University Of California, San Diego

NIH Research Projects · FY 2026 · 2014-08

Project Summary Cells respond to intracellular and extracellular challenges through intricate signaling pathways. An increasing number of studies revealed that the temporal dynamics of signaling pathways play crucial roles in controlling cellular processes. However, the basic mechanisms underlying the regulation and function of signaling dynamics remain largely unclear. Protein kinase A (PKA) is a highly conserved prototypic kinase that regulates many cellular behaviors, such as cell growth and stress resistance, through gene expression programs. Under physiological conditions, PKA displays various temporal dynamics of signaling activity. Defects in dynamic regulation of PKA activity can lead to disastrous diseases, such as neurodegeneration, cancer and heart disease. For over a decade, the research in our lab has been focused on understanding the regulation and function of PKA signaling dynamics upon stress. In this renewal, we propose to expand our research to study the mechanisms underlying dynamical regulation of PKA signaling in cellular aging and deterioration using budding yeast as a model system. Our preliminary results showed that PKA activity decreased in a fraction of aging cells and the interactions between PKA and the heme-activated protein (HAP) complex may drive this divergence of signaling dynamics among aging cells. Building upon these results, we will combine computational modeling with quantitative experiments to interrogate the regulatory circuit that drives the age- induced dynamic changes in PKA signaling. Specifically, in Aim 1, we will perturb the PKA-HAP circuit and evaluate the effects on PKA dynamics and their roles in regulating aging and lifespan. In Aim 2, we will evaluate the contributions of PKA dynamics to gene expression changes during aging. In Aim 3, we will investigate how PKA controls messenger ribonucleoprotein granules, e.g. processing bodies (P-bodies), to influence the posttranscriptional regulation of gene expression in aging. The completion of this project will further our basic understanding about the general mechanisms of PKA signaling in aging from a network perspective and will illuminate molecular causes of age-related diseases, setting the stage for new therapeutic strategies against them.

NIH Research Projects · FY 2025 · 2014-05

ABSTRACT: Nonalcoholic fatty liver disease (NAFLD), is a spectrum of liver disease ranging from steatosis (nonalcoholic fatty liver, NAFL) to non-alcoholic steatohepatitis (NASH) with fibrosis. Hepatic Stellate Cells (HSCs) play a critical role in the pathogenesis of NASH. In response to chronic toxic injury, quiescent HSCs (qHSCs) activate into aHSCs/myofibroblasts, that secrete the extracellular matrix to promote liver fibrosis. The mechanism of NASH-mediated activation of human HSCs is not well understood. Phenotypic changes in HSCs occur without a change in the DNA sequence but are regulated on an epigenetic level, e.g. specific modifications in the chromatin structure, which affect DNA accessibility of the regulatory transcription factors (TFs), causing transcriptional activation or repression of their target genes. We will analyze normal, NAFL, and NASH livers that have been declined for liver transplantation. We will compare our observations in human HSCs to the well characterized foz/foz mouse model of NASH. Our proposed study will integrate state-of-the- art single-cell-based technologies, a) Single cell (sc)RNA-Seq on purified human HSCs will identify major human HSC subsets; b) Single nuclei (sn)ATAC-Seq and snRNA-Seq will be performed using whole liver tissue to capture and characterize the areas of open chromatin and matching gene expression of individual HSCs; c) Transcriptional activity of the regulatory promoter/enhancer elements will be further accessed using PLAC-Seq followed by ChIP-Seq with H3K27ac, a mark associated with cellular activation (HiChIP-Seq). The transcriptome (AIM 1) and epigenome (AIM 2) of human HSCs, the genome-wide locations of the regulatory elements and their corresponding TFs that regulate distinct HSC phenotypes and drive NAFL®NASH progression, will be determined. Motif enrichment analysis of regions exhibiting characteristics of active enhancers in combination with gene expression data will enable inference of major classes of transcription factors critical for specific subsets of human HSCs. The factors that drive human HSC activation and thereby promote NAFL progression to NASH will be identified. Selected targets (AIM 3) will be evaluated using the experimental model of NASH in Western-diet (WD)-fed foz/foz mice, using ablation of individual aHSC subsets (via overexpression of Diphtheria toxin receptor (DTR) in Col1a1+ aHSCs in a Cre-loxP-dependent manner), or HSC-specific knockout of the key TFs. Specific factors that prevent or suppress HSC activation (for example, Etv1, E3F3, Egr2, NRF1, Tal1, Atf3) will be pharmacologically targeted, and the in vivo effect of treatment on Co1a1+ aHSC activation will be monitored in live WD-fed reporter LratCol1a1-Fluc foz/foz mice (that upregulate Col- 1a1-driven Luciferase in mouse aHSCs), or humanized patient-specific xenograft Rag2-/-gc-/- mice. Overall, we anticipate identifying new targets for the antifibrotic therapy of NASH.

NIH Research Projects · FY 2025 · 2014-05

Abstract The effects of alcohol use disorder are widespread throughout the brain. A fundamental knowledge gap in the field of alcohol research is the identification of the changes in whole-brain networks underlying the critical phases of the addiction cycle: intoxication, withdrawal, abstinence, and relapse. In the previous funding cycle, our lab started identifying these neuronal ensembles, their cellular phenotype, and their networks by focusing on a known region of interest, the central nucleus of the amygdala. While these efforts have been very successful, it has become clear that our research endeavor will benefit from moving away from the classical, biased region-of-interest-based approach to a modern, unbiased, whole-brain approach using single-cell whole-brain imaging. We believe that this innovative approach is required to identify the changes in whole- brain networks underlying the critical phases of the addiction cycle. The overarching goal of this renewal is to continue dissecting the neuronal ensembles of the volitional induction of alcohol dependence using single-cell whole-brain functional connectomics. We will use the rat model of ethanol vapor self-administration that we developed in the previous funding cycle to 1) identify the neuronal ensembles of alcohol intoxication, acute withdrawal, protracted abstinence, and stress-induced relapse and evaluate the contribution of CRF and dynorphin neurons to these networks, 2) compare pharmacological treatments on alteration of whole-brain functional networks and recruitment of CRF and dynorphin neurons, and 3) characterize the functional network of high-intensity binging in dependent rats. This project will provide unbiased brain atlases of the critical phases of the addiction cycle, identify the role of CRF and dynorphin neurons in these neuroadaptations, provide a mechanistic framework for the therapeutic effects of several pharmacological treatments, and identify the specific networks associated with high-intensity alcohol binging, an intensifying trend in alcohol drinking.

NIH Research Projects · FY 2026 · 2014-04

PROJECT SUMMARY The ventral tegmental area (VTA) is a core component of the neural circuitry that drives goal-directed behaviors and a primary target of drugs of abuse. Although generally regarded as a dopaminergic nucleus, many VTA neurons can signal through release of the neurotransmitters GABA and glutamate. These neurons are also targets of drugs of abuse but much less studied. Like VTA dopamine neurons, activity in VTA GABA and glutamate neurons can profoundly shape motivated behaviors. Furthermore, sub-populations of VTA neurons release more than one of these recycling transmitters, including neurons that co-release dopamine and glutamate, or glutamate and GABA. A major goal of this proposal is to identify how these three major transmitters released from VTA act on postsynaptic neurons to alter behavioral reinforcement. And to assess the contribution of non-dopamine VTA projection neurons and inputs to VTA in opioid-induced behaviors.

NIH Research Projects · FY 2024 · 2014-03

PROJECT SUMMARY A combination of entorhinal, hippocampal, and prefrontal pathology has a pivotal role in most neurological and neurodegenerative diseases and in the emergence of memory impairments that are associated with these diseases. Despite the knowledge that these brain regions are particularly vulnerable, the diversity of neural computations within and across these brain regions is only beginning to be revealed. For example, a key function of the hippocampus and medial entorhinal cortex (mEC) is to bridge events that are discontinuous in time, and entorhinal and hippocampal cells that are sequentially active (‘time cells’) have been proposed to be pivotal for memory retention over delay intervals of many seconds. In our previous work, we therefore investigated the firing patterns over the delay interval in a spatial working memory (WM) task. We unexpectedly found that hippocampal time cells were not a general mechanism for WM retention in hippocampus-dependent tasks. Rather, preliminary data indicate that information about past and future choices during the delay interval is evident during brief population bursts in hippocampus, while mEC cells may show memory-related activity that persists irrespective of brain state. We therefore hypothesize that memory retention does not require time-varying activity over the delay interval but is rather evident in sporadic population bursts in hippocampus and medial prefrontal cortex (mPFC) throughout the delay period and in firing patterns of entorhinal cells that provide continuity irrespective of brain state. To record activity during the delay period with varying brain states, we will – within each animal – use two variants of a spatial WM task, one with and one without forced running throughout the delay such that either theta or non-theta states are predominant. Aim 1 will focus on population bursts in hippocampus and determine whether they are only informative in non-theta states, as shown in our preliminary data, or a general mechanism across brain states. In addition, we will selectively interrupt hippocampal activity within the delay period to determine whether coding of future choices by population bursts and behavior are perturbed. Aim 2 will then perform recordings across deep and superficial layers and along the dorso-ventral axis of entorhinal cortex to identify how mEC contributes to WM. Finally, Aim 3 will carry out large-scale combined single-unit and LFP recordings in hippocampus and mPFC and in mEC and mPFC to reveal the coordination of mechanisms for WM retention across brain regions and brain states. Taken together, we will identify whether neuronal activity during population bursts is a general mechanism for memory retention over delay intervals irrespective of brain state, whether mEC supports WM retention with persistent activity, and how subregions of mPFC are coordinated with hippocampal and entorhinal subareas along the dorso-ventral axis. Identifying these memory computations will be the foundation for treatment approaches to restore memory functions when brain circuits deteriorate in neurological and neurodegenerative diseases.

NIH Research Projects · FY 2025 · 2013-09

Project Summary/Abstract Accurate, reliable and efficiently obtained structure determinations and annotations are of increasing importance in the current era of integrated omics research. Natural products continue to provide inspirational new structures for drug discovery programs, and genomic studies of microorganisms, including our work with marine cyanobacteria, have revealed that there are many times more secondary metabolites genetically encoded than are typically expressed in cultures. Building on this knowledge, new methods in elicitation and heterologous expression are successfully accessing this previously hidden genetic capacity for natural products biosynthesis. Metabolomic profiling interfaced with improved molecule annotations has richly informed on regulatory mechanisms of expression and the role of microbial interactions, especially including those involved in symbiotic interactions. This continuing NIH supported program has developed several of the most widely used annotation and analysis tools for MS and NMR natural product datasets, including GNPS, MASST, MassQL, SMART 2.1 NMR, DeepSAT, NP Classifier, NPOmix, SMART-Miner and PECAN, among others, and has led to 41 published papers in the previous 4-year grant cycle. These publicly available tools have changed the way that diverse scientists observe, organize, and make sense of complex metabolic profiling datasets from LC/MS and NMR analyses. For example, over the last 12 months, the GNPS ecosystem of tools has been used by >50K users from 156 countries and over the lifetime of the GNPS ecosystem, the community has analyzed over 25M LC/MS samples. SMART 2.1 NMR and DeepSAT have been used by 2.3K users in 66 countries. Collectively, these tools have democratized access to cutting edge computational approaches and are making an impactful difference in the way that researchers probe small molecule-based interactions of diverse life forms. In this next proposed grant period, we focus on machine learning and AI techniques to enhance the computational approaches we have pioneered and expand the breadth of problems within small molecule analysis. Cottrell and Gerwick will continue to develop annotation tools for even more precise predictions of structures and biological properties from NMR data, including improved mixture analysis. New member Wang will innovate on the integration of MS and NMR-based analyses to disambiguate closely related substances. Also, Wang will continue to leverage extensive computational infrastructure to ensure public and online access to the tools developed in this NIH program, democratizing their use and enhancing their impact. These overarching goals lead to five specific aims that will provide the community of diverse chemists and biologists a set of easily accessible tools for the improved identification of organic molecules and their properties by a variety of NMR and MS based methodologies. These tools will be based on the most current methods available for machine learning and artificial intelligence, thus successfully integrating computer science with analytical chemistry.

NIH Research Projects · FY 2025 · 2013-08

Project Summary Cardiomyocytes in the Drosophila melanogaster heart tube undergo progressive loss of their intercalated disc proteins and increased expression of extracellular matrix with age, resulting in reduced sarcomere lattice crystallinity and thus power stroke inefficiency; maintaining inter-myofilament spacing–by upregulating cardiac- specific expression of vinculin or downregulating matrix proteins–preserved heart wall velocity and extended life- and health-span in a mechanism conserved in non-human primates. Recently, we also showed that nuclei stiffen in aged hearts, reducing the amount of chromatin accessibility in key muscle transcription factors and leading to reduced cardiac output; when expression of the nucleoskeletal protein Lamin C–which decreases with age–is restored in aged cardiomyocytes, nuclei soften and become deformable again, prolong cardiac function, and maintain transcription factor expression, i.e., mechanogenetics. Most importantly in aged cardiomyocytes, we found that restoration of transcription factors alone was sufficient to maintain cardiac identity. While our prior studies have examined these structures separately, in this grant renewal application, we propose two further aims to investigate mechanical links between age-associated remodeling of sarcomere and nuclei. For aim 1, we will investigate age-associated changes in the structural connections between sarcomeres and nuclei, e.g., the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Preliminary data suggests that significant age-associated transcriptional and chromatin accessibility reductions in LINC genes MSP300 and Klar–which physically connect these structures–could underlie the maintenance of deformable nuclei, Lamin C and transcription factor expression with age, and the extension of lifespan. For a second aim, we have developed new engineering methods to ask a critical question of aging, namely: can “exercising” the sarcomeres restore transcription factor expression in aged hearts? Using mechanical and pharmacological perturbations, we will challenge aged hearts to maintain force transduction, which we hypothesize will maintain soft, deformable nuclei that will in turn preserve the chromatin accessibility of key muscle transcription factors. In light of the challenges that mechanical forces play on gene expression, we believe that this on-going work will continue to improve our understanding of how forces are required to maintain cardiac identity and ensure appropriate cardiac output in the aged heart.

NIH Research Projects · FY 2025 · 2013-07

This application responds to PAR-20-094 and seeks renewed R25 support for MH101072, UCSD’s “Psychiatry Residency Research Track” (RRT) to stimulate academic psychiatrists to conduct research into the causes and treatments of mental illness. This R25, first funded in 2013, was renewed in 2018 with a “10” priority score. In FY 6-9, 100% of the Residents who had “matched” into this RRT in FY1-5 graduated into “triple threat” positions: 6 are Faculty (5 with Career Development Awards plus significant private funding) and one is a PGY5 Fellow. Two “internal” trainees joined the RRT mid-Residency. This renewal application builds on the strengths of FY1-9 and the scientific expertise of UCSD faculty to sustain an RRT that is both substantive and innovative. In FY11-15, MH101072 will achieve the goals of the NIMH Council report, “Investing in the Future,” and of this FOA by expanding the pool of Psychiatric researchers - a key step towards improving treatment and outcome for mental disorders. Specific Aim: To recruit and educate the highest caliber future psychiatric researchers. A systematic and energetic recruitment process will match 2 outstanding young medical scientists per year into the RRT. These trainees will complete a 4-year program with increasing protected research time over the PGY2 (17%), PGY3 (40%) and PGY4 (70%), though core programs effectively extend these percentages. Clearly defined, developmentally appropriate “benchmarks” are set for each of the 4 training years and are achieved with careful mentorship from dedicated and accomplished full-time faculty. A primary research mentor is selected during the PGY1; the PGY2 starts by connecting trainees with their laboratory in a dedicated 2-block rotation, followed by longitudinal projects that span the PGY2. PGY3-4 seminars provide solid grounding in research design, data analysis, manuscript preparation and responsible conduct of research. Trainees learn academic survival skills and receive thoughtful supervision and personalized career mentorship. The core curriculum, detailed in this application, strengthens and sustains trainees' career connection with mental health research and the core missions of the NIMH. Procedures are in place for frequent, careful evaluations of trainees, mentors and all major aspects of the RRT program. Oversight of the RRT is provided by engaged internal and external advisory boards. In FY11-15, innovative changes will solidify trainee research funding, build vertical cohesiveness across the RRT and engage RRT graduates in early career mentorship activities. With renewed support, MH101072 will admit 10 new RRT trainees, graduate 9 trainees (5 current + 4 new), leaving 8 trainees “in the pipeline” to complete their training via renewed R25 or DoP support. This will bring the total yield of MH101072 (FY1-15) to 30 RRT graduates. This RRT will provide individualized oversight of each trainee in an innovative format that will accelerate their paths towards faculty positions and career research funding and thereby achieve the goals of this NIMH program.

NIH Research Projects · FY 2026 · 2013-05

PROJECT SUMMARY/ABSTRACT This application requests five additional years for the University of California, San Diego (UCSD) Training Grant in Rheumatology to continue to fulfill its goal of preparing pre- and post-doctoral fellows for careers as independent investigators in academia, conducting cutting-edge basic and/or translational rheumatology disease-related research. We are requesting 3 postdoctoral training slots (for MD, MD/PhD, MD/MPH, and/or PhD researchers in bench laboratory or clinical-translational investigation), and 3 slots for predoctoral students (for dual MD/PhD and/or single PhD students, all to be trained in basic-translational research in the UCSD/La Jolla Institute for Allergy and Immunology (LJI) Biomedical sciences Program in Immunology). The training program will consist of a research experience typically of 24 months, and up to 36 months. We re-imagined and re-energized our program by implementing a series of innovations for this renewal application that allow us to prepare our trainees for the research workforce of tomorrow. Rather than traditional research approaches, we will embed critical skills like team science, community engagement and dissemination and implementation science into each trainee project. The 48-participating faculty will build multi-disciplinary training teams that will include in this cycle a clinical or basic research mentor, one computational mentor, together with one Rheumatology clinician and one junior mentor-in-training for all trainees. This will emphasize collaboration, help nurture translational thinking and team science in the trainees, help access patient cohorts and samples, and develop the mentoring skills of junior faculty. This exposure to “team science” together with the mandatory coursework will help the trainees learn how to interact with colleagues, and share in the contributions and credit for interesting research programs. Of important note, to address health disparities and unmet needs, this program will focus on bi-directional communication between trainees and the community, will include a community member on the mentor team, will embed interactions with the community in planning studies and assessing impacts, as well as a Dissemination and Implementation plan. To promote creation of multi-disciplinary research teams, we have added new well-funded, outstanding primary mentors, with a strong emphasis on diversity and leaders focused on population science and community engagement. We have also re-organized our program around 2 major themes integrated with our central focus on translational rheumatic diseases research that represent strengths in our research community: (1) Translational Immunology, Joint Biology, Mucosal Immunobiology and Metabiomics, and (2) Epidemiology, Biomedical Informatics and Computational Biology. We will also emphasize recruiting and training outstanding independent researchers who help develop evidence-based treatments in routine care and expand collaboration with our community to improve health dissemination in the community.

NIH Research Projects · FY 2025 · 2013-01

Project Summary/Abstract The long-term goal of this project is to understand how DNA lesions are recognized and repaired in the actively transcribed genome. Harmful DNA lesions, caused by endogenous and environmental agents, must be promptly recognized and repaired in order to avoid deleterious threats to genome integrity. Transcription- coupled nucleotide excision repair (TC-NER) is an important DNA repair pathway as it removes DNA lesions within the transcribed genome. However, little is known about the molecular mechanism of eukaryotic TC-NER initiation. Cockayne Syndrome B protein (CSB), a master TC-NER coordinator, is recruited to the DNA lesion- arrested Pol II site and plays a key role in the initiation of eukaryotic TC-NER. Previously, we reported the first yeast Pol II-Rad26/CSB ternary complex structure, shedding new lights on this important process. However, there is still a fundamental knowledge gap in understanding what happens after CSB recruitment to the DNA lesion-arrested Pol II. Several long-standing questions in the field remain unanswered. First, how does CSB use its DNA translocase activity to remodel the DNA lesion-arrested Pol II and switch Pol II from the transcription elongation mode to the repair mode that leads to the initiation of TC-NER? Second, how is the DNA lesion-arrested Pol II moved away from the DNA lesion to allow the access of repair proteins during TC- NER initiation? Third, are there any missing TC-NER factors that remain to be discovered? If so, how do they fit into this decades-old puzzle? The objective of this proposal is to address these key mechanistic questions in TC-NER initiation. We propose to tackle these challenging questions with an innovative hybrid approach that combines X-ray crystallography, Cryo-EM, computational biology, biochemistry, genetic, and genomic methods. We hypothesize that CSB plays important roles in remodeling lesion-arrested Pol II and coordinates the displacement of elongation factors and Pol II with other repair factors to promote downstream lesion verification steps during the initiation of TC-NER. To test this hypothesis, we propose to investigate the functional interplays between lesion-arrested Pol II complex, Rad26/CSB, and other transcription/repair factors. We propose to elucidate the molecular basis of the enigmatic mechanism of TC-NER initiation. We expect to determine key protein complexes involved in the initiation of TC-NER. Our project has three Specific Aims: Aim 1: Determine the molecular basis of the interplay between Rad26/CSB, Spt4/5, and the DNA lesion- arrested Pol II complex. Aim 2: Elucidate the role of Elf1 in the initiation of TC-NER. Aim 3: Investigate how the lesion-arrested Pol II is displaced during TC-NER initiation. The proposed research is significant and groundbreaking because novel knowledge and structures obtained from this proposal will have a transformative impact on the field of DNA repair field. Ultimately, such knowledge will provide a framework for developing novel TC-NER targeting therapeutics against cancer and other human diseases.

NIH Research Projects · FY 2026 · 2012-12

Project Summary This proposal outlines plans for the next generation NIDDK Information Network (dkNET), whose goal is to provide a centralized resource that connects the National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK) research community to the growing number of biomedical resources (e.g., organisms, reagents, materials, protocols), data, and bioinformatics tools available to them through the multiple programs and centers established by NIDDK and the broader scientific community. dkNET has seen steady growth over the last project period and achieved significant success toward our mission, highlighted by 4 main areas of impact: 1) provided a novel Resource Information Network (RIN) that connects the NIDDK research community to the expanding universe of biomedical resources and data; 2) significantly improved the rigor and reproducibility of published research through its leadership in creating and promoting adoption of Research Resource Identifiers (RRIDs); 3) increased computational skills among the NIDDK workforce through educational programs, the dkNET Bioinformatics Pilot program, the Hypothesis Center, and the D-Challenge; and 4) brought FAIR (Findable, Accessible, Interoperable, Re-usable) data principles to DK researchers through educational materials, a webinar series, and the Summer of Data student program. dkNET tools, resources, and training materials assist researchers throughout the experimental process from hypothesis generation to supporting sound and reproducible science. Building on the success of dkNET (Resource Core) we will extend its services to the community through a new Computational Core that will bring powerful new AI/ML techniques and cloud computing resources to the NIDDK research community to fully leverage data assets cataloged by dkNET to explore and develop hypotheses. Our objective is to create a unified ML paradigm for performing a diverse range of analytical tasks. This paradigm will have the ability to process the various data types cataloged by dkNET; integrate data of multiple modalities from different sources; and incorporate domain-specific knowledge from public knowledge bases. The resulting knowledge model from this paradigm will facilitate hypothesis validation and recommendation by locating data that supports or contradicts a hypothesis/task and evaluating a given hypothesis’s likelihood.

NIH Research Projects · FY 2025 · 2012-09

Excessive alcohol drinking Initiated during adolescence is known to disturb typical neurodevelopmental patterns, increase the risk of developing alcohol use disorder (AUD), and accelerate involutional processes in adulthood. In response to RFA-AA-21-008, the Administrative Resource (AR) proposes to coordinate activities of the National Consortium on Alcohol and Neurodevelopment in Adolescence - Adulthood (NCANDA-A) to follow for the next 5 years a community sample of male and female participants recruited in three age bands (12-14, 15-17, 18-21 years old) as mostly no-to-low drinkers, tracked over the last 8 years across 5 sites (N=831; 93% retention rate). This consortium reflects seven applications: NCANDA - Administrative Resource (UCSD) and NCANDA - Data Analysis Resource (SRI), and five cross-national Research Project Sites, located at Duke University (Duke), Oregon Health Science University (OHSU), University of Pittsburgh (Pitt), SRI International (SRI), and UC San Diego (UCSD). Monitoring has involved annually multimodal neuroimaging (MRI, DTI, resting state fMRI, task fMRI), cognitive, clinical, behavioral, and biological data, collected in person or remotely by computer and our mobile app. These measures will now be complemented with new advanced neuroimaging and sleep and physical activity tracking. Our accelerated longitudinal design uniquely positions NCANDA-A (ages 18-34) to quantify transient or enduring alcohol-related disturbances in adolescent and early adult neural system growth trajectories and functional concomitants. NCANDA-A proposes four consortium-wide specific aims and two specialty project aims. In Aim 1, we investigate the impact of excessive alcohol drinking during adolescence and emerging adulthood on subsequent developmental trajectories of cognitive performance, brain structure and function, and psychopathology. Aim 2 identifies the extent to which alcohol’s effects on brain structure and function resolve or persist during desistance of binge drinking. Aim 3 identifies adolescent biological, environmental, and behavioral factors that forecast excessive drinking during early adulthood. Aim 4 quantifies the impact of a stressful time for many youths during 2020-2021 on alcohol use, stress, and wellbeing. Aim 5 (SRI and Pittsburgh sites) identifies interactions among alcohol use, sleep, and cardiac function. Aim 6 (UCSD, Duke and OHSU sites) determines the extent to which short-term (i.e., 4 weeks) alcohol use discontinuation results in acute improvement in cognition, affect, sleep and resting heart rate, and reversal of structural and functional brain effects of binge alcohol use. For each aim, sex differences will be tested. With longitudinal data collected into early adulthood during this renewal, NCANDA-A will provide novel information on the enduring and transient effects of adolescent drinking on adult functioning by discovering elements and mechanisms linking these dynamic processes and identifying modifiable risk factors.

NIH Research Projects · FY 2025 · 2012-09

Initiating excessive alcohol drinking during adolescence is known to disturb typical neurodevelopmental patterns, increase the risk of developing alcohol use disorder (AUD), and accelerate involutional processes in adulthood. In response to RFA-AA-21-007, this application proposes a Research Project Site of the National Consortium on Alcohol and Neurodevelopment in Adolescence - Adulthood (NCANDA-A) to follow for the next 5 years a community sample of male and female participants recruited in three age bands (12-14, 15-17, 18-21 years old) when most were no-to-low drinkers and tracked over the last 8 years across 5 sites (N=831; 93% retention rate). Monitoring has involved annually acquired multimodal neuroimaging (MRI, DTI, resting state fMRI, task fMRI), cognitive, clinical, behavioral, and biological data, collected in person or remotely by computer and our mobile app. These measures will now be complemented with new advanced neuroimaging and sleep and physical activity tracking. This cohort sequential design uniquely positions NCANDA-A to quantify transient or enduring alcohol-related disturbances in specific adolescent and early adult neural system growth trajectories and functional concomitants. NCANDA-A proposes four consortium-wide specific aims and two specialty project aims. In Aim 1, NCANDA-A will investigate the impact of excessive alcohol drinking during adolescence and emerging adulthood on subsequent developmental trajectories of cognitive performance, brain structure and function, and psychopathology. Aim 2 analyses will identify neurodevelopment patterns describing the extent to which alcohol’s effects on brain structure and function resolve or persist during desistance after binge drinking. Aim 3 will deploy data-driven analysis to identify adolescent biological, environmental, and behavioral factors (e.g., age of drinking onset) that forecast excessive drinking during early adulthood. In Aim 4, NCANDA-A will quantify the impact of a stressful time for many youths during 2020-2021 on life stress and social, emotional, and economic wellbeing and their relations with alcohol use patterns. In Aim 5, the SRI and Pittsburgh sites will identify interactions among patterns of alcohol use, sleep, and cardiac function. In Aim 6, the UCSD, Duke and OHSU sites will determine the extent to which short-term (i.e., 4 weeks) alcohol use discontinuation results in acute improvement in cognition, affect, sleep and resting heart rate, and reversal of the adverse structural and functional brain effects of frequent binge alcohol use. For each aim, sex differences in development, alcohol use patterns and history, impact of alcohol use on the brain, and sex-differentiating psychosocial factors will be tested. With the longitudinal data collected into early adulthood during this renewal, NCANDA-A will provide novel information to the public on the enduring and transient effects of adolescent drinking on adult functioning by discovering elements and mechanisms linking these dynamic processes and identifying modifiable risk factors.

NIH Research Projects · FY 2024 · 2012-08

Atherosclerosis is a multi-faceted vascular disease that involves maladaptation of several cell types in the arterial wall responding to systemic and local factors. During the last two funding cycles, we have used bioinformatics and system biology approaches together with in vitro and in vivo experimental validations to study the cellular and molecular mechanisms by which atheroprotective and atheroprone flows regulate the vascular endothelial cell (EC) in health and disease. Our results demonstrate the crucial roles of flow-regulated EC epigenomes and transcriptomes in the atheroprotective and athero-prone phenotypes. Emerging evidence suggests that the focal nature of atherosclerosis is linked to EC heterogeneity resulting from interplay between intrinsic EC properties and extrinsic shear forces. To further advance our understanding on EC heterogeneity in relation to atherosclerosis, we hypothesize that mediators (e.g., MED-1) coordinate with lineage-dependent transcription factors (LDTFs, e.g., KLF4) and signal-dependent transcription factors (SDTFs, e.g., SMAD2) to regulate the spatiotemporal networks of mechanotransduction. The five specific aims proposed to test this novel hypothesis are: Aim 1. To delineate the spatiotemporal changes in flow-mediated EC epigenomes and transcriptomes with single-cell resolution; Aim 2. To elucidate the effect of shear stress on interactions between ECs and vascular smooth muscle cells (SMCs) or macrophages (MØs) with spatial resolution; Aim 3. To characterize the transcriptomes and the regulating epigenomes in the arterial wall in vivo with spatial resolution; Aim 4. To employ system biology approaches to compute and integrate data for the construction of temporal and spatial regulatory networks; Aim 5. To validate the shear stress-regulated EC heterogeneity at the disease level using mouse atherosclerosis models and human artery disease specimens. With the use of multi-omics platform at single-cell resolutions, this renewal proposal will decipher the shear stress regulations of the EC heterogeneity and the consequential phenotypical changes of ECs and neighboring cell types (SMCs and MØs) relevant to atherosclerosis.

NIH Research Projects · FY 2025 · 2012-07

Project Summary One of the most frequently discussed challenges facing the auditory system is parsing the voice of a single speaker in a noisy acoustic environments comprising multiple other speakers, commonly referred to as the Cocktail Party Problem (CPP). Despite the significance of the CPP to illustrate the complex nuances of audition that unfold in real-world situations, remarkably little remains known about the supporting neural mechanisms at different levels of the auditory circuit. This gap in our knowledge has emerged, at least in part, due to a lack of a behavioral paradigm in animal models that both recreated the natural complexities and dynamics of natural cocktail parties, while at the same time affording experimental control to manipulate the acoustic scene. Here we seek to bridge this gap by leveraging a novel, multi-speaker behavioral paradigm in a series of complementary experiments to investigate the perceptual and neural mechanisms that are employed in the primate brain to resolve the CPP for natural communication in freely-moving common marmosets. Aim 1 establishes an innovative behavioral paradigm that recreates the natural dynamics of real-world cocktail parties by creating an acoustic scene comprising multiple computer-generated virtual monkeys (VM) whose individual identity and spatial position can be independently and systematically manipulated. Each marmoset placed in these cocktail parties naturally learn the identity of the interactive VM based on idiosyncrasies of their voice and vocal behavior and selectively engage them in conversations, while ignoring the distractor VMs engaged in conversations with each other. Experiments in Aim 2 involve performing simultaneous neurophysiological recordings of prefrontal cortex and hippocampus in marmoset monkeys as they communicate in a cocktail party described in Aim 1. Specifically, experiments are designed to test the respective and complementary roles of these neural substrates in the auditory representing the identity and spatial position of individuals in the CPE. Because of the respective role of prefrontal cortex in attention and hippocampus for allocentric representations of space, these substrates are hypothesized to be critical to resolving the CPP in primates. Aim 3 aims to build on the complementary conceptual and experimental innovations to more directly interrogate the perceptual and neural mechanisms that support communicating in a cocktail party. Specifically we will a cutting-edge closed-loop feedback paradigm that uses machine learning algorithms to optimize the conversational dynamics between the interactive VM and live marmoset in a multi-speaker cocktail party. This model will selectively manipulate targeted features within the ongoing natural conversations to directly interrogate the system and determine the functional role of behavioral parameters and related neural processes.

NIH Research Projects · FY 2025 · 2012-07

Abstract The San Diego Biomedical Informatics Education & Research (SABER) training program was awarded an NLM training grant in the summer of 2012 and renewed in 2017. In this renewal proposal, we describe our accomplishments for the past 8.75 years and our vision for the next five years. The SABER training program includes core faculty from the University of California San Diego (UCSD) Health Department of Biomedical Informatics, as well as multiple faculty across UCSD. Since the start of this program in 2012, we have hosted 78 long- and short-term trainees. Our pre-doctoral trainees enroll in competitive doctoral programs in Bioinformatics and Systems Biology (BISB), Computer Science, Cognitive Science, or Biomedical Sciences. Trainees complete a foundational curriculum in biomedical informatics (BMI), taught by our core faculty, in addition to core curricula from their respective degree programs. All of our trainees have been meeting their academic milestones and 11 have graduated. Our post- doctoral trainees enroll in a master’s program (Computer Science or Advanced Sciences), in addition to completing our foundational BMI curriculum. These trainees are embedded in projects at the medical center that help them contextualize the research methods they learn through coursework. All of our short-term trainees are under-represented minorities in science and participate in various research projects leading to presentations and publications. Most trainees who completed our BMI training have research positions in industry and academia— a few are continuing their training, two are employed in non-profit organizations, and several became assistant professors at various institutions (Carnegie Mellon, UCLA, UCSF, UCSD, Vanderbilt, etc.). Some highlights of our training in the past five years have been the high productivity of our trainees, who are pursuing innovative research in a wide spectrum of informatics areas of specialization, from translational bioinformatics to clinical research informatics to healthcare informatics. Additionally, the proportion of women (57%) and under-represented minorities (49%) is high in our overall trainee pool. We have filled all of our slots each year with highly qualified candidates from diverse educational and cultural backgrounds, who have already produced first-author publications in high impact journals (e.g., Cell, Science, JAMIA, PNAS) and conferences.

NIH Research Projects · FY 2025 · 2012-04

Lung cancer is one of the most lethal of solid tumors. The overall survival rate for patients with lung cancer remains low, at 21%. As 236,740 new cases of lung cancer and 130,180 deaths are expected in 2022, there remains a pressing need to advance research into new therapeutic approaches for the treatment of lung cancer. Solid tumors, such as lung tumors, are laden with abundant, tumor-promoting macrophages, monocytes and granulocytes. These innate immune cells promote tumor progression through profound inhibition of T cell recruitment and activation and through stimulation of tumor angiogenesis, stemness, drug resistance and metastasis. Strategies that reduce the accumulation of myeloid cells or alter their functional properties significantly slow or eliminate tumor progression in animal models of cancer and synergize with other therapeutic approaches to improve cancer outcomes. We previously discovered that a myeloid cell specific isoform of phosphatidylinositol-4,5-bisphosphate 3-kinase, PI3Kgamma (PI3Kg), controls both myeloid cell accumulation and immune suppressive polarization in tumors. Genetic or pharmacological inactivation of PI3Kg, but not other PI3K isoforms, reduces myeloid cell accumulation in tumors and alters the transcriptional profile of immune suppressive, pro-angiogenic tumor associated macrophages (TAMs) toward a pro-inflammatory, anti- tumor phenotype. We have found that PI3Kg inhibition synergizes with chemotherapy and with checkpoint inhibitors to activate memory T cells and reduce tumor growth in models of lung cancer, breast cancer, melanoma, head and neck cancer and glioblastoma. Based on our findings, the PI3Kg inhibitor, IPI-549 (eganelisib), was developed as an immune oncology therapeutic. We identified signatures of re-activated adaptive immune responses in tumor tissues from lung cancer patients who participated in IPI-549 Phase 1 clinical trials, indicating that PI3Kg blockade can re-awaken anti-tumor immunity in human tumors and thus may provide therapeutic benefit to lung cancer patients. However, we also identified pathways of resistance to therapy, indicating that further dissection of the roles that PI3Kg and its inhibitors play in lung tumors is warranted. The exact biochemical and cellular mechanisms by which PI3Kg inhibits anti-tumor immunity in murine and human lung cancer remain unclear. Deciphering these mechanisms will provide new therapeutic insights into the mechanisms of tumor immune suppression. Therefore, we propose studies to determine which molecular and cellular mechanisms are direct and indirect targets of PI3Kg mediated immune suppression, to identify and treat pathways of resistance to PI3Kg inhibitor therapy and to evaluate the impact of PI3Kg inhibition on immune responses in mouse and human models of NSCLC. The specific aims to accomplish these goals are: 1) To delineate molecular mechanisms by which PI3Kg regulates macrophage transcription; 2) To determine mechanisms by which PI3Kg controls lung tumor progression in vivo; 3) To discover and target mechanisms of resistance to PI3Kg inhibition in lung carcinoma.

NIH Research Projects · FY 2025 · 2012-03

Project Summary. Natural products from non-ribosomal peptide synthetases, polyketide synthases, and their hybrid pathways serve as therapeutics for infectious diseases, immunosuppression, anti-inflammatory regulation, antifungal and antiparasitic applications. Given their complexity and robustness, these metabolic pathways are excellent starting points for molecular design and production, particularly given the promise of synthetic biology for biomanufacturing new molecular entities. However, we do not fully understand the mechanics and organization that regulates these multi-modular and multi-domain catalytic machines. Using both model systems and clinically relevant biosynthetic pathways, our team will explore the use of peptidyl carrier protein (PCP) crosslinking enabled through recently developed chemical biology methods. We will focus on elucidating structural information about protein-protein interactions between PCPs and ketosynthase, condensation, and thioesterase catalytic domains to elucidate the molecular mechanisms and structural requirements that guide biosynthesis. Using in silico molecular modeling, we will apply these findings toward the in vitro evolution of new PCP-enzyme arrangements capable of catalyzing the biosynthesis of novel molecules. Our team combines chemical biological probe development with NMR, X-ray crystallographic and single particle cryo-EM structural biology to develop a computationally tested understanding of the protein-protein interfaces and mechanisms that guide substrate processivity within carrier protein dependent biosynthesis.

NIH Research Projects · FY 2024 · 2011-09

ABSTRACT Asthma affects over 300 million individuals worldwide. Uncontrolled asthma is associated with a doubling of direct costs, missed workdays, and school absenteeism; up to 80% of asthma patients may be uncontrolled with regard to asthma symptoms. Circulating microRNAs (miRNAs) are stable intercellular communicators that function to regulate gene expression and can serve as biomarkers for symptoms and disease. The overall objective of this study is to ascertain the role of circulating miRNA and miRNA genetics in control of asthma symptoms. In this competitive renewal proposal, “Genomics and Pharmacogenomics of Symptoms in Asthma”, we hypothesize that circulating miRNAs are functionally associated with asthma treatment guidelines- based symptoms control, symptomatic exacerbations, and symptoms response to therapy. Asthma symptoms will be defined as well-controlled, poorly controlled, and uncontrolled via Global Initiative for Asthma (GINA) guidelines and by frequency of severe exacerbations. The relevant miRNAs will be identified via three specific aims. The first aim seeks to identify differentially expressed miRNAs influencing asthma symptoms control through miRNA sequencing of serum samples from multiple asthma cohorts totaling over 4000 subjects. Cross sectional studies will identify miRNAs indicative of mechanistic differences between the controlled and uncontrolled asthmatic, while pharmacogenomic and longitudinal studies will identify miRNAs that provide biologic insights and may form the basis of a predictive biomarker test for identification and treatment of the difficult to control asthmatic. The second aim evaluates the role of miRNA genetics in asthma symptoms control through identification of polymorphisms that affect miRNA expression (via allele specific expression) and those affecting miRNA biogenesis. The salient variants will be identified via whole genome sequencing in over 3500 asthmatic subjects and through whole genome imputed genome-wide association data in over 30,000 asthmatic subjects. The genetic variants will be associated with symptoms control and assessed for ability to predict ongoing symptoms and drug treatment response. The final aim will functionally link the miRNA and genetic variants, including miR-130a-3p and let-7b-5p, from Aims 1 and 2 with physiologic and inflammatory changes in human airway epithelial cells, using miRNA mimics, miRNA inhibitors, and CRISPR-based gene editing. The success of this proposal will yield novel understanding into the pathogenesis of asthma symptoms control and asthma exacerbations and may provide direct future bedside clinical translation in the form of biomarkers to enhance guidelines based therapeutic recommendations and/or miRNA based therapeutics.

NIH Research Projects · FY 2025 · 2011-09

Project Summary Alcohol abuse and alcohol-related diseases are a major medical burden in industrialized countries. Chronic alcoholism is associated with changes in the intestinal microbiota, increases in intestinal permeability, and elevated systemic levels of bacterial products. We demonstrated that Enterococcus faecalis (E. faecalis) is sufficient to cause mild steatotic liver disease and to exacerbate ethanol-induced liver disease in mice. We identified cytolysin, a two-subunit exotoxin secreted by E. faecalis, to cause hepatocyte death and liver injury. Compared with controls, patients with alcohol use disorder or alcoholic hepatitis have increased fecal numbers of E. faecalis. The presence of cytolysin-positive (cytolytic) E. faecalis correlated with liver disease severity and mortality in patients with alcoholic hepatitis. How chronic alcohol use results in increased intestinal and hepatic numbers of cytolysin-positive E. faecalis is not known. Results from our laboratory suggest that increased intestinal numbers of E. faecalis are facilitated by changes in the intestinal glycocalyx and in particular by reduced (1,2)-fucosylation of glycoproteins on the apical membrane of intestinal epithelial cells. Alcohol- mediated suppression of Fucosyltransferase 2 (Fut2) allows intestinal colonization and bacterial translocation of E. faecalis. Furthermore, translocated E. faecalis is phagocytosed and eliminated by the complement receptor of the immunoglobulin superfamily (CRIg) on Kupffer cells. Patients with chronic alcoholic hepatitis have lower hepatic CRIg expression, which reduces E. faecalis elimination, prolongs exposure to E. faecalis and increases liver damage. Thus, ethanol associated changes in intestinal colonization and hepatic elimination of E. faecalis promotes alcohol-related liver disease. Our experimental approach is to use mouse models of ethanol feeding to investigate the role of Fut2 in limiting intestinal colonization of E. faecalis and reducing liver disease (Aim 1). We will also assess the functional contribution of the phagocytic protein CRIg to E. faecalis elimination and to liver disease (Aim 2). New strategies will be tested to prevent and ameliorate ethanol-induced liver disease in preclinical models. We believe these studies will provide novel insights into the contribution of the microbiota to alcohol-related liver disease.

NIH Research Projects · FY 2025 · 2011-07

PROJECT SUMMARY/ABSTRACT Interdisciplinary clinical research on the combined effects of substance abuse and HIV disease on the central nervous system (CNS) is of considerable public health importance. Despite this, few laboratories address this critical topic and the current training program is the only one dedicated to preparing the next generation of investigators to conduct this research. In this proposed second renewal of our NRSA T32 training grant, “Training in Research on Addictions in Interdisciplinary NeuroAIDS (TRAIN),” we aim to accelerate our contribution to the field. We will continue to recruit talented and diverse pre- and post- doctoral trainees whose academic development will be furthered by an accomplished multidisciplinary team of mentors and the extensive academic resources at UC San Diego. TRAIN has been highly successful in the current funding period–since 2016, the 8 trainees produced 55 research manuscripts (22 first-authored), 88 conference presentations, and 6 book chapters, as well as 2 extramural NIH-funded grants (1 under review, impact score=21, 2nd percentile), 3 supplements to NIH-funded grants, and 2 intramural pilot grants. In this second renewal, we plan to enhance our training grant by augmenting faculty expertise, didactics, and training in new methodologies to support our new complementary theme of the complex biological mechanisms underlying the CNS consequences of HIV and substance use. Factors of particular focus include the microbiome and gut brain-axis, systemic and CNS inflammation, neuroimmunology, and biomarkers. This focus will augment TRAIN continuity themes of neurobehavioral functioning (e.g., frontal systems behaviors, decision-making, memory) and health-related everyday functioning (e.g., treatment adherence, vocational outcomes). Given our previous success and the growing demands of the field, we propose to increase our steady state number of trainees to four pre- and three post-doctoral trainees. Our students and fellows will be actively engaged in individualized career development plans, such as applied research training, didactics, and targeted clinical experiences. TRAIN faculty, all with strong training interests and robust histories of collaborative research, consists of 25 primary mentors across multiple academic levels and disciplines, with considerable expertise across the aforementioned scientific themes. We also have made organizational enhancements (e.g., addition of internal TRAIN committees) to further ensure continued progress towards the accomplishment of trainee goals. TRAIN is led by R. Heaton, Co- Directors I. Grant and D. Moore (new to this role), and Associate Directors J. Iudicello and E. Morgan; this team will oversee all training, scientific, and administrative aspects of the program. TRAIN will be housed at the HNRP-CMCR within the Department of Psychiatry, which is a resource-rich research and training environment in substance abuse and neuroHIV research. Our central goal remains unchanged: to train, mentor, and guide the next generation of researchers on combined CNS effects of HIV and substance use.

NIH Research Projects · FY 2025 · 2011-03

Prion diseases are relentlessly progressive neurodegenerative disorders with death often within six months of the onset of neurologic symptoms. Pathologic features include widespread extracellular prion aggregates, spongiform degeneration, synaptic and neuronal loss, and severe astrogliosis and microgliosis. The structural determinants of the prion protein (PrP) and endogenous co-factors that drive aggregation, govern prion assembly, and impact aggregate spread through the central nervous system are unclear. A major goal of this application is to define when and how the endogenous co-factor, heparan sulfate (HS), promotes fibril assembly in the parenchyma and blood vessels and slows PrP clearance through the interstitial fluid using in vitro and in vivo model systems. We have previously pursued a range of approaches using cell-based prion conversion assays and newly generated transgenic and knock-in mouse models in collaboration with structural biologists to define the mechanisms that underlie PrP self-assembly and species barriers to prion conversion. We discovered using knock-in mouse models that N-linked glycans on PrP reduce spongiform degeneration, hinder plaque formation, and repel HS binding. Further, we found that plaque-forming prions were composed of poorly glycosylated, GPI-anchorless PrP bound to highly sulfated HS, underscoring the pivotal role of PrP post- translational modifications in driving the aggregate conformation and disease phenotype. We also found that reducing HS chain length decreases parenchymal plaque formation and prolongs survival. Finally, we identified highly amyloidogenic segments in the PrP sequence that control cross species prion conversion, as the number and location of glutamine and asparagine residues in PrP raise or lower the prion transmission barrier. In this renewal, we aim to determine the PrP-HS interactions that promote prion aggregate assembly and accelerate disease. We build on our long-standing observation that structural features of PrP, together with host glycosaminoglycans, drive efficient prion conversion. First, we will genetically manipulate neuronal, astrocytic, and endothelial HS chains and determine the impact on prion cell targets and survival using mouse models. Second, we will define how endogenous HS regulates PrP clearance through the interstitial fluid using conditional HS mouse models and radiolabeled PrP. Third, we will test the efficacy of antisense oligonucleotides (ASOs) targeting HS biosynthetic enzymes or Prnp mRNA in the early and mid-stages of prion plaque development in a prion disease model. We expect these mechanistic studies will (i) define how an endogenous co-factor, HS, accelerates and modifies prion disease, and (ii) determine whether reducing PrP interactions with this potential therapeutic target blocks prion spread.

NIH Research Projects · FY 2026 · 2010-05

PROJECT SUMMARY To ensure that each dividing cell receives a complete set of the correct genome, cell cycle checkpoints are in place throughout the cell cycle. Yet, less is known about whether similar checkpoints exist for division of the cytoplasmic components or organelles of the cell. The endoplasmic reticulum (ER) is a gateway for the secretory pathway, generating almost all of the secreted and cell surface membrane proteins as well as synthesizing cellular lipids. Previously, we identified a cell cycle checkpoint, termed the ER stress surveillance (ERSU) pathway, that ensures the inheritance of sufficient levels of functional ER in the model organism S. cerevisiae. In response to ER stress, the ERSU pathway (1) blocks the inheritance of the stressed ER into the daughter cell, (2) mislocalizes the septin ring from the bud neck, the site of cytokinesis, and ultimately, (3) leads to temporary cell cycle arrest at cytokinesis until ER functional homeostasis is re-established. Cells that lack components of the ERSU, and thus cannot mount the ERSU pathway, die upon ER stress, underscoring the importance of this checkpoint. The ERSU pathway is distinct from the well-studied unfolded protein response. We have found that levels of phytosphingosine (PHS), an early intermediate of sphingolipid biosynthesis, increase upon ER stress, setting in motion the ERSU hallmark events. Moreover, we defined a PHS binding motif that is found within two different transmembrane domains of reticulon family proteins (i.e., Rtn1 and Yop1), leading to the activation of the ERSU events. In the current proposal, in AIM 1, we will apply molecular and cell biological approaches to dissect how PHS binding to Rtn1 or Yop1 results in ERSU activation. In AIM 2, we will extend the scope of the ERSU by dissecting the impact of ER lipotoxic stress induced by ER morphological changes, on the ERSU molecular events. In AIM 3, we will investigate how ER stress impacts the mammalian cell cycle. As the nuclear membrane breaks down during mitosis in mammalian cells, the division of functional ER may be even more tightly choreographed with the nuclear mitotic mechanisms. Our preliminary result of a mammalian septin subunit holds great promise for the presence of regulatory events that may share similarity to the yeast ERSU. Thus, we will fully investigate the impact of ER stress on (A) the mammalian septin subunits and cytokinetic components, and (B) major mitotic cell cycle structural changes that involve the ER, such as ER clearing from the “mitotic exclusion zone” and nuclear disassembly and reassembly. Understanding the molecular mechanisms that integrate ER homeostasis with cell cycle events will provide unprecedented insights into human diseases caused by the failure of ER regulation. .

NIH Research Projects · FY 2024 · 2009-08

Project Summary / Abstract Natural products continue to provide important drug leads in medicine. While the majority of clinical antibiotics are derived from bacterial natural products, the emergence of antibiotic resistance emphasizes the need to discover new antimicrobial leads. This renewal application builds upon a productive collaboration between the Jensen (microbiology/bioinformatics) and Moore (biosynthesis/natural products chemistry) laboratories to address this need through the mining of microbial genomes and metagenomes for new bioactive compounds. We have prioritized two diverse groups of chemically gifted marine bacteria for study from a collection of >10,000 strains collected across the world’s oceans. We continue our efforts with the obligate marine actinomycete Salinispora, which produces the potent proteasome inhibitor salinosporamide A (Marizomib) that is presently in phase III trials to treat brain cancer. Here we capitalize on the recent identification of six new Salinispora species and 99 new genomes to expand our efforts in this unique taxon. We further expand our genome mining efforts to include the MAR4 lineage, a second chemically gifted group of marine bacteria for which we are uniquely situated to explore with 42 new genomes. This lineage shows the first evidence that marine adaptations are linked to natural product biosynthesis and includes at least six new species. We have identified hundreds of orphan biosynthetic gene clusters (BGCs) in these two groups and prioritized them as lead discovery targets. We have further taken this program into new directions by mining BGCs directly from environmental DNA (eDNA) using a nontargeted metagenomic approach that provides unbiased access to the biosynthetic potential of microbial diversity. These samples originate from both shallow tropical ocean sediments (1515 natural product BGCs already assembled) as well as deep sea sediments (down to 2000 meters) that are being collected for this program and have yet to be explored for natural products research. We will maximize access to these unique resources and employ innovative techniques in genome mining and synthetic biology to prioritize the targeted discovery of new antibiotic leads such as beta-lactone-containing products from eDNA that are predicted to inhibit protease virulence factors in Gram(-) bacteria. These genome mining efforts are governed by a logical workflow that prioritizes novel antibiotic discovery from poorly explored microbial resources.

NIH Research Projects · FY 2026 · 2009-07

ABSTRACT Persistent viral infections involve long-term equilibrium between the pathogen and the immune system. For this, immune cells must adapt in order to keep the pathogen in check while minimizing immunopathology. This immune adaptation is best exemplified by “exhausted” CD8 T cells (TEx), which were first described in a model of murine infection with the persistent lymphocytic choriomeningitis virus (LCMV) isolate Clone 13 (Cl13). In the same model, TEx were also first shown to progressively lose effector functions, express high levels of inhibitory receptors such as program-death-1 (PD-1) and be transcriptionally and epigenetically distinct from other CD8 T cell lineages. TEx were also shown to be a heterogeneous population, with TEx effector-like cells (TEff-like) and stem-like cells (TEx-Stem) emerging as competing differentiation paths within days after infection. TEff- like are short-lived and help to contain early viral spread but can also cause immunopathology. In contrast, TEx- Stem cells self-renew and maintain the TEx pool, being critical for long-term immunity and the success of PD-1- immunotherapy. Thus, understanding the factors that regulate TEx development and maintenance may offer unique insights into how we might exploit these TEx subsets to treat sustained viral infections. White adipose tissue (WAT) and brown adipose tissue (BAT) are both central regulators of whole-body metabolism. WAT stores energy which can be released via lipolysis and provision of free fatty acids (FFA) in times of need. In contrast, BAT is a highly metabolically demanding tissue that consumes great quantities of glucose to maintain core body temperature. Via untargeted metabolomics, we revealed a striking nutritional shift within the first ten days after LCMV Cl13 infection. In brief, we detected a profound systemic increase in FFA and a reduction in glucose, which coincided with WAT lipolysis and changes in BAT thermogenesis. Remarkably, mice with genetic inhibition of WAT lipolysis exhibited reduced FFA, decreased TEx-Stem and enhanced expression of the effector molecule granzyme B (GrzB). On the other hand, absence of BAT thermogenesis led to enhanced GrzB and CD8-T-cell-mediated death, while stimulation of BAT thermogenesis reduced effector CD8 T cells and delayed viral control. These results provide the first evidence that virus- specific CD8 T cells can be regulated by distant, non-immune, metabolically relevant tissues after an infection. Our overall goal is to leverage these exciting preliminary data and investigate the unique hypothesis that WAT (Aim 1) & BAT (Aim 2) regulate CD8 T cell responses during persistent infection through the provision and restriction of available nutrients (FFA and glucose, respectively), which in turn influence CD8-T-cell metabolism and differentiation, ultimately affecting the infection outcome. This work will establish the first foundational principles for a long-distance regulation of antiviral CD8 T cells by metabolically-relevant non-immune tissues, opening both a paradigm-shifting perspective to consider the host's bioenergetics when studying antiviral immunity and the possibility of targeting a patient's global metabolism to alleviate infectious diseases.