The long-term objective of the proposed research is to test the hypothesis that in systemic lupus erythematosus (SLE) there is an increase in the antigen driven autoimmune response through delayed clearance of DNA and nuclear-associated proteins due to an abnormally low C-reactive protein (CRP) response. This hypothesis is based on observations that in the SLE the CRP response during inflammatory episodes related to disease is markedly diminished compared to the degree of inflammation and tissue damage present. The mechanisms responsible for this reduced CRP response are not known. In addition, CRP has been shown to localize in the nuclei of damaged cells and to bind to chromatin. Given the inappropriate CRP-response and the characteristic antibody response to DNA and nuclear proteins in SLE, we, and others, hypothesize that CRP reacts with DNA associated components in damaged tissue and aids in their removal through interactions with the complement system and cells of the phagocytic system. We propose to use human CRP-transgenic (NZB X NZW)F1 mice to test our hypothesis. (NZB X NZW) F1 mice have been utilized as a model for SLE and display a well characterized anti-DNA and anti-nuclear antigen response. Therefore, transgenic (NZB X NZW)F1 mice which have the human CRP gene should be an exceptional tool for studying the effects of CRP on the development of anti-DNA and anti-nuclear antibodies in SLE. Specifically, we plan to determine the effects of human CRP on the development of the lupus like disease of (NZB X NZW)F1 autoimmune mice. We will construct transgenic CB6 mice which express human CRP in an acute phase manner, and then transmit the human CRP on the development of the lupus like disease of (NZB X NZW)F1 autoimmune mice. We will construct transgenic CB6 mice which express human CRP in an acute phase manner, and then transmit the human CRP gene to autoimmune mice through selective breeding. If there is a disease associated CRP response, the development of disease in the CRP transgenic (NZB X NZW)F1 mice will be compared to (NZB X NZW)F1 mice. If there is no significant, disease associated-CRP response in the (NZB X NZW)f1 transgenic mice, we will determine whether these mice have normal CRP expression in response to other stimuli and if the low response is due to some disease related phenomenon. In addition, we will examine the effect of constant expression of CRP on the development of disease in (NZW X NZW)F1 transgenic mice by introducing into the mice DNA constructs of CRP genomic DNA and the mouse metallothionein I enhancer/promoter sequences. The data from the proposed experiments will be an important addition to our knowledge of CRP's role in the development of the autoimmune response, as well as the regulation of CRP's expression in SLE. Finally, these studies will also provide valuable information on regulation of CRP expression within a normal background.


The development of ventricular fibrillation (VF) and sudden cardiac death include several key steps, such as initiation of reentry, degeneration to multiple reentrant waves, and maintenance of VF. issue heterogeneity has been traditionally considered as the major cause for these key steps leading to VF. However, recent studies indicate that dynamic wave instability operates synergistically with pre-existing tissue heterogeneity to promote wavebreak. Dynamic wave stability is regulated by multiple factors, including electrical restitution, intracellular Ca (Cai) cycling, cardiac memory, and electrotonic currents. The goal of this project is to use computer simulations with simplified models to developed theories which identify the critical parameters controlling the development of VF, as the theoretical basis for novel therapeutic strategies. The central task of this project is to investigate how voltage, Cai, and tissue heterogeneity interact to regulate vulnerability to rentry and maintenace of VF. The first aim is to develop simplified models to investigate the nonlinear dynamics of Cai cycling coupled to membrane voltage, in order to understand dynamical mechanisms of cardiac alternans. The second aim is to use physiologically-detailed AP models in homogeneous tissues to determine the physiological mechanisms by which voltage-Cai dynamics promotes spatially discordant alternans, dispersion of refractoriness, wave instability and arrhythmogenesis. The third aim is to determine how voltage-Cai dynamics interacts with tissue heterogeneities to regulate spatially discordant alternans, dispersion of refractoriness, wave instability and arrhythmogenesis. This project will develop general theories which will then be validated in more realistic settings in Projects 1, 3, and 4. The approach in this project will allow us to extensively explore the parameter space and systematically investigate the mechanisms of VF initiation and maintenance to identify the critical parameters essential for developing novel therapeutic strategies.


Mitochondrial diseases are devastating disorders for which there is no cure and no proven treatment. About 1 in 2000 individuals are at risk of developing a mitochondrial disease sometime in their lifetime. Half of those affected are children who show symptoms before age 5 and approximately 80% of these will die before age 20. The human suffering imposed by mitochondrial and metabolic diseases is enormous, yet much work is needed to understand the genetic and environmental causes of these diseases. Mitochondrial genetic diseases are characterized by alterations in the mitochondrial genome, as point mutations, deletions, rearrangements, or depletion of the mitochondrial DNA (mtDNA). The mutation rate of the mitochondrial genome is 10-20 times greater than of nuclear DNA, and mtDNA is more prone to oxidative damage than is nuclear DNA. Mutations in human mtDNA cause premature aging, severe neuromuscular pathologies and maternally inherited metabolic diseases, and influence apoptosis. The primary goal of this project is to understand the contribution of the replication apparatus in the production and prevention of mutations in mtDNA. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we have focused this project on understanding the role of the human pol gamma in mtDNA mutagenesis. Human mitochondrial DNA is replicated by the two-subunit gamma, composed of a 140 kDa subunit containing catalytic activity and a 55 kDa accessory subunit. The catalytic subunit contains DNA polymerase activity, 3'-5'exonuclease proofreading activity, and 5'dRP lyase activity required for base excision repair. As the only DNA polymerase in animal cell mitochondria, pol gamma participates in DNA replication and DNA repair. The 140 kDa catalytic subunit for pol gamma is encoded by the nuclear POLG gene. To date there are over 150 pathogenic mutations in POLG that cause a wide spectrum of disease including Progressive external ophthalmoplegia (PEO), parkinsonism, premature menopause, Alpers syndrome, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) or sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO). Presently, there are over 150 pathogenic disease mutations in the POLG gene that cause PEO, ataxia-neuropathy and Alpers syndrome. We described a novel POLG missense mutation (c.3218C>T;p.P1073L) that, in association with 2 previously described mutations, caused an Alpers-like hepatocerebral syndrome in 4 children. Four children, 2 related and 2 unrelated, with the novel p.P1073L mutation (all patients) and either the p.A467T (2 patients), p.G848S (1 patient), or p.W748S (1 patient) mutation presented with psychomotor delay, encephalopathy, and liver failure. Detailed clinical and laboratory examinations including brain magnetic resonance imaging, muscle biopsy, measurement of mitochondrial DNA, and sequencing of the POLG gene. All 4 patients had psychomotor delay, seizures, and liver disease. Three patients had severe gastrointestinal dysmotility, which may be associated with the new p.P1073L mutation. The heterozygous presence of the novel p.P1073L mutation in trans with another recessive POLG mutation causes a hepatocerebral disorder identical or very similar to Alpers syndrome. This adds to the already striking clinical heterogeneity of POLG mutations. In the Belgian patients, the familial occurrence without consanguinity is related to the high frequency of the recessive p.A467T and p.W748S mutations in northwestern Europe and reveals a pitfall for diagnosis and genetic counseling. The severity and dominance of many POLG disease-associated mutations are unclear, because they have been reported in sporadic cases. To understand the consequences of pol gamma disease-associated mutations in vivo, we identified dominant and recessive changes in mtDNA mutagenesis, depletion and mitochondrial dysfunction caused by 31 mutations in the conserved regions of the gene, MIP1, which encodes the Saccharomyces cerevisiae ortholog of human pol gamma. Twenty mip1 mutant enzymes were shown to disrupt mtDNA replication and may be sufficient to cause disease. Previously uncharacterized sporadic mutations, Q308H, R807C, G1076V, R1096H and S1104C, caused decreased polymerase activity leading to mtDNA depletion and mitochondrial dysfunction. We present evidence showing a limited role of point mutagenesis by these POLG mutations in mitochondrial dysfunction and disease progression. Instead, most mitochondrial defective mip1 mutants displayed reduced or depleted mtDNA. We also determined that the severity of the phenotype of the mip1 mutant strain correlates with the age of onset of disease associated with the human ortholog. Finally, we demonstrated that increasing nucleotide pools by overexpression of ribonucleotide reductase (RNR1) suppressed mtDNA replication defects caused by several dominant mip1 mutations, and the orthologous human mutations revealed severe nucleotide binding defects. Defects in mtDNA replication are the principle cause of severe, heritable metabolic disorders classified as mitochondrial diseases. In vitro analysis of the biochemical mechanisms of mtDNA replication has proven to be a powerful tool for understanding the origins of mitochondrial disease. Mitochondrial single-stranded DNA-binding protein (mtSSB) is an essential component of the mtDNA replication machinery. To facilitate ongoing biochemical studies, a recombinant source of mtSSB is needed to avoid the time and expense of human tissue culture. This chapter focuses on the subcloning, purification, and initial functional validation of the recombinant human mitochondrial single-stranded DNA-binding protein. The cDNA encoding the mature form of the human mtSSB protein was amplified from a HeLa cDNA library, and recombinant human mtSSB was overproduced in Escherichia coli. A procedure was developed to rapidly purify milligram quantities of homogenous, nuclease-free mtSSB that avoids DNA-cellulose chromatography. We show that, similar to E. coli SSB, human mtSSB assembles into a tetramer and binds single-stranded oligonucleotides in a 4-to-1 protein:oligonucleotide molar ratio. Missense mutations in the human C10orf2 gene, encoding the mitochondrial DNA (mtDNA) helicase, co-segregate with mitochondrial diseases such as adult-onset progressive external ophthalmoplegia (PEO), hepatocerebral syndrome with mtDNA depletion syndrome (MDS), and infantile-onset spinocerebellar ataxia (IOSCA). To understand the biochemical consequences of C10orf2 mutations, we overproduced wild type and 20 mutant forms of human mtDNA helicase in Escherichia coli and developed novel schemes to purify the recombinant enzymes to near homogeneity. A combination of molecular crowding, non-ionic detergents, Mg(2+) ions, and elevated ionic strength were required to combat insolubility and intrinsic instability of certain mutant variants. A systematic biochemical assessment of the enzymes included analysis of DNA binding affinity, DNA helicase activity, the kinetics of nucleotide hydrolysis, and estimates of thermal stability. In contrast to other studies, we found that all 20 mutant variants retain helicase function under optimized in vitro conditions despite partial reductions in DNA binding affinity, nucleotide hydrolysis or thermal stability for some mutants. Such partial defects are consistent with the delayed presentation of mitochondrial diseases associated with mutation of C10orf2.


X-ray crystallography and cryo-electron microscopy are to be used for structural studies of enveloped viruses and their self-assembled cores. The work will concentrate on alpha- (Sindbis, Semliki Forest, Ross River, Aura), rubella and flavi- (hepatitis C and yellow fever) viruses. Rubella virus is a major problem to the human fetus in pregnant mothers. Hepatitis C virus infections are a worldwide problem, affecting as much as 20% of the population in some countries. Alphaviruses can sometimes cause fatal diseases such as encephalitis fever and arthritis. Currently, little structural information is available about enveloped viruses. Structural studies are likely to provide understanding of the mechanisms by which these positive-stranded RNA viruses disassemble on cell entry and assemble prior to cell exit. Many laboratories have attempted to crystallize alphaviruses, but only very poorly diffracting crystals have been produced. We suspect that the probable cause for the crystalline disorder may be the presence of heterogeneous carbohydrate moieties. Hence, site-directed mutations are being made (in collaboration with Richard Kuhn) to eliminate or reduce the number of potential glycosylation sites on the surface glycoproteins of Sindbis virus. The most difficult and time-consuming part of the work will be in sample preparation involving site-specific mutagenesis to deglycosylate the surface glycoproteins to obtain homogeneous samples of in vitro assembled cores. Although the electron microscopic, image reconstruction and crystallographic studies will take time and effort, the technologies are well established. We have attained good expression of Sindbis virus, Ross River virus, rubella virus and hepatitis C virus capsid protein in E. coli, although crystallization of the capsid protein has so far been achieved for only Sindbis virus. Mutational and structural studies of these proteins will be pursued similar to those previously used in the analysis of Sindbis virus. Self-assembled core-like particles using various nucleic acids, including genomic RNA and a DNA 48-mer, have been obtained for all the above mentioned capsid proteins. Cryo-electron microscopy studies and crystallization trials are underway. Reproducible small crystals (less than or equal to 0.05 mm diameter) of the in vitro assembled Sindibis cores can be produced routinely.


The human gut microbiota provide physiologic attributes that we have not had to evolve on our own, including the ability to process otherwise indigestible dietary polysaccharides. Bacteroides thetaiotaomicron (B. theta), a prominent member of the normal human distal gut microbiota, has an expanded capacity to process both dietary and host-derived polysaccharides, a feature that likely enhances its fitness in the crowded gut ecosystem. A fundamental question is how this prototypic gut symbiont recognizes and adapts to changing carbohydrate availability. The B. theta genome encodes 50 extra-cytoplasmic function sigma (ECF-?) factors, 26 are located adjacent to genes encoding anti-sigma (anti-?) factors, which are predicted transmembrane proteins with periplasmic sensor domains. Most ECF-? /anti-? pairs (25/26) co-localize with a third gene, encoding a SusC-like outer membrane porin. SusC paralogs are implicated in polysaccharide binding and are predicted to interact directly with anti-? factors, comprising a series of cell envelope- spanning switches that transduce external cues (SusC and anti-?) to effect transcriptional changes (ECF-?). My results from in vivo and in vitro experiments indicate that B. theta adapts its physiology to utilize host-derived mucopolysaccharides via an ECF-? /anti-? dependent mechanism. I have identified four prototypic loci (44 genes), each containing an ECF-?/anti-? switch and polysaccharide catabolic functions. I propose to probe the function of these 4 systems by investigating their mechanism(s) and specificity of signal transduction as well as their contribution B. theta fitness in the mouse intestine. Aim 1 will test a working model of ECF-?/anti-? signal transduction through genetic disruption of its predicted components and subsequent assay of function. Yeast 2-hybrid analysis will be used to probe predicted protein-protein interactions between signaling components and, because these signaling components are expanded in B. theta, the potential for cross-talk between paralogs. Aim 2 seeks to chemically define mucopolysaccharide components that trigger ECF-?/anti-? switches and, in conjunction with a bioassay for locus induction, will provide valuable insight into the chemical language through which B. theta perceives its environment. Aim 3 addresses the hypothesis that mucopolysaccharide utilization by B. theta enhances in vivo fitness. The ability of B. theta to turn on these systems will be eliminated through genetic manipulations, and the colonization behavior of mutants evaluated in competition with wild-type B. theta in gnotobiotic mice. These studies will expand our understanding of how bacteria inhabiting the gut can shift their metabolism to coincide with changing availability of carbohydrate resources in the intestine. [unreadable] [unreadable] [unreadable]


Dithiobiuret (DTB), H2N-C-NH-C-NH2, is used in the synthesis of insecticides, rodenticides and resins and as a plasticizer and rubber accelerator. There is a paucity of studies but Astwood et al. (Science 102:196, 1945) reported skeletal muscle paralysis in rats after several days of treatment. The paralysis started in the hindlimbs and then rapidly ascended to involve forelimbs and head and neck muscles. If treatment was continued the animals died apparently of respiratory paralysis. The gross toxicologic effects appear similar to environmental agents that produce neuropathy and myopathy. Acrylamide produces muscle weakness by peripheral nerve damage and triorthocresyl phosphate through damage to motoneurons. Leptophos causes CNS-PNS distal axonopathy and paraboxon treatment myopathy. Thus, it is of interest to see if weakness produced by DTB is due to nerve and/or muscle damage. The objectives are to describe experimental conditions under which DTB treatment produces paralysis in rats and cats, see what tissues or organs are involved, attempt to determine its mechanism of action and develop antidotes or antagonists which in turn may give insight into its mechanism of action. Muscle weakness in the whole animal will be evaluated by a treadmill test, brain function by direct and indirect methods, spinal cord function by assessing spinal reflexes and neuromuscular function by nerve-muscle studies. Histological assessment of nerve and muscle tissue will be included. Metabolic activation will be assessed by seeing if a relationship exists between activity of the hepatic MFO system and delayed toxic effects. An indirect benefit may be establishment of DTB-treated animals as a model for the muscle weakness seen in either a myasthenic disease, myotonic muscular dystrophy or acute intermittent porphyria neuropathy in man.


Human studies show that cocaine dependence affects the microstructure of white matter, probably as a result of vasoconstrictive effects of the drug. Little is known, however, about the temporal development of the white matter injury and the changes in cerebral vasculature during the dependence period, or about the recovery of both with cessation of the drug use. Magnetic resonance imaging (MRI) provides tools that are highly suitable for investigating in a non-invasive manner the anatomical, structural and functional and chemical characteristics of the brain. In this application we propose to implement a set of MRI techniques for investigating the effects of the use of cocaine on monkey brain, in particular its effects on white matter microstructure on gray/white matter volumes, on the vascularity and perfusion of gray and white matter and on the neurochemical profile of the monkey brain. These techniques will be used to perform a preliminary study on a small and well-controlled population of monkeys. Aim 1. To develop a robust methodology for investigating structural, anatomical and functional measures in monkey brains. Aim 2. To collect preliminary data on the effects of drug use and its cessation on monkey brain. We hypothesize that: 1. Long term cocaine self-administration will result in brain changes in the monkeys similar to those seen in human studies. 2. Cessation of drug use will result in a normalization of brain measures. This will address the important issue of whether or not there is significant brain recovery following cessation of drug use.


The purpose of this study is to treat patients with metastatic brain tumors using Gadolinium Texaphyrin (PCI-0120) injection two to five hours before radiation treatments for ten consecutive days (excluding weekends and holidays); to establish an appropriate intravenous dose of Gadolinium Texaphyrin (PCI-0120) Injection; to determine the maximum tolerated total dose (MTD) of Gadolinium Texaphyrin (PCI-0120) Injection for palliative radiation therapy of metastases to the brain; to evaluate an additional 20 patients at the MTD to establish the safety profile of Gadolinium Texaphyrin (PCI-0120) Injection; to evaluate accumulation of Gadolinium Texaphyrin (PCI-0120) Injection in metastases and changes in tumor volume (i.e., response to treatment); to determine the dose limiting toxicity of repetitive doses of Gadolinium Texaphyrin (PCI-0120) Injection, where each dose is followed by a radiation treatment, in patients with metastatic cancer to the brain; to determine the biolocalization of Gadolinium Texaphyrin (PCI-0120) Injection in cancer (e.g., brain metastases) and normal tissue(s) (e.g., brain tissue), and the change in cancer volume, using MRI and the high (white) signal intensity Gadolinium Texaphyrin (PCI-0120) Injection produces on MRI scans; and to determine the in vivo pharmacokinetics of Gadolinium Texaphyrin (PCI-0120) injection in patients treated at the MTD.


Cellular senescence limits the proliferation of human cells and can be induced by a variety of cellular alterations, both intrinsic and extrinsic. Senescent cells accumulate in human tissues, including the prostate, with increasing age. These senescent cells have altered function, including increased expression of proinflammatory cytokines that can alter the function of adjacent cells. Benign prostatic hyperplasia (BPH) is the single most common pathology of aging men. Based on our published studies we hypothesize that senescence of a subset of epithelial cells in BPH tissue leads to release of cytokines and growth factors that, through direct and indirect actions, drives increased proliferation of adjacent non-senescent epithelial cells and stromal cells and ultimately prostatic tissue growth in aging men. We propose to characterize the mechanisms by which cellular senescence can promote the development of benign prostatic hyperplasia. Two Specific Aims are proposed. In Specific Aim 1, we will examine the underlying cellular alterations leading to prostatic epithelial senescence in vitro and in vivo;determine the types of cytokines and growth factors expressed by senescent epithelial cells in vitro;evaluate whether these same proteins are expressed at increased levels in BPH tissue in vivo and quantitatively evaluate the extent to which there is coexpression of these cytokines and growth factors at the cellular level in vivo with markers of senescence, including key cell cycle regulator proteins such as p21 and p16. In Specific Aim 2 we will use primary cultures of prostatic epithelial and stromal cells, the reactive stroma model system and transgenic models to examine the biological activities of the identified cytokines/growth factors and model potential autocrine and paracrine activities of those factors that are increased in BPH in vivo. In addition, we will establish a transgenic mouse model of epithelial senescence and examine the biological impact of epithelial senescence in this mouse model. Benign prostatic hyperplasia causes considerable morbidity in older men, with up to 30% of men requiring treatment for this condition, and with more than one billion dollars spent on the medical and surgical treatment of this disease annually. These studies will make a fundamental contribution to our understanding of the role of cellular senescence in the pathogenesis of this common disease and lead to more effective preventive treatments and medical therapies. PUBLIC HEALTH RELEVANCE: Benign prostatic hyperplasia causes considerable morbidity in older men by blocking the urinary tract and up to 30% of men will require treatment for this condition at some time in their lives, with more than one billion dollars spent on the medical and surgical treatment of this disease annually. We believe these studies will make a fundamental contribution to our understanding the causes of this common disease and by doing so lead to more effective preventive treatments and medical therapies.


Spinal muscular atrophy (SMA) is characterized by loss of motor neurons and atrophy of muscle. Proximal SMA is the second most common genetic cause of infant death. As in many neurogenetic disorders, the mutated protein SMN is ubiquitously expressed, yet only a particular type of neuron is affected. SMA is caused by loss of the SMN1 gene and retention of the SMN2 gene, leading to low levels of wild-type SMN, which is insufficient for motor neuron survival. Increasing SMN levels by increasing SMN2 copy number modulates the severity of the phenotype. In recent years the investigators, and others, have identified drug compounds that can induce SMN2 to produce more SMN protein. In addition, when the drug is given prior to motor neuron loss certain compounds can modify the SMA mouse phenotype. It has now become possible to perform newborn screening for SMA. However key questions remain concerning optimal deployment of therapeutics in SMA, including compounds that activate SMN2, oligonucleotides that stimulate incorporation of SMN exon7 by SMN2, or gene therapy. In this application the investigators will define the spatial and temporal requirement for high levels of SMN to correct SMA. Additionally, they will study modifiers of SMA and the effectiveness of combination drug treatments. These are essential components in order to optimize treatment regimes for SMA. In this application four aims are proposed using SMA mice. 1) Determine the importance of high levels of SMN in tissues other than neurons or motor neurons by using mouse lines that selectively create or correct the SMA condition in neurons or motor neurons. This will address if there is any benefit in SMA animals of increasing SMN levels outside the nervous system. 2) The temporal requirement for high levels of SMN to correct SMA will be determined using a SMN inducible system. This will allow determination of when SMN must be increased in SMA and whether induction of SMN must be continuous or just during a specific window of time. 3) Study the role of phosphorylation in SMN's ability to correct SMA. Additionally, they will determine if combinations of drug increase survival in SMA mice. The activity of current drugs must be increased in order to have a major impact in the SMA mouse. Combinations of drugs that activate SMN, as well as alteration of the phosphorylation state of SMN, may significantly impact the SMA phenotype. 4) Lastly, a modifier of the SMA phenotype, plastin3, has been reported. The investigators will determine if plastin3 is truly a modifier of SMA by asking whether increased plastin3 expression can correct SMA mice. The aims proposed here will indicate the temporal spatial requirement for SMN induction, identify the optimal compound combination for induction of SMN, and indicate the importance of a reported modifier of SMA in mice. This will result in optimization of therapeutics for SMA.


In this project, we will investigate the biomechanics that enable high-frequency (JHF) sound, greater than 10 kHz, to be transduced from middle-ear to cochlea in a group of terrestrial mammals. Anatomical studies indicate that in larger mammals, the malleus may-assume a twisting mode of vibration, rather than the classical hinging mode. Twisting would transfer sound energy more efficiently at HF because relative mass inertia is greatly decreased. At lower frequencies, the hinging mode is utilized due to the stiffness of the connecting ligaments and tendons. For smaller mammals, the impedance due to mass is not prohibitive at HF, so a twisting mode is not necessary. Accordingly, features of their anatomy seem to preclude twisting. ~ Here we propose an interspecies (human, cat, chincilla, and gerbil) investigation of the hypothetical twisting mode using physiologic measurement, morphologic observation and mathematical analysis. ~ SA1: Empirically determine impedance as a function of frequency for the hinging and twisting vibrational modes by inducing these motions through the malleus and measuring the cochlear output. Two magnetic voice coil motors will drive the anterior and posterior quadrants of the eardrum with independent phase. This causes hinging and twisting with equal and opposite phase respectively. A novel cochlea preparation that introduces a 'third window" will be created to measure the spiral ligament, which moves with stapes pressure, by laser Doppler. This gives a relative indication of cochlear output. SA2: Morphometrically quantify the primary anatomical structures of the eardrum and ossicles that could contribute to a twisting vibrational mode by multimodal imaging. Middle-ear morphometry will be Imaged by micro-CT. Collagen fiber structure at select areas in the eardrum will be imaged by multiphoton and transmission electron microscopy. Image analysis software will be used to obtain quantitative data from these Images. SA3: Analytically deduce the conditions required for a twisting vibrational mode by developing simple and finite element models of the eardrum and ossicles that can incorporate the methods and data acquired from SA1 and SA2. A simple model for the hinging and twisting modes will be formulated based on micro-CT data. This will guide the construction of a finite element model that can incorporate data from the other specific aims. Assessment of the parameters derived from the four species studied will allow us to formulate the fundamental physical and physiological basis for a twisting mode. ~ This research is relevant to current medicine because an improved understanding of eardrum and ossicle biomechanics is required to develop more effective technologies related to myringoplasty. This may be particularly important for post-surgical HF hearing preservation.


The Sickle Cell Scholar training program is an integral part of the education component of the Comprehensive Sickle Cell Center grant. The Sickle Cell Scholar will be chosen according to the guidelines in the RFA. Under the auspices of the Los Angeles Sickle Cell Center, the scholar will have the opportunity to do clinical and translational research related to the efficacy of L-glutamine in ameliorating the symptoms of sickle cell disease, the genesis of cardiac dysfunction in transfused sickle cell patients, the health services research related to provision of adult care, or translational research related to the role of viscosity in alteration of blood flow and oxygen delivery in humans and the use of engineering techniques to detect evidence of sickling. Unique to this program, the scholar will be supervised by the Center education director who is a cell biologist and doctoral level educator. This individual will be responsible for setting educational milestones and systematically monitoring the progress of the scholar to the training program. Also described here is a plan for the management of the summer high school student program that will also be run by the Center education director.


The search for the origins of diversity in primary vestibular afferent response dynamics has been a dominant theme of vestibular research and is the major subject of the present proposal. The long-term goal of the work is to study the role of transmitter systems in shaping afferent responses, and the immediate objective is to identify the impact of GABA and glutamate as hair cell transmitters. The central hypothesis of the research is that the crista performs a mathematical differentiation (differential calculus) of some velocity-sensitive input signals in processing convergent excitatory (glutamatergic) and inhibitory (GABAergic) hair cell synapses onto dendrites of single afferent neurons and that this push-pull input accounts, at least in part, for the wide range of observed afferent responses. Three specific aims will be pursued. These aims embrace a multidisciplinary approach utilizing techniques and experimental models selected for their particular suitability for the experiments, the research experience of the investigators, and the applicability of the results to understanding human vestibular disorders. Anatomical, physiological, pharmacological and mathematical tools are employed concomitantly to address the aims and test hypotheses from multiple perspectives. The first aim is to relate the chemical anatomy of the hair cells in the horizontal canal crista ampullaris to primary afferent response dynamics. The second aim is to identify the relationship between hair cell transmitter phenotype, hair cell morphology, and hair cell activation and response kinetics. The third specific aim is to evaluate the effects of pharmacological manipulation of GABA transmission on the horizontal canal nerve and on individual afferent fibers. The role of transmitter phenotype in shaping primary afferent activity is a fundamentally and clinically important aspect of vestibular function that is poorly understood. Most studies will be conducted in toadfish, which are experimentally advantageous because of their availability, the ease of exposing and extracting their labyrinths, and the similarity of vestibular endorgans throughout the vertebrate phyla. Additional studies in mouse are proposed in order to relate findings in toadfish to a mammalian vestibular system, thus establishing the generality and human health relevance of the results. The proposed research is unique, and will have direct bearing on the development of more effective therapeutic interventions, both medicinal and device-oriented, for peripheral vestibular disorders. [unreadable] [unreadable] [unreadable]


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. During the reporting period, we have completed the characterization of the prairie vole BAC library and published a paper describing the library. Using end sequencing of random BAC's we have identified more than 2000 SNPs in more than 300 loci. In addition, we have isolated BAC clones containing the genes for 20 behaviorally relevant genes (e.g. oxytocin, vasopress, CRF and their receptors, steroid receptor, etc), and a manuscript is in preparation describing these loci. Using the SNPs and a pedigree with more than 300 individual prairie voles we have created a first generation linkage map of the prairie vole genome. This map will be useful in assembling the prairie vole genome. The Broad Institute is currently sequencing the prairie vole genome.


Quantifying Environmental Variables Affecting Airborne Influenza Transmission In most temperate climates, influenza prevails in cold, dry winter months. However, in some temperate and tropical regions, influenza epidemicity is correlated with extremes of precipitation, not dryness, and can circulate at low levels essentially year-round or appear in uni- or bi-modal annual outbreaks. How environmental variables affect influenza circulation in the human population remains poorly understood, in part because the science behind airborne respiratory virus transmission crosses disciplinary boundaries between the biomedical and physical sciences, encompassing fields as diverse as virology, physiology, epidemiology, fluid mechanics, aerosol science, and climatology. Here we seek to understand how individual environmental variables - such as temperature, humidity, and airflow - cumulatively affect the transmission probability of influenza viruses in a representative mammalian experimental system. The theoretical framework behind these studies is a novel quantitative model, based upon data gathered in experimental guinea pigs, which attempts to characterize the impact of the environment on influenza virus transmission between infected and susceptible hosts. This project bridges the gap between virology and engineering in bringing together three co- investigators with relevant and complementary skill sets: Dr. Nicole Bouvier, a physician-scientist with extensive experience in the transmission of influenza viruses among guinea pigs; Dr. William Ristenpart, an engineer with expertise in the application of high-speed imaging technologies to investigations of complex fluid dynamics; and Dr. Anthony Wexler, an authority on aerosol transport who has developed novel techniques for high-resolution imaging of aerosol deposition in the rodent respiratory tract. Our preliminary theoretical modeling has generated innovative interpretations of the experimental data, yielding three testable hypotheses, which form the basis of this proposal: (1) Influenza virus transmission probability will decrease with increased airflow speed, (2) transmission probability will decrease with the degree of turbulence, and (3) transmission probability will increase with the time integral of the viral concentration within the inoculated animal. Rigorously controlled laboratory studies, designed to isolate a single variable for analysis while others are held constant, will provide a quantitative framework for understanding the cumulative effects of temperature, humidity, airflow velocity, turbulence, and position on the transmission of human influenza viruses in a relevant animal model. Quantifying these environmental variables, individually and cumulatively, will enable their extrapolation to larger environments and time scales, with the potential to transform our understanding of the epidemiology of seasonal and pandemic influenza.


Enabling diagnostic technologies that merge the use of nanoscale components and miniaturized detector systems could have a great impact in point-of -care diagnostics. In particular, diagnostic systems that are sensitive, robust, portable, low-cost, and can accurately quantify multiple molecular targets in parallel, would have a tremendous impact in biomedical research and molecular diagnostics. In this application, we propose to use a magnetic nanosensor technology, in conjunction with a portable magnetic relaxometer, to develop a sensitive diagnostic test for intracellular pathogens and toxins in clinical samples. Bacterial infections are on the rise in the United States and their economic impact on the healthcare sector is significant. The presence of enterohemorrhagic E. coli O157:H7 in tainted produce, the B. anthracis attacks in 2001, and the recent identification of drug resistant Mycobacterium tuberculosis have sparked public concern and underscore the need to develop sensitive and portable diagnostic technologies for fast and accurate detection of bacteria and toxins in food and clinical samples. Our approach utilizes "magnetic relaxation nanoswitches" (MRnS), i.e. magnetic nanoparticles that selectively change the spin-spin relaxation times (T2) of surrounding water molecules (NMR signal) upon specific molecular target interaction. The principal investigator of this grant application has previously shown that this technology can detect oligonucleotides in the femtomolemole range with extremely high molecular specificity [Nature Biotech. 2002;20:816-820], as well as other molecular targets such as proteins and viruses, without the need of target amplification. Most recently, we have developed magnetic nanosensors for the detection of a particular bacterium in blood and milk [Nanoletters 2007; 7, 380]. Also, with collaborators at the Army's Edgewood Chemical and Biological Center, we have gathered preliminary data to detect ricin toxin, using magnetic nanosensors. As a detector, we have used a newly developed minituarized and portable NMR relaxometer that allows substantial improvement in detection threshold and speed. The overall goal of this application is to extend research on magnetic nanosensors in an effort to develop highly sensitive, minituarized and portable detection systems to screen for the presence of a particular bacterium and toxin in clinical samples. We hypothesize that our magnetic nanoparticle system can provide a single homogeneous assay that can be used to detect the presence of intracellular pathogens and toxins in blood, without the need for target amplification. As an intracellular bacterial pathogen model, we will use Mycobacterium avium spp. paratuberculosis (MAP). As a toxin model, we will use anthrax toxin (AT), which is produced by another intracellular pathogen; B. anthracis. PUBLIC HEALTH RELEVANCE In this project, we will develop a diagnostic technology that merges the use of magnetic nanoparticles and a miniaturized detector to monitor the presence of an intracellular pathogen and toxin in clinical samples. This technology could facilitate quick, selective and sensitive bacterial detection to support clinical decision making. [unreadable] [unreadable] [unreadable]


Microarray-based information, now routine in most medical research communities, pervades most aspects of diagnostics, sensing, biotechnology, oncology and pathology. Detection limits and selectivities for DNA targets are far below theoretical performance limits, but very little information is reported or known about the chemical, physical or biological fate of full length DNA, cDNA or oligo-DNA probes immobilized on any of the diverse set of microarray surfaces. How immobilized DNA surface disposition influences subsequent hybridization efficiency, and reliability of array data interpretation and assay quantification is unknown. Quantitative interpretation of DNA microarray signal intensity is currently very difficult since factors influencing DNA probe-target interactions at microarray surfaces have not been analyzed with high-resolution surface analytical methods often applied to other biomedical surface problems. Our hypothesis is that DNA microarray target hybridization efficiency and diagnostic target detection limits in biomedical samples are correlated directly with the orientation, density, and immobilization efficiency of probe DNA on microarray surfaces. To investigate this hypothesis, we propose the following Specific Aims: 1. Establish a quantitative understanding of the correlation between immobilized probe DNA density on microarray surfaces and target hybridization efficiency in biological samples using radiometric 32P-DNA assay and optical imaging on several surface chemistry platforms and assess reliability and reproducibility issues in these strategies; 2. Develop reliable, quantitative methods for high-resolution surface analysis of DNA density and orientational populations on ss-cDNA and hybridized ds-DNA on arraying surfaces using modem biomedically relevant methods (XPS, ToF-SIMS and optical anisotropy of immobilized DNA). The overall objective is to correlate high-resolution surface analytical data on DNA arrays with radiometric measurements to establish a non-radiometric 'standard curve' to assess DNA immobilization on microarray surfaces more conveniently and accurately. Moreover, orientational information on immobilized DNA using a combination of innovative spectroscopy methods will be correlated to immobilized DNA density and hybridization efficiency in array formats. All methods will converge to produce a fundamental understanding of microarray surface & hybridization performance limitations currently not available.


Cocaine addiction continues to be a major health and social problem in the United States and other countries. Currently employed pharmacological agents for treating cocaine abuse have proved inadequate, leaving few treatment options. One alternative is to use protein-based therapeutics that bind cocaine, thereby blocking its effects, and/or degrade cocaine via hydrolysis of the benzoyl ester. This approach is especially attractive because the therapeutic agents exert no pharmacodynamic action of their own and therefore have little potential for side effects. The effectiveness of these agents, however, is limited by their inability to act directly within the central nervous system (CNS). We seek to address this deficiency by engineering filamentous bacteriophage, which have been shown to penetrate the CNS when administered intranasally, to display cocaine-sequestering proteins on its surface. Specifically, the aims of this proposal are: (1) construction of phagemid vectors that contain the genes of different cocaine-inactivating proteins as fusions with genes of phage coat proteins; (2) preparation and evaluation of phage displaying the various proteins of interest; (3) use of a locomotor activity rodent model to evaluate the ability of phage-displayed proteins to block the psychoactive effects of cocaine; (4) evaluation of phage distribution in the animals using immunohistochemical and molecular biological methods; (5) evaluation of a potential humoral immune response to the phage in the animals.


The overarching goal of this competitive revision application is to extend the mission of the University of Alabama at Birmingham (UAB)-University of California, San Diego (UCSD) O?Brien Center for Acute Kidney Injury (AKI) Research by defining the impact of AKI on the excess burden of chronic kidney disease (CKD) in the Southeastern US. The clustering of major CKD risk factors such as black race, obesity and diabetes in our region is a key driver of the high prevalence of CKD in the Southeastern US. As such, identifying potential interventions to reduce the incidence and progression of CKD in these vulnerable populations is critical for reducing the substantial social and financial costs associated with CKD and related health disparities (stroke, heart disease, death) in our region. AKI affects as many as 20% of hospitalized patients and is a recognized risk factor for the development of CKD, cardiovascular disease and death. These outcomes may be modifiable by care provided in the post-AKI period, yet optimum care for care of AKI survivors is not well defined and is often fragmented after discharge. Disadvantaged patients are at especially high risk for inappropriate follow-up and low quality of care, suggesting that disparities in AKI outcomes contribute to the excess risk of incident and progressive CKD in high-risk populations in our region. However, progress in this area is hampered by a lack of data on the degree to which differences in AKI outcomes contribute to CKD disparities in the Southeastern US, and potential interventions that may target these disparities. Accordingly, the primary focus of this application is to define the impact of AKI during acute hospitalization on incident and progressive CKD; how these associations differ by race, obesity and diabetes status; and pilot a dedicated AKI follow-up clinic as a potential intervention to address disparities in these outcomes. Specifically, Aim 1 will leverage an established, ongoing regional CTSA collaboration?the Southeastern Shared Health Research Information Network (SE-SHRINE)?to examine all hospitalized AKI patients within four centers (UAB, MUSC, UAMS, U Kentucky) and link them to the US Renal Data Systems to define disparities in AKI outcomes ([i] severity and duration of AKI; [ii] recovery of dialysis-dependent AKI or development of ESRD after discharge; and [iii] associated health disparities such as death and cardiovascular disease) by race, obesity and diabetes. Aim 2 will assess the feasibility and collect key efficacy measures of a dedicated AKI follow-up clinic for patients with AKI discharged to the community at three Southeastern CTSA hubs (UAB, Vanderbilt, U Kentucky). We will leverage the resources available in O?Brien Cores to integrate existing intellectual and technological resources of UAB and facilitate the success of this project. This project will help build a collaborative research network for AKI research that could serve as a platform for multi-center, transdisciplinary science and clinical trials focused on reducing the burden of CKD and related health disparities in the Southeastern US.


Institutional Racism is a process or set of policies which directly or indirectly produces differentially negative outcomes for minorities (Feagin, 1975). The result is an inequitable distribution of resources. Since the availability of resources and alternatives for problem solving is critical for coping with problems of living, Institutional Racism remains an important mental health concern with implications for individual, organizational, and community effectiveness. A workshop or convocation of scholars is planned to discuss and elaborate the unique perspectives and needs of Asian Americans, Afro-Americans, Latinos, and native Americans. In addition it will seek to explore current interventions for Institutional Racism and to exchange strategies of altering social systems in ways that make them more responsive to needs. Finally, following the workshop, a book will be published integrating the notions of Institutional Racism and Community Competence.


We intend to investigate and develop new, efficient and general synthetic methods for stereospecific and regiospecific carbon-carbon bond formation. The photochemistry and alkylation of vinyl sulfides, a potentially interesting, but generally overlooked, organic functionality, will be studied. The proposed photochemical ring annelation method, involving formation of a transient thiocarbonyl ylide followed by reaction with a dipolarophile could be a flexible and highly stereospecific technique for assemblage of a complex ring network. We hope to use this method for the construction of steroid and steroid-like molecules of potential biological interest. Oxidation of photogenerated dihydrothiophenes affords naphtho(2,1- b) thiophenes, derivatives of which have recently exhibited significant antimalarial activity. Regioselective alkylation of enolates derived from Beta- and gamma- keto-vinyl sulfides could be developed into a valuable synthetic method. Sulfur has been chosen as a temporary directing group because of its flexibility in recently reported synthetic operations. We hope to utilize these keto-vinyl sulfides as precursors to prostaglandins and prostaglandin-like intermediates. The undersigned agrees to accept responsibility for the scientific and technical conduct of the project and for provision of required progress reports if a grant is awarded as a result of this application.


DESCRIPTION: (Adapted from the application) Why do some people die at 60, more (in most developed countries) at 80, and a few at 100? Why is the chance of dying at 80 rather than 60 increasing and the chance of dying at 100 rapidly increasing (albeit from a very low level)? How important are genetic vs. environmental, behavioral, and medical factors in determining how long an individual will live? It might be expected that the answers to these questions--and the determinants of longevity more generally--are well understood. Surprisingly little, however, is known. The continuation of this program of demographic research on the oldest-old focuses on mechanisms and determinants of survival and longevity. The theoretical foundation that underlies the research and the conceptual framework that ties the various projects together are derived from the perspectives and methods of demography. The research program emphasizes demographic research on the genetic and non-genetic determinants of longevity, including research on the interaction between fertility and mortality and research on why age-specific mortality decelerates with age. Data from longitudinal surveys of thousands of elderly Danish twins and more than 10,000 Chinese oetogenanans, nonagenanans, and centenarians will be gaffiered and analyzed. Biodemographic experiments on the mechanisms and determinants of longevity will be conducted on several model species, inncluding Ceratifis capitam, Drosophila melanogaster, Melonoplus sanguinpes, Saccharomyces cerevisiae, and Plantago lanceolata. New demographic and statistical methods of survival analysis will be developed and applied to analyze these data. The data will be released as public-use files.


This proposal is designed to define the molecular basis of sickle cell adhesion to the endothelium. Vasoocclusion is believed cause the painful, reoccurring crisis suffered by sickle cell patients. Recent evidence suggests that this vasoocclusion may be precipitated by sickle cell adhesion to the endothelium. The molecules involved on the sickle cell surface, the endothelial cell surface, and any intermediary ligands have not been clearly identified. Our working hypothesis is that sickle cells and possible normal reticulocytes contain a receptor or receptors that bind to a ligand such as unusually large von Willebrand factor (ULvWF), which in turn binds to an endothelial cell receptor(s), precipitating the vasoocclusive crises in sickle cell disease. The specific aims of this grant are fourfold. First, we propose to identify and characterize the receptor(s) on the sickle cell surface that mediate adhesion to endothelial cells. This will involve a variety of approaches such as flow cytometry and sorting, immunoelectron microscopy, monoclonal antibody production, and adhesion assays. Second, we will identify and characterize the receptor(s) on the endothelial cell surface that mediate adhesion to sickle cells, using techniques similar to those listed above, and by testing specific receptor-deficient mutants in an endothelial-like cell line. Third, we will evaluate the role that ULvWF and other potential ligands play in the adhesion of sickle cells to endothelial cells. Finally, we will examine the contribution of activated platelets and platelet secretory products to the adhesion of sickle cells to endothelial cells. Completion of these studies should result in a greater understanding of the molecular events that contribute to the damaging, painful events in sickle cell disease and should provide a rationale for future treatment of this disease.


The overall goal of the proposed studies is to define the mechanisms by which MD-2, a co-receptor to TLR4 participates in LPS-induced signaling, to investigate the regulation of MD2 promoter and to characterize the expression, function and regulation of an alternatively spliced version of MD2 (MD2-S) that occurs in human but not mouse tissues. MD-2 is a secreted surface protein, which is required for LPS/TLR4 signaling. A fundamental gap in our understanding of LPS/TLR4/MD-2 signaling is the exact function of MD-2 and the molecular mechanisms, which make MD-2 a critical component of the LPS/TLR4 signaling complex. The regulatory mechanisms of the LPS/TLR4/MD-2-mediated inflammatory signaling at the level of MD-2 and the role for alternative splicing of human MD-2 in this regulation are also unknown. We have observed that an alternatively spliced version of MD-2 exists in human tissues. The studies proposed in this application will seek to characterize and define the functional role and the regulation of the alternatively spliced form of human MD-2 and will investigate the role of MD-2 as signaling molecule and the regulation of the human MD-2 promoter. The hypothesis: Based on our preliminary data, we hypothesize that altematively spliced isoform of MD-2 (short MD-2 or MD-2 S) is unable to transduce LPS/TLR4 signaling and that it may play a negative regulatory role in LSP/TLR4/MD2-induced inflammatory signaling at the level of MD-2. Based on preliminary data, we further hypothesize that MD-2 participates in LPS signaling, and is tyrosine phosphorylated upon LPS stimulation and internalization and that mutations of the tyrosine sites in MD-2 diminishes it's function to transduce LPS/TLR4 signaling. We propose that MD-2 may provide a potential regulatory step in TLR4/MD-2-mediated inflammatory signaling. We have recently cloned the promoter site of MD-2 and we propose to investigate the regulation of MD-2 expression by investigating the regulation of MD-2 promoter. Thus, MD-2 may provide an additional potential regulatory step in LPS/TLR4/MD2-induced inflammatory signaling. The Specific Aims are: 1- To characterize and determine the tissue expression and function of alternatively-spliced MD-2 isoform (MD-2 S) and expression of recombinant MD-2 S to investigate its function in LPS/TLR4 signaling in in-vitro and in-vivo experiments. 2-To define the molecular signaling mechanisms for the involvement of MD-2 as a signaling molecule in LPS/TLR4-mediated NF-kB activation. Investigate the role of Tyrosine phosphorylation of MD-2 upon LPS signaling. 3-To investigate the regulation of MD-2 promoter, and to determine cytokine responsive elements in the promoter. Significance: Improved understanding of the molecular mechanisms by which MD-2 participates in LPS/TLR4 signaling and the understanding of the functional role of alternative splicing of MD-2 and of the regulation of MD-2 promoter may provide new targets for intervention and prevention of inflammatory diseases where TLR4/MD-2 signaling plays a major role, including gram-negative sepsis and endotoxic shock.


[unreadable] Our goal is to improve the versatility, safety and effectiveness of E. coli as a platform for commercial production of DNA, proteins and metabolites. The natural E. coli genome will be reduced to its essentials by removing unnecessary, detrimental and potentially harmful genes through our Scarless Genome Reduction (SGR) technology. The result will be a robust, benign, environmentally friendly organism which can be modified for production as needed. DNA applications include vaccines, gene therapy and RNAi based therapeutics. Protein applications include drugs, hormones, industrial enzymes and some antibiotics. Metabolites include many antibiotics, vitamins, amino acids as well as industrial chemicals and fuels such as ethanol and hydrogen. As the technical ability to manipulate genomes grows, it will be possible for reduced genome [unreadable]. coli to take on many applications previously reserved for other organisms or chemical processes. In the Phase I work, the E. coli genome was reduced to the point that there were no more IS transposable elements. These elements have the capacity to hop around the genome and inactivate genes including the genes for products. The new IS-free strains have remarkably improved properties, including a greater tolerance for high level product expression, greater cloning ability, and better stability. IS hopping is one of the ways E coli defends itself from the stress of production which, from the bacterial point of view, resembles an infection. For production of medicines, the cleanness of the genome is a benefit because purification of product is less difficult and good quality assurance and quality control are more readily attainable. For injectable DNA preparations, the elimination of IS hopping is a tangible safety benefit, which will avoid the risk of transposition into a chromosome of a patient. Finally, the cells grow robustly to high density in industrial fermentation conditions using inexpensive minimal salts media, alleviating concern about prion contamination from bovine-derived media additives. This Phase II project will explore a variety of commercial applications which have been brought to our attention by companies interested in our product. [unreadable] [unreadable] [unreadable]


We propose to study the mechanism by which several restriction enzymes recognize, interact with and hydrolyze specific nucleotide sequences. This study will involve a) the physical-chemical characterization of restriction enzymes as proteins; b) the kinetic characterization of DNA hydrolysis; and c) the characterization of the enzyme-DNA binding reaction in the absence of hydrolysis. In addition, we will attempt to grow crystals of at least one restriction enzyme bound to its specific recognition sequence for analysis by x-ray diffraction techniques. One of the major goals of the proposed research will be to develop a method for the physical mapping for restriction enzyme binding sites on a DNA molecule by the electron microscopic visualization of the position of the bound enzyme. This technique would provide a simple and rapid analysis of the positions of specific nucleotide sequences, and would have a wide variety of applications in studying nucleic acid structure and function. We will ultimately attempt to extend this method to an analysis of DNA-RNA hybrid molecules of biological significance, such as reverse transcriptase products of RNA tumor virus genomes. Our proposed study of restriction enzyme mechanisms requires an analytical method capable of resolving and quantitating all reaction intermediates and products. We have developed agarose gel electrophoresis to a degree of resolution which allows us to start with a closed circular DNA in the molecular weight range 3 to 10 million, and to resolve from each other all conformational isomers of a given size, the full-length linear and circular single strands, and all linear intermediate and final cleavage products generated by a given restriction nuclease. We propose to further develop this method for studying DNA-protein interactions, for restriction enzyme analysis of intermediates in DNA replication and recombination, and for other applications requiring ultra-high resolution.


The Jackson Laboratory Cancer Center (JAXCC) is an organizational unit with dedicated administrative oversight that is strategically integrated with the parent organization's administration. Dr. Edison Liu, JAXCC Director, heads the Cancer Center administration, working with other members ofthe JAX executive committee, the Senior Management Team; Drs. Braun, (Center Deputy Director), Donahue (Director of Genetic Resource Science and co-Project Leader Cancer Models Development Resource) and Hewett (JAX Executive Vice President and Chief Operating Officer). Dr. Barbara Tennent, Associate Director for Research Administration provides dedicated administrative support to the JAXCC and reports to Dr. Liu. Two committees comprise the formal administrative structure ofthe JAXCC. The Operations Committee, chaired by Dr. Liu comprises Drs. Braun, Hewett, Nair and Tennent, and works to ensure that the overall direction ofthe JAXCC operations anticipates and supports the needs of JAXCC members. The Shared Resources Management Committee, which comprises Ms. Valerie Scott, Senior Director of Scientific Services, Ms. Yu-Hui Rogers, Site Director, JAX Genomic Medicine, and Drs. Donahue and Tennent coordinate the operations and development of CCSG-supported shared resources across campuses. Funds are requested for partial support of Dr. Tennent's administrative responsibilities. She oversees the necessary planning, policy development, budget development and research developmental activities for the ongoing growth ofthe JAXCC cancer research focus. Dr. Tennent is the primary coordinator for scientific and administrative management of the JAXCC. She is an ex officio member of the Scientific Executive Committee and serves on the Operations Executive Committee and the Shared Resources Committee. She works closely with all members ofthe administrative team to coordinate the activities ofthe JAXCC, including organization of the External Advisory Board meetings, management ofthe pilot project process, and membership records. Dr. Tennent is the primary administrative contact for NCI Cancer Center program officials and is responsible for management ofthe renewal, progress reports and requests from NCI as required. Institutional funds support dedicated effort for Ms. Scoff, who oversees the four institutional scientific services for which CCSG support is requested. She is responsible for advocacy, policy development and application, budget management, operations, quality assurance, technical staffing and development, planned technology acquisition and implementation as well as user communications. Institutional funds also support Ms. Roger's together in implementing processes, procedures and management structure that facilitate efficient resource sharing and access to CCSG-supported resources. The JAXCC is strongly supported by institutional administrative offices including the Office of Sponsored Programs for pre and post award administration, compliance, and financial management of the CCSG; Financial Services for budget development and account management; and Scientific Program Development, for securing external funding from foundations and federal agencies for research, education, and resource programs.


CORE COMPONENT G: ADMINISTRATION The administrative core supports all administrative and budgetary aspects of the Program Project. These support services include: 1. Clinical Coordinator Team - a group of physicians and a clinical specialist who are responsible for the initial contact with referring physicians and patients, patient financial counseling, scheduling and protocol assignments. 2. Protocol Management Team - a group of individuals responsible for coordinating protocol development, review, renewal and monitoring. 3. Grant Coordinator - the individual responsible for financial management of this Program Project grant. 4. Research Nurse Facilitator- a research nurse specialist responsible for facilitating the initiation and conduct of clinical trials. 5. Word Processing andAdministrative Services - a group of individuals responsible for word-processing manuscripts and submitting manuscripts for publication;updating citation and grant databases;creating presentation materials for internal investigator meetings and external scientific conferences;scheduling lectures, visitors and conferences. Relevance to Public Health: By providing essential administrative support, this core assists all projects on this grant in achieving the goal of making allogeneic hematopoietic cell transplantation safer, more effective, increasingly available, and applied more appropriately in the course of disease so as to maximize benefit and minimize risk.


The menisci are C-shaped fibrocartilage disks that occupy the periphery of the knee joint and serve a number of significant mechanical functions including load distribution across articular cartilage, and joint stabilization. In the face of such mechanical demands, it is not surprising that the meniscus can become damaged. Unfortunately, surgical options for the treatment of damaged menisci are limited, primarily due to the poor vascularity and healing capacity of the central two thirds of the meniscus. Allograft implantation is complicated by the threat of transmission of infectious diseases, the difficulty of matching meniscal shape and material properties, and problems with maintaining cell viability. While meniscectomy (the complete or partial removal of the affected tissue) which is the most common orthopedic procedure performed in the United States can lead to the early onset of osteoarthritis. Providing a meniscal substitute engineered to function much in the way of the native tissue could delay or even avoid the onset of osteoarthritis. Meniscal substitutes, whether cell-seeded degradable scaffolds or synthetic implants, should ideally help to distribute loads across adjacent articular cartilage much in the way of the native tissue, thereby protecting the underlying articular cartilage. However, the properties of a substitute that are required to ensure that this functional capability is met are unclear. This is caused in part by the absence of a robust, comprehensive and physiologically relevant model within which the effect of substitute design variables can be parametrically studied. This lack of such a test, or series of tests, makes the design and evaluation of candidate scaffolds impossible, retards the regulatory pathway to commercialization, and leads to a situation where scaffolds are implanted in humans with little information about their ability to perform mechanically in the highly-loaded environment of the knee joint. Our goal is to define the relationship between meniscal substitute material and structural properties and joint contact mechanics under multiple physiological activities across a range of patient populations. The main element of the framework required to achieve this goal is a statistically-based, rapidly computable interpolator that will be built using results from experimentally-validated knee specific computational models. We will gather information about factors that most markedly affect the functional performance of the native meniscus and use this information as a template to identify the design space into which substitutes should fall if they are to function appropriately. Our goal is not to design a meniscal substitute; rather, we will demonstrate that our approach can establish a workable design space for use by those developing solutions for meniscal repair. Our efforts will culminate in the development of a new paradigm for the development and screening of any complicated tissue substitute, whether a non-degradable implant or a cell-seeded degradable scaffold prior to initiating more time consuming and costly animal and clinical trials. PUBLIC HEALTH RELEVANCE: Our goal is to define the relationship between meniscal material and structural properties and joint contact mechanics under multiple physiological activities across a range of patient populations. The main element of the framework required to achieve this goal is a statistically-based, rapidly computable interpolator that will be built using results from experimentally-validated computational models of the intact and meniscal substituted knee. Our approach will serve as a tool for rapid evaluation and design of functional substitutes and provide a new paradigm for the development and screening of any complicated tissue substitute, whether a non- degradable implant or a cell-seeded degradable scaffold.


Success in science requires a variety of professional "survival" skills as well as the background and research experience normally provided by pre- and post-doctoral training programs. Thus, ultimately most researchers must learn to communicate effectively (orally or in writing), teach, supervise and mentor, obtain a job and funding, and behave responsibly. Over the past 15 years the PIs have developed a course to provide students with explicit training in these subjects. In addition, they now offer annual trainer-of-trainers workshops in which faculty obtain the instruction and materials necessary to establish such courses at their own institutions. In this proposal, funding is requested to extend their efforts at disseminating their model for providing training in survival skills and ethics. There are three specific aims: (l) To increase the implementation of courses in survival skills and ethics at other institutions. This would be accomplished by providing 36-40 scholarships that will cover the cost of participating in the trainer-of- trainers workshop and provide faculty with small start-up grants for establishing courses in survival skills and ethics at their institutions. (2) To promote the sustainabilitv of courses implemented. This would be done by continuing to assist faculty who attend future (or have attended former) trainer-of-trainers workshop in sustaining courses and workshops in survival skills and ethics by providing them with on-going consultation and support services including updates of materials. (3) To strengthen and expand the evaluation of project effectiveness. This would be done through surveys of participants efforts to implement courses. In these ways the PIs seek to improve graduate and postdoctoral education, promoting explicit instruction in many of the skills that are necessary for success as a professional, yet are often not formally taught in graduate training programs in the sciences.


The research described in this project is designed to ask and provide answers to basic questions about the structure and functions of the outer membrane of Gram-negative bacteria. Specifically, it will yield knowledge about membrane protein tertiary and quaternary structure, provide specific information about how an important class of transmembrane proteins (the porins) form hydrophilic channels through the membrane, and give insights into the relationships between the various components of the outer membrane. Gram-negative bacteria are important enteric and systemic pathogens. The outer membrane and its related structure play key roles in colonization, immunogenicity, viability and the antibiotic response of these organisms. A better knowledge of the structure and function of the outer membrane will aid in the development of antibiotics and treatments for pathological conditions caused by these organisms. The main thrust of the proposed research is the in-depth use of genetic and biochemical approaches to isolate and characterize Escherichia coli mutants which have specific altered properties of the outer membrane. One approach that will be used is to select for the uptake of substrates which normally do not traverse the outer membrane barrier. This selection yields mutants which have altered porin proteins. These alterations impart a hypersensitivity to detergents and antibiotics. The hyperpermeable mutants will be analyzed phenotypically and the exact nature of the lesions in the gene for the porin proteins will be determined at the DNA level. The hyperpermeability phenotype exhibited by these mutants will be used to develop additional selections for the isolation of novel mutants which have acquired regulatory or compensatory mutations which reduce or eliminate the hyperpermeability phenotype. The genetically and biochemically analysis of the many mutant types isolated in this project will help to achieve the goals of the project.


Principal Investigator/Program Director (Last, first, middle): Cole, Ronald, A. RESEARCH & RELATED Other Project Information 1. * Are Human Subjects Involved? l Yes m No 1.a. If YES to Human Subjects Is the IRB review Pending? l Yes m No IRB Approval Date: Exemption Number: 1 2 3 4 5 6 Human Subject Assurance Number 00007232 2. * Are Vertebrate Animals Used? m Yes l No 2.a. If YES to Vertebrate Animals Is the IACUC review Pending? m Yes m No IACUC Approval Date: Animal Welfare Assurance Number 3. * Is proprietary/privileged information m Yes l No included in the application? 4.a.* Does this project have an actual or potential impact on m Yes l No the environment? 4.b. If yes, please explain: 4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an environmental assessment (EA) or environmental impact statement (EIS) been performed? m Yes m No 4.d. If yes, please explain: 5.a.* Does this project involve activities outside the U.S. or m Yes l No partnership with International Collaborators? 5.b. If yes, identify countries: 5.c. Optional Explanation: 6. * Project Summary/Abstract 1582-MENTORAbstract_SUMMARYPhaMseim_1e_Tanypde_:2a[1p]p.plidcfation/pdf 7. * Project Narrative 3538-MENTOR project_narrative[1].pdf Mime Type: application/pdf 8. Bibliography & References Cited 3318-MENTORNIHApr06References_r3[M1]i.mpdefType: application/pdf 9. Facilities & Other Resources 5600-MENTOR facilities.pdf Mime Type: application/pdf 10. Equipment 7794-MENTOR EQUIP.pdf Mime Type: application/pdf Tracking Number: Other Information Page 5 OMB Number: 4040-0001 Expiration Date: 04/30/2008 Principal Investigator/Program Director (Last, first, middle): Cole, Ronald, A. Project Summary Millions of children in the U.S. are at risk for learning to read well despite major advances in our understanding of the causes and cures for poor reading. In this Fast- Track proposal we propose to address this significant need and opportunity with a computer-based reading program that combines the power of one-on-one tutoring with the benefits of self-assessing, individualized, scientifically-based reading instruction. We plan to position this product, Foundations to Literacy", as a service over the Web, so that it is accessible in classrooms and resource rooms, community learning centers and homes. The goal of the Phase I work is to demonstrate the technical feasibility of delivering an immersive, individualized one-on-one tutoring experience to children in organizations and homes using a scalable, cost-effective Application Service Provider (ASP) delivery model. Success in the Phase I project will be measured by satisfactory learning experiences by 16 children connected to a single server who are using the program simultaneously. The goal of the Phase II research and development effort is to enhance the functionality of the Foundations to Literacy program by incorporating a new set of learning activities into the program that teach children to read text fluently with good comprehension. Improving reading comprehension is a national priority, and research has shown that good comprehension requires sufficient vocabulary and the ability to read words in context fluently and automatically. We propose to develop a novel set of learning activities that facilitate acquisition of basic oral vocabulary-the meanings of spoken words-and also teaches children to recognize printed versions of these words accurately and rapidly. In addition, using speech recognition technology to measure the


Diabetic patients are at high risk of a range of comorbid complications, including cardiovascular disease (CVD). The increased risk of cardiovascular events seen in patients with type 2 diabetes is associated with a cluster of risk factors for cardiovascular and metabolic disorders that tend to coexists in these patients. Despite successful implementation of evidence based strategies, many individuals are not identified as high risk before their first event and others continue to experience cardiovascular events despite optimal management. While much of this risk is attributable to the presence of conventional risk factors, such as hyperglycemia, hyperlipidemia, hypertension, a substantial burden of this risk remains unexplained. Inflammatory mediators and growth factors are increasingly recognized as playing important roles in the development of atherosclerosis. Therefore the overall objective of this proposal focuses on performing longitudinal assessment to define the role and contribution of a novel biomarker, connective tissue growth factor (CTGF), in the initiation/ progression of macrovascular and microvascular disease in subjects with type 2 diabetes and to determine whether increases in CTGF in the presence of microvascular disease will predict development of macrovascular disease. Our preliminary findings, based on cross-sectional data generated from a cohort of type 2 diabetic patients, suggest that the occurrence of cardiovascular events is independently linked to elevations in CTGF level. Our results demonstrate that diabetic patients with a prior history of myocardial infarction (MI) or coronary artery disease (CAD) have significantly higher levels of plasma CTGF than patients who have not had a prior cardiovascular event. We also uncovered a strong association between circulating levels of plasma CTGF and retinopathy ETDRS scores as well as albumin excretion rate (AER) in these type 2 diabetic subjects. In addition, our findings demonstrate that CTGF expression is induced in aorta of ApoE-/- knockout mice with atherosclerosis compared to control mice. Therefore, based on the preliminary data we generated, we hypothesize that higher levels of circulating CTGF reflect ongoing vascular damage from hypertension, endothelial dysfunction and inflammation, and that elevated CTGF levels will predict greater risk for future cardiovascular events and progressive retinopathy and nephropathy. To test our hypothesis we propose the following specific aims: 1) Determine whether plasma CTGF levels predict future cardiovascular events and progressive retinopathy and nephropathy in individuals with type 2 diabetes. 2) Determine the role and contribution of CTGF to the initiation and progression of diabetic vascular disease. The proposed studies should establish CTGF as a pathologically-important risk factor for diabetic vascular disease that will form the basis for defining new targets for interventional therapy.


Several lines of evidence have suggested that certain strains of Escherichia coli may produce an enterotoxin with physiologic properties similar to cholera-toxin. In this grant we propose to develop an in vitro assay system to measure E. coli enterotoxin. Purification of the toxin is continuing with the goal of producing an immunogenic product suitable for vaccine usage. The toxin assay system will also be employed in further clinical studies. E. coli strains from children with acute diarrhea will be tested for enterotoxin activity. Their serum will be examined for antitoxin activity based on neutralization of the toxin assay system.


The MBRS-IMSD Program at the University of California, Irvine (UCI) started in 1996 as a comprehensive effort to increase the number of underrepresented minority (URM) undergraduate and graduate students pursuing biomedical research careers. During the last funding cycle (2004-07), the program has increased the rate at which undergraduate participants enter PhD programs following graduation and the number of URM students completing PhD programs at UCI. Independent paid research conducted under the direction of faculty mentors serves as a core element to induce MBRS students to pursue graduate school and research-focused careers. Over 100 faculty with funded research programs serve as preceptors of MBRS trainees. The program offers a series of components to increase the interest, motivation and academic preparedness of undergraduates (freshmen to seniors) to enter PhD programs in biomedical sciences, including a peer tutoring/mentoring program of science classes, a seminar series, workshops on laboratory methods, scientific communications, GRE preparation and application to graduate school. The graduate component of the program is designed to provide a comprehensive training for URM Ph.D. students to excel in graduate school. The program provides summer research training for incoming URM PhD students to prepare them for the core graduate classes, a workshop on extramural funding, counseling and orientation about the graduate studies in a non-departmental setting, a workshop to prepare oral exams after the first year in the PhD program and preparation for advancement to candidacy and dissertation research. Public Health Relevance: The inclusion of underrepresented minorities in the biomedical scientific workforce is critical to address the need of improving the health of the people of the United States and eliminating health disparities in the nation. This project will improve the quality and quantity of undergraduate and graduate students from underrepresented groups being trained as the next generation of biomedical research scientists.


The present proposal aims to analyze various physiological and pharmacological properties of adult and developing mammalian extraocular muscles (EOM) and to study some aspects of the ultrastructure of rate EOM using serial sectioning. The study of the electrical properties of rate EOM fibers with intracellular microelectrodes will be continued with emphasis on the slow multiply-innervated fibers and the synaptic transmission. In rabbit EOM, the possibility that the morphological variations described along the multiply innervated fibers are accompanied by functional variations will be explored. The analysis of the different types of contractile responses of rat EOM subjected to various treatments, will be continued to establish the contribution of the different types of fibers to the total tension and to determine the mechanism of action of cholinergic drugs. The changes of the contractile and electrical properties of rate EOM during the first three weeks after birth will be studied to define the differentiation process of these muscles.


Project Summary The expansion of access to ART has the potential to greatly benefit the physical, social and economic well being of individuals and households, as well as produce unintended negative consequences with regard to increased risk behavior and social disruption which can compromise its effectiveness. Drawing on Social Cognitive Theory, the proposed 4-year study will examine (1) how depression influences multiple health outcomes of patients and their households including economic output, household constellation, children's school attendance, and sexual risk behaviors, both directly and as a moderator of impact of ART; and (2) the impact of antidepressant treatment in mitigating the negative consequences of depression on these health outcomes in the context of ART scale-up in Uganda. The study will consist of two phases. In Phase 1, semi- structured qualitative interviews with ART patients, clinic providers, administrators, and support service organizations will be used to explore (1) the impact of HIV and ART on patients' physical, mental, social, economic and sexual health; (2) how mental health and depression influence these ART outcomes; and (3) attitudes towards and acceptability of depression treatment and antidepressants. Findings will help develop and refine hypotheses and measures for use in Phase 2. In Phase 2, patients entering HIV care and starting ART will be followed for 12 months in a longitudinal prospective cohort study. Those diagnosed with depression will be offered antidepressant treatment. Data from our two other just-funded observational cohort studies of ART and non-ART patients in Uganda, which include a subgroup of patients who are depressed but not receiving depression treatment, will provide important additional comparative data for evaluating the effects of depression and antidepressant therapy. These findings could provide critical information for the development of interventions and public health policy aimed at maximizing the benefits of ART in limiting HIV transmission and the social and economic consequences of the epidemic in the developing world, including the prioritization of improved diagnosis and treatment of depression.


We have discovered the first experimental system for in vitro assembly of gap junctions. Assembly of gap junctions is pre-conditioned by disruption of cytoskeletal elements and proceeds even in the presence of inhibitors of protein synthesis. We now want to investigate the role of temperature in the assembly process, the ontogenetic and structural relationship between tight and gap junctions and the role of lipid molecules in the structure of the connexon (its building unit). This project has been interrupted for lack of personnel and aacess to freeze-fracture equipment.


Modern biomedical research relies on interdisciplinary approaches such as bioinformatics that synthesize knowledge and methods from other disciplines to provide an integrated framework for solving biomedical problems. The rapid advancement of high-throughput technologies for measuring biological systems has generated a significant demand at Dartmouth College and other research institutions across Northern New England for interdisciplinary approaches in the quantitative sciences (e.g. bioinformatics, biostatistics, genomics, mathematical biology, proteomics, and systems biology). Integrating high-dimensional research databases with clinical databases from medical schools and hospitals across the region will be needed for translational medicine to become a reality. Unfortunately, the research institutions in Maine, New Hampshire, and Vermont are in a largely rural setting have not kept pace those in larger metropolitan areas such as nearby Boston or New York. The goal of this COBRE program is to establish a Quantitative Biology Research Institute (QBRI) that will support and enhance quantitative biology research across the region and facilitate its integration and synergy with experimental and observational biology. This will be accomplished by 1) establishing a Quantitative Biology Research Institute (QBRI) focused on developing, supporting, and enhancing quantitative biology research in Maine, New Hampshire, and Vermont that will become nationally and internationally recognized, free standing, and will foster meaningful collaborations with experimental biologists thus improving the ability of investigators in the region to compete for NIH funding, 2) recruiting talented tenure track quantitative biologists to Maine, New Hampshire, and Vermont, 3) mentoring the development of four junior quantitative biologists across the region and 4) promoting synergistic collaborations between quantitative biologists and experimental biologists through four research projects, an Administrative Core and an Integrative Biology Core. The scientific focus of the four research projects is gene-environment interaction within the context of environmental health and toxicology. This provides an important unifying and synergistic theme for the COBRE.


Parathyroid hormone (PTH) is a key regulator of bone metabolism and is used as anabolic pharmacotherapy for treatment of osteoporosis. Recent evidence suggests that lipids may affect bone metabolism and that intermittent PTH therapy may fail in the presence of hyperlipidemia. This is clinically important because hyperlipidemia is prevalent in patients with osteoporosis even accounting for age. In the hyperlipidemic condition, bioactive derivatives of low-density lipoproteins (LDL) are generated in the subendothelial space of tissues, triggering chronic inflammatory responses including oxidant stress and expression of cytokines and chemokines. We have found that these inflammatory lipoproteins/lipids are also present in bone and that they inhibit osteoblastic differentiation. In additional studies, we and other investigators demonstrated that hyperlipidemic mice have reduced bone density compared to WT mice. Our preliminary studies, both in vitro and in vivo, now show that lipids inhibit PTH-induced immediate early genes, including Nurr1, a transcriptional regulator of osteoblastic genes, by attenuating cyclic AMP production and that hyperlipidemia blunts PTH-induced osteoanabolism in vivo, primarily in cortical bone. These findings strongly suggest that hyperlipidemia induces PTH resistance. Whether PTH resistance is at the molecular and/or tissue level remains to be determined. Since osteoporosis and hyperlipidemia remain widespread despite treatment, understanding effects of lipids on basal (endogenous) and intermittent (exogenous) PTH may provide new approaches to osteoporosis. We hypothesize that inflammatory lipoproteins, which are increased in hyperlipidemia, reduce PTH anabolic effects. Based on our preliminary studies, in Specific Aim 1, we will test in vitro whether the inhibitory mechanism of lipids on PTH-induced cyclic AMP production is at the level of PTH receptor expression, receptor trafficking, downstream at the level of G-protein activation, or further downstream at the level of adenylate cyclase activation. In Specific Aim 2, we will identify the level at which lipid-induced PTH resistance occurs in vivo: at the level of differentiation of marrow progenitors toward osteogenic vs. adipogenic lineages; anabolic responses of mature osteoblasts/osteocytes; and/or transient expression of osteoclastogenic factors by osteoblasts. We will generate Ldlr-/- mice that express green fluorescent protein targeted to osteoblasts and osteocytes. In Specific Aim 3, we will test whether reducing hyperlipidemia or inhibiting lipid oxidation will reverse PTH resistance in vivo by measuring bone density, histomorphometric parameters and bone turnover markers in the hyperlipidemic (Ldlr-/-) mice that are treated with liver X receptor agonists or that overexpress the anti-oxidant enzyme, paraoxonase-1. These proposed studies will provide insights into how inflammatory lipids inhibit PTH-induced osteoanabolism, pinpoint the site of inhibitory action within the PTH signaling pathway, and demonstrate approaches to reverse lipid-induced PTH dysregulation.


Summary/Abstract Despite ~10% of the human genome being comprised of secretory proteins, little is known about the physical states of granule lumenal and membrane proteins before and during fusion. This proposal is based upon the hypothesis that the mobility characteristics of granule lumenal contents and of granule membrane proteins shape the secretory response. The proposal will provide fundamental new insights concerning secretory granule structure and function in exocytosis and will provide the first quantitative measures of the rotational and translational mobility of granule lumenal and membrane proteins of individual granules. Our research focuses on events in the exocytotic pathway that occur in the highly specialized domain of the plasma membrane-cytoplasm interface. This region is superbly imaged by total internal reflection fluorescence microscopy (TIRFM), a core technique that we use extensively in our studies. The proposal is supported by strong preliminary results demonstrating distinct mobilities of different lumenal proteins, NPY-Cerulean and tPA-Cerulean. tPA-Cerulean, which has a much lower mobility, is released much more slowly upon fusion of individual granules and the fusion is associated with much slower fusion pore expansion. The rotational and translational mobility of lumenal and granule membrane proteins and of individual granules will be measured by novel combinations of TIRFM, polarization, fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). There are several related goals in the proposal: 1) To understand the MOBILITY CHARACTERISTICS OF PROTEINS IN THE GRANULE LUMEN and reveal their influence in determining the rates of protein and catecholamine release, 2) To determine the translational MOBILITY OF GRANULE MEMBRANE PROTEINS and whether the mobility permits their recruitment by diffusion to the fusion site on the granule membrane, 3) To determine the ROTATIONAL MOBILITY OF ENTIRE GRANULES in order to better define the tethered and/or caged state of the granules before fusion, 4) To determine whether the increase in granule travel immediately before fusion reflects a combination of translational and rotational motion that permits the granule to `ROLL' into a fusion competent interaction with the plasma membrane, and 5) To measure for the first time the ABSOLUTE DISTANCE BETWEEN THE GRANULE AND THE PLASMA MEMBRANE using supercritical angle emission, thereby determining the timing of engagement of the granule with the plasma membrane before fusion. In addition, it is anticipated that the new techniques will not only be applicable to secretory cell biology, but more generally to the multitude of cell biological issues at the plasma membrane- cytosol interface.


DESCRIPTION: Colorectal cancer (CRC) is a leading cause of cancer-related death in the U.S. Screening is a cost-effective strategy for reducing both CRC incidence and mortality but has yet to have a major impact on disease burden partly because of poor patient adherence. Shared decision-making is a potentially effective yet unproven strategy for increasing CRC screening rates. The overall objective of this proposal is to develop an interactive, web-based decision aid and evaluate its impact on shared decision-making and patient adherence to CRC screening recommendations. During Phase 1 (Year 1), an interactive, web-based decision aid will be developed that employs state-of-the-art technology to educate patients about the five recommended CRC screening strategies, heighten risk perception, and empower patients to participate in the decision-making process. Content will be developed using a critical review of relevant literature and existing decision aids, expert opinion, and decision-making theory. A personalized risk assessment tool plus pre-/post-test modules to assess demographics, knowledge, beliefs, desire, barriers, and patient preferences will be incorporated. Tailored feedback will also be provided. Once developed, the aid will be subjected to external review by an outside advisory committee for content validity and pre-tested for usability. During Phase 2 (Year 1), an optimal strategy will be defined for implementation of the decision aid in two ambulatory care settings. The research team will interact with physicians and office staff to identify potential logistical barriers and to conjointly develop a tailored strategy for implementation at the targeted ambulatory care sites. Issues to be addressed include resource needs, space allocation, patient identification and scheduling, and tracking. Physician and staff educational seminars will be conducted before implementation. During Phase 3 (Years 2 to 5), a randomized controlled clinical trial will be conducted to evaluate the impact of the decision aid on shared decision-making and adherence to CRC screening recommendations. Eligible patients will be randomized to review the decision aid, the personalized risk assessment tool alone or a control condition. The primary outcome will be patient adherence to CRC screening recommendations using computerized tracking systems; secondary outcomes will include patient knowledge and patient satisfaction using evaluation components of the decision aid or validated instruments.