Jun Zhu - US grants

DESCRIPTION (provided by applicant): Many species of bacteria can exchange chemical signals to help them monitor their population densities, a phenomenon referred to as quorum sensing. Vibrio cholerae, the causative agent of the epidemic diarrheal disease cholera, has been shown to have dual quorum sensing systems, one of which is similar to that of V. harveyi. Preliminary results show that quorum sensing is involved in biofilm formation and pathogenesis in V. cholerae. However, quorum-regulated genes remain largely unidentified in V. cholerae. The goal of this research is to identify quorum sensing regulated genes in V. cholerae by using whole genome microarray expression analysis. The first aim of these experiments is to identify a novel AI synthase and components required for AI signal transduction in V. cholerae by genetic screening. The second aim is to identify quorum-regulated genes using microarray analysis. RNA isolated from wild-type and autoinducer synthase mutants grown under different conditions will be subjected to microarray analysis. The next aim is to characterize these quorum-regulated genes and understand how quorum sensing affects V. cholerae pathogenesis. The final aim is to study the expression patterns of quorum-regulated genes in various V. cholerae biotypes to determine if there is any correlation between the prevalence of quorum sensing systems and the emergence of epidemic strains.

The overall purpose of this research is to explore the effects of, and the interactions between, herbivory on stems and herbivory on roots of red pine trees. Interactions between below-ground and above-ground processes such as herbivory are widely recognized as important but are poorly understood components of terrestrial ecosystems. In red pine, the group of herbivores that feeds on stems includes bark beetles and other boring insects; a different group of organisms feeds on tree roots. Previous results suggest that root-colonizing beetles also bring fungi into a few trees in new stands. These organisms do not kill mature trees, but they interfere with transport of materials (water, nutrients, carbon) between stems and roots. This interference reduces trees' ability to resist infection by bark beetles. At the same time that bark beetle infestation is increasing, fungi spread through roots and contribute to tree mortality. As infected trees die, different types of plants colonize the resulting empty space, leading to major vegetational changes in the forest. The specific objectives of the current study are to 1) study the effects that herbivory on roots has on susceptibility to stem herbivores, 2) measure movements of root beetles, bark beetles and their predators among trees and among forests, and 3) conduct experiments to determine the relative importance of stem-colonizing organisms, root-colonizing organisms, and their predators in tree mortality and gap (open forest space) formation. Because bark and root insects cause significant losses in commercial and natural forests, results from this study will aid forest management and public land management. The study will provide a better understanding of the scale at which crop diversity can reduce losses. The research will also contribute to science training by emphasizing interdisciplinary education at the undergraduate, graduate and postgraduate levels.

MicroRNAs (miRNAs) are extremely small (21-23 nucleotide) RNAs that do not encode proteins but serve as important post-transcriptional regulators. Specifically, miRNAs target messenger RNAs to which they are perfectly complementary for degradation, and those to which they can base pair with internal mismatches for translational repression. It is widely believed that miRNAs are embedded in an extensive array of gene regulatory circuits, thereby playing essential roles in establishing and maintaining cellular functions during development and differentiation. However, direct evidence in support of this proposal is lacking. Given the importance of miRNAs in gene regulation, one key question is how miRNA biogenesis itself is regulated at the levels of transcription and processing. To date, little has been learned about transcriptional regulation of miRNA genes, mainly due to the lack of information about the precise location of miRNA promoters. Mapping the transcriptional regulatory elements that comprise miRNA promoters is critical for determining the sequence motifs present and the factors with which they interact to turn on and off miRNA synthesis. The goal of this research project is to (1) use high throughput methods to globally determine the transcription start sites of miRNA genes in human cells; and (2) to use computational strategies to identify the sequence elements and regulatory factors involved in miRNA transcriptional control. Therefore, this study will serve as an initial yet critical step towards dissecting the molecular mechanism underlying regulated miRNA expression, bringing us one step closer to fully understanding miRNA-mediated gene regulation at the network/system level.
Broader Impacts: The proposed project provides unique training opportunities for undergraduate, graduate and postdoctoral fellows in genome science, including but not limited to genome technology development and computational biology. In addition, the research program will promote curriculum development for graduate and undergraduate courses with an emphasis on genome biology. By taking advantage of the existing infrastructure and outreach programs at Duke University, in particular at the Institute for Genome Sciences & Policy and its associated NIGMS National Center for Systems Biology, the project will reach out to scientists of diverse backgrounds as well as educate the general public.

DESCRIPTION (provided by applicant): Tobacco smoking prevalence among the HIV-positive population is approximately 50%-70%, which is 2 to 3 times higher than that in non-HIV population (Centers for Disease Control and Prevention, 2005). Nicotine (NIC), the primary reinforcing agent in tobacco, stimulates the mesolimbic dopamine (DA) system through activation of nicotinic acetylcholine receptors (nAChRs) in the brain. The DA projection from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) plays a critical role in NIC-mediated behaviors that may contribute to NIC craving in humans. Compared to non-HIV individuals, HIV-positive smokers are more likely to develop NIC dependence, suffer from depression and experience more difficulty to quit smoking. Taken together, tobacco smoking presents an elevated health hazard to HIV-positive individuals, and HIV infection may increase the risk of NIC dependence. Currently, little is known about the neurobehavioral mechanisms through which HIV-positive individuals show increased vulnerability to NIC dependence. Infection with HIV is associated with a variety of neurological impairments that result from the presence of the viral proteins. HIV-1 trans-activator of transcription (Tat) protein is essential for efficient viral replication and plays a crucial role in pathogenesis of HIV-1-associated dementia and synergistic neurotoxicity in the dopaminergic system. Our recent studies indicate that intra-striatal infusion of Tat decreases K+evoked DA levels in rats (Ferris et al., 2008) and that intra-accumbal Tat alters the acute and sensitized response to cocaine (Harrod et al., 2008). The preliminary results show that in vitro exposure to the Tat protein decreases DA transporter (DAT) function in rat striatum. Thus, the major experimental question of this proposal is: Does microinjection of Tat into either the NAc or VTA produce neural changes that alter sensitivity to acute and/or repeated intravenous (IV) NIC administration? The proposed research will test the following hypothesis: HIV-1 Tat protein alters functioning of the mesolimbic dopamine system, thereby resulting in NIC-mediated behavioral changes. The experiments proposed here are designed to focus on two specific aims: 1) To determine the effects of microinjected Tat on IV NIC-mediated locomotor sensitization, 2) To determine the effects of microinjected Tat on DAT activity and nAChRs expression in rats with acute or repeated IV NIC administration. The long-term experimental goal of the present research proposal will be to elucidate the underlying neurobiological mechanisms of Tat-induced dysfunction of mesolimbic DA system contributing to NIC dependence. Such research will provide new insights into developing effective smoking cessation programs in HIV-positive population. PUBLIC HEALTH RELEVANCE: These results will provide new insights into the underlying neurobehavioral mechanisms through which HIV- positive individuals show increased vulnerability to NIC dependence. Understanding this mechanism will have the potential to facilitate the development of effective smoking cessation programs in HIV-positive population.

DESCRIPTION (provided by applicant): Tobacco use is the number one preventable cause of death in the world. Nicotine (NIC), the most abundant alkaloid in tobacco, is the primary reinforcing agent in tobacco smoking. Many factors contribute to susceptibility to NIC. One such factor is the environment. The environment animal model is that rats are raised in one of three different conditions: an enriched condition (EC) containing novel objects and social partners, a social condition (SC) containing social partners only, or an impoverished condition (IC) without objects or partners. This animal model identifies environmental influences that may alter individual vulnerability to drug abuse. Environmental stimuli-induced alterations in the behavioral response to psychostimulants are mediated through differential modulation of dopaminergic neurotransmission. Activation of the dopamine (DA)/D1 receptor/cAMP/protein kinase A (PKA)-regulated signaling pathway leads to phosphorylation of the DA and cAMP-regulated phosphoprotein-32 (DARPP-32). This postsynaptic signaling pathway is known to be essential for cellular plasticity in response to repeated exposures to drugs of abuse (Greengard et al., 1999). Although evidence shows individual differences in the susceptibility to NIC addiction, little is known about the molecular consequences of environmental enrichment on DARPP-32 phosphorylation, and the potential relationship between these molecular changes in DARPP-32 and behavioral responses to NIC compared to environmentally impoverished rats. This proposal will test overall hypothesis that environmental enrichment changes DA receptor-mediated cAMP/PKA signaling, in turn modifying the effects of NIC on DARPP-32, and these changes will contribute to differences in NIC-induced behavioral sensitization observed between EC and IC. The hypothesis that drives this grant attempts to elucidate the underlying neurobiological mechanisms of the environmental influences on NIC addiction. The experiments proposed here are designed to focus on two specific aims: 1) To determine the NIC-induced behavioral sensitization in EC, SC or IC rats following repeated NIC injection, 2) To determine the correlation between environmental enrichment-mediated changes in DARPP-32 phosphorylation and behavioral sensitization following repeated NIC injection in EC, SC or IC rats. An understanding of mechanisms by which environmental enrichment alters DA signaling will have the potential to facilitate the development of therapeutic programs for tobacco dependence, and will contribute to the effectiveness of prevention and treatment intervention strategies in adulthood. These results will elucidate the underlying neurobiological mechanisms of the environmental influences on nicotine addiction. Understanding this mechanism will have the potential to facilitate the development of therapeutic programs for tobacco dependence, and will contribute to the effectiveness of prevention and treatment intervention strategies in adulthood.

Under the leadership of Dr. Alan Michelson and the steering committee (Drs. Mark Knepper, Warren Leonard and Keji Zhao), the DSC has become in shape within the last 5 months. Several areas we have been working on in order to meet the increasing demands of intramural investigators for high-throughput sequencing. (1) Equipment acquisition: The core has acquired the latest Illumina sequencing platform, HiSeq-2000. The instrument has been installed and extensively validated against the manufactures specifications. With a full-bloom run, it can generate up to 200 GB raw sequences (or two human genomes with 30x coverage) within one week. Other equipments have also been installed including Corvaris ultrasonicator, CryoPrep and ABI StepOnePlus Real-time PCR system, Agilent Bioanalyzer. Lastly, the core has acquired a Linux cluster, which is essential for data acquisition, analysis and storage. (2) SOP development: Another key function of the DSC is SOP development. Working closely with NHLBI investigators, high priority was set for technologies that are beneficial to multiple users or expected to facilitate the broad applications of high-throughput sequencing platform. We have developed and optimized protocol for (a) RNA-seq with limited starting materials;(b) mitochondria sequencing (C) genome-wide mapping transcriptional start sites and polyadenylation sites. In addition, we have been developing computational pipelines and workflow for systematic identification of SNPs, splicing variants and other regulatory events in gene expression network. (3) Consultation and data acquisition: While the DSC is still in early stage to reach its full capacity, we had offered consultations and sequencing services for intramural investigators with diverse research interests, including miRNA deep sequencing in LDL biology (Dr. Alan Remaley), expression profiling of iPS cell with RNA-seq (Drs. Manfred Boehm and Chengyu Liu), identification of mitochondria mutations (Dr. Hong Xu), genetic and epigenetic gene regulation in immune system (Drs. Keji Zhao and Warren Leonard). We also work closely with Drs. Alan Michelson, Bob Balaban, Adrian Wiestner, Mark Knepper, Robert Kotin, Nalini Ragharachari for their specific projects in basic and translational research. (4) Collaboration with Framingham Heart Study: the core was also involved in initiating a collaborative project with Dr. Chris O'Donnell at Framingham Heart Study. The initial goal is to obtain transcriptome profiles of a cohort of MI patient samples, which are selected with the guidance of existing GWAS data. The resulting gene expression profiles will be further integrated with other genome-wide data (e.g. exome sequencing, exon array, etc) to gain a better understanding of pathophysiology of myocardial infarction.

Clostridium difficile is a spore-forming bacterium that is the leading cause of antibiotic-associated diarrhea (CDAD) and is a problematic hospital-acquired pathogen. Despite the fact that patients are infected via ingestion of the spores yet only the vegetative form of C. difficile can produce the toxins that result in the pathological effects of CDAD, the molecular basis of germination of C. difficile spores in the intestinal mucosal environment has not been examined. Through our prior work with Vibrio cholerae, we have developed a simple, flexible, and effective ex vivo model that recapitulates the conditions found in the mammalian intestine, representing a major new tool in the study of intestinal pathogens that promises to pave the way for developing virulence-targeted drugs and vaccines. This model will be used to determine the rate of germination of spores as well as aid in the identification of host- or bacterial flora-derived signals that induce germination. In addition, the ex vivo model will be used to identify genes induced upon germination using a genetic screen. Identification of signals that affect spore germination and knowledge of how genes are regulated during germination may provide potential therapeutic targets for blocking C. difficile infection. Once spores have germinated into vegetative cells, it is not known how they evade the host immune system. Our preliminary data indicate that cells grown in vitro, but not those grown in the presence of intestinal tissues, can bind to the mucosal antibody, secretory IgA. The immune evasion strategies employed by C. difficile will be examined, including whether C. difficile modulates the expression of its surface proteins in response to the host environment and what host signal is responsible for this. In addition, the consequences of S-lgA binding to C. difficile will be examined, including the fate of cells bound to S-lgA and possible avoidance strategies used by C. difficile. Identification of immunogenic cell surface factors and/or understanding the mechanisms of immune evasion could lead to novel treatment options for CDAD.

Under the leadership of Dr. Adrian Wiestner and the steering committee (Drs. Mark Knepper, Warren Leonard, Keji Zhao), the DIR DNA Sequencing and Genomics Core (DSGC) has been providing a wide spectrum of high-throughput genomics services to facilitate basic and translational research at NHLBI and across the NIH. (1) Consultation and data acquisition: the DSGC has provided the state-of-the-art genomics services for DIR investigators in a cost-effective and timely fashion. Diverse projects have been carried out including whole-genome sequencing, exome sequencing, transcriptome sequencing (RNA-seq and small RNA sequencing), MitoRCA-seq, targeted sequencing, ccf DNA sequencing, methyl-seq as well as single cell transcriptome analysis based on Fluidigm platform (2) In-depth data analysis: The DNA Sequencing and Genomics Core has explored and implemented a wide range of open-source and commercially available software packages for primary and secondary NGS data analysis. In addition, project-specific data analysis is further achieved by in-house software and algorithm development. As the result, it provides diverse tools for DIR investigators to convert the high-throughput data into biological meaningful findings for further characterization. (3) Training and education: The DSGC has taken all possible venues to promote broad dissemination of NGS technologies. Consultations are provided for experimental design and data analysis. The core also offers routine one-on-one training for library preparation and data analysis. (4) Research and development: Several R&D projects were carried out in collaboration with the DIR investigators. High priority was set for technologies that are beneficial to multiple users or expected to facilitate the broad applications of high-throughput sequencing platform. We have developed and optimized protocol for (a) mitoRCA-seq to identify single nucleotide mutations and indels related to aging and heart diseases (b) targeted sequencing assays for myeloid leukemia, pheochromocytomas, congenital heart disease (c) single-cell transcriptome analysis with Fluidigm C1 platform and an improved SPA-seq procedure; (d) cell-free DNA analysis for transplantation rejection and early detection of tissue damage. In addition, we have been developing computational pipelines and workflow for systematic identification of SNPs, alternative polyadenylation, global intron retention and other regulatory events in gene regulatory network (GRN).

DESCRIPTION (provided by applicant): In cancer biology it has become evident that requirements for specific gene activities can vary widely across cancer types. These differences presumably arise from context-specific molecular constrains intrinsic to the tissue or cell type of origin and to the process of cancer development itself. Our current lack of knowledge of cancer-specific gene requirements and the processes driving their requirement has hampered development of targeted therapeutic strategies for cancer. However, in the past five years significant progress has been made in the development of culture systems for patient cancers that allow unprecedented access to cancer- evolved molecular pathways and cellular phenotypes. Over the past three years, we have developed a strategy for defining the nature of gene requirements in patient cancer samples. Our approach integrates data from functional genetic screens in patient derived cancer stem cells with network models constructed from cancer- omics data sets to make gene requirement predictions. In proof of concept studies for Glioblastoma multiforme (GBM), an incurable form of brain cancer, we have demonstrated the existence of GBM-lethal genes, which when inhibited render patient GBM tumor cells sensitive to cellular stresses that arise as a consequence of cellular transformation. In this application w use this cancer-lethal prediction paradigm to address Provocative Question 8: Why do certain mutational events promote cancer phenotypes in some tissues and not others? We test the hypothesis that GBM-specific requirements for gene activities arise from one of three context-specific constraints: (a) the tissue of origin (i.e., neural-specific activity); (b) a GBM-specific evolution process; or (c) cellular transformation process in general. Our experimental approach will combine data from functional genetic screen in human GBM stem cells (of multiple subtypes) with pre-existing Bayesian network models for GBM and other cancers including, breast, lung, ovarian, and prostate (generated from The Cancer Genome Atlas patient data sets). If successful, these studies will reveal the extent and origin of GBM-specific requirements for gene activities in GBM patient samples. In addition to providing key insight into brain tumor biology, these studies will significantly aid in identifying new targeted therapeutic strategies fo GBM and other cancers with standard of care therapies suffering from poor therapeutic windows.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) affects half of the US population over the age of 85 and causes destruction of select networks and cell groups within the brain. AD manifests initially as mild cognitive decline, but gets progressively worse and is always fatal. Despite significant progress identifying susceptibility loci for AD in genome-wide association and whole exome sequencing studies, to date, a predictive risk score for AD that achieves clinical utility on an individual basis given DNA variation information alone has been elusive. This proposal aims to develop a multiscale-network approach to elucidating the complexity of AD. Multiscale network models causally linked to AD will be developed based on existing AD-related large scale molecular data and the high-impact, high-resolution complementary datasets generated through this application. Using brain slice cultures, iPS-cell-derived mixed cultures of human neuronal, oligodendroglial, and astrocytic cell systems, and fly models of AD, we seek to reconstitute the AD-related networks discovered in the multiscale analysis in these living systems and then employ high-throughput molecular and cellular screening assays to not only validate the actions of individual genes on molecular and cellular AD-associated processes, but also validate the molecular networks we implicated in the disease. Our initial multiscale studies have implicated the microglial protein TYROBP as one key driver of AD pathogenesis, a hit we have partially validated, but that we will further validae along with other hits using iPSC-derived mixed cultures of different brain cell types, murine brain slices and AD fly models. We will analyze the potential ability for network-derived hits like TYROBP to modulate standard AD pathology involving A¿ and tau as well as its ability to shift networks in those same systems in such a way as to reflect the behavior of networks discovered in the multi-scale analysis. Importantly, the model building and validation will be iterated to produce updated/refined models based on validation results that, in turn, will be mined to generate updated lists of prioritized targets for validation. In this way, through the course of th grant, as new knowledge accumulates externally and as we generate increased amounts of data including validation data, our models will take into account the most up to date information to produce the most predictive models of AD. As a service to the AD research community, we will provide dramatically improved general access to large-scale, multidimensional datasets, together with systems level analyses of these datasets.

DESCRIPTION (provided by applicant): HIV-1 associated neurocognitive disorders (HAND) remain highly prevalent in the era of effective antiretroviral therapy. HIV-1 infection within CNS system plays a central role in the development of HAND. Drugs of abuse, such as cocaine, have been shown to increase the incidence and exacerbate the severity of HAND by enhancing viral replication. However, the mechanistic links between cocaine and HAND progression remain undefined. Although changes in many neurotransmitter systems may contribute to HAND, the central dopamine (DA) system plays a crucial role in the development of neurocognitive dysfunction in HAND patients and in the control of psychostimulant action of cocaine. The interplay of HIV-1 Tat protein with cocaine augments synaptic DA level and Tat release within dopaminergic brain regions. Long lasting exposure to elevated DA and Tat eventually lead to DA deficit that potentiates severity and accelerates progression of HAND. Antiretroviral agents cannot prevent the production of HIV-1 viral proteins, such as Tat protein, in proviral-containing brain cells. It is unclear how the DA system is altered in HIV-1 positive cocaine abusers. Therefore, there is a pressing need to define the molecular mechanism(s) by which the impaired DA system by HIV-1 infection affects the progression of HAND in concurrent cocaine abusers. Presynaptic DA transporter (DAT), which is critical for neurocognitive function, is a major molecular target for both Tat and cocaine to impact the DA system. In this application, we hypothesize that Tat, via allosteric binding sites in the DAT, potentiates inhibitory effects of cocaine on DA transport, which is the key to DA system dysfunction occurred in HAND patients. Our proposed experiments will investigate how Tat and cocaine interact with the human DAT through their recognition binding sites on human DAT, thereby leading to dysfunction of the DA system. Our strategy encompasses creating a dynamic 3D computational model to predict potential Tat and cocaine binding pocket residues of human DAT, validating these residues via site-directed mutagenesis, and analyzing the consequent functional changes of the Tat and DAT interaction in neuronal cells and primary neurons. The completion of this application will identify molecular targets on the DAT for developing compounds that specifically block Tat binding site(s) in DAT and stabilize physiological dopaminergic tone, which should be beneficial to the preservation of neurocognitive function in patients with HAND in concurrent cocaine abusers.

Because of the slow pace of terrestrial ecosystem processes, including the slow generation time, growth rate, and decomposition rate of trees, the impact of changing climate and disturbance on forests plays out over hundreds of years. For this reason, terrestrial ecosystem models are used to anticipate the centennial scale projections of forest response to environmental change. Current terrestrial ecosystem model predictions vary widely and results have large statistical uncertainties. Furthermore, testing and calibration of these models relies on short term (sub-daily to decadal) data that fail to capture longer term trends and infrequent extreme events. The capacity of ecosystem models for scientific inference and long-term prediction would be greatly improved if uncertainties can be reduced through rigorous testing against observational data. PalEON is an interdisciplinary team of paleoecologists, statisticians, and modelers that have partnered to rigorously synthesize longer term paleoecological data and incorporate into ecosystem models to provide a deeper understanding of past dynamics and to use this knowledge to improve long-term forecasting capabilities.

Funds are provided to address four objectives and associated research questions: 1) Validation: How well do ecosystem models simulate decadal-to-centennial dynamics when confronted with past climate change, and what limits model accuracy? 2) Initialization: How sensitive are ecosystem models to initialization state and equilibrium assumptions? Do data-constrained simulations of centennial-scale dynamics improve 20thcentury simulations? 3) Inference: Was the terrestrial biosphere a carbon sink or source during the Little Ice Age and Medieval Climate Anomaly? and 4) Improvement: How can parameters and processes responsible for data-model divergences be improved? The data synthesis will include wide range of ecosystems, encompasses past climate variations that were large enough to affect tree growth rates, disturbance regimes, and forest demography, and leverages available paleodata. The synthesis will include 1) fossil pollen and Public Land Survey data to reconstruct forest composition, 2) sedimentary charcoal, stand-age and firescar indicators of past disturbance regimes, 3) tree-ring records of tree growth rates, and 4) multiple paleoclimatic proxies and paleoclimatic simulations. Bayesian hierarchical statistical models will be used to reconstruct key ecological variables and their associated uncertainty estimates. A standardized model intercomparison involving 13 ecosystem modeling groups will be used to evaluate the robustness of the modeling approach.

Three areas will be emphasized for PalEON's broader impacts. Community Building: The PalEON research community has doubled over the past 10 months, with more than 60 participants now. It is anticipated to nearly another doubling over the next five years, and the funds will allow the ongoing community-building via annual large meetings and task-oriented workshops. Interdisciplinary Training and Mentoring: A new generation of researchers will be trained to naturally conceptualize large spatial and temporal scales and to approach ecological forecasting as an integrative activity spanning data collection to model prediction. Eight postdocs and seven graduate students will be involved in proposed PalEON research, with multiple opportunities for cross-training. Additionally, the PalEON Summer Short Course provides an intensive cross-training experience for young scientists in all areas encompassed by PalEON. The 2012 course will be followed by courses in 2014 and 2016. Building Scientific Infrastructure: All PalEON datasets will be made publicly available upon publication, as will our new data-assimilation methods and model intercomparison protocols. Tools will be developed for optimal site selection (given the goal of reducing the integrated prediction uncertainty about past vegetation and climate over space and time) and will distribute a publicly available webtool version that will be linked directly to the Neotoma Paleoecology Database.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) affects half of the US population over the age of 85 and causes destruction of select networks and cell groups within the brain. AD manifests initially as mild cognitive decline, but gets progressively worse and is always fatal. Despite significant progress identifying susceptibility loci for AD in genome-wide association and whole exome sequencing studies, to date, a predictive risk score for AD that achieves clinical utility on an individual basis given DNA variation information alone has been elusive. This proposal aims to develop a multiscale-network approach to elucidating the complexity of AD. Multiscale network models causally linked to AD will be developed based on existing AD-related large scale molecular data and the high-impact, high-resolution complementary datasets generated through this application. Using brain slice cultures, iPS-cell-derived mixed cultures of human neuronal, oligodendroglial, and astrocytic cell systems, and fly models of AD, we seek to reconstitute the AD-related networks discovered in the multiscale analysis in these living systems and then employ high-throughput molecular and cellular screening assays to not only validate the actions of individual genes on molecular and cellular AD-associated processes, but also validate the molecular networks we implicated in the disease. Our initial multiscale studies have implicated the microglial protein TYROBP as one key driver of AD pathogenesis, a hit we have partially validated, but that we will further validae along with other hits using iPSC-derived mixed cultures of different brain cell types, murine brain slices and AD fly models. We will analyze the potential ability for network-derived hits like TYROBP to modulate standard AD pathology involving A¿ and tau as well as its ability to shift networks in those same systems in such a way as to reflect the behavior of networks discovered in the multi-scale analysis. Importantly, the model building and validation will be iterated to produce updated/refined models based on validation results that, in turn, will be mined to generate updated lists of prioritized targets for validation. In this way, through the course of th grant, as new knowledge accumulates externally and as we generate increased amounts of data including validation data, our models will take into account the most up to date information to produce the most predictive models of AD. As a service to the AD research community, we will provide dramatically improved general access to large-scale, multidimensional datasets, together with systems level analyses of these datasets.

? DESCRIPTION (provided by applicant): Biological systems employ multiple levels of regulation that enable them to respond to genetic, epigenetic, genomic, and environmental perturbations. Advances in high throughput technologies over the past several years have enabled the generation of comprehensive data sets measuring multiple aspects of biological regulation (such as genetics, epigenetics, transcriptomics, metabolomics, glycomics, proteomics, etc.). Many databases, such as TCGA (The Cancer Genome Atlas) database and the LGRC (Lung Genome Research Consortium) database, have been created for depositing diverse types of omics data and for sharing data for public dissemination. However, data errors, including sample swapping, mis-labeling, and improper data entry, during large-scale data generation and data management are inevitable. Our preliminary results indicate that sample labeling errors frequently occur in every database we examined. Data quality control (QC) is critical for all public databases. Data errors need to be identified and corrected before data is released for data analysis and data mining. Analyzing error infested data wastes public resources. Importantly, wrong data could lead to wrong scientific conclusions. And, sample errors could have a large impact on statistic power. To maximally utilize genetic, genomic, and other omics data in public databases, it is critical to properly match different types of data pertaining to the same sample or individual before applying integrative analyses. There is an urgent need for developing methods that can identify data labeling errors in large databases and properly connect diverse types of omics data pertaining to the same individual. In respond to the Big Data to Knowledge (BD2K) initiative, we will develop computational methods to address the topic area Data Wrangling. Here we propose to develop a sample mapping procedure called MODMatcher (Multi- Omics Data matcher) to simultaneously QC multiple types of omics data (Aim 1), and to develop a suite of predictive models based on multi omics data to identify inconsistency between clinical data and omics data (Aim 2). Our proposed methods will be used to clean data, identify and correct data annotation and metadata attribute errors in large databases, which are all within the scope of the Data Wangling.

? DESCRIPTION (provided by applicant): HIV-1 Tat protein within the central nervous system plays a pivotal role in the neurotoxicity and cognitive impairment evident in HIV-associated neurocognitive disorders (HAND). Converging lines of clinical observations, supported by imaging, neuropsychological performance, and postmortem examinations, have implicated that dysregulation of dopamine (DA) signaling is a risk determinant of HAND. Drugs of abuse, such as cocaine have been shown to exacerbate the severity of HAND by enhancing viral replication. Tat and cocaine synergistically increase synaptic DA levels by directly inhibiting DA transporter (DAT) activity, ultimately leading to dopaminergic neuron damage. Therefore, an intervention for HIV infection-induced dysfunction of DA system has the potential to improve neurocognitive function in patients with the early- stage of HAND. Currently, it is unclear how Tat influences the DA system in the brains of HIV- infected patients with HAND, thereby producing neurocognitive impairment. Our studies will identify the intermolecular interactions between Tat and human DAT (hDAT) and explore how Tat potentiates cocaine-induced inhibition of DA transport, leading to DA dysregulation for both mechanistic and interventional purposes. Recently, we identified key residues for hDAT interacting with Tat, which are critical for Tat-induced inhibitio of DAT uptake. For example, we found that DAT Tyrosine88 replaced by Phenylalanine (Y88F) displays normal surface DAT expression and DA uptake but attenuates Tat-induced inhibition of DA transport. Moreover, we found that Y88F partially attenuates the allosteric modulatory effects of SRI-20041, a novel DAT allosteric modulator, on DAT function. These studies led us to the novel hypothesis that Tat acts via the unique tyrosine88 site to perturb the DAT regulatory network that normally sustains concentrative DA transport, resulting in DA-linked neuropsychiatric dysfunction prominently featured in HAND. We will generate DAT Y88F knock-in mouse line harboring a doxycycline- inducible Tat transgene, and determine the impact of DAT Y88 on Tat-induced inhibition of DA transport and associated cognitive/behavioral deficits in Tat transgenic mice (Aim 1). Moreover, we will determine whether microinfusion of SRI-20041 into prefrontal cortex alleviates cognitive and behavioral deficits in Tat transgenic mice (Aim 2). This exploratory research will uncover critical functions of DAT important for Tat-induced cognitive/behavioral deficits and provide a novel mechanistic basis to identify targets on the DAT for developing compounds that specifically block Tat binding site(s) in hDAT, thereby stabilizing physiological DA transmission.

Perturbation of dopaminergic transmission is implicated as a risk factor of HIV-1 associated neurocognitive disorders (HAND). Dopaminergic system plays a causal role in drug rewarding and modulation of the brain function including cognition. Prefrontal cortex is an important brain region for higher cognitive function, where norepinephrine transporter (NET) is the primary mechanism of homeostatic regulation of stable synaptic dopaminergic tone. HIV-1 Tat protein and cocaine synergistically increase synaptic dopamine levels, thereby producing neurocognitive impairment. Our initial findings show that in vitro exposure to recombinant Tat1-86 inhibits dopamine and norepinephrine reuptake by dopamine transporter (DAT) and NET and Tat binds to DAT and NET through a direct protein-protein interaction. We have demonstrated that Tat-induced inhibition of DAT function is mediated by binding to allosteric binding site(s) on DAT, not by interacting with the DA uptake site. Accordingly, attenuating Tat binding to DAT would be expected to have minimal influence on physiological DA transport. Indeed, our recent findings show that a novel quinazoline series of allosteric modulators decrease cocaine potency for inhibition of DA uptake and attenuate Tat-induced inhibition of DA reuptake and cocaine binding by DAT. We hypothesize that Tat, via the unique allosteric modulatory sites, perturbs the DAT and NET regulatory network that normally sustains concentrative DA or NE transport and potentiates cocaine?s effect on DAT and NET, resulting in DA/NE-linked neuropsychiatric dysfunction prominently featured in HAND. We will (Aim 1) Identify the binding sites for Tat in human NET, and explore allosteric modulation of this transporter by Tat and cocaine; (Aim 2) determine the pathogenic role of DAT/NET-mediated dopaminergic transmission in inducible Tat transgenic mice by assessment of Fast-scan cyclic voltammetry and whole cell patch clamp recording; and (Aim 3) perform proof of concept studies using novel allosteric modulators to establish their potential for therapeutic application in HAND using integrated computational modeling, pharmacological, and behavioral approaches. Our long-term goal is to explore new ways to target DAT/NET for therapeutic interventions to improve neurocognitive dysfunction of HAND in concurrent cocaine abusers.

SUMMARY A system-level understanding of the dengue virus (DENV) host relationship, in particular, the network structure and dynamics, can be derived from experimental data with computational analysis across data sets and modeling. The host response to infection is a complex process involving entire networks of RNA transcription, protein signaling, and metabolism that complementarily influence cellular, tissue, and whole organism behaviors. This complexity demands a systems biology approach for understanding immune response, since investigation of single pathways is unlikely to explain changes taking place across the entire network. The Data Analysis and Modeling Core (Core E) will not only perform standard multivariate analyses on each dataset to find reliable biomarkers for differentiating outcomes of infection, but will furthermore integrate them with the full range of public network and pathway data to construct a multiscale, holistic network model of biologically meaningful DENV-host interactions. Because this model is quantitative and mathematically defined, it is well suited for training advanced classifiers that can predict both individualized clinical outcomes with more accuracy than biomarkers alone and novel ?key driver? biomolecules that can be validated with ex vivo siRNA screens (Project 3). These data should further inform on the synergy among multiple interrelating molecular pathways and networks that underpin the differences in phenotype between individuals. The scale of our proposed model for DENV is unprecedented, spanning the genomic, transcriptomic, proteomic, intercellular signaling, and immune cell subpopulation levels?and only with this scale of modeling will superior unbiased, data driven models that address the key biological questions in each of the Projects emerge, explaining the diverse subtleties of host response to DENV infection and vaccination that affect clinical outcomes.

1. Intron retention coupled with RNA degradation as a prevalent mechanism in modulating gene expression We embarked on the T-cell activation model system because of an initial observation that differential gene expression between resting and activated human CD4 T cells is discordant with changes in RNA Pol II and other well-established transcriptional initiation and elongation marks. This finding is largely unexpected, suggesting a transcription independent mechanism. Using DeLi-seq, a strand-specific RNA-seq strategy developed by our group, we found that a large number of introns are retained in the resting T cell transcriptome, and that intron retention is significantly reduced in the activated cell state. In fact, >600 genes were identified whose expression is predominantly regulated at the intron retention level16. These genes are highly enriched in the proteasome complex, which is required for proper T cell proliferation and cytokine secretion. Furthermore, by integration of RNA-seq and ChIP-seq data, we acquired evidence that intron-retained (IR) transcripts are unstable, likely degraded by the RNA surveillance complex. Our data demonstrated that intron retention coupled with RNA degradation may serve as an on/off switch to precisely control gene expression. Under the permissive condition (e.g. T cell activation), intron-retained transcripts can be converted into productive isoforms for protein synthesis. This mechanism allows for a shortened response time in gene regulation by bypassing the requirement for de novo transcription. As a follow-up of our previous study, we employed BruChase-seq53, a high-throughput sequencing method based on pulse-chase labelling of nascent transcripts, to monitor RNA stability in resting and activated T cells. We employed a first-order mathematical model to fit the BruChase-seq data and took into account of both intron-retained and fully spliced transcripts. Consistent with our working hypothesis, the results provided unambiguous evidence that IR transcripts are significantly less stable than spliced transcripts. While the half-lives of IR transcripts are comparable between resting and activated T cells, we found that the degradation rates of fully spliced transcripts are dramatically skewed between the two conditions with more transcripts became stabilized than destabilized upon T cell activation. By integrating transcription, intron retention and RNA stability data, we further classified differentially expressed genes into distinct categories. One such group consists of genes whose steady-state expression level is solely regulated by RNA stability. These genes are highly enriched in the NFkB pathway, including NFkB1, REL and RELB. We further demonstrated that LARP4 directly binds to the 3' UTR of NFkB1 transcripts in an activity dependent manner. CD4 T cells derived from LARP4 knockout mice show defects in cytokine secretion in response to T cell activation. (2) Genome technology development and collaborative research As a technology savvy group, we have collaborated with many IRP investigators in diverse areas of gene regulation and human diseases -- including transcription memory in innate immunity, alternative polyadenylation during EBV infection, long noncoding RNA mediated gene regulation in metabolism, contribution of mitochondrial heteroplasmy to aging/hypertension, as well as cell free mitochondria methylation status in sickle cell diseases.

HIV Associated Neurocognitive Disorder (HAND) is one of the most common and clinically important complications of HIV infection. Neurotoxicity is due to the accumulation of HIV-encoded Trans Activator of Transcription (Tat) and other viral proteins released from infected cells. Neuronal damage in HIV-infected patients is significantly exacerbated by drug abuse. Thus, there is an urgent and increasing need for effective therapeutic strategies for HAND. We utilized a computational approach in our NIH-funded work to uncover DEAD Box RNA Helicase 3 (DDX3) as a potential target in HAND. DDX3 was recently established as a target protein for cancer therapy. A selective small molecule inhibitor of DDX3 helicase activity, RK-33, has recently been developed and tested in animal models of lung cancer. However, DDX3 has never been proposed to be involved in HAND development, and RK-33 has never been tested in models of HAND or other neurodegenerative diseases. We therefore analyzed the effects of the DDX3 inhibitor RK-33 on the viability of Tat/cocaine-treated primary mouse cortical neurons. Our preliminary results show that RK-33 protects these neurons against the damage caused by a combined insult of Tat and cocaine, and that the effect was quite robust. Thus, the hypothesis of this proposal is that the activity of DDX3 promotes drug-induced HAND-associated neuropathology via pathological Stress Granules (SGs), and the inhibition of DDX3 enzymatic activity alleviates this neurotoxicity by interfering with pathological SG formation. The main goal of the proposal is to establish DDX3 as a target for anti-HAND drug development, validate that pharmacological inhibition of DDX33, can be a treatment for HAND, and also uncover important mechanistic links in HAND neuropathology. Aim 1 will determine the mechanism of RK-33 protection of cortical neurons from the combined neurotoxicity of Tat and cocaine. Published evidence suggests that Tat interacts with DDX3, and that this interaction depends on the enzymatic activity of DDX3. We propose that Tat interacts with DDX3 to facilitate Tat/cocaine compounded neurotoxicity, and the protective effects of RK-33 result from inhibition of DDX3?s enzymatic activity. Aim 2 will establish the function of SGs as a central regulator of the protective effects of RK-33 inhibition against Tat /cocaine-induced neurotoxicity. DDX3 is a core component of SGs and is believed to directly participate in their assembly. The accumulation of pathological SGs is a noticeable feature of amyotrophic lateral sclerosis and other neurodegenerative diseases. We propose that DDX3 inhibition by RK-33 protects the neurons from Tat/cocaine by impairing pathological SG assembly and dynamics. Over the course of this project, the role of DDX3 in the pathology of HAND will be better understood. Determining a connection of DDX3 to pathology associated with SGs could have much broader implications for many other neurodegenerative diseases. Going forward, pharmacological DDX3 inhibition will be validated in vivo, pursued with drug development efforts, and could ultimately lead to new clinical treatment options for otherwise untreatable neurodegenerative diseases.

Immune checkpoint inhibition (CPI) with anti-PD-1/PD-L1 antibodies has changed the treatment landscape for several types of cancers including bladder cancer. In patients with advanced bladder cancer, CPI can induce unprecedented durable responses. However, only a subset of patients responds to such treatment necessitating a better understanding of mechanisms of primary resistance to facilitate the identification of predictive biomarkers and rational combination approaches. We used an integrative approach to identify biomarkers in a large clinical trial cohort of patients with metastatic bladder cancer that are independently associated with response/resistance to CPI beyond tumor mutational burden (TMB) alone. We identified three key gene modules derived from pre-treatment tumor transcriptomic data: a ?good immune? module associated with response to CPI, a stromal module associated with resistance to CPI, and a ?bad immune? module also associated with resistance to CPI, but that appears to mediate the negative impact of the stromal module upon outcomes with CPI. Using single cell RNA sequencing data generated from fresh bladder cancer specimens to dissect the cellular components and molecular interactions responsible for expression of these 3 gene modules, we determined that the ?good immune? module emanates largely from T- cells and NK cells, the stromal module emanates from cancer-associated fibroblasts (CAFs), and the ?bad immune? module emanates predominantly from monocytes-macrophages (M?). We hypothesize that myeloid cells drive primary resistance to CPI in bladder cancer. We further hypothesize that comprehensive cellular and molecular maps of bladder cancer will facilitate identification of precise monocyte-M? subpopulations, and CAF-myeloid-T-cell interactions, that can be leveraged to define novel biomarkers and therapeutic targets. With access to a unique set of clinical trial cohorts that will provide tumor tissue, blood and urine specimens as well as outcome data to CPI, we are uniquely positioned to: (Aim 1) Dissect gene modules associated with response/resistance to CPI using high resolution maps of the cellular and molecular landscape of muscle-invasive bladder cancer; Aim 2: Define the role of ?bad immune? module genes in governing monocyte-M?-mediated immune suppression; Aim 3: Refine and validate a monocyte-M?-related gene signature as a biomarker of CPI resistance in clinical trial cohorts. This proposal comprises an integrated network of multi-disciplinary collaborative investigators (GU oncologists, urologists, immunologists and bioinformaticians) to accelerate translational research and maximize future clinical benefits. These efforts will lead to the discovery of biomarkers, and potential therapeutic targets, to extend the benefits of CPI beyond the 15-20% of patients with advanced bladder cancer that respond to treatment.

PROJECT SUMMARY More than 37 million people are living with Human Immunodeficiency Virus (HIV) infection worldwide which continues to be a global public health problem. Despite the widespread use of antiretroviral therapy (ART), up to 70% of HIV-positive individuals suffer from cognitive and behavioral deficits collectively known as HIV-associated neurocognitive disorders (HAND), for which no therapeutic options are currently available. Converging lines of evidence indicate that the HIV-1 transactivator of transcription (Tat) protein plays a crucial role in causing neurotoxicity and cognitive impairment in HAND. HIV-1 Tat exerts its neurotoxicity through interaction with crucial proteins, such as the monoamine transporters in the central nervous system (CNS). The dysregulation of dopamine (DA) neurotransmission in HAND occurs through direct interaction of Tat protein with the DA transporter (DAT) which is essential for maintaining DA homeostasis and a target of cocaine. While most ARTs cannot efficiently cross the blood-brain barrier, Tat-induced increase in DA levels accelerates viral replication in the brain. Moreover, drugs of abuse, such as cocaine, exacerbate neurological impairments. Our published work has demonstrated that Tat-induced inhibition of DAT is mediated by binding of Tat to allosteric binding site(s) on DAT, not by interacting with the DA uptake site. This provides a basis for a novel approach to address the problem by developing compounds to attenuate Tat binding to DAT by an allosteric mechanism. Our recent studies with small molecule allosteric ligands of DAT reveal that these compounds are capable of attenuating Tat-mediated effects on DAT, thus providing a potential opportunity to develop therapeutic interventions for the treatment of HAND. The research effort proposed herein is to explore the hypothesis that disruption of Tat-DAT interactions with small molecule allosteric ligands of DAT, with minimal disruption of normal DA uptake, will have therapeutic potential for prevention of neurocognitive dysfunction in HAND. The primary goal of our research is to optimize lead compounds and perform proof-of-concept pharmacological studies in animal models. To this end, the specific aims to be pursed in the proposed effort are to: (1) design and synthesize novel allosteric ligands with improved physicochemical and pharmacokinetic properties using in silico property predictions and computational docking studies with DAT-Tat complex models, (2) characterize the allosteric interaction of the compounds with human DAT in vitro to identify optimized compounds with improved physicochemical properties that can be used to alleviate Tat-induced dysfunction of DAT, and (3) determine the efficacy of selected compounds in attenuating Tat-mediated cognitive deficits and rewarding effects of cocaine in inducible Tat transgenic mice in vivo. This collaborative effort involves investigators with complementary expertise in medicinal chemistry, drug design, and biochemical and behavioral studies with the long-term goal of developing drugs for the treatment of HAND in HIV-positive patients.