care so that they can assist patients in becoming active in their healing process. We propose to develop a comprehensive medical resource for physicians on Complementary & Alternative Therapies for Cancer. This resource will consist of CME courses for Physicians and Practioners, a customized syllabus program and Discussion Forums, which will be distributed in internet and CD-ROM/DVD formats to facilitate distance learning. A resource will be developed for the physician and medical professionals, based on research from nationally recognized experts. Our Phase I objectives are to study the feasibility of developing the physician resource, and to develop and test an internet web site prototype of one module through interviews with physicians and medical professionals. In Phase II, we will develop the full courseware for all C&A Therapies for the web site and the CD-ROM/DVD products based on Phase I results. Effectiveness of the two phases will be measured through online testing/scoring, extensive user traffic analysis reports and interviews with physicians and medical professionals. PROPOSED COMMERCIAL APPLICATION: The complementary & alternative resources products and content produced from this grant can be sold, promoted, or licensed through outlets such as; medical clinics, wellness clinics, healthcare organizations, catalog distributors, and other not yet identified professional outlets. Additional revenue can be generated through web links, web registration, and corporate sponsorship of CME courses. Successful testing of the program will lead to the next step, the delivery of a product designed to serve up custom information based upon the students current knowledge of the subject matter. This has significant implications for the medical education market. Why should professionals need to review materials with which they are already familiar? Why should healthcare practitioners need to watch 10 minutes of video to arrive at the 7 minutes in which they have interest? The next step for this product will be to continue the expansion of the software instruction set with the goal of producing a "shrink-wrap" product. This product will allow the user to customize his/her syllabus so that each student is able to learn at his/her own speed.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Degenerative disc disease is an epidemic, ultimately resulting in untenable pain and immobility. In advanced cases, spinal fusion is performed where vertebrae are surgically fixed with a mechanical device and an osteogenic material (bone substitute) is bridged between them in an attempt to induce fusion. Of the 600,000 yearly procedures performed in the US, the most common is posterolateral lumbar arthrodesis but the failure rate can reach 25-40% with standard commercial bone substitutes. The reason for failure rests in part with limited biocompatibility of synthetic bone substitutes, inconsistencies with processed cadaveric bone substitutes and in some cases, health complications caused by supraphysiologic doses of bone morphogenic protein. Autologous bone grafts are much more effective, but the approach is associated with donor site morbidity and the volume of available graft is limited. Compromising strategies that employ bone substitutes and bone marrow aspirate (BMA) are becoming common, but efficacy continues to be limited by the bone substitute. The fact that spine related disability is a growing global problem and standard of care interventions have an unacceptable failure rate clearly demonstrates the need for implants that safely and effectively promote bone fusion. The successes and failures of past spinal fusion strategies indicate that a bone substitute that mimics autograft will meet this need. Therefore, the goal of this proposal is to develop a 3D printed biomimetic bone graft substitute (the scaffold) by an innovative combination of stem cell biology, matrix biology, and biomedical engineering. The scaffold will consist of a tough, porous and flexible nanoengineered hydrogel consisting of gelatin methacrylate (gel-MA) coated with extracellular matrix (ECM) purified from osteogenically enhanced human mesenchymal stem cells derived from induced pluripotent stem cells (OEihMSCs). By mimicking the composition of bone matrix, OEihMSC-derived ECM is highly osteogenic. The gel-MA will be further enhanced by addition of novel silicate nanoparticles that impart stiffness and further stimulate osteogenesis. The scaffold will be designed to drive fusion with efficacy equivalent to autograft, but it will be manufactured from a standardized and sustainable source of materials. To achieve this goal, we will: optimize methodology for the generation of various forms of scaffold with a range of gel-MA, nanosilicate and ECM formulations with variations in macroporosity and stiffness (Aim1), optimize attachment, distribution, viability and osteogenesis of cells on the scaffold using in vitro 3D cell culture assays based on rotating wall bioreactor technology (Aim2) and finally, test the optimized scaffold with human BMA in a rodent posterolateral fusion model, incorporating imaging and biomechanical testing approaches (Aim 3). The rationale for this approach is that it has the capacity to satisfy a need for safe and effective autologous bone repair scaffolds for a rapidly growing population. With the current disposition of the FDA favoring autologous and minimally manipulated cytotherapeutic preparations, this strategy is well suited to clinical translation.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The long-term goal of this study is to understand the molecular mechanism of mammalian Hedgehog (Hh) signal transduction. Aberrant Hh signaling has been associated with various human cancers and birth defects. Recent evidence indicates that several aspects of Hh signaling are not conserved between invertebrates and vertebrates. Not only have the roles of some of the main players changed in vertebrates, but also their genetic interactions and the mode of signal transduction have been modified. Further, primary cilia appear to play a major role in mammalian and not Drosophila Hh signaling. These findings highlight the importance of elucidating the functions of mammalian Hh pathway components directly in order to further understand vertebrate Hh signal transduction. This proposal focuses on one critical player in mammalian Hh signal transduction, Suppressor of Fused (Sufu), and will define the mechanism of Sufu in regulating the functions of Gli transcription factors, the sole mediators of Hh signaling during mammalian development. We propose the following specific aims: 1) Define the role of Sufu in controlling ciliary localization of Gli2. 2) Elucidate the molecular mechanism of Sufu-mediated Gli3 protein stabilization. 3) Delineate the requirement of primary cilia in Sufu- or SPOP-mediated Gli protein function. 4) Test the hypothesis that Sufu interacts with Kif7, a kinesin family member, in modulating mammalian Hh signaling. A combination of cell-based assays, genetic studies using knockout mice and protein biochemistry will be employed to address these central issues. These studies will provide new mechanistic insights into how the mammalian Hh signal is transduced in responsive cells 7. PUBLIC HEALTH RELEVANCE: Aberrant Hedgehog (Hh) signaling is associated with cancers and birth defects. Elucidating how the Hh signal is transduced is critical to understanding the underlying mechanisms of Hh-related diseases as well as identifying potential targets for diagnosis and rational therapies. [unreadable] [unreadable] [unreadable]
|
{
"pile_set_name": "NIH ExPorter"
}
|
Funds are requested to help defray the expenses of the fifth biannual Neurotrophin Gordon Conference, which will take place in June, 2001. The goal of the Neurotrophin Gordon Conference is to provide a forum for the exchange of new information about neurotrophic factors and to facilitate the dissemination of new tools and approaches for studying these important proteins. Neurotrophic factors are polypeptide growth and differentiation factors that influence the properties and functions of neurons and non-neuronal cells within the nervous system. The identification and characterization of neurotrophic factors has had a major impact on modern research in developmental biology. During vertebrate development, these factors play essential roles in regulating neuronal survival, proliferation, differentiation, axon growth and guidance, and synapse formation. In the mature nervous system, neurotrophic factors regulate the expression of neurotransmitters and their receptors, and modulate synaptic function. In animal models, the loss of particular neurotrophic factors has been shown in some cases to have dramatic effects on nervous system development, and in other cases, to affect mature functions of the brain, such as learning, memory, and behavior. Several neurotrophic factors have been found to suppress the loss of neurons that occurs in response to a variety of toxic insults and in animal models of human neurodegenerative disease. Research on neurotrophic factors has facilitated the characterization of neural stem cells, a population of cells that may eventually be used in efforts to repair the injured or diseased brain. Efforts to employ neurotrophic factors in experimental treatments of Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Parkinson's Disease, stroke, and spinal cord injury continue. Clinical trials examining possible beneficial effects for these factors in the treatment of sensory neuropathies and pain are fairly advanced. The best characterized neurotrophic factors are the neurotrophins, which include Nerve Growth Factor and its close relatives BDNF, NT-3 and NT-4. Over the last decade, many additional soluble and membrane-bound polypeptides that promote the growth and development of cells of the nervous system have been identified, including neuropoietic cytokines, such as CNTF and LIF, members of the TGF-beta superfamily, the ephrins, netrins, and slits. Current research in this field aims to define the diverse biological actions of the many neurotrophic factors and to characterize the signaling mechanisms that mediate the effects of these factors. This research effort is being enhanced by a variety of novel strategies for the discovery and functional characterization of neurotrophic factors including newly developed molecular, electrophysiological, and imaging technologies, and through the innovative use of genomics and proteomics. The latest findings and innovations in the neurotrophic factor field and the possible therapeutic potential of these neurotrophic factors will be the subject of intense discussion and debate at the upcoming Gordon Conference.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The first project area explores metabolic pathways that have been proposed based on in vitro studies to be important in non-replicating (NR)-MTb. We are exploring the importance of the biosynthesis of the cofactors biotin, coenzyme A and pyridoxal, peptidoglycan turnover, the role of putative F420-binding and genetically annotated pyridoxal-generating enzymes, beta-oxidation and iron acquisition and validating these by chemical and genetic means in non-replicating (NR)-MTb. We have shown that Rv2607 is the canonical pyridoxine phosphate oxidase of MTb and have enzymatically characterized this enzyme. In contrast, Rv1155, which is also annotated as a pyridoxine phosphate oxidase family protein has been expressed, purified, crystalized with its F420 cofactor, biophysically characterized with and without bound cofactor and we are attempting to identify the natural substrate of this protein by analyzing shared chemotypes with known metabolites from fragments identified as binders to this protein. Another F420-dependent enzyme, Rv2991, has been crystalized and fragments chemically similar to known metabolites of flavoenzymes analyzed for binding to Rv2991 with and without F420. By analyzing common pharmacophores between known metabolites and the binders identified by this fragment-based approach, we are attempting to probe the enzymatic function of this unknown protein We have also demonstrated the importance of biotin synthesis for the viability of MTb in vitro and in vivo. We have reported that conditional downregulation of pantothenate synthase makes Mtb hypersusceptible to inhibitors of coenzyme A biosynthesis and are using this approach to identify vulnerable targets in this metabolic pathway. Our studies of mycobacterial cell wall synthesis using meropenem as probe have allowed us to track the formation of the various layers of the mycobacterial cell wall during its assembly using a combination of cryo-electron, transmission and scanning electron microscopy. We have shown that the dual action of meropenem on both the D,D-carboxypeptidases as well as the transpeptidases on newly synthesized peptidoglycan leads to the observed polar lysis of cells. The second major focus area of this project starts from a different perspective and uses compounds that are in clinical development (PA-824 and SQ109) which are known to possess activity against replicating as well as NR-TB. We capitalized our recently determined crystal structure of Ddn, the nitroreductase responsible for the bioreductive activation of PA824 to understand the differences in binding of the enzyme to nitroimidazoles and the relationship of this binding to the formation of the reactive nitrogen intermediates responsible for killing of Mtb. We are attempting to understand what the natural substrate is for the Ddn, since this will allow us to probe the enzymatic processes that are important during non-replicating persistence. Preliminary studies have identified menaquinone as a substrate for this enzyme. For SQ109 we were able to demonstrate that the mechanism by which this drug kills Mtb is by inhibition of the MmpL3 protein which we identified as a trehalose monomycolate transporter. To further unravel the key events in cell wall mycolyl-arabinogalactan synthesis, we have enzymatically characterized the three mycolyl transferase enzymes (Antigens 85 A, B and C). We have found that the enzymes are kinetically distinct with Ag85C being enzymatically the most active and that certain amino acid residues residing in a secondary ligand binding site control rates of acyl transfer by affecting protein confirmation in a helix connecting the two ligand binding pockets. The third major focus of this project involves global approaches to understanding the metabolism in NR-TB. Using a chemostat model of MTb combined with metabolomic studies, we demonstrated that the NADH/NAD+ ratio changed as a function of oxygen concentration, that the direction of the TCA cycle reverses under hypoxia with concomitant extracellular succinate accumulation which is consistent with a model of oxygen-induced stasis in which an energized membrane is maintained by coupling the reductive branch of the TCA cycle to succinate secretion. An essential non-redundant step in this process is fumarase and we have initiated studies to validate the role of the forward as opposed to reverse TCA cycle in vitro as well as in vivo by using structure-based design based on the fumarase crystal structure to design inhibitors of this target. Co-crystal structures of Mtb fumarase with bound inhibitors, enzymatic as well as in situ demonstration of fumarase inhibition have corroborated our model with further inhibitor optimization being required for in vivo studies. In a fourth approach, we are identifying inhibitors of metabolism by high-throughput screening approaches performed under a variety of in vivo relevant environmental conditions. Hits from these screens have provided a useful tool to map metabolism of MTb as a function of carbon source, oxygen concentration or presence of low pH in the presence or absence of nitrosative stress and are currently being studied to identify the target. In the process of target identification, parallel studies are done to rapidly progress the hits to in vivo proof of concept studies so that the importance of the target for in vivo pathogenesis can be validated early on in the drug discovery process. We are studying some of the hits that were identified from a 35,000 compound BioFocus collection in collaboration with various researchers in South Africa. In addition, hits from a 100,000 compound library screen from a collaborator have yielded 12 different scaffolds that are being pursued. The scaffolds that gave us evidence of a specific target based on SAR studies were taken further into target identification by a combination of approaches including resistant mutant generation followed by whole genome sequencing to identify single nucleotide polymorphisms, transcriptional profiling, macromolecular incorporation assays and metabolomics studies. For 2 chemically different scaffolds, the same target in mycobacterial cell wall synthesis was identified and efficacy studies confirmed that inhibition of some cell wall biosynthetic genes in vivo, led to a mild bacteriostatic effect. The targets of eleven other scaffolds were identified. For several other scaffolds, mutations in MmpL3, a protein we previously identified as the SQ109 target, conferred resistance suggesting that this transporter is promiscuous in its ability to bind diverse ligands. For several scaffolds, generation of resistant mutants was impossible and in several of these cases, inability to generate resistant mutants was correlated with mammalian cytotoxicity suggesting a non-specific mechanism of action. One class of compounds was shown to target oxygen-dependent respiration in Mtb. We have demonstrated that the coupling of respiration to energy generation in a vulnerable point in NR-Mtb based on inhibitors identified in a screen against anaerobically persisting Mtb.The precise point in inhibition of respiration is currently being explored by analysis of respiratory knockout mutants, biochemical assays and complementation studies. Resistance to another hit mapped to an enzyme in folate metabolism. We have been able to show that this drug functions as a metabolic poison by its ability to mimic substrates and become incorporated into folate-like metabolites by a combination of metabolomics and biochemical analyses. With collaborators at Weill Cornell Medical College, we have used this inhibitor as well as other known inhibitors of folate biosynthetic enzymes to explore how perturbation of folate-dependent reactions leads to inhibition of Mtb replication.
|
{
"pile_set_name": "NIH ExPorter"
}
|
DNA topoisomerases are the enzymes responsible for maintaining and controlling the topological state of DNA in the cell. They play an important role in replication, transcription and genetic recombination. Despite their importance, their structure and detailed mechanism of action are still unknown. This project is concerned with the study of the structure of Escherichia coli DNA topoisomerase I. The enzyme works by forming a transient phosphotyrosine covalent bond with DNA that allows for one DNA strand to pass through the other. In this way it changes the DNA linking number in steps of one. No external energy source is required as the bond energy is conserved. This project is relevant not only to the studies of topoisomerases but also to the studies of those proteins involved in re-arrangements of DNA and proteins that form covalent bonds to DNA. The long-term goals of this project are to study the structure and function of E. coli DNA topoisomerase I and its complexes with DNA. In particular, to study the active site atomic environment, the nature of the covalent bond with DNA, and the DNA-protein interactions. The objective of these studies is to obtain a detailed model for the catalytic mechanism of E. coli DNA topoisomerase I and the way it modifies the topology of DNA. To accomplish these goals the specific aims of this proposal are i) to study the structure of a 35 kD fragment of the enzyme containing the catalytic tyrosine and responsible for the cleavage of single stranded DNA. ii) To characterize the 35 kD fragment, study its DNA binding properties and sequence dependence of the cleavage reaction. We will study the formation of covalent complexes of the fragment with short oligonucleotides. iii) to study the structure of the same fragment complexed with short oligonucleotides. iv) To study the structure of the intact protein both with and without DNA. The structural work will be based mainly on x-ray crystallographic studies. Crystals of the 35 kD fragment have already been obtained and diffract to at least 2.8 A resolution. We will continue our studies of the single stranded DNA cleavage reaction by both the intact protein and the 35 kD fragment; we are particularly interested in obtaining covalent complexes with short oligonucleotides that are suitable for crystallographic work. The work on attempting to crystallize the intact protein will continue.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Schizophrenia affects 1% of the population worldwide;neuroleptic (antipsychotic) medications are essential for treatment despite an incomplete understanding of the mechanisms through which palliative symptomatic remediation is exerted. Though different in the degree of antagonism for the dopamine 2 receptor (D2R) and extradopaminergic effects, both 'typical1 neuroleptics (e.g. haloperidol) and 'atypical1 neuroleptics such as clozapine are effective at long-term remodeling of the corticostriatal synapse, a primary site of disease pathophysiology and pharmacotherapeutic activity as demonstrated by in vivo imaging and postmortem tissue analyses. Previous study has been impeded by an inability to experimentally separate a heterogeneous cell population in the striatum, and by inadequate resolution from the soma of dendritic processes. BAG transgenic mice expressing eGFP under the dopamine 1 receptor (D1R) and D2R promoters will be used to distinguish dichotomous principal neuron populations in the striatum, in combination with 2-photon laser scanning microscopy (2PLSM) to gain insight into dendritic function. As adaptations to chronic neuroleptic administration may be different in normal versus diseased states, an RGS4 knockout mouse will be employed as a schizophrenic-like model in parallel to wildtype strains;RGS4 has been implicated as both a schizophrenia susceptibility gene in cases of demonstrable hereditary transmission and a gene sensitive to altered dopamine transmission in the striatum of Parkinsonian animal models. In summary, this proposal aims to characterize the compound effect by which pharmacotherapeutic improvement in clinical symptomatology is effected. Whole-cell patch clamp electrophysiology, combined with molecular transgenic, pharmacologic, and 2-photon imaging modalities will be used to study the remodeling of neuronal subpopulations in the dorsal striatum of wildtype and RGS4 knockout mice following subchronic typical (i.e. haloperidol) and atypical (i.e. clozapine) neuroleptic administration. Specific Aim 1 will examine the intrinsic, morphologic, and dendritic properties of medium spiny neurons of the dorsal striatum. To assess functional connectivity between prefrontal cortical and striatal neurons, Specific Aim 2 will target presynaptic and postsynaptic adjustments in the corticostriatal synapse.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Members of the Section have determined the CTNS mutations responsible for benign (ocular) and intermediate cystinosis, and have reviewed the worlds reported CTNS mutations. They have developed a multiplex PCR amplification method for diagnosing the presence of the 57-kb deletion present in 56% of 216 cytinosis alleles tested. The Section continues to treat over 100 patients with oral and topical (eyedrop) cysteamine. 2. In collaboration with NHGRI investigators, a new gene adjacent to CTNS was discovered. The gene, called CARKL, presumably produces a carbohydrate kinase and may distinguish cystinosis patients bearing the 57-kb deletion from nondeletion patients. It has a 1434 bp open reading frame with 7 exons encoding a 478 amino acid protein. Expression of a 3.9 kb transcript occurs prominently in liver, kidney, and pancreas. 3. The fifth patient in the world with sialuria has been characterized. This 7-year old Portuguese girl carries a single copy of a UDP-GlcNAc 2-epimerase gene containing an R266Q mutation, and the resulting enzyme is inhibited only 26% by 100 micromolar CMP-sialic acid (normal, 79%). 4. More than 95 patients with Hermansky Pudlak syndrome (HPS) have now been examined at the NIH Clinical Center, and the pulmonary, dermatologic, and ophthalmologic aspects of the disorder have been described. Members of the Section have also identified and clinically characterized two HPS patients who are compound heterozygotes for mutations in the beta-3A subunit of AP-3, an adaptor protein complex responsible for vesicular trafficking and cargo sorting. - Cystinosis, sialuria, Hermansky-Pudlak syndrome, vesicular trafficking, mutation analysis - Human Subjects
|
{
"pile_set_name": "NIH ExPorter"
}
|
Nazia Raja-Khan, M.D. is an endocrinologist with a long-term career goal of establishing an integrative medicine research program for Polycystic Ovary Syndrome (PCOS) aimed at integrating evidence-based non-conventional complementary therapies with conventional medical therapies for PCOS to optimize psychological and cardiometabolic health in PCOS. PCOS is a common endocrine disorder that affects 5-10% of reproductive-aged women. Women with PCOS are at increased risk for psychological distress, which may contribute to their increased risk for diabetes and cardiovascular disease. However, there is a lack of effective strategies for managing psychological distress in PCOS. Mindfulness-Based Stress Reduction (MBSR) reduces psychological distress in various patient populations and a pilot study showed that MBSR lowers glucose and blood pressure. Therefore, MBSR could significantly impact PCOS by reducing psychological distress and cardiometabolic risk; however, MBSR has not been investigated in PCOS. MBSR may have similar benefits for overweight or obese women who do not have PCOS (non-PCOS) as stress is also believed to play a major role in obesity. Our objective is to conduct a pilot randomized controlled trial investigating the feasibility and effects of MBSR in PCOS and non-PCOS women who are overweight or obese. Ninety eight (98) women will be randomized to 8-weeks of MBSR or health education control and followed for 16 weeks. The overall hypothesis is that MBSR will reduce cardiometabolic risk and psychological distress and improve quality of life in PCOS and non-PCOS women. Specific Aims are to: 1) Determine the effects of MBSR compared to health education control on changes in mindfulness, primarily assessed with the Toronto Mindfulness Scale; 2) Conduct a preliminary evaluation of the effects of MBSR compared to health education control on hemoglobin A1c, mean arterial pressure (MAP), quality of life, and overall psychological distress; 3) Explore physiologic mechanisms potentially mediating the effects of MBSR, including salivary cortisol and insulin resistance measured by homeostasis model assessment (HOMA-IR). This career development award will provide Dr. Raja-Khan with the skills, experience, and preliminary data to develop a competitive R01 proposal to more comprehensively investigate the effects and mechanisms of MBSR in PCOS. This research will inform the development of novel integrative mind-body approaches for PCOS, diabetes, and cardiovascular disease.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The goal of the Career Development Program of the Northwestern University-University of Chicago SPORE in Prostate Cancer is to recruit talented investigators to a career in translational prostate cancer research. In the current project period the SPORE Career Development Program (CDA) funded 7 CDA investigators from a pool of 20 applicants. These investigators were successful in obtaining 14 grant awards and they published 66 papers related to their CDA projects. The fact that the faculty come from a variety of departments has provided a source of diversity and scientific expertise to the investigators in the Program. Special efforts have been placed to emphasize the recruitment of women and minorities to the SPORE CDA. The Career Development Program has demonstrated flexibility in terms of offering awards and the length of the award. In the current funding period, the Prostate SPORE has maintained a consistent and highly structured process for advertisement of CDA positions, selection of CDA investigators and a good tracking system for monitoring and evaluating their progress. In the last SPORE submission, the CDA received an assessment of "Excellent". In this revised application, we have updated information of all CDA investigators and we again propose CDA funding for two investigators, one is to a woman (Hou) and the other is to a minority applicant (Posadas
|
{
"pile_set_name": "NIH ExPorter"
}
|
It is proposed to obtain realistic and credible risk estimates for breast-cancer mortality due to clinical mammographic imaging examinations. Given the increasing emphasis on clinical mammographic screening for breast cancer, it is of societal importance to provide realistic risk estimates - with realistic confidence bounds - for breast cancer induction from routine mammographic X rays. Concern about this risk is a major factor, both from the perspective of the patient and the family physician, in non compliance with suggested guidelines for routine screening. Previous estimates of this risk have a) not accounted for the significantly increased risk from the very low-energy X rays used in screening mammography and b) based their estimates on only a single projected breast-cancer risk model. Direct studies of mammographic screening programs have insufficient power to quantitate risks of mammographic screening, so estimates of the risks must be based on extrapolations from other cohorts (A-bomb survivors, medical irradiation). These other cohorts, however, were exposed to far higher- energy X or gamma rays than the very low-energy X rays used in mammograms; dose-for-dose, low-energy X rays are considerably more radiobiologically damaging than high-energy X or gamma rays. So it is likely that this risk estimation process underestimates risks of screening mammograms - preliminary estimates are by a factor of 1.5-2. State-of-the art radiobiological techniques will be used to extrapolate from those situations where radiation-induced breast-cancer risk estimates are better known (A-bomb survivors, medical irradiations), to risks of a screening mammograms. Experimental microdosimetric measurements will be used to characterize the energy-deposition characteristics of various mammographic X-ray beams in current or projected use. Based on these data, the biological effectiveness of these mammographic beams will be estimated relative to those for which breast-cancer risks have been estimated (e.g. A-bomb survivors, TB patients). The bottom line of these studies will be realistic estimates of the risk from routine screening mammograms, together with a sensitivity analysis of the confidence limits that may reasonably be associated with these risk estimates.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The development and implementation of the mass spectrometry core is essential to the research of the listed[unreadable] principal investigators. In particular the core will provide mass spectral analysis using the proposed bottomup[unreadable] and top-down methods employing both Q-TOF technology as well as FTICR high-resolution analysis.[unreadable] Both a top-down (intact protein identification) and a bottom-up (protein digestion) approach will be used to[unreadable] identify proteins of the various ribosomal complexes. Differences in post-translational modifications will be[unreadable] determined for ribosome complexes which have been subjected to infection by various viruses (hepatitis c,[unreadable] dengue, etc). eif3 factors associated with the ribosome will be analyzed and investigated for the presence of[unreadable] phosphorylation. Both the high-resolution capabilities of the exisiting FTICR and the sensitivity and flexibility[unreadable] of the Q-TOF, in combination with well established sample preparation methods such as IMAC, will allow for[unreadable] the unambiguous determination of protein constituents and associated post-translational modifications.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The Molecular and Cellular Basis for Obesity Core is a broadly based research and technology center that emphasizes the genes, proteins and metabolites linked to obesity and obesity-related disease. Dr. David Bernlohr, PhD, is the Core Director. Dr. Bernlohr is Professor and Head of the Department of Biochemistry, Molecular Biology and Biophysics at the University of Minnesota. He is the recipient of a Distinguished McKnight University Professorship and holds the Cargill Chair in Systems Biology of Human Metabolism. Dr. Bernlohr has served the scientific community in a number of capacities. He has served on the editorial boards for the Journal of Biological Chemistry, Protein Science and the Journal of Lipid Research. He finished his second term as a permanent member ofthe Integrated Physiology of Obesity and Diabetes (IPOD) Study Section and was (ihair of IPOD from 2008-2009. Dr. Bernlohr holds multiple NIH awards and has studied adipose biology his entire professional career. His primary research themes involve the role of fatty acid binding proteins and fatty acid transporters in adipocyte and macrophage metabolism, oxidative stress and mitochondrial metabolism and the role of inflammation in obesity. He uses a combination of genomic, proteomic and metabolomic methodologies in his studies and uses a variety of cell based and animal models (knockout and transgenic) in his work. He is ideally suited to be Core Director for his experimental expertise utilizes all the Core technologies and he is able to address questions plus serve as a mentor for others interested in basic obesity research. Dr. Bernlohr will devote 5% of his effort to directing the Core and will be responsible for: [unreadable] Overseeing all budgetary and scientific activities of the Core [unreadable] Representing the Core on the Center Executive Committee [unreadable] Preparing all Core reports and providing information on Core activities to the Center Director [unreadable] Evaluating Core usage and needs and planning for future development [unreadable] Organizing the Core Research Club [unreadable] Assisting in the oversight of the Gene Expression and Analysis Subcore, especially with regard to development and use of lentiviral vectors. The Core will be organized into three primary subcores with each one having a subcore leader:
|
{
"pile_set_name": "NIH ExPorter"
}
|
The primary goal of the Biostatistics Shared Resource (BioSR) is to facilitate the peer-reviewed research of members of the Comprehensive Cancer Center at Wake Forest University (CCCWFU). We collaborate with members from all four programs (Cell Growth and Survival, Clinical Research, Cancer Prevention and Control, and Cellular Damage and Defense) throughout all phases of cancer-related research projects. Major responsibilities are assumed for methodological, statistical, and computer-related issues including study design, sampling, statistical aspects of clinical trial monitoring, interim reviews, and final analysis. Specifically, the BioSR supports the following five areas 1) Protocol Development, i.e., developing appropriate statistical designs, preparing statistical sections, and reviewing the statistical content of all protocols submitted for review at the CCCWFU; 2) Protocol Monitoring, i.e., monitoring patient accrual and performing interim analyses when needed; 3) Statistical Analyses and Publications, i.e., collaborating with CCCWFU investigators from all programs to provide statistical analyses of data and interpretations of results to assist in publications of findings; 4) New Grant Development, i.e., meeting with CCCWFU investigators to discuss the design and analysis plans for potential grants as well as conducting regular grant assistance planning sessions to brainstorm with CCCWFU investigators about potential grants; and 5) Leadership and Training, i.e., participating in CCCWFU committees responsible for scientific and administrative decisions and conducting a monthly seminar series in topics in research methods for CCCWFU members. The BioSR is a highly utilized and cost efficient resource for the CCCWFU. From Nov. 1, 2009 through Oct. 31, 2010, the BioSR provided over 5,000 hours of support for cancer related work. During the same time period, the BioSR assisted over 115 different investigators (71 Scientific members) from all Programs and Centers of Excellence within the CCCWFU. In addition, 20 investigator-initiated institutional protocols were opened, 30 investigator-initiated institutional protocols were monitored, 20 manuscripts (in 2010) were published and over 30 grants submitted with the assistance of the BioSR during this time. For the coming grant period we request at total of 2.45 FTEs of support divided into statistical, administrative and programming support to provide these continued services.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Abstract The circuits in the brain that mediate our perception of reward, known collectively as the "reward system," couple pleasure with the essentials of life: food and reproduction. The reward system also lies at the root of some of the most tragic, harmful, and costly behaviors in our society. These include addiction to substances of abuse, obesity-related behavior, dangerous thrill seeking behavior, and aberrant sexual behavior. Research that I performed on altruistic behavior in honey bees has led me to a new insight about the reward system. Finding that the same neurochemical system that modulates selfish behavior in solitary insects modulates altruistic behavior in the highly social honey bee, I conclude that not only is the reward system vulnerable to "hijacking"- -coupling to stimuli with negative consequences-- over the course of a lifetime as mentioned above, but it also is vulnerable to hijacking in evolutionary time. I propose to use 'omic technologies (high-throughput sequencing, transcriptomics, epigenomics, proteomics, and metabolomics) to understand in molecular terms how to "flip" the reward system, from selfish to altruistic behavior. These analyses will be performed on a carefully selected set of closely related species of bees, some social (with altruistic behavior) and some solitary (without). The insights gained from this novel synthesis of systems biology, neuroscience, and evolutionary biology will transform our understanding of drug addiction and other diseases of the reward system and provide the foundation for new forms of therapeutic intervention.
|
{
"pile_set_name": "NIH ExPorter"
}
|
There is limited knowledge about the relationship between the GABAA receptor subunit composition and the synaptic, extrasynaptic and other subcellular localization of this receptor. This is in spite of the importance of the GABAA receptor in brain function. It has been estimated that 40% of the brain synapses are GABAergic. The long-term objective of this proposal is to understand the molecular mechanisms involved in clustering and anchoring GABAA receptors at the postsynaptic membrane. The aims of this proposal are: 1) To identify the amino acids of various subunits involved in the synaptic clustering of the GABAA receptors; 2) To study the synaptic and extrasynaptic localization of the gamma 3 and alpha 5 subunits and 3) To biochemically separate and characterize synaptic and non-synaptic GABAA receptors from various subcellular fractions isolated from the brain. Already available subunit-specific GABAA receptor antibodies in combination with immunocytochemistry at the light microscopy and electron microscopy levels will be used for revealing the cellular, subcellular and synaptic localization of various GABAA receptors in the rat brain and in hippocampal cultures. The same antibodies will be used for the biochemical characterization of the GABAA receptors in the subcellular fractions isolated from the brain. Techniques such as siRNA treatment of cultured hippocampal neurons and the expression of mutated GABAA receptor subunits in these cultures and in the brain will also be used aiming to elucidate the mechanisms involved in the synaptic clustering of GABAA receptors. These studies are relevant for understanding the mechanisms involved in the formation, maintenance and plasticity of the inhibitory GABAergic synapses in the brain. It is predicted that the inappropriate synaptic and extrasynaptic localization of GABAA receptors affects GABAergic synaptic function and brain development, leading to neurological and mental disorders. These studies will also be relevant for designing GABAergic therapeutic drugs with higher clinical specificity and fewer side effects.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The neural crest is a population of multipotent progenitor cells that forms at the border of the developing central nervous system and epidermis. During early development, neural crest cells migrate from this neural border domain to form diverse derivatives throughout the embryo, including bones and cartilage in the head, and portions of the cranial ganglia and heart. Disruptions in neural crest formation cause numerous human developmental abnormalities in the head, face, heart, and nervous system, as well as neural tube closure defects. The critical first step in neural crest development is the segregation of the neural border from adjacent neural and epidermal ectoderm. This process has been shown to involve Wnt and BMP signals secreted from adjacent ectoderm and underlying paraxial mesoderm. These signals induce neural border formation by activating the expression of the 'neural border specifier' genes, Zic, Pax3/7, and tfap2. While Wnts and BMPs are essential for neural border induction, how these pathways are integrated to drive expression of the neural border specifiers is unknown. Wnt and BMP signals can regulate transcription through activation of their canonical downstream effectors, TCF/LEF and SMAD1/5/8 transcription factors, or through several non- canonical effectors. Thus, neural border specifier expression could involve direct regulation by TCF/LEF and SMAD1/5/8, direct regulation by non-canonical effectors, or indirect regulation by unknown intermediate factors. To evaluate these possibilities, we are characterizing 8 evolutionarily conserved cis-regulatory elements (CREs) from the Pax3, Pax7, Zic2/5, Zic3/6 and tfap2a loci that recapitulate early neural border expression of these genes. Preliminary analyses of these CREs support a general mechanism for neural border induction in which the Wnt and BMP signaling pathways are integrated directly on evolutionarily conserved CREs through TCF/LEFs and SMAD1/5/8. The proposed work will test this model using mRNA injections, mutagenesis, and chromatin immunoprecipitation (ChIP). The experimental aims of the proposed work are; 1) Test for regulation of the neural border specifiers by TCF/LEFs and SMAD1/5/8 using hormone- inducible, activated forms of these factors, and mutagenesis of evolutionarily conserved TCF/LEF and SMAD binding sites and, 2) Test for direct binding of the TCF/LEF-ss-catenin complex and SMAD1/5/8 to CREs using ChIP. In addition to testing our model, the proposed work will validate ss-catenin and SMAD antibodies for use in zebrafish embryo ChIP, and establish strategies for rapidly testing the BMP and Wnt responsiveness of novel CREs. Future work will leverage these results to test if neural border induction via direct, combinatorial action of canonical BMP and Wnt signaling pathways is a genome-wide mechanism for achieving neural border gene expression. These studies will also identify novel direct targets of BMP and Wnt signaling in the neural border, adding both breadth and depth to our understanding of neural border formation. The proposed work will support the training of one postdoctoral scholar for two years. PUBLIC HEALTH RELEVANCE: This research is relevant to public health because neural border formation is critical for neural tube closure and normal development of the head, face, and heart. Thus, understanding how the neural border forms can help us understand birth defects and other diseases which occur when neural border and neural crest development is impaired. In addition, the genetic similarly of neural crest cells to cancer and stem cells means that this research will likely shed light on the biology of these cell types, including the genetic processes that lead to multipotency and migratory ability.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The Molecular Imaging Reporter Core (IVIIRC) is a central facility (See GENERAL RESOURCES AND ENVIR0NI\/1ENT). David Piwnica-Worms, PSO PI, also serves as Reporter Core Director. Julie Prior, B.S., is the Senior Core Manager. Ms. Prior is a seasoned Medical Research Lab Manager with 15 years experience in biomedical research/molecular imaging projects, and has been a part ofthe PSO ICMIC program here at Washington University since its inception. She has extensive experience with cell biology, tracer assays, biochemistry and all aspects of bioluminescence animal and cellular imaging as well as lab management. She is the primary technical investigator and directs two FTE's engaged in active experimentation, cloning of MIRC reporter genes, engineering constructs, and managing a catalog of vector systems for general use in reporting the regulation of genes of interest for scientists involved with ICMIC activities. Julie also actively participates in discovery research projects. The MIRC not only develops and generates new reporter constructs, cell lines and genetically engineered animals, but also provides expertise, materials and collaborative assistance for design and execution of the biological aspects of molecular imaging. The MIRC serves investigators with a wide range of resources and experience in niolecular biology, mammalian cell culture, and small animal experimentation. One of the most important service activities of this core is dissemination of our newly developed molecular imaging reagents and genetically encoded reporters to investigators within our institution, to other PSO program sites and to cancer biology investigators throughout the world (Figure 1). The MIRC has been and continues to be one of our most productive and comprehensive cores, impacting a broad range of programs throughout the world through our discovery research activities.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Evidence abounds that inflammatory mononuclear phagocyte iMP; microglial cells, perivascular and brain macrophage) secretory processes are centrally implicated in the neuropathogenesis of HIV-1-associated dementia (HAD). Whether this can be harnessed to positively affect neurodegenerative processes is less apparent. Thus, this program project (PPG) seeks support for investigating the specific immunologic basis of HAD and linkages between it and other neurodegenerative disorders. The focal point of disordered immunity for HAD is the microglia, the cell that links all PPG research efforts. The studies will utilize, as its foundation, a well-developed research infrastructure within the Center for Neurovirology and Neurodegenerative Disorders (CNND) of the University of Nebraska Medical Center. The CNND consists of investigators of diverse expertise who maintain a unified focus for their research on how MP biology affects both neurodegeneration and neuroprotection. This group of scientists has significant expertise in areas of neurotoxicology, cellular immunology, neuropathology, neurophysiology, neuropharmacology and molecular biology. "State of the art" technologies in magnetic resonance imaging/spectroscopy, electrophysiology, gene arrays and proteomics are being developed and strengthen this proposal. Currently, ties between innate and acquired immunity for brain MP function serve to bridge all of the proposed projects. It is our hypothesis that inflammatory mechanisms cause or intensify tissue damage during neurodegenerative processes. Most importantly, we believe these can be reversed through drug or immune manipulations. We purport that the interplay between the peripheral immune system and the brain serves to counter neuronal death caused as a result of viral infection. The balance between MP trophic and toxic activities may, therefore, underlie the process of dementia. The strength of this proposal is its well-developed cross-disciplinary program of young, energetic investigators whose scientific expertise spans the gantlet of MP-virus-neural interactions. Working under one roof, in one organizational structure with a strong track record of collaboration and joint publications should serve the PPG well in its quest to merge research initiatives designed to address pathogenic and therapeutic aspects of HAD. Results of these works should have implications for monitoring disease and in developing novel therapeutics (for example, Alzheimer's disease) which now has few treatment options.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Tryptophan synthase. The structure, function, and subunit interaction in the multienzyme complex are being investigated. The alpha subuunit contains 2 independently folding domains which can be separated after limited proteolysis. Circular dichroism studies show that the guanidine hydrochloride-induced unfolding of the alpha subunit is stepwise: the smaller C-terminal domain unfolds at low concentrations of denatuant to yield a partially unfolded intermediate; the larger N-terminal domain unfolds at higher concentrations of denaturant. The sites of interaction between the alpha and beta subunits and between the alpha domains are being mapped b chemical modification experiments. An active site arginine in the beta subunit has been modified by phenylglyoxal and is being located in the sequence. The stereochemistry of the sodium borohyhdride reduction of Schiff bases of the coenzyme, pyridoxal phosphate, with amino groups of a substrate and of an active site lysine, has been determined.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Schistosomiasis is by far the most important helminth parasitic disease of humans. Vaccines are unavailable, the only effective treatment involves repeated dosing with a single drug (praziquantel), and now drug resistance is a major concern. Schistosomes require aquatic snails for transmission. Understanding the molecular mechanisms by which snails and schistosomes interact is key for new strategies to interrupt transmission. Decades of painstaking research on the molecular basis of snail-schistosome compatibility have yielded just a handful of candidate genes or mechanisms. Using a genome-wide association mapping approach, we recently identified a small region of the genome of the snail, Biomphalaria glabrata, in which allelic variation at an unknown gene has a very strong effect on resistance to Schistosoma mansoni. This region contains 10 putative coding genes, none of which was previously known to be immune relevant in molluscs. The goal of this proposal is to unambiguously identify which of the genes in this region is causal. Firstly, candidate genes will be ranked by their likelihood of being the causal gene. Ranking will be based on whether or not alleles on the resistant versus susceptible haplotypes (versions of the region) differ in (a) expression levels or (b) amino acid sequence, together with information on putative gene function. Then, for each remaining candidate in ranked order, we will functionally test whether allelic variation at that locus actually controls resistance. This will be accomplished using RNA interference (RNAi) and allele-specific RNAi (i.e. knock down one allele or the other in heterozygotes). These complementary approaches allow one to evaluate causality for alleles that differ in either expression level or amino acid sequence. Innovation: Association mapping through functional identification of a causal gene illustrates a fresh new approach in the field of Biomphalaria genetics. The use of inbred lines with RNAseq (whole-genome expression) data, RNAi and allele-specific RNAi in a hypothesis testing framework is also novel. Significance: Identifying new resistance pathways will indicate new ways to potentially interfere with parasite transmission (i.e. how do some snails block schistosomes' ability to detect, penetrate or successfully develop within a host?). Identifying resistance genes in snails is also essential for evaluating whether genetic manipulation of snail populations might become a viable approach for blocking transmission. Understanding resistance in snails should also aid the search for genes in the parasite that control host specificity. Finally, molluscs are intermediate hosts for many diseases of medical and economic importance worldwide. None of the genes in the region of association have been previously identified as immune-relevant in molluscs. Thus, whichever gene turns out to be causal, it will identify a new mechanism of disease resistance in this important group of disease-transmitting organisms.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Trachoma is the world's leading cause of preventable blindness. This disease, caused by Chlamydia trachomatis, is endemic in many parts of the developing world. Several years ago in a project called Azithromycin in Control of Trachoma (ACT I) we evaluated the use of community-wide treatment with oral Azithromycin. This approach resulted in clinical improvement and dramatic reduction in prevalence of chlamydial infection through a 1- year follow-up. We now propose to return to these villages and do clinical surveys to assess trachoma activity, and to test conjunctival swabs for the presence of C. trachomatis by ligase chain reaction (LCR). The villages will include the previous treatment sites (oral) azithromycin versus topical tetracycline) as well 2 new villages that have not had any prior treatments. Thus we will determine the longterm (5 year) effects of the azithromycin, and follow them for 3 years. We will compare single-dose treatment with the 3 doses used in ACT I to determine the efficacy of this simpler, less expensive regime. All LCR positive specimens from the ACT I villages will have the major outer membrane gene amplified and sequenced. The genovars will be mapped for location within villages and families and then their distribution will be followed over time, after treatment to provide a better understanding of the epidemiology of the infection. Results of the study will be used as data input for the generation of mathematical model to predict whether community-wide retreatment (or alternate strategies) will be needed, and the optimal timing for such retreatment. In sum, this study should provide a rational approach to use of community-wide azithromycin treatment to eliminate blinding trachoma as a public health problem.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Mechanical loading plays a fundamental role in tissue remodeling by regulating cell behaviors in a manner dependent on the modality, frequency, and amplitude of loading. However, the mechanisms by which cells are able to differentiate dynamic signals in vivo remain unknown. By altering cytoskeletal-receptor-matrix interactions within focal adhesions in response to mechanical stimuli, cells may sense and respond to dynamic loading in a manner dependent on magnitude and frequency and specific substrate-integrin pair involved. To test this hypothesis, the objectives of this proposal are to: (1) develop a single fiber force-measuring device with bound extracellular matrix proteins to apply dynamic loads to receptors on a cell surface in vitro, (2) quantify the assembly of focal adhesions at the surface of fibers using GFP-paxillin and measure the development cytoskeletal reinforcements in response to loading protocols over a range of physiologic frequencies, and (3) determine the effects of different ligands on focal adhesion formation and the development of reinforcements at the surface of fibers. An understanding of cellular response, in terms of number and distribution of focal adhesions and force generation resulting from cytoskeletal reinforcement, to dynamic loads applied through various extracellular matrix fibers will elucidate the mechanisms by which cells are able to differentiate mechanical loading of various frequencies and amplitudes in vivo.
|
{
"pile_set_name": "NIH ExPorter"
}
|
In this reporting period we have continued a collaboration with Dr. David Margulies on the characterization of interactions of proteins constituting the peptide loading complex, specifically the TAPBPR protein and its interactions with MHC class I molecules in the presence of different peptides. While we are largely employing analytical ultracentrifugation at the current state, this project involves SPR and fluorescence anisotropy studies and may potentially evolve into a model application for global multi-method analysis of multi-protein interactions. In collaboration with Dr. Mark Mayer, we have continued to study the homo- and hetero-oligomerization of different isoforms of glutamate receptor amino terminal domains. Since the quaternary structure of these receptors controls their ion gating properties, their interactions are of great biological importance. In addition, for us they provide challenging model systems for the development of techniques for studying high-affinity interactions with fluorescence-detected sedimentation velocity. To broaden our experience in this technique, we have also applied it collaboratively to the study tubulin dimer dissociation (with Dr. Dan Sackett), and the self-association properties of several new fluorescent proteins (with Dr. George Patterson). We have also continued our collaboration with Dr. Patrizia Farci applying surface plasmon resonance biosensing to examine the interaction of anti-core antibodies isolated and cloned from livers of patients HBV-associated acute liver failure with their HBV core antigens. The determination of binding epitopes, affinities and kinetics of these antibodies against the homologous core and wild type may provide new insights into the role of these antibodies in the pathogenesis of ALF. We have intensified our long-term collaboration with Dr. Lawrence Samelson on the study of multi-protein interactions in signaling particles after T-cell activation. In more detail, we have explored the hydrodynamic properties of protein constructs that will enable us to study four-component complexes of adapter proteins, and characterize their architecture with regard to the binding stoichiometries and potentially affinities and cooperativity by using multi-signal sedimentation velocity and calorimetric techniques.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The long-term goal of this project is to apply advances in immunogenetic knowledge to achieve prolongation of organ survival after transplantation. Newer matching techniques that provide information about the HLA-D system will be combined with long-term preservation of kidney allografts. In addition to the classical typing for the HLA-A, B, C loci antigens and the serological cross-match, we shall use the following approaches. (1) We shall measure HLA-D region compatibility using primed lymphocytes. (a) Lymphocytes of the recipient will be primed to a pool of stimulating cells, and this pool-primed LD typing (pPLT) reagent will be used with cells of potential donors. (b) Primed LD typing (PLT) cells will be used to define the D region antigens on cells of the donor and recipient as the PLT reagents become available. (2) We shall use lymphocytes primed to the cytotoxic target antigens for cytotoxic T lymphocytes in a pool of sensitizing cells to obtain a cellular cross-match for these target antigens. This test, the pool-primed CML (pCML) procedure, will hopefully complement the serological typing for A, B and C locus antigens. These tests will be done prospectively for all kidney allografts. In addition the immunological status of the recipient posttransplantation will be evaluated and monitored with specific attention to changed reactivity to donor LD (in matched leukocyte culture test) and CD (in cell-mediated lympholysis tests) alloantigens. New developments, both with respect to organ preservation and donor-recipient matching, will be added to the protocols as indicated during the grant.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Demonstrate that the administration of oral magnesium hydroxide will decrease iron absorption after a supratherapeutic dose of iron sulfate in humans.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Diabetes mellitus is one of the major health problems of the growing elderly population. The overall goal of the Diabetes Care for Order Adults Project (DCOAP) is to develop and test models of care for older adults with diabetes mellitus that can be translated into community practice. The initial phase of the DCOAP was carried out during the current funding cycle of the MDRTC as a randomized clinical trial comparing a program of insulin therapy combined with an intensive education and social support intervention with conventional physician directed patient care management. Preliminary results demonstrate substantial improvement of blood glucose control in the experimental intervention group with no detectable negative effects on patients' quality of life. The proposed activities for the next funding cycle of the MDRTC include follow-up and completion of the initial phase of the DCOAP and will emphasize two new specific objectives designed to aid in the translation of the initial phase of the DCOAP into clinical practice. One project is designed to determine if the successful results of the initial phase of the DCOAP can be replicated using a less resource intense care system that could be realistically adapted in community settings. A randomized clinical trial will be carried out comparing intensive team care management of elderly patients with diabetes, using the initial phase DCOAP model, with patient management by a diabetologist supported only by a standard hospital outpatient diabetes education program. If comparable outcomes are obtained, a key economic barrier to community translation of an intensive management program for older adults with diabetes mellitus will be eliminated. Because our experience with the initial phase of the DCOAP indicated that there is a substantial number of older adults with diabetes mellitus who are unwilling or unable to participate in an intensive treatment program emphasizing use of insulin, a second project is proposed to determine whether a management program which is based on patient-directed behavioral change and which minimizes use of insulin can improve blood sugar control in older adults with diabetes mellitus. To address this objective, a second randomized clinical trial will be carried out to compare outcomes of elderly diabetic patients participating in a management program based on patient-directed behavioral change with those of a control group of patients receiving standard care. Successful completion of the initial phase of the DCOAP plus the two new proposed translation projects will provide the basis for establishment of effective management strategies for older adults with diabetes mellitus that can be implemented in community practice.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Vitamin A is an essential nutrient required not only for vision but also for the proper differentiation of many cell types in the body. This proposal seeks to define and study the molecular apparatus of the intestinal absorptive cell that carries out the necessary metabolism and movement of vitamin A during its transit of that cell during absorption. Particular elements under study are the enterocyte-specific retinol-binding protein, cellular retinol-binding protein II [CRBP(II)], a brush-border retinyl ester hydrolase, a brush-border retinol transporter, microsomal retinal reductase, and microsomal lecithin-retinol acyltransferase (LRAT). The specific aims are to: 1.) Purify the brush-border retinyl ester hydrolase for physical and biochemical characterization and for production of antiserum for radioimmunoassay and immunolocalization. Its ontogeny and distribution along the gastrointestinal tract in rat and other species will be determined. Its physiological role will be tested by comparing its properties to the abilities of rat everted gut sacs and the human intestinal Caco-2 cell line to utilize retinyl esters. 2.) Characterize retinol transport in both rat everted gut sacs and the Caco-2 cell. Possible coupling of the putative retinol transporter with both the brush border retinyl ester hydrolase and with CRBP(II) will be tested. Evidence for specific uptake mechanisms for retinol bound to several retinol binding proteins will be sought. 3.) Purify and characterize microsomal retinal reductase and produce antiserum for immunolocalization. Its physiological function will be tested by comparing its developmental pattern and the stereospecificity of its reduction of retinal to the abilities/properties observed for everted gut sacs and Caco-2 cells. 4.) Purify and characterize LRAT and produce antiserum for radioimmunoassay and for immunolocalization, with particular reference to the localization of retinal reductase. Possible channeling of retinoid from the reductase to LRAT will be tested. The source of substrate lecithin will be examined by testing the potential role of phospholipid transfer proteins as substrate carrier.
|
{
"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. Attention to asbestos-related diseases (ARD) has intensified due to the exposure of Libby, MT residents to asbestos-contaminated vermiculite. Vermiculite distribution to 200+ sites nationwide and the long latent period for disease development make ARD a continuing public health issue. Exposures to asbestos fibers by inhalation cause multiple ARD including asbestosis, lung cancer and mesothelioma. This investigation will focus on asbestos-caused fibrosis by studying the role of SPARC in this process, since we found increased expression of SPARC in mouse lungs exposed to several types of asbestos fibers. SPARC is a matricellular protein involved in the regulation of extracellular matrix (ECM)[unreadable]cell interactions through modulation of growth factor activity. These previously identified functions make SPARC an exciting candidate for involvement in asbestosis. The investigations proposed here will elucidate the role of SPARC in asbestosis, specifically targeting how inhibition of SPARC expression can regulate ECM production. The goals of this project are to delineate the involvement of the matricellular protein SPARC in the regulation of ECM deposition in response to crocidolite and the Libby amphibole. The central hypothesis to be tested in our studies is that expression of SPARC is a significant step in the development of lung fibrosis through the modulation of ECM production. To test the hypothesis, we propose the following aims: 1) Establish the in vivo expression of SPARC and ECM proteins in Sparc-null and matched wild-type (WT) mice after exposure to saline, the Libby amphibole, and crocidolite asbestos. Key elements of this aim include the use of Sparc-null mice constitutively lacking the protein as well as RNA interference (RNAi) as a mechanism to control SPARC expression in WT mice after fiber exposure. 2) Characterize the response of primary lung fibroblast cultures isolated from Sparc-null and matched WT mice to saline, the Libby amphibole, and crocidolite asbestos. Key elements of this aim include analysis of ECM proteins as well as changes in gene expression after amphibole exposure. As a result of the proposed studies, we hope to achieve a better understanding of the role of SPARC in the development of lung fibrosis as measured by ECM production after amphibole exposure. We will also study the potential for blocking SPARC expression as a therapeutic measure to reduce the fibrotic response. By investigating a critical question concerning fibrosis development, this proposal will also begin to address the potential for using RNA interference to control SPARC expression as a treatment for fibrotic diseases of the lung. Because of the health risks faced by thousands of asbestos-exposed individuals, the ultimate goal of these studies is to identify a direction for the development of novel therapeutic targets for these and other similar environmentally-caused lung diseases. In addition, this potential therapy has implications for treatment of all forms of fibrosis, including IPF.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Retinal degeneration is one of the leading causes of visual loss in the world. The mechanism of retinal cell death in different forms of retinal degenerations is not fully understood. There is evidence that a subset of patients with retinopathy may have an autoimmune component to the disease. One possibility is that autoantibodies associated with retinal dysfunction and retinal degeneration could cause retinal damage. Based on clinical and in vitro observations, we propose the hypothesis that autoantibodies may participate in the development of some forms of retinopathies in patients with retinal dysfunction. Some types of human acquired retinopathy may be caused by the action of autoantibodies, functioning through the activation of cell apoptotic responses after prolonged exposure to autoantibodies specific to retinal proteins. When antibodies gain access to the retina through the blood-retinal barrier and penetrate retinal cells, they initiate photoreceptor death by apoptosis. A massive death of retinal cells may lead to significant tissue damage, to visual loss, and finally, to blindness. Antibody penetration into living retinal cells will then constitute a new mechanism of immunologically mediated retinal degeneration. Because the lack of availability of tissue for a direct demonstration of apoptotic cell death in affected retinas of patients, studies using an in vitro model and animal models provide better understanding of the mechanism of cell death induced by immunoglobulins. We propose to use cultured retinal cells and an animal model to study the involvement of autoantibodies in retinal damage. Our long-term goal is to define the mechanism of induction and retinal damage in those autoimmune retinopathies and develop a new in vitro model to analyze the effect of antibody action on retinas. To achieve our goals we propose the following specific aims: (1) Determine whether autoantibodies associated with retinal dysfunction and degeneration are cytotoxic for retinal cells by investigating the relationship between specificity, antibody cell penetration, and cytotoxicity; (2) Define the role of anti-retinal autoantibodies in retinal damage by examining the mechanism of cell entry and the effect of autoantibodies on cell function; (3) Examine the mechanism of cell death in antibody-induced retinal damage by defining the mechanism of antibody-induced apoptosis in retinal cells leading to retinal damage and studying the sensitivity of different retinal cells to antibody-induced apoptosis.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The development of impaired renal function during sepsis predicts a poor outcome and increases the risk for mortality. The pathogenic mechanisms that underlie renal tubule dysfunction during sepsis are poorly understood and the identification of effective therapeutic approaches remains a priority. In preliminary studies, we demonstrate that absorption of HCO3- by the medullary thick ascending limb (MTAL) is inhibited by lipopolysaccharide (LPS). These studies provide the first evidence that bacterial molecules act directly through Toll-like receptors to impair the transport function of renal tubules, thereby identifying a new pathophysiological mechanism contributing to renal tubule dysfunction during bacterial infection. Monophosphoryl lipid A (MPLA) is a detoxified derivative of LPS that retains the parent compound's beneficial immunomodulatory activities without the proinflammatory side effects. Our preliminary studies show that treatment with MPLA attenuates kidney dysfunction in a mouse model of polymicrobial sepsis induced by cecal ligation and puncture (CLP) and prevents sepsis-induced impairment of MTAL HCO3- absorption. Accordingly, the Specific Aims are: AIM I. Identify the transport and signaling mechanisms through which basolateral LPS inhibits HCO3- absorption in the MTAL. These experiments will test the hypothesis that basolateral LPS decreases HCO3- absorption by inhibiting the apical NHE3 Na+/H+ exchanger through activation of the ERK signaling pathway, mediated through a novel interaction of Toll-like receptor 4 (TLR4) and TLR2. AIM II. Identify the transport and signaling mechanisms through which lumen LPS inhibits HCO3- absorption. These experiments will test the hypothesis that lumen LPS decreases HCO3- absorption by inhibiting basolateral NHE1 through activation of the PI3K-mTOR signaling pathway, mediated through TLR4. AIM III. Determine mechanisms by which sepsis decreases HCO3- absorption in the MTAL. These experiments will test the hypothesis that CLP sepsis impairs MTAL HCO3- absorption through a novel two hit mechanism, involving the combination of a decrease in baseline transport capacity and potentiation of inhibition by LPS. We propose that sepsis-induced transport inhibition is mediated through activation of ERK and results in increased urinary HCO3- excretion that contributes to sepsis-induced metabolic acidosis. AIM IV. Determine mechanisms by which MPLA protects against sepsis-induced MTAL transport inhibition. These experiments will test the hypothesis that MPLA stimulates the PI3K-Akt pathway through TLR4 and Trif, which prevents sepsis-induced inhibition of HCO3- absorption through downregulation of ERK. The studies proposed in this application will use a multidisciplinary approach to examine cellular and molecular mechanisms through which the bacterial molecule LPS impairs the transport function of the MTAL, the importance of these mechanisms in the pathogenesis of MTAL dysfunction during sepsis, and mechanisms through which the novel therapeutic agent MPLA protects against sepsis-induced alterations in MTAL function. PUBLIC HEALTH RELEVANCE: Bacterial sepsis is a major cause of mortality in critically ill patients, accounting for more than 200,000 deaths per year in the United States and consuming considerable health resources. Acute kidney injury is a frequent and severe complication in human sepsis and the risk for death doubles when impaired kidney function accompanies sepsis. The goal of our research is to understand mechanisms by which sepsis impairs the function of renal tubules and to determine how the therapeutic agent monophosphoryl lipid A protects the kidney from sepsis-induced injury.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Biomedical Big Data has become a sign of our times in this new genomics era, marked by a major paradigm shift in biomedical research and clinical practice. Advances in genomics have led to the generation of massive amounts of data. However, the usefulness of these data to the basic scientist or to the clinical researcher, to the physician or ultimately to the patient, is highly dependent on understanding its complexity and extracting relevant information about specific questions. The challenge is to facilitate the comprehension and analysis of big datasets and make them more user friendly. Towards this end we propose to develop a Massive Open Online Course (MOOC), Demystifying Biomedical Big Data: A User's Guide, that aims to facilitate the understanding, analysis, and interpretation of biomedical big data for basic and clinical scientists, researchers, and librarians, with limited/no significant experience in bioinformatics. This course will be a resource freely availabl to users, at no cost. Given the continuous progress in the field, our plan for the course is to be living resource, regularly revised and updated to maintain its relevance.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Objectives: The proposed project will refine and validate a computerized self-assessment of patient symptoms and health-related quality of life (HRQL) for common gastrointestinal (Gl) disorders. The goals are to improve patient care, to support research, and to ensure the quality of care. Work is being conducted using CarePrep, a Web application for patient self-reporting of health data. RESEARCH PLAN: CarePrep systematically captures data directly entered by the patient regarding symptoms and relevant psychosocial variables, and measures condition-specific HRQL. A prioritized, problem-oriented summary is generated for the clinician. Specific aims are to develop and refine assessment and HRQL content for the targeted Gl disorders {acid reflux, dyspepsia, irritable bowel syndrome, inflammatory bowel disease, colon cancer, and common hepato-biliary and pancreatic diseases); pilot test and perform preliminary psychometric analysis; develop problem management features for clinicians; enhance administrative and research functions; implement revisit functions; perform psychometric validation; assess feasibility and impact on care. In our work thus far, a model-driven assessment strategy has been developed for common Gl disorders, installed in CarePrep, and pilot tested with excellent acceptance from patients and clinicians. METHODOLOGY: Item development will use standard methodology, with review of published instruments and input from experts and patients, the latter using focus groups and cognitive interviews to ensure that issues of relevance for patients are covered. HRQL assessment will use a generic core with condition-specific modules. Pilot testing will gather data for psychometric analysis and item reduction. After necessary revisions, validity and clinical reliability will be tested. The impact of CarePrep on clinician-patient communication and on clinician effectiveness will be compared to usual care. CONCLUSIONS: The refinement and validation of this system will demonstrate the value of an integrated bio-psychosocial instrument for condition-specific assessment of the targeted Gl disorders.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Behavioral tolerance is a hallmark of exposure to most drugs of abuse. It is characterized by reduced drug effects over time, either via altered metabolism of the drug, or functionally, whereby the effects of the drug decrease in spite of unaltered concentration. Indeed, the presence of enhanced acute behavioral tolerance in humans can serve as a marker for the likelihood of a future development of alcoholism. The large conductance potassium ion channel (BK) has become increasingly recognized as an important target of alcohol action, both in the intoxicating actions of the drug, as well as in adaptation to the drug (tolerance). This proposal is based upon exciting new data which identifies the BK channel beta4 subunit as an important mediator of alcohol tolerance and consumption, and provides an animal model to examine the relationship between molecular and behavioral tolerance, and consumption. We will make use of genetically engineered mice, in which the beta4 subunit is either present or knocked out. In Aim 1, we test the hypothesis that BK beta4 influences acute tolerance via a mechanism involving kinases and associated signaling pathways. Electrophysiological experiments will probe the role of kinases in BK acute tolerance in expression systems and in neurons of the striatum, which is important in drug reward and addiction. In Aim 2, we test the hypothesis (based upon preliminary data) that beta4's influence on rapid tolerance will not be as pronounced as in acute tolerance. These studies will utilize electrophysiological, molecular biology, and imaging techniques, and will probe at the molecular, cellular, and behavioral levels. Aim 2 will include studies on the role of beta4 in the finding, described in our preliminary data that in striatal neurons, there is a switch which is activated after 3 to 6 hrs of alcohol exposure, which radically changes the duration of tolerance after alcohol withdrawal. This has implications for the development of alcohol dependency as a function of drinking habits. Finally, in Aim 3, we test the hypothesis that beta4's influence on alcohol consumption will reflect influences on behavioral parameters such as alcohol preference and reward, both known to influence consumption. A two bottle choice variant of the drinking in the dark (DID) paradigm will be utilized to measure ethanol vs. water preference in beta4 KO and WT mice. In addition, we will measure the rewarding properties of ethanol in WT and beta4 KO mice using a conditioned place preference (CPP) assay. Together, these experiments will help identify the role of the beta4 subunit and acute tolerance in motivational behaviors underlying alcohol consumption. PUBLIC HEALTH RELEVANCE: Alcoholism represents one of the most important problems in the public health arena. Tolerance, which develops with alcohol use, may be related to the development of drug dependency and addiction. In this proposal, we pursue exciting new data which indicates a role for the BK channel beta4 subunit protein in the control of alcohol tolerance and consumption.
|
{
"pile_set_name": "NIH ExPorter"
}
|
APPLICANT'S ABSTRACT: Independent Scientist Award-KO2: As Co-Director of an Alcoholism Research Center, I have supported my salary since 1991. In July, 1998, the VA will terminate this national program. At that time, I will assume substantial clinical responsibilities unless I find an alternative mechanism to support my salary. This K02 Award would enable me to sustain my research and develop further expertise in areas critical to my research program. My research has focussed on characterizing the clinical significance of glutamate and GABA dysregulation in alcoholism. With NIAAA support, I conducted a study that indicated that the NMDA antagonist, ketamine, had ethanol-like effects and altered reward valence in alcoholic patients. These patients showed blunted dysphoric responses and increased euphoric responses. Thus, NMDA receptor dysregulation may alter the NMDA antagonist component of ethanol response in patients in a manner that promotes relapse. To build towards pharmacotherapy, I have proposed to compare responses to a partial agonist of the strychnine-insensitive glycine (SIGLY) site of the NMDA receptor in recently detoxified alcoholics and controls (Years 1-3) and to determine whether drugs acting at this site modulate ethanol cue-induced craving in patients (Years 4-5). To facilitate these objectives, I will pursue additional training in multivariate statistics and I will work collaboratively to develop methods for assessing craving that are independent of self-report and would be applied to studies in Years 4-5. My other major research focus has been the study of reductions in GABA function in alcoholics using 133I iomazenil single photon emission computerized tomography (SPECT) and magnetic resonance spectroscopy (lH-NMR). Respectively, these techniques measure benzodiazepine/GABA-A receptor density and GABA levels. I have proposed a study that employs both SPECT and 1H-NMR in a longitudinal assessment of the recovery of GABA systems with sobriety. If funded, this study would cover years 1-4 of this K02 period. In year 5, I propose to characterize the activity of the GABA synthetic enzyme, glutamic acid decarboxylase, in alcoholic patients using a 13C-glucose infusion technique. In this work, I focus my career development on gaining further expertise in 1H- and 13C-NMR. As a result, I have developed an educational program designed to facilitate my development in these areas. In summary, this K02 award would foster my academic development and enable me to better contribute to the building of pathophysiologic models of alcoholism that can guide medications development for this disorder.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Project Summary An appropriate routing of information in the brain based on the current task demands is essential for successfully dealing with a complex, changing environment. Such routing mechanisms seem to be impaired in disorders like schizophrenia. Unfortunately, we currently do not understand how the brain is able to regulate the flow of information. A recent theory suggests that synchronous activity plays an important role. However, it is still fiercely debated in the neuroscience community whether synchronous activity and precise spike timing in cortex have a functional significance and, if so, what role they play. A major obstacle to answering these questions is the lack of suitable experimental techniques for artificially manipulating brain activity with a precise timing relationship to currently ongoing neural activity. Techniques that allow the experimental manipulation of neural activity play an instrumental role in establishing a causal link between brain activity and its functional significance. The goal of the proposed project is therefore to develop a closed-loop stimulation technique that allows the measurement of currently ongoing neural activity in the form of a local field potential, analyzes it in real time, and can trigger a stimulation device like, for example, electrical microstimulation or optogenetic stimulation in such a way that artificially injected neural activity is phase-locked to currently ongoing oscillations. This includes the development of an algorithm that reliably tracks the instantaneous phase of a dominant component of the local field potential as well as finding a suitable implementation of this algorithm for controlling a stimulator in real time. Making the proposed technology available to the scientific community is expected to provide major breakthroughs in understanding the functional significance of synchronous activity and precise spike timing in the brain.
|
{
"pile_set_name": "NIH ExPorter"
}
|
SUMMARY A fundamental advance in Neuroimaging in the last 10 years has been the development of methods to derive regional parcellations of the cerebral cortex, or `parcellated connectomes', from structural and/or functional MRI data. Within the Conte Center, the Neuroimaging core will focus on providing single subject and group connectomes of the brain in humans and macaques. Specifically, connectomes will allows for: (1) the localization of recording sites in relation to independently localized human cortical regions and networks; (2) standardization of recording site localization across centers, which will in turn provide a way to compare results across experiments; (3) definition of a macaque monkey cortical parcellation atlas for the comparison, sharing, and meta-analysis of physiological studies within our consortium, but also the wider non-human primates physiological community. The Neuroinformatics core will facilitate data sharing across the Conte Center sites and the neuroscience community through an open source web interface.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The infectious microorganisms that mosquitoes may carry and pass to humans via injection of saliva, during the taking of a blood meal, can be a very serious health hazard. The most serious of these disease are malaria, yellow fever, encephalitis, and dengue fever. Of these, encephalitis can be contracted from mosquitoes in Montana. In Montana and by US citizens throughout the world, DEET, N,N-diethyl-m-toluamide is the most frequently used mosquito repellent. Materials of plant origin, most of which are grown in Montana, have shown potential for increasing the effectiveness of DEET and for replacing it entirely. MBRS students will collaborate with scientists in an ongoing research project which identified natural materials that are effective when applied to clothing and skin (forearm) surfaces of humans. Students will be involved in maintaining secure mosquito rearing facilities, observing adult mosquito landing behavior, introducing human subjects to the project, and in testing the material with human subjects. The long term objective of this project is to suggest to the private sector ideas derived from traditional wisdom, such as that of medicinal and pest management plants used by Native Americans cultures, that have modern technological potential.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Experiments in choloralose-anesthetized dogs indicated that the degree of respiratory variations in pulse interval (VHP) correlated linearly with the degree of parasympathetic cardiac heart rate control (PC), when the latter was defined as the change in pulse interval caused by the elimination parasympathetic efferents while keeping sympathetic efferent activity to the heart unaltered. The overall goal of the project is to determine under what physiological conditions this relationship holds. If it is shown that the relationship holds under a wide range of conditions, then the noninvasively measurable VHP could be used as an indicator of PC, otherwise obtainable invasively only. The objectives for the current year of this project are to concentrate on chronically prepared animals and to determine the relationship between VHP and PC not only during rest (as primarily done during the first year) but also during exercise. The effect of sympathetic blockade on the relationship between VHP and PC is also to be investigated, and the measured slope between the two variables is to be compared with the slope predicted by theoretical considerations that involve respiratory timing.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The applicants proposed to build an ultra-low-cost and high-quality prototype Prepolarized MRI scanner for diagnosing head tumors. The overall goal of this research is to develop a new and innovative imaging modality that shows great potential for cancer screening, diagnosis and image-guided treatment. The specific aims are to design and build both a homogeneous, low-field electromagnet and a lowfrequency receiver system optimized for head imaging. The final aim is to integrate these subsystems with our working Prepolarized MRI system and to obtain an image of a normal volunteers head. This research is intended to demonstrate that Prepolarized MRI system contrast and SNR are equivalent to a conventional 0.5 T MRI system; and that this high-quality Prepolarized MRI system will cost less than $50,000 in capital costs. The successful development and integration of this innovative technology will require the application of several principles of engineering and science. For example, a homogeneous electromagnet design method draws from both electromagnetics and linear programming. A low-noise receiver subsystem design designed by the applicants relies on the principles of optimal RF receiver electronics. Pulse sequence design relies on the principles of MRI physics.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Recent research has shown that naive listeners have significant success in identifying the regional dialect of unfamiliar talkers. This finding suggests that listeners encode detailed sociolinguistic information in memory and can draw on these representations to make explicit judgments about new talkers. The proposed project extends this line of research by exploring the interaction between this social-indexical variation and phonological processing. First, the role of linguistic experience in the activation and suppression of phonological dialect subsystems will be explored in a series of speech perception and production tasks. Second, the role of phonological similarity between two linguistic varieties in spoken language processing will be explored in the same series of perception and production tasks. Finally, an exemplar-based model of the representation of dialect variation will be developed to account for how linguistic information is perceived, encoded, and represented in long-term memory with respect to phonetic, phonological, and lexical categories.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Project Summary Genome wide association studies (GWAS) have successfully identified thousands of loci likely affecting human health. To translate these findings into therapeutic targets and disease treatments, we need to understand the cellular context and underlying biological mechanisms through which each disease associated variant disrupts function. Large scale, information rich datasets are being generated across multiple modalities including transcriptomics from single cell RNA-seq studies, traits and phenotypes from the UK Biobank and germline genetic variation from exome sequencing studies. Here, we propose to develop methods to integrate these amazing resources towards understanding the identifying biological and cellular mechanisms that are leading to disease. The objectives will be accomplished with the following specific aims: 1) Integrate population scale biological datasets including UK Biobank and single cell transcriptomics data to construct gene modules with the goal to recapitulate biological pathways. 2) Develop a statistical framework to measure mutational burden across each of the cell type specific gene modules. Together, this research proposal will increase the power in interpreting human genetic variation and help better understand the mechanism through which they act. These methods are being developed around an IBD dataset and will derive substantial molecular information about the mechanisms driving IBD. The lessons and methodological advances from this work will be directly applicable in many complex disease contexts.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The overall goal of this multiple PI application is to understand the basic cellular and molecular mechanisms underlying maternal, fetal and newborn vascular adaptation in response to high altitude, long-term hypoxia during gestation. Hypoxia is one of the most common and severe stresses to an organism's homeostatic mechanisms, and hypoxia during gestation has profound adverse effects on maternal health and developmental plasticity. Gestational hypoxia is associated with high incidence of clinical complications including preeclampsia and fetal intrauterine growth restriction (IUGR). Both human and animal studies have revealed a causative role of increased uterine vascular resistance and lowered uterine blood flow in preeclampsia and IUGR. Our recent studies revealed that high altitude hypoxia suppressed pregnancy-induced uterine arterial adaptation and increased uterine vascular resistance and systemic blood pressure in pregnant sheep. Infants born at high altitude show significantly increased risk of persistent pulmonary hypertension. We demonstrated that gestational hypoxia at high altitude elevated pulmonary vascular resistance and increased pulmonary artery pressure and pressure response to acute hypoxia in newborn lambs. In addition, fetal hypoxia negatively impacts cerebral vascular development and increases the risk of intraventricular hemorrhage in newborns. Hypoxic-mediated responses are highly integrated across many cell types; nonetheless, they are tissue specific. In many respects these responses differ significantly between the fetus and newborn, as well as between non-pregnant and pregnant states. Much remains unknown of the molecular mechanisms underlying programming of maternal, fetal and newborn vascular response to long-term hypoxia in gestation. The proposed study is broadly based, multidisciplinary, integrated project using physiological, pharmacological, cellular, biochemical, and molecular approaches to investigate the mechanisms underlying maternal uterine, and fetal and newborn pulmonary and cerebral vascular response to long-term hypoxia in gestation. Based on >25 years of research by our group, the proposed study will be conducted in sheep acclimatized to high altitude (3801 m/12,470 ft). The overall hypothesis is that high altitude, long-term hypoxia during gestation increases micro RNA-210 and endoplasmic reticulum (ER) stress, differentially regulating spontaneous transient outward currents (STOCs) in programming of maternal, fetal and newborn vascular response, impacting developmental plasticity and the subsequent risk for disease. The proposed study has strong scientific premise with a novel concept and an innovative and mechanistic approach. It will provide new insights into the understanding of fundamental mechanisms underlying programming of maternal, fetal and newborn vascular dysfunction caused by gestational hypoxia, impacting maternal health and developmental plasticity. Given that STOCs are fundamentally important in regulating vascular tone and blood flow in virtually all vascular beds, revealing molecular and epigenetic regulation of STOCs function in programming of vascular response to hypoxia will have broad impact in the comprehensive understanding of the mechanisms in vascular physiology and pathophysiology.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Rhesus monkeys that were exposed to PCB three years previously and still exhibiting ill effects from this exposure will be evaluated for a 4th year to determine if these deleterious effects persist, particularly in their offspring. In addition to reduced birth weights and neonatal growth rates, these infants have shown inhibited learning and behavioral development. A second group of rhesus monkeys that have been fed PCBs for 1 year at levels that are close to what heavy fish-eating humans may consume, will continue the exposure for a second year. Emphasis will be placed on the cumulative effects that may arise from long term, low level exposure in the adult monkey, as well as the injurious effects that may arise in their offspring. In other experiments, infants born to rhesus mothers that were exposed to levels of PCB (Aroclor 1016) as low as 25 ppb for one year will be evaluated psychologically for learning and behavioral development. Since these infants have shown gross signs of PCB intoxication, the potential effects on the development of the central nervous system is of prime importance. The question invariably arises as to the difference in response that may be experienced by man and lower animals to commercial preparations of PCB vs. PCB that is present in the food chain. Fish oil containing PCB will be extracted from fish and placed in the diet of rhesus monkeys at levels similar to that utilized in experiments where commercial PCB preparations were used. These animals will be evaluated as to the effects this diet has on the adult monkey and its offspring. These data will then be compared with that obtained in experiments that have used commercial PCB mixtures in the diet.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The broad, long term objective of this application is to understand how regulatory proteins of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) control viral and cellular gene expression during lytic replication. Lytic gene products are likely to have a role in pathogenesis of KSHV, contributing to the ability of KSHV-infected cells and adjacent cells to proliferate, survive and avoid immune responses. Understanding the mechanisms by which lytic gene expression is regulated is therefore relevant to KSHV- related pathogenesis which continues to be a significant cause of morbidity and mortality, particularly in areas with high endemic rates of HIV and KSHV infection. The ORF57 protein of KSHV is expressed early during KSHV replication and is a member of a family of herpesvirus proteins that plays an essential role in herpesvirus replication. ORF57 has unique regulatory properties, post-transcriptionally enhancing expression of many intronless genes. In addition, ORF57 enhances expression of specific cell genes. ORF57 is likely to exert many of its effects by physically binding to mRNA and modulating its stability and nuclear export. Much remains unknown about the mechanisms by which ORF57 affects RNA processing and how its specificity of action is determined. We therefore propose three integrated aims to investigate the role of ORF57 in KSHV biology. In the first aim, we will define the mechanisms by which ORF57 binds mRNA and how ORF57 specificity is determined. In the second aim, we will identify the major cellular proteins that are important for ORF57 function, especially with regard to nuclear export and mRNA processing. In the third aim, we will determine which functions of ORF57 are essential for KSHV replication using molecular genetics. We will also determine the specific effects of ORF57 on gene expression in endothelial cells and B lymphocytes. KSHV is a virus that is linked to the development of cancer, particularly in those people who have weakened immune systems, such as those with HIV infection. Studying how the virus reproduces is important to understand how it affects the cells it infects and converts them to tumor cells. Such studies also have the potential to identify targets for new drugs against the virus that may help prevent or treat infection. [unreadable] [unreadable] [unreadable]
|
{
"pile_set_name": "NIH ExPorter"
}
|
Inflammatory pain is a very common clinical phenomenon that causes great individual suffering and expense to society. Some of the most severe forms of pain, particularly chronic inflammatory pains, are partly or wholly intractable to currently available therapies. The investigators have shown that the cAMP/PKA second messenger pathway plays a major role in hyperalgesia induced by prostaglandin E2 (PGE2) and recently that nitric oxide (NO) also contributes via a guanylyl cyclase-independent mechanism. They now propose to study the cellular mechanism(s) underlying an additional novel action of NO, guanylyl cyclase-dependent hyperalgesia. They will also investigate the role of PKCepsilon in hyperalgesia and nociceptor sensitization produced by other inflammatory mediators. The investigators will also study mechanisms of mechanical transduction in nociceptors. They have recently been able to demonstrate a mechanically-induced whole cell current in cultured dorsal root ganglion neurons. They propose to fully characterize this current in order to establish an in vitro electrophysiological model for the study of nociception of mechanical stimuli. The investigators will also investigate changes in second messenger pathways involved in chronic hyperalgesia, focusing on a novel phenomenon they recently discovered in which the hyperalgesic response to an inflammatory mediator is enhanced for a period of weeks following recovery from the inflammatory hyperalgesic state. These experiments should provide significant novel information about mechanisms of inflammatory pain and hyperalgesia and cellular mechanisms of mechanical transduction in primary afferent neurons that may be important in chronic pain states in humans. These investigations should yield insights into potential pharmacological targets for the treatment of chronic inflammatory pain.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The purpose of the present work is to elucidate the pattern of lipid metabolism occurring in the axon and the nature of the membranes involved. Studied will be conducted to determine how this metabolism is altered under those conditions in which nerve impulse propagation and/or axonal transport are blocked. Axonal transport of phospholipids will be studied in order to understand the relationships in the metabolism of the perikaryal supplied lipids to those formed locally. An independent study will be undertaken to determined if axon-associated enzymes appear earlier in development in spinal nerves than Schwann-cell and myelination-related enzymes. Squid giant axon will be studied and comparisons will be made between lipid metabolism occurring in this invertebrate axon and mammalian peripheral nerve axons. These above studies will utilize autoradiographic and biochemical approaches already employed in this laboratory. The autoradiographic approaches will be extensive, employing a wide variety of potential lipid precursors, yet quantitative. Quantitative analyses will be aimed at determining the extent of axonal lipid formation (both with respect to overall level and to fiber population) and the fate of axonally formed lipids, e.g. turnover from synthetic sites. Under conditions in which axonal functions are impaired, changes in lipid metabolism will be characterized both as to radioactive products formed (biochemical) and sites of formation and subsequent metabolism (autoradiographic). Biochemical studies, with rat sciatic nerve (nerve ligation paradigm) and squid giant axon as enzyme sources, will be conducted to determined the capacities of the axon for phospholipid metabolism and the specific nature of the membrane sites where this metabolism is located. These studies, employing enzyme assays, will complement the autoradiographic studies in the localization of lipid products to axon will elicit experiments to confirm that responsible enzyme are present in axoplasm and/or axolemma.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Abstract: In this proposal, we describe a plan to build artificial platelets from biological components. The vision is based on the belief that the critical accumulation of our knowledge about individual biomolecules will enable us to integrate them into a system in a meaningful way to create cellular devices. We have identified platelets as a tractable target of such emulated biological system. Our design strategy necessitates an understanding of the functionalities of natural platelets so that the artificial platelets can confer the essential functions of natural platelets. Platelets are the first cellular responder to injured vasculature. It binds to the exposd collagen that is otherwise protected by endothelial cells. Platelets couple binding to activation via the expression of several cell surface receptors. Importantly, activated platelets attain predominantly negatively charged membranes by increasing phosphatidylserine (PS) on the outer lipid leaflet. Activated platelets then trigger a series of biochemical events known as the coagulation cascade that results in the formation of cross-linked fibrin network that forms a plug to block the injured vasculature. The artificial platelets will mimic the essential functions of natural platelets. They will be made as lipid vesicles that have defined lipid and protein composition. Microfluidic jetting, a technique recently developed that is akin to blowing bubble from a soap film, will be used to make the vesicles. This technique allows the ability to create asymmetric lipid bilayer with PS on the inner leaflet as well as incorporation of membrane proteins. The vesicles will be decorated with anti-collagen antibody that will bind to the exposed collagen during vascular injury. Our platelet activation strategy will utilize the large mechanosensitive channel (MscL) that is gated by membrane tension. When the artificial platelets attaches to the substrate, shear flow will activate MscL, this would then causes calcium in the blood serum to enter the vesicles. Scramblase will be part of the artificial platelet such that upon calcium entry, it becomes activated and exposes PS to the outer lipid leaflet. When PS is exposed, factors V and X become activated and subsequently stimulate thrombin activity. Thrombin then cleaves fibrinogen into fibrin, which assembles into a durable and cross-linked network. At each stage of our artificial platelet assembly, the design strategy can be tested and validated in a microfluidic platform that mimics the injured vasculature. In summary, the proposed research describes our vision for how synthetic biology can potentially bring about novel cellular devices that would have tremendous benefits in medical settings. Public Health Relevance: Platelets are the first cellular responder to injured vasculature, but it is constantly under shortage in the blood bank due to its short shelf life, susceptibility to contamination, and challenges in storage condition. Using biological components, we propose to build artificial platelets that mimic the functionalities of natural platelets. We envision such cellular devices will bring tremendous benefits in medical settings.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The objective of this proposal is to determine the molecular basis for the regulation of pre-mRNA alternative splicing. Alternative splicing plays a crucial role in differentiation and development, and expression of specific alternatively spliced variants is characteristic of many human diseases. Despite the importance of alternative splicing to normal cellular function, little is known about the mechanisms that direct tissue-specific regulation. This proposal continues our investigation of the cardiac troponin T (cTNT) pre-mRNA in which inclusion of a cassette alternative exon predominates in embryonic striated muscle and skipping predominates in the adult. Exon inclusion in embryonic muscle requires conserved intronic elements located upstream and downstream of the exon. Multiple copies of individual elements promote muscle-specific inclusion of a heterologous exon in embryonic striated muscle. The mechanism of regulation is complex involving antagonist activities of positive and negative-acting factors which bind adjacent sites within these elements. A role for PTB as a repressor of cTNT exon inclusion in muscle and nonmuscle cells has been established. The positive-acting factors include members of a novel family of RNA-binding proteins expressed in different tissue-specific patterns. These proteins activate element-dependent exon inclusion when coexpressed with cTNT minigenes in nonmuscle cells. Element-dependent inclusion has been reconstituted in a cell-free splicing assay using recombinant proteins. Changes in the expression of one of these proteins correlates with changes in cTNT splicing during skeletal muscle differentiation and heart development. The main goals of this proposal are to: (i) characterize the regulatory functions of the positive-acting factor expressed during striated muscle differentiation and development. (ii) determine the molecular mechanism of element- dependent exon inclusion using the established in vitro splicing assay. (iii) isolate and identify the components of the RNP complexes associated with exon skipping and exon inclusion. Genetic and biochemical approaches will be used to identify the critical interactions that link muscle-specific auxiliary splicing elements to the basal splicing machinery. Insights gained from these studies will be directly applicable to basic molecular mechanisms that affect human health.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Each year ~1.5 million American women between ages of 45 and 55 enter into the perimenopause. Yet to be discovered is the impact perimenopause has on key brain regions involved in cognition and known to be vulnerable to Alzheimer's disease (AD). Women have a higher lifetime risk of developing AD and represent more than 60% of the Alzheimer's disease population. The mission of our Perimenopause in Brain Aging and Alzheimer's Disease Program Project is to discover the biological transformations that occur in the brain during the perimenopausal transition that can result in phenotypes at risk for development of Alzheimer's pathology. We seek to identify the mechanisms by which these changes occur and to translate these discoveries to determine the optimal timing and strategies for preventing conversion to the perimenopausal at-AD-risk phenotype. To achieve our mission, we have developed a focused research center model with an integrated set of four Projects and three Cores. Projects 1, 2 and 3 are basic, mechanistic and preclinical translational science investigations of the perimenopausal brain utilizing our program developed rodent models of human perimenopause. Project 4 uses the results of Project 1, 2 and 3 to inform its ancillary association analysis of existing data and samples from the NIA-funded clinical trial Early versus Late Intervention Trial with Estrogen (ELITE) (R01AG-024154). To achieve our Program mission, each project has a unique but complementary research focus with all Aims of all projects and cores addressing Program wide aims. Innovative and integrative features of our Program include: bidirectional translational research; rodent models of human perimenopause transition; custom designed gene arrays that include GWAS identified AD risk factor genes and bioenergetic, inflammatory and AD system pathways, shared customized data and document management. Program-wide gene array and bioinformatic analyses across all projects. Discovery of at-AD-risk phenotypes and the underlying mechanisms of phenotype development could potentially lead to the early identification of those at greatest risk for developing AD and mechanistically inform interventions to prevent the disease. Program research addresses NIA Strategic Goals A and C.
|
{
"pile_set_name": "NIH ExPorter"
}
|
PROJECT SUMMARY Despite advances in the diagnosis and treatment of cardiovascular disease, the prevalence of heart failure continues to rise at epidemic rates. In response to varied injuries, the heart undergoes a complex process of remodeling, marked by upregulation of a fetal expression program and corresponding switching of metabolic activity to a fetal-like state, in close association with the development of cardiomyopathy. Over the course of the initial K08 funding period the Applicant trained in technical mass spectrometry based metabolomics and computational sciences, and their collective application to interrogate substrate utilization in cells in culture, metabolic measures of organ level physiology, and shifts in metabolic enzyme expression. Moreover, Applicant has applied these approaches to characterize the metabolic remodeling that occurs during the pathogenesis of heart failure, identifying previously unrecognized metabolic activities in the heart. These metabolic studies, however, have been limited to rodent models of myocardial injury, and it remains unclear to what extend these models recapitulate the development of human heart failure, particularly given critical differences in cardiac size, physiology, and bioenergetics between human and rodent cardiomyocytes. The development and advance of human induced pluripotent stem cells (iPSC) technologies has transformed the study of human disease. Human iPSCs may be readily differentiated into ventricular cardiomyocytes (iPSC-CMs) in vitro at high efficiencies and have been found to recapitulate in vivo human phenotypes across a number of genetic cardiomyopathies, with impaired calcium handling, decreased force generation, and re-expression of the metabolic fetal gene program. In this R03 proposal, the Applicant now aims to extend upon the initial K08 award, and apply comprehensive metabolomics to the study of human cardiomyopathy, in an effort to decipher the metabolic underpinnings of human heart failure. Using iPSC-CMs derived from patients with sequence- verified familial cardiomyopathies (dilated and hypertrophic cardiomyopathy) the Applicant will comprehensively define the metabolic changes that occur with cardiac pathology through state-of-the-art liquid chromatography - mass spectrometry. These proposed studies will shed tremendous insight into the metabolic basis for human cardiomyopathy, distinguish new metabolite biomarkers for impaired contractile activity, and identify metabolic pathways amenable to pharmacologic and dietary intervention, as well as will form the foundation for the Applicant's transition to an independent, R01 funded investigator.
|
{
"pile_set_name": "NIH ExPorter"
}
|
It is proposed to investigate the biogenesis of cellular membranes in a variety of cell types in which proliferation of membranous organelles takes place. Tissues for study include the livers of phenobarbital treated rats, alloxan diabetic rats and neonatal rats, the hepatocytes of which exhibit marked proliferation of smooth endoplasmic reticular membranes compared with control livers, the rat pancreas, the acinar cells of which show an increased formation of Golgi membranes on stimulation of secretion, and the salt gland of the duckling, the secretory cells of which exhibit marked growth of plasma membranes in response to salt stress. Using both electron microscopy, including morphology and cytochemical approaches, and biochemical techniques, including cell fractionation, enzyme assays, and analyses of membrane components, it is proposed to investigate the site of synthesis of the phospholipids and marker enzymes of newly formed membranes, and to investigate the time course of incorporation and the site of assembly of these components into new membrane during proliferation. The phospholipid patterns of different membranes and the relationship of this to the increased specific activity of marker enzyme will also be investigated. On the basis of the results of these studies, investigations of the control of membrane biogenesis will be made. In initial studies actinomycin D, puromycin, and cycloheximide will be used to determine the possible involvement of mRNA synthesis or protein synthesis in the process. In addition, the role of non-genetic controls will be investigated, the individual experiments being dependent on the tissue studied and the stimulus of membrane proliferation.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The goal of this proposal is to better understand the molecular mechanisms by which the retrieval of declarative/episodic memory occurs. The proposal focuses on the time-limited role that norepinephrine (NE) and [unreadable]1-adrenergic signaling play in the hippocampus during memory retrieval. The first aim will determine in which hippocampal cell(s) [unreadable]1 signaling acts to promote memory retrieval. Contextual fear, which depends to NE and [unreadable]1 signaling, will be used to assess hippocampus-dependent memory. Cued (tone) fear, which does not depend on NE or [unreadable]1 signaling, will be used to test whether manipulations affect performance or memory. Imaging of immediate-early gene induction in mice will be employed as a marker of neuronal activity following exposure to salient and neutral contexts, [unreadable]1 signaling in the mice will be manipulated pharmacologically and genetically so that it is blocked or activated either, systemically, only in the dorsal hippocampus, or in specific subfields of the hippocampus. Because [unreadable]1 signaling activates the cAMP / protein kinase A pathway, the second aim will determine whether this pathway is required for memory retrieval. Dorsal hippocampal infusions of agents that block this pathway will be performed in control mice, and infusions of agents that stimulate this pathway will be performed in mutant mice that lack [unreadable]1 signaling. The relationship between NE and activation of extracellular signal-regulated kinase and phosphatidylinositol 3- kinase, which are also required for retrieval, will be determined. Further, one of the most prominent physiological effects of [unreadable]1 signaling in the hippocampus is reduction of the slow afterhyperpolarization (sAHP) that mediates accommodation of firing. The third aim will determine whether calcium influx through voltage-dependent calcium channels influences retrieval, and whether pharmacologic block of the sAHP rescues retrieval in mutant mice lacking [unreadable]1 signaling. This aim will also determine whether there is a transient reduction in the sAHP that can be observed in brain slices of mice after fear conditioning but not pseudoconditioning and, if so, whether the reduction depends on [unreadable]1 signaling. Finally, a key hypothesis explaining why NE is required for some but not all memory retrieval will be tested in the fourth aim. Relevance: Dysfunction of adrenergic signaling may contribute to symptoms of depression and post- traumatic stress disorder that include difficulties with memory retrieval in the former and unwanted, intrusive retrieval of traumatic memories in the latter. Results from this proposal may also be relevant to potential cognitive side effects that might arise when treating heart failure, hypertension and performance anxiety with drugs that block [unreadable] receptors. Finally, results should also be relevant to understanding how dysregulation of neuronal Ca++ homeostasis in the elderly may lead to memory deficits.
|
{
"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. This research is studying body cooling begun in newborns after 6 hours of age to reduce death and improve outcomes of infants with Hypoxic-Ischemic Encephalopathy [HIE]. HIE results from decreased blood flow and oxygen to all organs in the body including the brain. In term infants [babies born near their due dates] certain signs of HIE follow a sudden lack of oxygen. Some of the causes include acute infections and problems with the placenta and/or the umbilical cord. The risk of death and the risk of handicaps [like cerebral palsy, blindness, or deafness] in infants who survive moderate and severe encephalopathy [effects of decreased oxygen in the brain] is 50%. If a baby has seizures shortly after birth, there is a high risk of disability. At this time, infants with HIE diagnosed at less than six hours of age are cooled on a cooling blanket to decrease their body temperature to 33-34o C [91.4-93.2o F] for 72 hours. Mildly decreasing the baby's body temperature [induced hypothermia] appears to be the most promising treatment to protect babies'brains that has come from studies of animals, adults, and babies to date. Safety studies done in newborn babies who were cooled in the first 72 hours of life have shown no bad side effects. There is information to suggest brain injury occurs over a long period of time [days to weeks following HIE]. It is unknown if induced hypothermia is of benefit when started at a later age [after 6 hours]. Some infants with HIE have not yet arrived at a hospital where induced hypothermia is offered, or may not show signs of moderate to severe HIE until after they are 6 hours old. It is also not known if a longer period of induced hypothermia [more than 72 hours] may be beneficial. The purpose of this research study is to evaluate whether induced hypothermia with body cooling begun between 6 and 24 hours of age and continuing 96 hours in infants who were born within 4 weeks of their due date and who show signs of HIE will reduce death and improve outcomes at 18 months of age.
|
{
"pile_set_name": "NIH ExPorter"
}
|
During T cell development, thymocytes that are able to recognize self MHC are signaled to mature (positive selection), whereas those capable of self reactivity die (negative selection). Many of our studies deal with these selection processes that result from interactions that occur between T cell receptors (TCR) on developing T cells with MHC on antigen presenting cells in the thymic microenvironment. Experiments have been performed in order to determine how and when T cells commit to the CD4 and CD8 lineages. Since CD4 and CD8 T cells arise from a common CD4+8+ precursor, coreceptor (CD4 or 8) expression in mature T cells appears to be linked to MHC class recognition by TCR. Experiments involving TCR and CD8 transgenic mice were used to test whether lineage commitment occurs by a stochastic process or is directed by the class of MHC that the TCR and coreceptor coengage during the course of thymic selection. Our studies show that at least some T cells can be selected on more than one MHC and imply that positive selection is a multi-step process involving a stochastic event that randomly modulates one of the coreceptors on CD4+8+ immature T cells. In other studies, we find that at least a few T cells can be rescued with an inappropriate coreceptor (inappropriate for the MHC specificity of the TCR on the same T cell) when the appropriate coreceptor is expressed as a transgene. Most of the results suggest that there is a stochastic component regulating coreceptor expression in T cell development; however, the data do not as yet establish whether this is a major pathway for the bulk of T cells and/or whether the stochastic process actually mediates lineage commitment. Collaborative studies with W. Leonard of NHLBI have established that IL-2Rgamma is expressed in the thymus. Studies are under way to explain how mutation of this gene could account for the failure to generate mature T cells that is observed in human severe combined immunodeficiency disease (SCID). Collaborative studies are in progress with F. Alt using terminal deoxynucleotidyl transferase (TdT)-deficient mice in order to establish whether T cells that lack N regions in their TCR variable regions undergo normal thymic development and selection and/or mediate normal antigen recognition.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Under the leadership of Dr Fine,the NOB Lab has collaborated with pharmaceutical companies and academic institutions,and the NCI Developmental Therapeutics Program in the preclinical and clinical development of a number of new anti-glioma agents.The first step in the development pipeline is screening of the agent through the ABTC.The ABTC provides the professional service for screening these agents both in vitro and in vivo using both standard subcutaneous and stereotactic intracranial models.Since 2005,a large number of anti-glioma agents have been screened.Of those,25 new agents showed significant enough promise to warrant extended evaluation through the ABTC.These extended studies involved stereotactic-based intracranial models looking at various dose and administration schedules as well as combination trials of the new drug with other agents.Furthermore,ABTC provides experimental and technical support to other investigators both within and outside of the NOB for evaluating newly developed therapeutics.For example, the role of stem cell factor (SCF) in glioma angiogenesis; Notch-1 in glioma cell survival and proliferation;Stathmin in the resistance of malignant gliomas to DNA alkylating agents in vivo. Systemic as well as neurotoxicity (behavior) is also monitored by routine animal screening.In addition,a number of newer drug delivery technologies including intracarotid administration, delivery with or without selective or gross blood-brain barrier disruption, convection delivery, etc.have been evaluated in animal models within the ABTC.For example, the ABTC in collaboration with the SNB of NINDS and in collaboration with the private sector, has used convection-enhanced drug delivery (CED) to directly administer various genetic vectors into the brains of immundeficient animals harboring human glioma xenografts.Many of the new classes of anti-tumor therapeutics will have cytostatic rather than cytotoxic properties.Evaluating which of these agents will have biologic activity in humans in small,early clinical trials is a challenge since the standard response criteria are based on the determination of cytotoxic responses.The only truly valid clinical parameter available for evaluating the activity of a truly cytostatic agent is patient survival or tumor progression-free survival.These,however,are not useful parameters for screening drug activity in small,early phase clinical trials.Thus,if surrogate markers of biologic activity could be identified,one could utilize these as early endpoints for screening out agents with little or no clinical activity.Toward that end,the ABTC is actively working to develop surrogate markers of drug anti-tumor activity that can be utilized and validated in clinical trials,which includes three major areas:1)Imaging;2)Gene expression profiling;3) Proteinomics/Serum markers. For example, in collaboration with investigators in NOB, NINDS and the Clinical Centers program of experimental imaging science,noninvasive MR imaging has been used to image magnetically labeled endothelial progenitor cells in vivo to directly identify vasculogenesis in a glioma model.The core has provided the technical support for this project which involved the MRI tracking of in vivo Ferumoxides-Protamine Sulfate (FE-PRO) complex-labeled endothelial progenitor cells incorporating into the vasculature of established intracranial mouse gliomas.The ABTC has also successfully generated the preclinical toxicology data required by the FDA for preparation of our IND for the clinical trial of using ferrodex-labeled endothelial progenitor cells as MRI trackable markers of angiogenesis in patients with gliomas.Additionally,we have collaborated with Dr. Robert Innis(NIMH)for attempting to adapt PET scanning into a monitoring system for real time imaging of drug permeability through the BBB and following the administration of inhibitors of the multiple drug resistance (MDR) protein.This work is being extended to use the ABTC tohelp evaluate novel PET ligands that bind to the peripheral benzodiazepine receptors (PBR) which is highly overexpresed in gliomas.A major effort of the core is to generate the RNA for gene expression profiles using microarray technology from given glioma cell lines treated with a specific class of agents.If characteristic patterns could be identified that correspond with anti-tumor activity,then clinical trials can/will be devised to administer one of these agents to patients with brain tumors immediately prior to biopsy/surgery in order to attempt and identify a similar genetic profile clinically.In collaboration with the NOB Lab and the GMDI team,gene expression signatures are being generated in all of glioma cell lines and GIC/GSCs for all compounds tested within the ABTC.Finally,the ABTC stores representative tumor, tissue and serum samples from animals treated with each new compound tested with the expectations that new candidate tissue and/or serum-based protein markers of drug activity, tumor activity and/or some tumor biological process(i.e. angiogenesis)may be found.This will be an invaluable preclinical resource for validating such claims in the future.A major effort of the NOB is to develop human glioma cell lines that more closely model primary human gliomas both biologically and molecularly.The ABTC is actively involved in the generation of primary human glioma cell lines and GIC/GSC lines from fresh surgical specimens for every glioma patient operated on at the NIH.The ABTC staff works closely with the cancer stem cell biologists in the Fine laboratory for the growth, propagation and characterization of each of these cell lines and animal xenografts. The ABTC uses these well-characterized cell lines as screens for two major categories of drugs;1)The most promising of the drugs that have made it through the first levels of in vitro and in vivo screens using the more conventional established glioma cell lines;2)drugs that target pathways that may not be well represented by the biology of standard glioma cell lines but are reproduced in the GIC/GSCs.The cores expertise with these cells, and the large resources of different GIC/GSC lines, are a potent enticement for potential partnerships between NCI and the pharmaceutical/biotechnology community given their growing appreciation of the limitation of standard cancer cell lines and the promise of cancer stem cells for better representing the human disease.Finally, given the hundreds of requests we receive each year for these valuable GIC/GSC lines,the ABTC serves a vital function as the group designated to expand,freeze and distribute various cell lines to investigators both within and outside of the NIH.In doing so,the staff of the ABTC spends a significant amount of time teaching other investigators from within and outside of the NIH how to grow GIC/GSCs and how to perform stereotactic implantation of tumor cells into mice and rats.Evidence of the success of the ABTC is the fact that we have activated 11 clinical trials as a direct result of translational work performed within the NOB,all of which had preclinical animal studies performed within the ABTC.Even more to the point,we have identified 12 compounds solely through the ABTC preclinical screening program that have since been brought forward to clinical trials at the NIH (AZD6918,RO4929097,AZD8005,MLN-518, ZD6474,LY317615,sunitinib,CC5013,Talampanel).The potential power of the ABTC is well documented by our demonstration of being able to take an agent sent to us for preclinical evaluation by one of our pharmaceutical collaborators and generate preclinical data supportive of clinical trials that resulted in NOB sponsored (two) phase I trials,(two)phase II trials and a NOB-chaired phase III worldwide randomized registration clinical trial; all the while discovering a novel mechanism of action of the drug (GSK3 inhibition).
|
{
"pile_set_name": "NIH ExPorter"
}
|
The applicants have been investigating associations between clinical depression and immunity. This research, which has been supported by the NIMH Small Grants Program, 1 R03 MH 37774-01MSMB, demonstrated that responses to T cell and T-dependent B cell mitogens as well as the absolute number of lymphocytes and T and B cells were significantly lower in drug free hospitalized patients with major depressive disorder than in apparently healthy matched controls. We also found that a group of less severely depressed outpatients with major depressive disorder had no changes in mitogen responses but did have a decrease in the number of lymphocytes and that hospitalized schizophrenics did not differ from matched controls on any of the immune measures. These findings suggest that depression is associated with altered immune function. The proposed research will investigate whether altered immunity is specific to the state of being depressed and if it is related to severity of the illness. Immune function will therefore be assessed in drug free patients with mild and severe depressive disorders compared with matched controls during depression and in clinical remission. Studies of alterations of immunity in depression may provide information about underlying neurobiological processes in depressive states. The immune changes may be related to other biological systems which can influence the lymphocyte and other immunocompetent and immunoregulatory cells. Neuroendocrine function and specifically cortisol secretion will therefore be investigated in relation to altered immunity in depressed patients. To further elucidate the biological processes which may link depression and the immune system, a comprehensive investigation of immunity will be undertaken, including: antigen as well as mitogen responses, T-independent B cell mitogen responses, numbers of T, T helper, T suppressor and B lymphocytes, natural killer cell function, and phagocyte activity. These studies may help to elucidate the pathophysiology of depressive states manifested in patterns of dysregulation of neurotransmitter, neuroendocrine and immune systems. The investigation of specific alterations in the immune system of depressives and their behavioral and neurobiological correlates may also provide a biologic basis for precise diagnosis of affective diseases as well as of risk factors. The findings may ultimately suggest strategies for intervention and treatment of depression.
|
{
"pile_set_name": "NIH ExPorter"
}
|
We have established and characterized 87 human lung cancer cell lines. Resected primary tumors and aspirates or biopsies of metastitic lesions were cultured in non-selective serum supplemented medium or in partially or fully defined selective media (HITES for small cell carcinoma, SCLC, and Rheinwald's or LA3 for non-SCLC cancers). Success rates were 62/92 (69%) for SCLS and 25/55 (45%) for non-SCLC. Cell lines were characterized by histology of xenografts in athymic nude mice, by DNA content analysis, and by cytological, biochemical and ultrastructural examination. Clonogenic and dye exclusion assays were used for in vitro drug sensitivity studies. SCLC lines, from untreated and previously treated patients consisted of classis lines (45) having typical morphology and neuroendocrine markers, or variant lines (17) having atypical morphology and selective loss of markers. Non-SCLC lines, from untreated patients, consisted of: adenocarcinomas (8), well or poorly differentiated; bronchioloalveolar (3) of Clara cell or type II pheumocyte varieties; squamous cell (3), moderately or poorly differentiated; large cell (3); adeno squamous (2); mesothelioma (3); and other (4), consisting of mucoepidermoid, carcinoid and oncocytoma. In most cases, the lines retained the morphological and biochemical features of the original tumors. Drug sensitivity assays demonstrated considerable differences in the relative sensitivity of the lines, and also in the effects of individual drugs. The cell lines demonstrate the great diversity of lung cancer types and they provide a comprehensive panel that is suitable for biological and drug sensitivity studies.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Current understanding of the cellular processes implicated in the maturation, homeostasis and repair of the human heart is extremely deficient and the need for basic information is striking. Findings in nematodes, fruit flies, zebra fish and rodents have often been translated to human beings with little caution, emphasizing the necessity to study the fundamental principles that regulate the plasticity of the myocardium during the lifespan of women and men. Moreover, the mechanisms modulating the response of the female and male heart to ischemic and non-ischemic myocardial injury and the principal factors conditioning end-stage heart failure and death in humans are at present unknown. This may explain why the astonishing advancements made in cardiac biology experimentally have had so far little impact on the management of the human disease. Thus, the major objectives of this application are: a) To define the contribution of human cardiac stem cells (hCSCs) to the physiological growth of the heart postnatally; b) To establish the rate of myocyte and non-myocyte turnover mediated by hCSC activation and differentiation, together with the analysis of the functional properties of myocytes, in the developing, adult, aging and failing heart; and c) To identify the role of hCSCs in the aging myopathy and heart failure to answer the question whether ventricular decompensation is a stem cell disease. To achieve these goals, we will employ five distinct protocols: a) Retrospective 14C birth dating of cardiac cells to establish the average age of myocytes and non-myocytes; b) A mathematical model of age- structured cell populations to define the age distribution of myocytes and non-myocytes; c) A mathematical model of hierarchically organized cells to assess the rate of formation of myocytes and non-myocytes by lineage commitment of hCSCs; d) hCSC and myocyte senescence by the expression of p53 and p16INK4a, and the accumulation of DNA damage and telomere dysfunction induced foci (TIFs) to determine the integrity of the telomere-telomerase axis; and e) The mechanical, electrical and calcium transient characteristics of myocytes to evaluate the effects of parenchymal cell physiology on ventricular hemodynamics. These five sets of complementary data will offer a novel comprehensive perspective of the cellular processes which govern the lifespan of the human heart. This information is critical for the recognition of the mechanisms that control the dynamics of the human heart, its reserve, adaptation to stress and failure. PUBLIC HEALTH RELEVANCE: The documentation that the human heart contains a population of stem cells that regulate myocardial growth in health and disease may have important clinical implications. Stem cell therapy may be prospected as a novel therapeutic strategy for the failing heart.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Two new projects on primate play, utilizing the squirrel monkey as a model, were begun this year. Data collection on both was completed in FY86. A. Social Environment Effects on Play. This study investigates possible differences in the frequency and/or style of play when young squirrel monkeys are experimentally restricted to particular social companions from whom they differ with respect to age, size, or sex. Our basic strategy is to allow subject youngsters the opportunity to play only with particular partners with known play profiles. This new approach promises to yield differences in play experience without producing the devastating effects of depriving animals of social contact with peers, a flaw in previous studies performed elsewhere that has made their results ambiguous at best. The primary objective of this study is to learn the effect of different play experiences on later social outcomes. The importance of reciprocity (role reversal) in sustaining mutually beneficial play relationships may be reflected in the later development of normal and disordered cognition and behavior. B. Vocalizations Used in Play. Squirrel monkeys are one of only a few species (including man) having a prominent and specific play vocalization, providing a good window for investigation of motivational changes occurring during play. Two main call types, one with four variants, were identified by sound spectographic analysis. Vocalization rate varied with the type of ongoing behavior and with play bout duration, an association considered to be indicative of motivation to play. Structural differences also varied with bout duration, in that longer play bouts were associated with longer and more complex calls. In addition to providing direct information about motivation, vocalizations may also carry a metacommunicative message to nearby adults that the play activity is relatively harmless.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The Clinical Research in ALS and related disorders for Therapy Development (CReATe) Consortium is a Rare Diseases Clinical Research Consortium (RDCRC) that forms part of the National Institutes of Health Rare Diseases Clinical Research Network (RDCRN). CReATe comprises a multi-disciplinary group of clinicians, scientists, educators, patient advocacy groups and other strategic partners, and aims to advance therapeutic development for patients with amyotrophic lateral sclerosis (ALS) and related disorders including progressive muscular atrophy (PMA), primary lateral sclerosis (PLS), frontotemporal dementia (FTD), multisystem proteinopathy (MSP), and hereditary spastic paraplegia (HSP). We recognize that the obstacles to therapeutic development are multifactorial and include: (a) etiological and biological heterogeneity; (b) phenotypic heterogeneity; (c) limitations of existing clinical outcome measures for use in early-to-mid phase clinical trials; (d) a paucity of biomarkers that have been validated as ?fit for purpose?); (e) the relatively late stage at which symptoms appear, diagnosis is made, and treatment is initiated; and (f) variable (often low) rates of patient participation in clinical research studies. CReATe investigators are engaged in a range of clinical trial readiness activities that aim to overcome these obstacles, and to thereby advance therapeutic development for this group of rare diseases. In addition, CReATe aims to promote and support collaborative research in the field of ALS and related disorders; develop and disseminate resources such as the CReATe Biorepository and our electronic health record-based ALS Toolkit that are of value to the broader scientific community; lower barriers to patient participation in research; cultivate and enhance the careers of young clinical investigators and translational scientists dedicated to the study of this group of rare diseases; and engage both lay- and scientific-community stakeholders in a partnership that fosters efforts to develop treatments for patients afflicted with these rare diseases.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Central pattern generators (CPGs) are rhythmically active neural networks that underlie many stereotyped and repetitive voluntary muscle movements such as whisking or fluid licking. Fluid licking in rodents is an example of a promising, simple model of a CPG: It is a highly stereotyped behavior characterized by rhythmic tongue and jaw movements, and it is thought to be controlled by a neural substrate distributed in the medullary reticular formation in the brainstem. In preliminary observations, we have demonstrated that mice are a superb species choice for investigating the genetic basis of the licking CPG- the common inbred strains C57BL/6J (B6) and DBA/2J (D2) have a robust, non-overlapping phenotypic difference in lick rate. The identification of genes underlying differences in lick rate should have a huge payout for the study of CPGs. Mice can then created with gene-targeted deletions or insertions, allowing for specific physiological or anatomical investigation. We propose to take advantage of a newly augmented resource for our genetic dissection of licking: The BXD advanced recombinant inbred (Rl) strain set. These mice will allow for precision genetic mapping of quantitative trait loci (QTL) that underlie behavioral traits. We propose to use a novel high-throughput assay for lick rate to test a set of -80 BXD Rl strains, and map quantitative trait loci (QTLs) with high precision that underlie strain variation in lick rate. Relevance: Complex animals such as humans possess a number of motor responses that are stereotypic and/or repetitive in nature. Comparison of the genes and mechanisms underlying stereotyped repetitive movements vs. plastic behaviors will be a source of information relevant for a number of human CMS disorders involving abnormal repetitive movements.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The proposed work for the immediate future will be on: (1) the effect of purified enzymes including lysosome enzymes on polymorphonuclear migration; (2) the possible release of Beta-lysin from platelets and lysozyme from neutrophils during delayed hypersensitivity reactions; (3) the source of Beta-lysin in the blood platelets; (4) the measurement of Beta-lysin concentration by use of an isothermal calorimeter.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Early changes in AD brain include loss of synapses. AB, considered to have a central role in the pathogenesis of AD, bind to dendritic spines and cause synaptic dysfunction. However, the mechanisms responsible for AB-induced synaptic dysfunction and spine loss are not firmly established. Notably, synaptic terminals have abundant mitochondria which play an indispensable role at these sites. Along this line, mitochondrial dysfunction is an early prominent feature of AD neurons. Mitochondria are dynamic organelles that undergo continual fission and fusion events which are regulated by a machinery involving large dynamin-related GTPase that exert opposing effects, e.g., dynamin-like protein 1 (DLP1) and Fis1 for fission, and mitofusins (Mfn1 and Mfn2C) and OPA1 for fusion. These mitochondria fission and fusion proteins control not only mitochondrial number and morphology but also mitochondrial distribution and function. Indeed, defects in the mitochondrial fission/fusion balance and thus, the morphology and distribution have the potential to cause localized energy and calcium imbalance, which is especially damaging to polarized cells such as neurons, resulting in cellular dysfunction and death. Our preliminary studies suggest that the normally strict regulation of mitochondria morphology and distribution is impaired in AD neurons and fibroblasts which may be caused by differential expression of mitochondrial fission/fusion proteins induced by AB. Our central hypothesis is that AB induces mitochondrial dysfunction and synaptic abnormalities via its toxic effect on mitochondrial fission/fusion. Four aims will be pursued: Aim1) ADDLs induce mitochondrial dysfunction and synaptic abnormalities via its toxic effect on mitochondrial fission/fusion in vitro; Aim2) To Explore the Mechanisms of ADDL-induced DLP1 Reduction; Aim3) mutant PS1 causes mitochondrial abnormalities and neuronal dysfunction at least in part through its interaction with DLP1 and impaired balance in mitochondrial fission/fusion; Aim 4) DLP1 reduction underlies mitochondrial abnormalities and synaptic loss in vivo. PUBLIC HEALTH RELEVANCE: AB-caused synaptic dysfunction and spine loss is an early change and the most robust correlate of AD- associated cognitive deficits, however the underlying mechanism is not firmly established. It is known that mitochondria play an indispensable role in synaptic terminals and the balance of mitochondrial fission/fusion is critical for mitochondrial distribution and function. Our preliminary studies suggest the potential involvement of an impaired balance of mitochondrial fission/fusion in the pathogenesis of AD, in this application, we propose to investigate whether AB cause synaptic dysfunction and mitochondrial abnormalities via its toxic effect on the balance of mitochondrial fission and fusion.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.