University Of California, San Diego

NIH Research Projects · FY 2025 · 2021-08

Project Summary/Abstract All eukaryotes harbor host-associated microbiomes. Determining what regulates host-microbiome function has the potential to revolutionize our approaches towards maintenance of host health. Host genetics and the environment are two key factors that contribute towards host-microbiome composition and function. We aim to advance our understanding of the relative roles of these two factors in regulating assembly of microbial communities, short-term changes in these communities through ecological succession, and long-term changes through evolutionary processes. Further, microbiomes are complex biological networks. Understanding the underlying structure of ecological interactions within these networks can improve predictions for when and how microbiomes might confer beneficial versus deleterious functions associated with disease. Our lab aims to advance fundamental understanding of host-microbiomes by leveraging the microbiomes of microbes. Specifically we employ single-celled eukaryotic phytoplankton as a highly-tractable experimental system. To further these goals we will focus on the following three themes over the next five years. (1) We will couple the unparalleled diversity of phytoplankton with bacterial –omics approaches to test how microbiomes assemble in response to host genetics. By assessing bacterial gene expression responses to host genetics, in tandem with fluctuating environmental conditions, this work will lend insights in to the host genetic x environmental forces that drive microbiome assembly of eukaryotic microbiomes. (2) We will evaluate mechanisms of microbiome change for maintenance of host homeostasis in fluctuating environments, including ecological shifts in bacterial taxonomic composition, shifts in bacterial gene expression, and bacterial strain evolution. It is important to understand the relative roles of these mechanisms because each occurs over different timescales and their effects can have varying degrees of permanence on their host. (3) We will leverage classical community ecology theory in biological networks with recent advances in flow cytometry bacterial fingerprinting to characterize traits of transient versus stable microbiome networks. We will quantify bacteria-bacteria interaction strengths within naturally assembled and engineered microbiomes to understand how network structure contributes to transitions between host health and disease states. Additionally, our research program will elucidate the implications of declining microbial diversity on eukaryotic host health. We will study host- microbiome co-evolutionary mismatches, such as those caused by humans consuming processed diets and living in human-built environments that differ from those of our evolutionary history. Ultimately, our work will leverage a highly tractable experimental system to advance our understanding of the microbiomes that modulate human health.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY/ABSTRACT: There is critical need to develop novel therapies for neuroblastoma (NB). NB is the most common extracranial pediatric cancer and even after aggressive multimodal treatments, high-risk patients succumb to progressive disease. One of the reasons underlying failure of these therapies in NB is the highly immunosuppressive tumor microenvironment generated by tumor associated macrophages (TAMs) that inhibit both innate and adaptive immune responses. The long-term goal is to better understand the signaling mechanisms by which TAMs mediate tumor immunosuppression and inhibit anti-tumor immune responses in high-risk NB. The overall objectives in this particular application are 1) to determine the role of spleen tyrosine kinase (Syk) in macrophage (MΦ)-mediated immunosuppression in MYCN and non-MYCN amplified (MYCN-NA) NB tumors and 2) to test whether Syk inhibitors combined with immune checkpoint inhibitors or standard care of therapies can improve anti-tumor immune responses in NB. The central hypothesis motivating this research is that Syk in immunosuppressive TAMs inhibits T cell responses and promotes resistance to checkpoint inhibitors in mouse models of NB. This hypothesis has been formulated on the basis of evidences generated in our laboratory utilizing Syk inhibitors and Syk-/- murine models for immuno-oncology. The rationale for the proposed research is that understanding molecular mechanisms by which Syk promote immunosuppression in MYCN and non- MYCN amplified (MYCN-NA) tumors has the potential to identify an immunological signature which will predict responsiveness to Syk inhibitors in NB tumors driven by a Syk-MΦ-dependent immunosuppressive TME. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: in Aim 1, we will evaluate the effects of myeloid Syk deficiency on NB tumor microenvironment and infiltrating immune populations. In Aim 2, we will investigate molecular mechanisms by which Syk regulate immunosuppressive MΦ polarization in MYCN and MYCN-NA NB tumors. In Aim 3, we will determine whether Syk inhibitors in combination with checkpoint blockade or current therapies can augment anti-tumor immune responses in NB. The significance of our proposal lies in our capacity to develop novel combinatorial therapy of Syk inhibitors with immunotherapy or standard of care therapies that is more effective than current therapies in NB.

NIH Research Projects · FY 2025 · 2021-08

Project Summary/Abstract    Eukaryotic cells organize their interior into a set of membrane-­enclosed compartments, referred to as organelles  or the endomembrane system, that enable cell growth and viability. Each organelle exhibits a unique biochemical  composition,  complex  dynamics,  and  a  distinct  morphology.  How  organelles  are  shaped,  how  their  shape  is  linked to their functions, and how individual organelles engage in specific contacts are important open questions  that are the focus of the proposed research.  Understanding  of  the  spatial  organization  of  human  cells  is  currently  experiencing  a  revolution,  with  the  realization that components of the cytoplasm can undergo a “liquid-­liquid” phase separation from the rest of the  cytoplasm, forming dynamic and functionally specialized domains that lack any membrane. Our recent research  raises the novel possibility that membrane-­containing organelles are structured by a two-­dimensional variation  of  this  principle,  in  which  ‘rod-­like’  proteins  (‘golgins’  and  golgin-­like  proteins)  self-­assemble  into  lamellar  liquid  geometries. We aim to test and develop this new organizing principle in context of the organization of the early  secretory  pathway,  where  two  organelles,  the  ER  and  the  Golgi  stack,  form  a  ‘synapse-­like’  interface  that  is  conserved across taxa. It is poorly understood how the spatial organization of the ER-­Golgi interface is achieved,  and why this specific organization is required. It is also a mystery how this junction can exhibit structural integrity  while resisting a high throughput of material, yet exhibit dynamic properties under specific regulatory cues.   Our  goal  is  to  understand  the  mechanisms  that  establish  the  specific  morphology  of  the  ER-­Golgi  interface  in  order to enable efficient processing and sorting of cargo within this space. Our motivating hypothesis is that this  interface  represents  a  dynamic  membrane  contact  site  organized  by  local  phase  separation  proteins.  We  will  employ a ‘bottom-­up’ approach in which we purify individual components to homogeneity and probe them in a  model  membrane  environment,  seeking  out  the  minimal  components  and  mechanisms  needed  to  reconstitute  morphology  and  function.  We  will  complement  this  approach  with  super-­resolution  and  electron  microscopy,  genetic  perturbations  and  functional  assays  in  both  human  cell  lines  and  in  the  model  organism  C.  elegans.  Through  this  dual  strategy,  we  expect  to  elucidate  the  principles  by  which  the  ER-­Golgi  interface  is  formed,  maintained, and how its spatial organization impacts cellullar functions.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Lipids represent a diverse class of biomolecules that are the building blocks of cell membranes. In recent years, alterations in lipid composition have been identified as hallmarks of numerous diseases, ranging from type 2 diabetes to neurodegenerative disorders. However, understanding the functional roles of bulk membrane lipids has long been a challenge, in part due to the difficulties of manipulating and imaging them in cells. Our laboratory applies genetic and chemical tools to study lipid function and develop biophysical models for membrane- associated cellular processes. The proposed research program will carry out this approach to identify how two disease-associated lipid perturbations alter the behavior of cellular compartments. In the first thrust, we will use effects of saturated phospholipids on membrane viscosity to uncover how structure and dynamics control respiratory metabolism. Specifically, we will engineer inner mitochondrial membrane composition in both yeast and mammalian cell lines and use this perturbation to dissect the contributions of diffusion and supramolecular assembly to the electron transport chain. This effort will uncover the function of conserved features of mammalian mitochondria, such as respiratory supercomplexes, and test how increases in saturated lipids caused by metabolic disorders could directly contribute to mitochondrial dysfunction. In the second thrust, we will use a genetic system to interrogate the function of 1-deoxysphinglipids, non-canonical products of serine- palmitoyltransferase that have been associated with several genetic and metabolic disorders. We will focus on how synthesis of 1-deoxysphinglipids dysregulates the endomembrane system in retinal pigment epithelium cells, which have been linked to adult-onset blindness caused by 1-deoxysphinglipid accumulation. Development of new imaging approaches will broaden the impact of this thrust to the emerging biomedical roles for these enigmatic lipids. If executed, the research program will thus generate models for two sets of lipids molecules and their cellular points of action in both healthy and diseased cells. Our long-term goal is to understand how changes in lipid composition across organelles, cells, and tissues arise and function, and use this knowledge to uncover the molecular mechanisms underlying membrane biology.

NIH Research Projects · FY 2025 · 2021-08

A higher epigenetic age relative to chronological age, described as ‘epigenetic age acceleration (EAA),’ indicates that an individual is biologically older than their years. The investigative team recently showed that EAA is associated with lower cognitive function (e.g., episodic memory, phonemic fluency) and lower white matter and total brain volumes, supporting a role of EAA in cognitive and brain health. Yet, while age is the strongest risk factor for Alzheimer’s disease and related dementias (ADRD), the association of EAA with mild cognitive impairment (MCI), ADRD, and brain aging is vastly understudied. The NIA’s 2020-2025 Strategic Directions for Research goals include understanding how “molecular bases of changes associated with aging contribute to the development and course of age-related dementia” and identifying “biological and clinical markers for early detection of cognitive decline, MCI, and AD.” Towards these goals, the overall objective of this project is to clarify the association of EAA with MCI, ADRD, successful cognitive aging, and brain aging in a nested case-cohort (N=2,836) within the deeply phenotyped, racially and ethnically diverse, NIA-funded Women’s Health Initiative Memory Study (WHIMS). With 25 years of follow-up that will continue at least through 2021, WHIMS contains detailed data on longitudinal measures of cognitive function; 1,336 incident cases of rigorously ascertained MCI and ADRD; genome-wide genotyping; and longitudinal neuroimaging measures of brain health. As an innovative aspect of this study, novel genome-wide DNA methylation data using stored blood DNA from two study visits 14- 18 years apart will be generated to examine changes in EAA. The Aims are to: 1) determine the extent to which EAA is associated with higher risk of MCI and ADRD and lower likelihood of survival to age 90 without cognitive impairment; 2) determine the extent to which EAA is associated with decreased total and regional brain volumes and increased total ventricular and white matter lesion volumes; and 3) identify epigenetic signatures across the genome associated with these cognitive and brain outcomes in epigenome-wide association studies (EWAS). Moderation of associations by race/ethnicity, APOE ε4 carriage, polygenic risk for AD, and cardiovascular disease will be explored. Findings will be replicated and extended in well-phenotyped, independent cohorts of men and women with data on genome-wide DNA methylation and cognitive and brain outcomes. Given that the pathophysiological ADRD process can begin up to 20 years before symptom onset, understanding the role of EAA as an aging biomarker identifying older adults early in the disease course is imperative to potentially prevent irreversible cognitive and functional decline. This study also presents a unique opportunity to identify age- associated epigenetic mechanisms of MCI, ADRD, and accelerated brain aging with potential to act as therapeutic targets to promote preserved cognition in late life. Importantly, the novel genome-wide DNA methylation data will enrich the existing WHIMS data resource and enable future examination of EAA and EWAS in relation to diverse phenotypes of aging, thereby having a broad and lasting scientific impact beyond this study.

NIH Research Projects · FY 2025 · 2021-08

Abstract The occurrence of CRC in the United States shows a large disparity among recognized races and ethnicities, with African Americans demonstrating the highest incidence and mortality from this disease. We have observed a novel “loss of function” phenotype for the DNA mismatch repair protein MSH3 that is induced by pro-inflammatory interleukin-6 (IL6) to shuttle MSH3 from the nucleus (where it normally repairs DNA microsatellites and double strand breaks) to the cytosol, where it no longer can repair DNA with coincident accumulation of tetranucleotide microsatellite frameshifts (termed EMAST, elevated microsatellite alterations at selected tetranucleotide repeats). These inflammation-associated microsatellite alterations are observed in 50% of all sporadic CRCs and is associated with advance-staged disease and poor patient survival. This inflammation-induced somatic MSH3 defect is observed in twice as many African American than Caucasian rectal cancers, and is associated with poor patient outcome. In this proposal, we hypothesize that MSH3 disruption contributes to the consequence of advanced stage and poor survival in African American CRC patients. Our preliminary data demonstrates clear evidence that MSH3 participates in Homologous Recombination repair of DNA double strand breaks as well as prevents aneuploidy. We have identified 6 unique somatic deleterious MSH3 mutations among African American CRCs that have not been reported in public databases. And we have characterized that chromosome 9p24.2 loss of heterozygosity (LOH) is associated with EMAST, and dramatically modifies survival of patients whose primary CRC demonstrates EMAST and 9p24.2 LOH. In this proposal, we will examine the central role of MSH3 dysfunction in its contribution to the survival outcome of African American CRC patients. Our aim is to assess the role of MSH3- disrupted double strand break mis-repair among African American CRCs, determine the functionality of 6 unique MSH3 mutations observed in African American CRCs, and ascertain the contribution of MSH3- deficiency with chromosome 9p24.2 LOH in the aggressiveness of African American CRCs. Overall, this proposal examines the role and contribution of defective MSH3 protein that likely contributes to the poor phenotype associated with African American CRC patients.

NIH Research Projects · FY 2024 · 2021-08

Project Summary It is increasingly clear that bacteria play an important role in human health. While it is natural to focus on how intestinal bacteria affect disease, intriguing findings have elucidated the extent to which bacteria inhabit solid tumors. Microbes have been detected in lung, pancreatic, breast, oral, gallbladder, ovarian, liver, and colorectal cancers. Localization has been ascribed to several mechanisms, including preference for anaerobic or facultative anaerobic bacteria to grow in the hypoxic core of tumors, presence of bacterial nutrients, lack of immune surveil- lance, and leakiness of the often poorly structured vasculature surrounding neoplastic tissue. This tendency for localization to solid tumors suggests that bacteria could be engineered for precise and robust drug production and delivery from within the solid tumor environment. This dovetails with 20 years of progress in synthetic biology, which has tended to focus on microbial engineering. However, information on how the tumor microenvironment affects bacterial growth is largely unknown. The microenvironment will affect bacterial gene expression that ul- timately underlies the functionality of engineered therapies, and it is difficult to imagine a predictive framework for engineered bacterial therapies without a quantitative understanding of how bacteria react to the environment of a growing tumor. We will use a probiotic strain of E. coli with an established safety record to develop a novel class of biosensors to noninvasively investigate bacterial growth in the tumor microenvironment. Initially, we will develop lysis-based biosensors that respond to specific tumor environment targets: hypoxia, pH, glucose, and lactate (Aim 1). We will also engineer an inducible quorum sensing (QS) system that enables external control of bacterial population dynamics, including the ability to eliminate a specific strain whenever desired (Aim 1). Together these strains will allow us to modulate and monitor population dynamics in vivo, enabling both sens- ing of the local environment and maintenance of an external control switch. We will test these strains using an established in vitro organoid model (Aim 2) and in two clinically relevant animal models for solid tumor growth. Additionally, we will use our previously developed dynOMICS technology to screen tumor extract from the two animal models and construct a second suite of biosensors for monitoring the tumor environment (Aim 2). These biosensors will then be tested in the animal models. We will visualize bacterial populations in a colorectal tumor model with bacteria that are engineered to produce luciferase in order to monitor colony dynamics using our es- tablished methods (Aim 3). We will also build on recently reported technology whereby bacteria are modified for use with ultrasound through addition of gas vesicles that permit high resolution imaging of the engineered bac- teria. We will use the ultrasound method to investigate NASH-induced hepatocellular carcinoma (HCC) where a high-fat diet is used to induce HCC at 20 weeks in mice (Aim 4). This project will quantitatively characterize how bacterial strains sense, respond, and grow in the tumors. The results will establish a platform for future exploration of therapies that are produced and delivered by bacteria that grow within solid tumors.

NIH Research Projects · FY 2025 · 2021-08

Abstract Sepsis, a heterogeneous syndrome characterized by whole-body inflammation caused by the body's response to an infection, is the most expensive and deadly condition treated in hospitals, with over 270,000 cases of sepsis-related deaths in the U.S. alone. Untreated sepsis may result in dilated and leaky blood vessels and severe hypotension requiring vasoactive medications (aka septic shock), and eventual injury to kidneys, lungs, and liver (aka organ injury) with mortality rates in excess of 40%. Successful prevention and management of sepsis, septic shock, and organ injury rely on the ability of clinicians to anticipate and estimate the risk, and administer the right life-saving treatments (e.g., antibiotics, fluids and vasopressors) at the right time. In recent years, data-driven modeling has been shown to enable early prediction of sepsis and to reveal clusters (or phenotypes) of sepsis, which may help with personalizing therapeutic interventions. However, crossing the translational chasm between clinical research and improving patient care also requires addressing 1) `data deserts' at different levels of care through better data integration, smarter lab ordering, and utilization of continuous monitoring wearable sensors; 2) interoperability and portability of clinical data and analytics; 3) principled dissemination and implementation studies; and 4) education of the next generation of caregivers to effectively utilize advanced analytical tools. The proposed research program builds upon PI's K01 early career development award focused on multicenter development and validation of sepsis predictive analytic algorithms (including hourly EHR data spanning ED and inpatient encounters from over 500,000 hospitalized patients across five district healthcare systems). Drawing insights from recent advances in domain adaptation and multi-task learning (sub-fields of machine learning), this project aims to discover generalizable dynamic phenotypes that are directly relevant to the prediction and management of sepsis, septic shock, and downstream organ injury. We propose to augment EHR-based analytics with high-resolution data from bedside devices (e.g., monitors, ventilators, dialysis, and IV pumps) and wearables (e.g., continuous blood pressure and lactate sensors) to address existing gaps in monitoring. Additionally, this program aims at advancing FHIR (Fast Healthcare Interoperability Resources) and OMOP (Observational Medical Outcomes Partnership) interoperability standards through the implementation of specific resources for high-resolution data sources. Finally, this research program will be conducted in close collaboration with our dissemination and implementation and hospital quality improvement teams to ensure early assessment of usability, barriers to implementation, and effective education to maximize the potential for clinical impact.

NIH Research Projects · FY 2025 · 2021-07

Project Summary In the cell, the RNA made by RNA polymerase (RNAP) folds into its functional three-dimensional shape while it is being synthesized by RNAP. The kinetics of RNA synthesis, which determine the RNA folding outcome, are influenced by myriad factors, such as intracellular temperature, pH, concentrations of small molecules and proteins in the cell, and the exact sequence of DNA being transcribed into RNA. Transcription is not a continuous process: RNA synthesis by RNAP is interrupted by sequence-dependent pauses, during which RNAP remains bound to the nucleic acids without active nucleotide addition occurring. These pauses create windows of time for regulation of transcription to occur. Our research program will address the mechanisms of pausing at the atomic level and the contribution of pausing to co-transcriptional events, such as folding of RNA, in bacteria and human mitochondria. The first direction of the program aims to develop tools for capturing and visualizing RNA folding intermediates during transcription and to understand the effect of pH on the kinetics of RNA synthesis by RNAP, and thus the RNA folding pathway. The resulting tools will be of broad interest to the RNA community because they can be applied to follow folding of other biologically important RNAs. A second direction will apply those tools to map the differences in co-transcriptional RNA folding of “healthy” and mutated human mitochondrial transfer RNAs (mt-tRNA), thus providing the structural basis for disease-causing mt-tRNA mutations. We will assess the contribution of mitochondrial RNAP (mtRNAP) pausing to the differential folding of unmutated vs. disease-variant mt-tRNA. Additionally, we will test another hypothesized function of mtRNAP pausing: coupling of transcription of mitochondrial DNA (mtDNA) to its replication, which is critical for maintaining enough mtDNA copies for production of protein components of the oxidative phosphorylation machinery. Finally, a third research direction will address how the balance between transcription of mtDNA and its packaging is achieved to cater to the ever-changing cellular needs for energy. The completion of the proposed research will be transformative to the understanding of basic principles governing gene expression, the molecular mechanisms of diseases linked to mtDNA, and to the applications of RNA-based tools in synthetic biology.

NIH Research Projects · FY 2024 · 2021-07

PROJECT SUMMARY Dysfunction of the striated external urethral sphincter is the strongest predictor of stress urinary incontinence, defined as involuntary loss of urine due to increased intraabdominal pressure in the absence of bladder contraction. Stress urinary incontinence affects approximately 1 in 2 women at some point in their lives. The primary inciting event behind the development of chronic stress urinary incontinence has been unequivocally identified as injury to the urinary continence mechanism sustained by women during vaginal deliveries. This astoundingly prevalent condition dramatically decreases quality of life, causes significant morbidity, and is associated with large economic burden to the individuals and society. Despite this, preventative strategies are almost non-existent, and the available treatments are delayed and compensatory as they do not directly target the underlying pathophysiology. The above is largely due to the fact that our understanding of the pathways that lead to failure of the intrinsic muscular components of the external urethral sphincter following birth injury remains limited. Furthermore, the prevailing preclinical studies do not utilize biologically relevant pregnant animal model of birth injury. To address the existing unmet clinical need and knowledge gaps, we assembled a cross- disciplinary team with diverse but complimentary expertise to execute the current project at the interface between basic science, biomaterial development, and translational medicine. We will use a validated and biologically relevant pregnant pre-clinical model to investigate structural, molecular, and cellular events at multiple time points across a recovery continuum of the striated external urethral sphincter following birth injury. These basic processes will inform the development of and the critical time to deliver new, minimally invasive tissue- engineered therapy for the prevention and treatment of urethral muscle dysfunction. Specifically, we will test a novel pro-regenerative skeletal muscle-specific injectable extracellular matrix hydrogel, derived from decellularized porcine skeletal muscles, in preventing and reversing maladaptive recovery of the external urethral sphincter following birth injury. Collectively, this innovative study will provide fundamental knowledge of the biological processes involved in the regulation of external urethral sphincter muscle regeneration, and comprehensive functionally relevant assessments of the role of low-cost acellular minimally invasive regenerative therapy to counteract the existing epidemic of urinary incontinence.

NIH Research Projects · FY 2025 · 2021-07

Mood, anxiety, and traumatic stress disorders are common psychiatric conditions - affecting over 40 million U.S. adults - and are leading causes of disability worldwide. People with these conditions are commonly plagued by difficulty controlling distressing personal thoughts and memories, collectively referred to as repetitive negative thinking symptoms. Models suggest that repetitive negative thinking is driven by executive functioning deficits, such that cognitive resources are insufficient to downregulate unwanted thoughts. Executive functioning deficits could be a promising treatment target but are not typically addressed with existing interventions. The long-term goal advanced by this proposal is to develop effective, mechanistic cognitive training programs that can improve cognition and reduce symptoms associated with mood, anxiety, and traumatic stress disorders. The objectives of this proposal are first to determine the optimal dose of a cognitive training program designed to improve executive functioning in this population using behavioral and neural outcomes (R61). If the cognitive training tested in the R61 successfully improves executive functioning (go/no-go decision), we will evaluate the relationship between change in executive functioning and change in clinical symptoms (R33). Our central hypothesis is that repeated training exercises will enhance executive functioning and will lead to a reduction of repetitive negative thinking in mood, anxiety, and traumatic stress disorders. The hypothesis will be tested by pursing two specific aims: Aim 1 is to identify the cognitive effects and optimal dose of cognitive training. Aim 2 is to evaluate the clinical effects of the optimized cognitive training program relative to a sham condition. We will also conduct an exploratory aim to determine generalization of cognitive training to real-world cognitive performance. The R61 phase will test Aim 1 by randomizing participants with depression, anxiety, and/or traumatic stress disorders to one of two doses of cognitive training or a no-treatment control condition. We will examine executive functioning change with cognitive task performance and functional neuroimaging assessments. The R33 phase will randomize participants to the dose decided by the R61 or a sham condition. The R33 intends to replicate the impact on executive functioning and assess the relationship between change in executive functioning and clinical symptoms. We will explore how cognitive training helps people in their daily lives by including assessments given with a smartphone mobile cognitive testing app. The research proposed is innovative because it aims to address symptoms by intervening on a cognitive target thought to generate symptoms across multiple disorders, thus explicitly testing the interactions of cognitive and emotional symptoms. Outcomes derived from the proposed research will include a novel treatment program and information about its utility for reducing clinical symptoms. Knowledge from this proposal will advance our treatment options by targeting a specific cognitive system, providing a foundation for neuroscience-based therapeutic alternative for symptoms that span mood, anxiety, and traumatic stress disorders.

NIH Research Projects · FY 2025 · 2021-07

ABSTRACT Epilepsy is a devastating neurological illness that affects over 50 million people worldwide. Approximately one-third of patients do not respond to anti-seizure medication (ASM) and require additional diagnostic work-up, including consideration for surgery. Structural neuroimaging plays a pivotal role in the diagnostic evaluation of epilepsy, identifying visible lesions in many patients that co-localize with the seizure focus. However, up to 40% of patients have normal-appearing MRIs and this number is growing. As a result, there is increased interest in identifying subtle brain network abnormalities that could help to delineate the epileptogenic network and aid in the prediction of treatment response (i.e., response to ASMs and surgical outcomes). Unfortunately, methods for reliably identifying which patients will be drug-responsive versus drug- resistant, and which patients will achieve successful versus unsuccessful surgical outcomes are lacking. A major barrier to progress in this field has been obtaining quantitative imaging, including structural MRI (sMRI) and diffusion-weighted imaging (dMRI), clinical, and genetic data on large, geographically diverse samples of patients in whom different treatment outcomes can be evaluated. In the past, sample sizes have been insufficient to detect subtle, but reliable, brain abnormalities in patients with focal or generalized epilepsies that are genuinely associated with epilepsy and not with vicissitudes related to small or geographically restricted samples. A new, large-scale data initiative, ENIGMA4-Epilepsy, coupled with technological advancements that enable improved data harmonization are now lifting these barriers and allowing us to combine multi-site sMRI/dMRI, clinical, genetic data to predict important clinical outcomes, and making the results generalizable to a global epilepsy community. In this grant, we will leverage data collected through ENIGMA-Epilepsy—a consortium of 24 epilepsy centers from 14 countries (more than 2,250 patient and 1,727 healthy control sMRI/dMRI datasets) and the Human Epilepsy Project (HEP). We will include new network models (i.e., individualized connectomes) and polygenic risk scores (PRS) to test whether a combination of imaging, clinical, and genetic risk can accurately predict two clinical outcomes: drug-resistance and post-operative seizure outcome. Our scientific premise is that MRI-based assessment of whole-brain network properties, in combination with clinical data and PRS derived from genetic data, are able to predict (i) drug response in recently diagnosed epilepsy cases and (ii) postsurgical outcomes in individuals with drug-resistant epilepsy. This R01 addresses NIH's call for more reproducible studies by introducing a highly-powered design capable of capturing variability across patients with diverse clinical characteristics and treatment outcomes. This grant is also directly aligned with NINDS's 2020 Epilepsy Benchmarks (IIIB), which encourage the identification of genetic, clinical, and imaging biomarkers capable of predicting treatment response in epilepsy.

NIH Research Projects · FY 2025 · 2021-07

SUMMARY HIV infects the brain soon after transmission, but it is unknown how infected brain cells contribute to HIV persistence and whether these cells release viable virus that can seed cells outside the brain. It is also unclear how HIV persistence leads to local cellular damage, although inflammatory and external factors (like antiretroviral [ARV] penetration and opioids) likely impact such damage. Such new knowledge could be important for HIV cure strategies and ways to improve brain health in persons with HIV (PWH). This project will address stated objectives of RFA-MH-20-701, Role of Myeloid Cells in Persistence and Eradication of HIV-1 Reservoirs from the Brain, by: (i) mapping HIV reservoir size, composition, and activity in brain myeloid cells (BMC) in relation to cellular density and levels of ARV and opioids, (ii) determining the role of BMC in HIV dispersal within the central nervous system (CNS) and across the body in the setting of ARV treatment (ART) and after treatment interruption, and (iii) defining how HIV reservoir size and activity in BMC is associated with local inflammation and cell damage. Our goal is to examine the role of BMC in HIV persistence, local inflammatory-induced damage and as a source of viruses that can egress from the CNS to re-seed peripheral organs. The rationale for this project is supported by literature demonstrating that brain macrophages and microglia can harbor HIV that persists during modern ART. The low turnover of these macrophages and microglial cells (from months to years) make them unique reservoirs for HIV. While HIV in resting T cells has been extensively characterized, the role of BMC as a source of rebound upon cessation of ART is yet to be determined. Further, HIV in BMC likely triggers immune responses, even during ART, causing local damage. Our overall hypothesis is that BMC (primarily microglial cells) contribute to HIV persistence in the CNS with regional heterogeneity. HIV harbored in these BMC likely also causes inflammation-associated brain damage and contributes to viral dispersal when ART is stopped. We also hypothesize that HIV persistence, local damage and viral dispersal are influenced by local ARV and opioid levels. To address these open questions, our study will collect and analyze tissues throughout the CNS (white and grey matters of frontal cortex, thalamus, hippocampus, basal ganglia, cerebellum, spinal cord), ileum, spleen, blood and cerebrospinal fluid (CSF) of altruistic PWH enrolled in the Last Gift cohort, an ongoing rapid autopsy study. Some participants (n=15) will remain virally suppressed until the time of death, while others (n=5) will want to stop their ART before death. Half of the population will use prescription opioids. These studies will be important for PWH because they will provide new insights for the development of strategies to clear HIV infection and lessen inflammatory-dependent microglial-induced neurological damage.

NIH Research Projects · FY 2025 · 2021-07

Project Summary/Abstract The accurate clinical diagnosis of Lewy body diseases (LBD: Parkinson’s disease (PD), dementia with Lewy bodies (DLB)), and Alzheimer’s disease (AD) is challenged by overlapping clinical features. Novel biofluid biomarkers including real time quaking induced conversion (RT-QuIC) offer the ability to diagnose patients with greater accuracy during life by identifying pathologic α-synuclein (a-syn) or tau seeds. However, in LBD, approximately 50% of cases will harbor significant AD pathology, and in AD up to 50% harbor limbic a-syn which can complicate interpretation of biomarker signatures. Therefore, comprehensive pathological validation studies are essential to understand the utility of these new assays and their association with histopathologic burden and clinical correlations. If RT-QuIC metrics relate to pathological burden or cognitive outcomes, they could be used as an objective biomarker in LBD, an ongoing critical need. The aims of this proposal are to test whether a-syn and tau RT-QuIC is associated with 1) histopathological features and 2) cognitive outcomes in LBD and AD. The hypotheses are that a-syn and tau RT-QuIC will be able to predict primary and co-pathologies in LBD and AD and that higher degrees of RT-QuIC activity will associate with greater neuropathological burden and worse cognitive outcomes. The proposed experiments will leverage pre-existing autopsy cohorts with antemortem CSF and cognitive testing collected at the University of California San Diego (UCSD) and the University of Pennsylvania with the RT-QuIC expertise of the NIH/NIAID Rocky Mountain Laboratories. The K23 candidate is an Assistant Professor of Neurosciences at the UCSD, having previously completed movement disorders fellowship and a NIH TL1-supported Masters of Translational Research. He has a history of productivity, having conducted translational and clinical research in neuroscience, recently focusing on the neuropathology of PD, DLB, and AD. The candidate is committed to a career in translational research and proposes a comprehensive five year plan of mentorship, formal training, self-directed learning, and research. This K23 career development award will support Dr. Coughlin’s short-term goals, including 1) developing a detailed understanding of RT-QuIC and its association with histopathological and cognitive outcomes, 2) acquisition of skills for multimodal tissue characterization using immunohistochemistry and Western blotting techniques, 3) learning the neuropsychology of dementia with a focus on LBD and AD, and 4) learning the statistical methods to carry out these and future studies. Dr. Coughlin will meet these objectives under the guidance of a mentorship team, including Dr. Douglas Galasko, an expert in biofluid biomarkers, Dr. Robert Rissman PhD, an expert in the molecular pathology of neurodegenerative disease and Dr. David Salmon PhD expert neuropsychologist with a long research track record in LBD and AD. This award will support Dr. Coughlin’s development towards becoming an independent clinician-scientist with expertise in biomarker-pathological validation studies and a unique research program in translational neuropathology.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY/ABSTRACT Understanding peripheral tolerance and the maintenance of immune system homeostasis are vital in the control of human diseases. We have previously demonstrated that anti-inflammatory cytokine IL-37 participates in immune tolerance by generating semi-mature tolerogenic dendritic cells (DCs) in antigen- specific adaptive immune responses. IL-37 is one of eleven IL-1 family members and the only known member to be broadly anti-inflammatory. In our recent project, we found IL-37 levels were elevated in multiple human immune cell types, specifically in regulatory T cells (Tregs) cells. Further analysis revealed that human Treg cells express the highest IL-37 levels among all T-cell subsets and that intracellular expression of IL-37 correlates with the expression of master transcriptional regulator, FOXP3, in human Treg cells. Our current project hypothesizes that elevated IL-37 expression stabilizes Treg cells and induces potent immune suppression by controlling FOXP3 expression. IL-37 is not expressed in mice, but using transgenic mice and peripheral blood T cells from human donors, we generated strong preliminary data to support our hypothesis. In this grant proposal, we will use human primary Treg cells and T cell lines overexpressing IL-37 and its mutant form to elucidate the biological and molecular mechanisms of IL-37 in controlling human Treg cell function. Since our proposal uses human T cells, the results could be easily translated into clinical medicine and patient care. Our findings will have an immense translational impact on many human diseases such as autoimmunity and transplantation.

NIH Research Projects · FY 2024 · 2021-07

PROJECT SUMMARY Mitochondria are essential for cardiomyocyte (CM) differentiation and cardiac morphogenesis. Mutations in genes encoding mitochondrial proteins frequently result in congenital heart disease, highlighting the need to elucidate key molecular pathway(s) in mitochondrial homeostasis during heart development. Protein Tyrosine Phosphatase localized to the Mitochondrion 1 (PTPMT1) is a dual-specificity mitochondrial phosphatase encoded by nuclear DNA. PTPMT1 is exclusively localized to mitochondria, being anchored to the inner mitochondrial membrane. PTPMT1 is expressed in CMs throughout several developmental stages. To determine the role of PTPMT1 in CMs, we generated a Ptpmt1 constitutive CM-specific knockout (cKO) mouse model. Our preliminary data revealed that Ptpmt1 cKO mice display embryonic lethality. Ptpmt1 cKO mice displayed thinner compact zone myocardium, with decreased CM proliferation. We also observed significantly decreased mitochondrial respiration rate and abnormal mitochondrial morphology in Ptpmt1 cKO hearts, demonstrating that PTPMT1 plays a critical role in developing CMs and in maintaining normal mitochondrial homeostasis. We also examined previously described PTPMT1 substrates in Ptpmt1 cKO hearts relative to controls, but could find no evidence to support them being direct substrates of PTPMT1 in CMs. To gain further insight into pathways affected by loss of PTPMT1, we performed RNA-seq analysis of Ptpmt1 cKO hearts. Bioinformatics analysis revealed that loss of PTPMT1 significantly activated the Activating Transcription Factor 4 (ATF4) pathway. ATF4 controls expression of a wide range of adaptive genes that allow cells to survive periods of mitochondrial stress. However, under persistent stress conditions, ATF4 promotes induction of cell- cycle arrest, apoptosis and senescence. Notably, reduced expression of ATF4 in global Atf4-haplodeficient and smooth muscle-specific Atf4 knockout mice attenuates ER stress and reduces medial and atherosclerotic calcification, highlighting new opportunities afforded by favoring a stress-relief adaptive effect over a maladaptive effect by modulating ATF4 activation. The foregoing evidence leads us to the hypothesis that PTPMT1 plays an essential role in cardiac development through modulation of specific substrates, and that partial loss of ATF4, activated in response to mitochondrial stress in Ptpmt1 knockout CMs, may ameliorate, but complete loss of ATF4 may exacerbate, Ptpmt1 cKO phenotypes. Accordingly, our specific aims are to: 1. Elucidate the role of PTPMT1 in CM mitochondrial homeostasis and cardiac development and function by analyzing Ptpmt1 cKO mice, and to identify endogenous substrates of PTPMT1 in CMs by performing unbiased lipidomics and phosphoproteomics analyses; and 2. Determine the consequences of partial or complete loss of ATF4 in CMs on phenotypes of Ptpmt1 cKO mice by analyzing CM-specific Ptpmt1 knockout/Atf4 heterozygous (hcKO) knockout mice and CM-specific Ptpmt1/Atf4 double knockout (dcKO) mice.

NIH Research Projects · FY 2025 · 2021-07

ABSTRACT. The goal of the Molecular Biophysics Training Grant (MBTG) at the University of California San Diego is to provide a rigorous and strongly interdisciplinary training program for graduate students, with the aim of training the next generation of scientific leaders striving to solve important biological problems using Molecular Biophysics approaches. To accomplish this goal, trainees with strong quantitative backgrounds and a demonstrated aptitude, commitment, and passion for research are selected at the end of their first year from a diverse group of applicants from 32 well-funded training faculty labs. The MBTG faculty are highly interdisciplinary and many have cross-campus appointments in several departments. Trainees come mostly from two graduate programs, Chem/Biochem and Biomedical Sciences but a few come from Physics, Biology and Bioengineering. During their first year, all students engage in research rotations and choose a research lab. At UCSD, students are free to rotate with any faculty across campus and to choose any faculty for their thesis advisor. First year students receive training from their respective graduate programs in critical reading of literature, exposure to important unsolved biological problems, ethics and graduate school survival skills. MBTG Trainees are appointed and choose their thesis committee at the end of their first year. All MBTG trainees take two rigorous core courses in Molecular Biophysics and will have an intensive week-long Statistics workshop. During their second year, they write and defend their thesis research proposal. Required trainee activities include a full-length seminar presentation of their research at the monthly student seminar, monthly discussions of rigor and reproducibility, a monthly journal club, a yearly retreat, career workshops and outside mentors. Trainees may apply to be reappointed for a maximum of two years of support. All trainees present their research every year at the annual retreat, with alumni trainees giving lightning talks each year until graduation. Throughout their graduate training, trainees take advantage of career development opportunities and mentorship, including yearly IDPs and group and individual engagement with outside mentors. The program emphasizes the development of creative independent thinking, strong quantitative skills with a focus on rigor and reproducibility, scientific communication, and mentoring. The MBTG provides the critical interdisciplinary “home” at UCSD for students and faculty interested in Molecular Biophysics. The high quality of student publications, postdoctoral fellowships, faculty positions, and industry positions obtained by our trainees are strong evidence of successful training outcomes. Given the depth, quality, and diversity of our student pool and our demonstrated training outcomes, we request 12 students be supported each year.

NIH Research Projects · FY 2025 · 2021-07

Project Summary/Abstract This proposal details a five-year research and career development plan for Jeremy E. Orr, M.D., a specialist in pulmonology and critical care medicine, and Assistant Professor of Medicine at the University of California, San Diego. His research has been supported by a National Research Service Award fellowship and currently an American Thoracic Society Foundation award. The overall focus of his research is understanding the importance of breathing issues during sleep in patients using chronic opioid medications, and identifying treatment strategies for these complex breathing issues that will improve health. This K23 award will provide necessary support for Dr. Orr to gain expertise in patient-oriented clinical research, applied physiology of sleep disordered breathing, and clinical trials. Dr. Orr has assembled a comprehensive team of mentors to support his research and career development. His primary mentor, Dr. Atul Malhotra, is a world expert in sleep disordered breathing (SDB) and applied respiratory physiology, with a strong track record of mentoring and promoting junior faculty to become independent investigators. Dr. Robert Owens is a NIH-funded physician-scientist with expertise in advanced techniques to measure SDB physiology, and will serve as a “hands-on” co-mentor. Additional members of the team are Dr. Sonia Jain (statistics and trials), Dr. Mark Wallace (opioids and pain), Dr. Shamim Nemati (signal analysis), and Dr. Frank Powell (control of breathing). Patients with chronic pain who use chronic opioids are at increased risk for poor health including ongoing pain, poor sleep, decreased quality of life, and an increased risk of mortality. Opioids are known to have effects on breathing which may lead to SDB, an under-recognized factor potentially contributing to adverse outcomes in these patients. SDB contributes to sleep disruption and impairments in oxygen levels, but has been little studied in this high-risk group of patients using opioids. In a broad group of subjects with chronic pain who use chronic opioids, this research will determine whether treatment of SDB with continuous positive airway pressure (CPAP) leads to improved sleep quality, as well as investigating other symptoms including pain in these patients (Aim 1). CPAP treatment may not be effective in some patients due to unstable breathing (due to opioids), so techniques to identify such individuals will be investigated, including new measures of breathing instability. For patients with persistent SDB despite CPAP, treatment options are limited. The research will evaluate whether a medication (acetazolamide) that helps to reduce the instability of breathing will help to resolve SDB (Aim 2). This research will provide Dr. Orr with a strong foundation to become an independent investigator studying the impact, pathogenesis and treatment of SDB in those using chronic opioids.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY This is an initial submission of a K23 application by Dr. Alison Potok, under the mentorship of Dr. Dena Rifkin, at the University of California San Diego (UCSD). This proposal will establish Dr. Potok as an independent investigator, and will evaluate the clinical applications of the difference in estimated glomerular filtration rate by cystatin C (eGFRCys) vs. creatinine (eGFRCr), for prognosis in the long-term and drug dosing in the short-term. Candidate: Dr. Potok’s training objectives and career goals through this proposal include: 1) to become an expert in geriatric nephrology and proficient in pharmacology and drug dosing in the elderly; 2) to develop skills in advanced statistical methods, epidemiology, manuscript and grant writing; 3) to learn the necessary skills to design and conduct a clinical study and develop a research team. She has assembled a multidisciplinary mentorship team comprised of a primary mentor, Dr. Rifkin, an established leader in geriatric nephrology, and the following additional co-mentors and collaborators: Dr. Ix, an expert in nephrology clinical trials; Dr. Moore, an authority in geriatric medicine and aging research, Dr. Gutierrez, an expert in kidney disease with extensive experience and insight to the REasons for Geographic and Racial Differences in Stroke (REGARDS) study utilized for Aim 2; Dr. Katz, the Director of Biostatistics at the University of Washington, who has worked extensively with Drs. Rifkin, Ix, and Potok; Dr. Hallan, Professor of Medicine with expertise in decision curve analysis and extensive experience in the Norwegian Nord-Trondelag Health Study (HUNT) utilized for Aim 1. Research: Most patients with chronic kidney disease (CKD) will not progress to end stage kidney disease (ESKD) due to the competing risk of death. Frailty may increase the risk of death vs. the risk of ESKD. The current kidney failure risk equation (KFRE) and mortality risk equation in kidney disease (MREK) do not account for frailty. In preliminary work, Dr. Potok has showed that the difference in eGFR by cystatin C vs. creatinine (eGFRDiff defined as eGFRCys – eGFRCr) is associated with risk of incident frailty and death. Moreover, Dr. Potok’s preliminary results show heterogeneity across the spectrum of eGFRDiff regarding which marker between cystatin C vs. creatinine is the best surrogate for true kidney function. The overall hypothesis is that eGFRDiff can be used to guide clinicians on whether to start preparing patients for renal replacement therapy and with medication dosing. In Aim 1, she will determine whether the inclusion of eGFRDiff, as a marker of frailty within the KFRE and MREK will improve assessment of the competing risk of ESKD vs. death in older adults. This Aim will be conducted in participants aged >65years with CKD of the HUNT study, an exclusively White European population. In Aim 2, she will explore the competing risk of ESKD vs. death in a biracial American population in REGARDS. In Aim 3, Dr. Potok will examine whether eGFRDiff could be used to determine those in whom eGFRCr should not be trusted for drug dosing, and eGFRCys should be used instead, with vancomycin as the prototype medication.

NIH Research Projects · FY 2024 · 2021-07

Project Summary Atrial fibrillation (AF) is a cardiac arrythmia that affects over 5 million individuals in the US and quintuples the risk of stroke. AF is a critical disease state to measure the effects of the COVID-19 pandemic on non-COVID disease because every aspect of stroke prevention in AF is vulnerable to disruption: 1) Patients with new onset AF may be more likely to remain undiagnosed. 2) Established AF patients may have complications that remain undetected and worsen without treatment. 3) Patients newly diagnosed with AF may be less likely to initiate stroke prevention therapy with oral anticoagulation (OAC). 4) Established OAC users may have increased difficulty adhering to therapy. 5) Patients on warfarin, an OAC agent that requires routine blood tests, may have less frequent monitoring. Our goal is to measure the impact of the COVID-19 pandemic on diagnosis, therapy initiation, therapy adherence, monitoring, and health outcomes for patients with AF. We will determine whether pandemic disruptions of AF care have exacerbated health disparities. We will also assess the role of telemedicine, whose uptake has been catalyzed by the pandemic, in offsetting decreased access to in-person care during crises. We will use 2015-2022 claims data for Medicare fee-for-service beneficiaries and Optum® Integrated claims- electronic health record data for commercially insured and Medicare Advantage beneficiaries. We will construct interrupted time series analyses to measure changes in outcomes after pandemic start and pandemic end. To determine whether the pandemic has exacerbated disparities, we will test whether the degree of change in outcomes differed by age, sex, race/ethnicity, and area-level measures of urbanization, socioeconomic status, deprivation, racial composition, and segregation. In aims 3 and 4, we will use marginal structural models to estimate the association between telehealth visits and outcomes. We will achieve four specific aims: (1) quantify changes in the incidence rate of new AF diagnoses in 2016-2021, including new AF diagnoses manifesting as stroke; (2) determine whether the COVID-19 pandemic was associated with decreased OAC initiation among newly diagnosed AF patients; (3) quantify changes in adherence and monitoring of OAC therapy among established AF patients; (4) quantify changes in the incidence rates of stroke, bleeding, cardiovascular hospitalization, and death among established AF patients. Our quantification of pandemic effects on AF care will have major implications for the provision of chronic disease care during emergencies. Our identification of populations disproportionately affected by the pandemic and our determination of the ability of telemedicine to offset decreased access to in-person care will inform clinical guidance and policies that prevent care avoidance during health emergencies, optimize models for the delivery of chronic disease care during major crises, and protect vulnerable populations.

NIH Research Projects · FY 2026 · 2021-07

PROJECT SUMMARY In the United States, individuals living with HIV face challenges maintaining adherence to antiretroviral therapy (ART), which is critical for achieving viral suppression. A subset of this population also reports adverse adulthood experiences and behavioral health challenges that can further complicate sustained ART adherence. Digital interventions offer a scalable approach to improving treatment engagement in the context of limited in-person resources. To address this gap, we developed and pilot-tested a four-month web-based behavioral program integrating structured video sessions and psychoeducational content. Preliminary results demonstrated feasibility, acceptability, and promising improvements in ART adherence, stress-related symptoms, coping self-efficacy, and treatment engagement. Building on this pilot, we propose a randomized controlled trial of the digital intervention model, referred to as SHINE, involving 360 participants recruited from California. Participants will be randomized to either (a) the intervention arm (n=180), which includes a four-month digital program with individual video-based navigation sessions, group psychoeducation modules, and access to a static resource website, or (b) the control arm (n=180), which includes a single self-care session and website access only. Participants will complete web-based assessments and provide biospecimens at baseline, 4-, 8-, and 12-months post-randomization. The primary aim is to assess the intervention’s impact on ART adherence. Secondary aims include evaluating changes in emotional regulation and post-treatment stress indicators. We will also examine potential mechanisms of change, including self-efficacy and engagement with educational and behavioral content. If successful, this trial will inform future strategies for broad implementation of scalable digital interventions to enhance HIV care outcomes in high-need populations.

NIH Research Projects · FY 2025 · 2021-07

Project Summary Homeostasis is a fundamental feature of physiological control in all cells. In the past five years, a new homeostatic principle co-discovered by us has been changing our view of cell-size control. This principle, known as the “adder”, states that cells add fixed size between birth and division irrespective of the cell size at birth. The adder principle is therefore distinct from many biological controls due to its passive nature, as the adder-like cells do not employ any apparent size sensing or feedback mechanisms to trigger division when they reach a fixed critical size. The proposed program is an extension of our success in cell-size control research to understand a broader class of physiological controls. First, we will study the mechanisms responsible for the precision and robustness of physiological processes. We will focus on replication initiation in bacteria as it can serve as a tractable model to solve these long- standing problems. Another important aspect of physiological control is how cells allocate their resources to growth. The current paradigm based on E. coli is that cells balance supply and demand of amino acids to maximize growth rate under all growth conditions. These models are important because they have been able to explain the tradeoff between production of cellular energy vs. production of proteins that is pertinent to cancer. However, we have obtained experimental results in B. subtilis that directly challenged this E. coli centric view of growth control. We will thus seek more general principles of cellular resource allocation that encompass both E. coli and B. subtilis, and ideally beyond bacteria. Finally, we will extend our previous work on cell-size control to investigate how cells ensure physiological equilibrium when proteins and organelles partition asymmetrically, which is important in the context of inheritance and cellular aging. These questions require multidisciplinary approaches from physiology to development of novel technologies. To this end, we will work with collaborators who are leaders in their research fields. Furthermore, over a decade we have been making major efforts to democratize technologies to the research community. We expect the knowledge and technology that will be generated from our proposed research to open exciting new research avenues and facilitate other important discoveries in physiology and cell biology.

NIH Research Projects · FY 2025 · 2021-07

Graduate Training Program in Bioinformatics Program Abstract Biology is increasingly becoming an information-driven science. To harness the opportunities of the post- genomic era in furthering health sciences research and improving health care, there is an enormous demand for biologists who are trained in mathematics and computer science and can think quantitatively. However, current disciplinary graduate training programs are not designed to accommodate these rapid changes in the biological research perspective. This need serves as the motivation for the development of specialized graduate training programs that will train students at the interface between biology, engineering and computer science. To address this need, UCSD established an interdisciplinary Graduate Program in Bioinformatics in 2001 under the directorship of Dr. Shankar Subramaniam. In 2008, it was renamed Graduate Program in Bioinformatics and Systems Biology and reorganized. The current program directors Drs. Trey Ideker, Euegene Yeo, and Theresa Gaasterland work closely with the Training Grant co-PIs Dr. Subramaniam, Vineet Bafna, and Theresa Gaasterland, and with an active steering committee containing representative faculty from all five participating UCSD schools and academic divisions. The primary objectives of this (renewal) application of the Training Grant by the three co-PIs are to continue and expand this premier Graduate Program, and support the highest quality students in their truly interdisciplinary training which blends biomedicine, computer science and engineering. The Program will continue to evolve the curriculum (including online offerings) and develop and offer electives that will prepare students for the challenges of big data and computational biomedical research. The program will continue its mode of training that begins with a set of research rotations in laboratories of faculty members, and continues through doctoral research work under the supervision of a PhD advisor and co-advisor who provide complementary interdisciplinary expertise. The Program will also continue a recently established weekly Colloquium, the student Journal Club, and annual retreat. In the course of their training, program students have contributed important discoveries and impactful advances in health sciences research. Alumni of the program are placed in leading positions in Academia and industry. Given the extraordinary number and quality of applicants, the capacity and eagerness of the Program faculty to train the Program’s students, and the institutional support for the Program, this application seeks to increase the number of trainee slots to 12. Following Training Grant support of Graduate students during their course work education and initial research training, all graduate students will be supported by their thesis advisors for the duration of their PhD studies. The Proposal outlines our past success in training students and discusses significant novel strategies for enhancing the training program. The new inception of the UCSD Halicioglu Data Sciences Institute provides great synergy for our Graduate Program and offers the trainees an opportunity to become leaders in the emerging areas of biomedicine that are heavily becoming data science disciplines.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY There is a vast repertoire of species within cells for which we have a poor understanding of their function and biomolecular interactions. These species can be referred to as the “dark matter” of biology, as their mechanism of action is hidden from conventional observation. Our laboratory seeks to illuminate the function of cellular “dark matter” through the development of new chemical technology. The proposed research program will pursue two major research thrusts. First, we plan to develop tools for site-specific RNA modification, and apply these tools for the manipulation, imaging, and isolation of disease relevant RNA protein complexes. We will create tools for use in live cells and develop the ability to covalently recruit proteins to RNA, forming RNA-protein macromolecular conjugates. The technology will be applied to study RNAs implicated in disease. Specifically, we are interested in characterizing the pathways of pathogenicity for the C9orf72 nucleotide repeat expansion RNA, which is thought to play a major role in genetic amyotrophic lateral sclerosis (ALS). In the second thrust, we will carry out the in situ synthesis of lipid species within living cells, with the goal of uncovering the molecular mechanism by which enigmatic lipid species affect cell behavior. We plan to develop approaches enabling the selective and bioorthogonal delivery of sphingolipids to living cells. Building upon technology previously developed in our lab, we will deliver cell permeable lipid precursors which will spontaneously assemble into functional lipids within the cell. Leveraging this approach, we will create photoaffinity probes for the pulldown of sphingolipid-interacting proteins, with the goal of elucidating the protein partners of the non-canonical deoxysphingolipid 1-deoxydihydroceramide, which is cytotoxic and implicated in several diseases. Realization of our research program goals would improve our knowledge of cell biology and lead to the development of new tools for interrogating RNA and lipid species. Our long-term vision is to create and apply technology that enables improved mechanistic understanding of biomolecular interactions, leading to an increased understanding of human disease, and accelerating the development of possible therapeutic interventions.

NIH Research Projects · FY 2025 · 2021-06

Malignant gliomas are the most common and deadly primary brain tumor. Despite current therapeutic approaches, most glioma patients die within one year of diagnosis. Genomic instability coupled with aberrant regulation of cell-fate decisions in progenitor cell populations has been linked to glioma, leading to the view that a convergence of genetic mutation and developmental context lead to tumorigenesis. Recent findings demonstrate that a large set of developmental transcription factors are activated in gliomas. These studies suggest that the gene regulatory programs governing glial cell formation are reutilized during glioma formation, pointing to common transcriptional requirements for glial development and tumorigenesis. Therefore, it is critical that we leverage our understanding of glial cell development to gain valuable insights into the biology and treatment of gliomas. We have previously identified a gliogenic transcriptional complex – Sox9/Brn2– that is important for the initiation of gliogenesis. We showed that in a mouse model of malignant glioma disruption of the ability of the complex to bind DNA regulatory elements leads to decreased expression of the glial initiating factor Nuclear Factor-IA (NFIA) and reduced tumorigenesis. Our preliminary data demonstrate that a protein Med12 (Mediator Complex subunit 12), which is expressed during glial cell development, linked to chromatin conformations, and implicated in cancer tumorigenesis, associates with the Sox9/Brn2 complex. Further, reduction of Med12 expression in cortical astrocyte cultures compromises DNA chromatin conformation at the Nfia locus. Therefore, we propose to explore the parallels between embryonic glial development and tumorigenesis by delineating the transcriptional circuitry and regulatory landscape governing glioma tumorigenesis. Analysis of these mechanisms is expected to identify molecular targets important in gliomagenesis. We focus this proposal on the function of a transcriptional complex, important for glial cell differentiation, -Sox9/Brn2/Med12- and its role in coordinating transcriptional mechanisms through gene regulatory elements, and chromatin conformations during gliomagenesis. We will investigate the role of Med12 in glioma formation and tumor cell biology using a wide range of in vitro and in vivo techniques in a novel mouse model of malignant glioma. We will interrogate the mechanisms by which Med12 functions in mediating enhancer/promoter interaction during glioma formation and progression by exploiting recent technological advances that allow for examination of long-range chromatin interactions (i.e. 3C Assays). We will functionally validate downstream target genes of the Sox9/Brn2/Med12 complex that may influence glioma formation. Together, these studies will define how developmentally relevant transcriptional mechanisms including gene regulatory elements and chromatin architecture interface to influence glioma biology and reveal novel gene targets potentially regulating gliomagenesis.