Ut Southwestern Medical Center

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Current oral antivirals used against SARS-CoV-2 infection such as protease inhibitors have limitations such as absence of anti-inflammatory effects and rebound COVID-19 after a 5-day course of outpatient antiviral treatment. Novel antiviral therapies against SARS-CoV-2 are urgently needed that ideally also limit inflammatory responses that contribute to morbidity from COVID-19. Reactive oxygen species (ROS) impair cellular functions and drive pro-inflammatory signaling and pathogenesis of infections with RNA viruses like coronavirus. Mitochondria are the main source of energy and ROS (mito-ROS). Mito-ROS are regulated by the antioxidant Nrf2 pathway that mediates pathogenesis and tissue damage of viral infections. We showed that MitoQ, a mitochondrial antioxidant and Nrf2 agonist, inhibits in vitro and in vivo viral replication of several SARS-CoV-2 variants of concern (VOC) and associated inflammatory response and apoptosis in airway epithelial cells through the Nrf2 pathway. Given that MitoQ is a diet supplement that is safe and immediately available to humans for use as possible therapeutic, we also showed antiviral efficacy of MitoQ in humans in a carefully designed open label clinical trial of post exposure prophylaxis (PEP) of MitoQ, compared to no MitoQ (control group), to prevent development of SARS-CoV-2 infection after high-risk exposure to a person with confirmed SARS-CoV-2 infection. MitoQ was well tolerated. We hypothesize that MitoQ attenuates the SARS- CoV-2-induced aberrant mito-ROS and Nrf2 pathway in epithelial cells and ultimately tissue injury that drives development and progression of severe COVID-19. The goal of this application is to determine if MitoQ can be used as novel oral safe outpatient post-exposure prophylaxis treatment against development of severe COVID-19. We propose the first proof-of concept randomized, double-blind, placebo-controlled Phase II efficacy study (RCT) with oral MitoQ use in adults at high risk for development of COVID-19, to further establish the safety and the efficacy of MitoQ against development of SARS-CoV-2 infection. We will include 2 treatment groups (MitoQ 20 mg daily, placebo) and we aim to recruit a total of 112 participants in total (50 per group accounting for 10% attrition). The primary outcome of efficacy will be the development of SARS- CoV-2 infection after high-risk exposure. The primary outcome of safety will be the proportion of participants exhibiting adverse effects of any grade. Given that attenuation of detrimental host responses that propagate viral replication may impose a higher barrier to generation of resistant viruses, this study will directly test MitoQ as a novel oral, safe, potent therapeutic strategy for COVID-19 against emerging SARS-CoV-2 VOCs. Our proposal will set the foundation of larger clinical trial that will advance use of MitoQ for COVID-19 to supplement existing treatments.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY / ABSTRACT Voice behavior in daily life is assumed to be a primary factor in the development and persistence of phonotrauma (e.g., vocal fold nodules, polyps, etc.), which is one of the most prevalent behavioral voice disorders. The shortage of prospective, longitudinal investigations into the ambulatory voice behaviors in high-risk individuals limits the differentiation between factors that are causative versus compensatory to phonotrauma, and therefore impacts optimal risk-assessment, mitigation, and therapeutic decision-making. Recent work has found that individuals with phonotrauma had higher levels of personality traits related to social dominance and impulsivity and higher speaking vocal doses and increased speaking vocal intensity during voice use in daily life. Individuals without current vocal pathology had strong relationships between those same personality traits and vocal behaviors, suggesting that personality traits and speaking voice use in daily life could be robust factors in the development of phonotrauma and could play a role in the prognosis for recovery. The primary objective of this project is to determine the enduring role that personality traits and daily voice use have on the development of phonotrauma, changes in vocal fold kinematics, and on the ability to change damaging vocal behavior using prospective, longitudinal methods. Aim 1 seeks to identify personality and vocal behavior factors involved in the development of phonotrauma. This will be achieved through initial assessment using a personality trait inventory, ambulatory voice monitoring, and laryngeal assessment via high-speed videoendoscopy of vocally asymptomatic singers who are freshmen in a college voice concentration music program. We will conduct these assessment procedures bi-annually, beginning in the singer’s freshman year and ending at the conclusion of their junior year. Aim 2 will assess which ambulatory and vocal fold kinematic parameters are the most predictive of phonotrauma. Aim 3 will investigate the impact that personality has on compliance with therapeutic/behavioral recommendations in daily life through personality assessment and ambulatory monitoring before and after participation in voice therapy. These studies will be the first to use ambulatory monitoring to (1) prospectively observe factors related to the development of phonotrauma and (2) measure the impact that personality traits have on the ability to change individual vocal behaviors. It will also be the first to use high-speed videoendoscopy to observe development of phonotrauma over time. This project will support advancement of the PI’s long-term career objective, which is to implement a program that uses psychosocial and behavioral factors to improve the prevention and treatment of phonotrauma in high-risk populations. This career objective can only be achieved with adequate training in longitudinal research methods, advanced statistical analyses, and acquisition and analysis of high-speed videoendoscopy. Results from this study will build a framework to ultimately improve early identification of phonotrauma risk, develop risk mitigation strategies, and inform treatment approaches.

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT This proposal describes a mentored training and research plan that will facilitate the development of Elie Abu Jawdeh, M.D., Ph.D., to an independent clinical and translational investigator in neonatal respiratory control and intermittent hypoxemia (IH). Dr. Abu Jawdeh will complement his current background as a neonatologist and a clinical scientist to attain new knowledge in basic science methods and biomarker science. The goal of learning basic science techniques and methods is imperative for Dr. Abu Jawdeh to make the leap into understanding mechanisms of injury from IH and develop treatments in the future. He will achieve these goals through structured mentorship, rigorous hands-on laboratory experiences, formal classwork, and skills training. A team of mentors with an established track record in clinical, translational, and basic sciences will oversee Dr. Abu Jawdeh’s development and progress to research independence. Despite significant progress in neonatal intensive care, preterm infants continue to suffer from significant morbidities and neurodevelopmental impairment. The societal cost of prematurity is approximately 26 billion dollars with the cost of care for an extremely preterm infant being 20 times that of a late preterm infant. Major consequences of prematurity are apnea and lung disease that lead to repeated occurrences of IH; episodic drops in blood oxygen saturation. These IH episodes, occurring up to hundreds of events/day, have a cumulative effect on neonatal outcomes. While the evidence linking IH to impairments is mounting, the degree of IH associated with injury and pathophysiological mechanisms for IH’s contribution to injury remain unclear. This knowledge gap in the mechanistic understanding of IH creates a critical barrier to improving clinical outcomes. Our goal for this proposal is to better understand mechanisms and biomarkers of injury from IH in order to discover and titrate treatments in the future. Through both a prospective clinical cohort of preterm infants and in vitro cell culture experiments, we will first test the hypothesis that IH increases systemic circulating ligands (bio-mediators) that injure brain cells using in vitro cell culture system of oligodendrocyte progenitor cells (OPC). Identifying bio- mediators and pathways of cell death and injury from IH will help develop treatments that we will also explore in vitro. Then, we will investigate a promising biomarker Neurofilament-L (NfL) for IH-related brain injury and predictor of early neurological outcome. Identifying biomarkers of injury will allow prognostication of neonatal outcomes and monitoring of IH treatment effectiveness and titration in the future. We have pilot data to support both our Aims and hypotheses. This will be the first time such studies and mechanisms are investigated in preterm infants with IH.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Lung cancer cells express high levels of PD-L1, a ligand of the PD-1 receptor on T-cells, allowing tumors to directly suppress T cell activity. Anti-PD-1/PD-L1 antibodies induce potent anti-tumor immunity and have been approved as a first-line therapy for lung cancer. However, only ~20% of all non-small cell lung cancers (NSCLCs) benefit from checkpoint blockade. A further understanding of the mechanisms that regulate the immune checkpoint in lung cancer is therefore needed. To address this, my laboratory used CRISPR-based screening to identify regulators of PD-L1 in lung cancer cells, revealing potent induction of PD-L1 upon activation of the integrated stress response (ISR) pathway. Mechanistically, ISR activation resulted in enhanced PD-L1 translation and suppression of anti-tumor immunity. We further demonstrated that ISR- dependent translation of PD-L1 requires the alternative translation initiation factor eIF5B. The canonical role of this GTPase is to catalyze ribosomal subunit joining, ensuring 80S ribosome assembly and efficient start codon selection. eIF5B overexpression and/or amplification is frequent in human lung cancers and is associated with poor prognosis. Remarkably, eIF5B overexpression is sufficient to induce PD-L1 protein levels even in the absence of ISR activation. These findings uncovered a new mechanism of immune checkpoint activation and suggested that eIF5B may be a novel target for lung cancer intervention. We further demonstrated that enforced expression of eIF5B accelerates proliferation in lung cancer cells, in mouse syngeneic models, and in human bronchial epithelial cells suggesting that it also promotes tumor growth in a cell-autonomous manner. We propose to elucidate the role of eIF5B in lung tumorigenesis by testing the following central hypothesis: EIF5B functions as an oncogene in human lung cancer by inducing PD-L1 translation and by driving a tumor-promoting translational program. Three Specific Aims will be pursued in order to test this hypothesis: In Aim 1, we will dissect the mechanisms through which eIF5B promotes translation of PD-L1 and additional oncogenic mRNAs in lung cancer. In Aim 2, we will functionally evaluate the oncogenic activity of eIF5B using a novel transgenic mouse model with conditional eIF5B overexpression. In Aim 3, we will characterize the effects of eIF5B loss of function on cell autonomous tumor growth versus its effect on T cell responses in the KrasLSL-G12D; Tp53 fl/fl mouse model using a newly generated conditional floxed knockout allele and a heterozygous germline knockout mouse. These aims will take advantage of our expertise, and that of our collaborators, to evaluate eIF5B oncogenic activity, and elucidate the mechanisms through which this translation initiation factor promotes lung tumorigenesis. We anticipate that these studies will provide new insights into mechanisms of translational control in tumor progression and immune evasion.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Gliomas represent 80% of the 26,000 newly diagnosed cases of malignant brain and central nervous system tumors in the United States each year and are among the most lethal and treatment-resistant human cancers. Hot-spot, mono-allelic, gain-of-function mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 are present in more than 70% of certain subtypes of gliomas, thus representing the genetic hallmark of these malignant brain tumors. The ‘oncometabolite’ (R)-2-hydroxyglutarate [(R)-2HG], produced by IDH mutant enzymes, modulates the activities of certain 2-oxoglutarate (2OG) dependent DNA and histone demethylases, which subsequently promotes neural cell transformation. Accordingly, broad changes in histone and DNA methylation are strongly associated with IDH mutations in glioma. Despite these discoveries, the precise molecular mechanisms linking oncometabolite-dependent chromatin remodeling with gliomagenesis remain obscure. I propose to directly address this knowledge gap and test a new conceptual model to explain oncometabolite-driven tumorigenesis. In Aim 1, I will perform time-resolved single cell and bulk RNA-seq and ATAC-seq analyses of the molecular and cellular changes that occur during brain tumor initiation in a novel genetically engineered mouse (GEM) model of glioma to reveal key mechanisms of (R)-2HG dependent malignant transformation. In Aim 2, I will conduct integrative analyses of sequencing datasets from human glioma samples and murine glioma samples from our GEM models to discover functional targets of (R)-2HG in IDH mutant gliomas. In Aim 3, I will leverage novel mouse brain organoid models to investigate the impact of oncometabolite activity on neural cell specification during brain development. If successful, my work will provide a new conceptual framework for understanding the deterministic and stochastic functions of oncometabolite signaling to chromatin and their influence on cell fate. This advance would deepen our understanding of how metabolic alterations signal to chromatin in cancer and other diseases. Furthermore, identifying the molecular processes that functionally link oncometabolites with brain tumor initiation may reveal new therapeutic targets to combat this disease.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY This application seeks to understand how circadian rhythms control T cell immunity to influenza infection through signals delivered by the sympathetic nervous system. Circadian rhythms are biological clocks that are entrained by light/dark cycles, and in mammals, those rhythms are synchronized systemically throughout the body via signals delivered by sympathetic and parasympathetic neurons. Our recent studies uncovered an unexpected role for the β2-adrenergic receptor in cytolytic responses of CD8+ T cells to a virus infection. Further, we found that selective deletion of the Adrb2 on CD8+ T cells profoundly disrupted circadian gene expression and oscillation frequencies as a function of light/dark entrainment. This proposal will test the hypothesis that the signaling through the ADRB2 in CD8+ T cells regulates circadian rhythm genes, and that those genes are required for orchestrated T cell responses to influenza infection. Aim 1 will distinguish the roles of the Adrb2 and downstream core clock in CD8-mediated lung inflammation during influenza infection. Aim 2 will determine how jet-lag impacts CD8+ T cell responses to influenza. This proposal tests a very straight- forward hypothesis, which if supported, will be the first to connect a circadian rhythm entrainment receptor to downstream T cells responses to influenza. The outcomes of this research will significantly advance our understanding of environmental pathways that regulate immune responses to influenza and improve mouse models of viral pathogenesis and vaccine design.

NIH Research Projects · FY 2024 · 2023-07

Project Summary The human intestine is colonized by a diverse array of almost 100 trillion bacteria that are critical for health. Intestinal mucus is the critical interface between the host and the gut microbiome; it is a barrier between humans and pathogenic microbes, and it is also an attractant for beneficial microbes, supporting vital commensal microbes with nutrients and attachment sites. The main components of intestinal mucus are highly O-glycosylated mucins and other glycoproteins. Inflammatory bowel diseases, such as ulcerative colitis and Crohn’s, are characterized by gut inflammation that results in dysbiosis of the microbiome and alterations in intestinal mucus glycan structures. The molecular structure of mucus has functional significance. Fucosylation is abundant in mammalian intestinal mucus, and it has been shown to regulate commensal microbe colonization and maintain host-microbe symbiosis. Additionally, fucosylation has been shown to alter the quality and quantity of intestinal mucus in ulcerative colitis patients. Mucin sulfation has also been implicated in ulcerative colitis. β1-3-N-acetylglucosaminyltransferase 7 (B3GNT7) is an O-glycosyltransferase present on the Golgi apparatus membrane that transfers GlcNAc to glycan substrates and participates in polyLacNAc chain biosynthesis. Importantly, these polyLacNAc chains can go on to be further modified by fucose and/or sulfate. We previously reported that that IL-22, a cytokine critical for maintaining intestinal epithelial homeostasis, promotes B3GNT7 expression, increases fucosylated O-glycans, and increases polyLacNAc chains on a model of the human intestinal epithelium. Furthermore, we found that overexpression of B3GNT7 is sufficient to increase cell surface fucosylation. The research outlined in this proposal that B3GNT7 functions to maintain healthy intestinal mucus and support beneficial commensal gut microbes. This proposal will 1) Define the impact of B3GNT7 on the biophysical properties of intestinal mucus in cell lines and mice; and 2) Determine the effect of B3gnt7 expression on host-microbiome interactions. Regulation of intestinal mucus and the microbiome is poorly understood, and the experiments outline above will elucidate how changes in glycosylation regulate the microbiome at the molecular and tissue level. Importantly, it will lay the foundation for future studies of how glycosylation of intestinal mucus contributes to both human health and disease.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Genomic instability is a hallmark of cancer and can drive high rates of chromosome segregation errors during mitotic cell division, which can generate abnormal structures called micronuclei that entrap mis-segregated chromosomes. Micronuclei are susceptible to massive DNA damage, triggering the catastrophic pulverization of the entrapped chromosome into small DNA fragments, a process termed chromothripsis. In addition to generating genomic rearrangements that drive cancer development, DNA fragments from micronuclei can also mis-accumulate and persist in the cytoplasm. These cytoplasmic DNAs are detected by the cytosolic DNA sensor cGAS, resulting in the cell-autonomous activation of the STING pathway to trigger an innate immune response. Although the cGAS-STING signaling mechanisms are well defined, the fate of cytoplasmic DNAs following recognition by cGAS remains a critical knowledge gap. Here I propose to investigate non-cell autonomous roles of cytoplasmic DNAs, which hold critical implications in how genomically unstable cancer cells can elicit inter- cellular responses with the tumor microenvironment. I hypothesize that cytoplasmic DNAs derived from fragmented chromosomes in micronuclei become exported for uptake by neighboring cells. To test this hypothesis, I will leverage an experimental system enabling chromosome-specific induction of micronuclei and cytoplasmic DNA followed by tracking of specific cytoplasmic DNA fragments that harbor a selectable marker. In Aim 1, I will determine whether and how cytoplasmic DNAs are released into the extracellular environment to facilitate non-cell autonomous activation of the cGAS-STING pathway in adjacent cells. I will further determine whether extracellular DNA derived from the cytoplasm of host cells can be taken up by recipient cells, which will be monitored by live-cell imaging using a dCas9-based cytoplasmic DNA reporter. In Aim 2, I will track the incorporation of cytoplasmic DNAs into recipient cell nuclei and determine whether these fragments can integrate into the host genome. This will be studied using a combination of cytogenetics and whole-genome sequencing to investigate the possibility of lateral DNA transfer between human cells. Despite occurring frequently in bacteria, inter-cellular DNA transfer has been a longstanding challenge to test in the context of human cancer. These studies have potential for broad impact by advancing our understanding of cancer cell interactions, including the transfer of oncogenes and/or mutations from chromosomally unstable cancer cells to non-cancer cells in the tumor microenvironment.

NIH Research Projects · FY 2024 · 2023-07

Project Summary Cells use post-translational modifications (PTM) to modulate protein function by conjugating and deconjugating modifiers to protein targets. Imbalance in these reactions leads to human disease. Conjugation of ubiquitin-fold modifier 1 (UFM1) to protein targets (UFMylation) has been linked to many cellular processes at the endoplasmic reticulum (ER). In contrast, much less is known about the roles of UFM1 deconjugation (de-UFMylation), the process of removing UFM1 from UFMylated proteins. Defects in UFSP2, a de-UFMylation enzyme, were identified in patients with skeletal and neurodevelopmental disorders. Notably, patient-derived fibroblasts harboring UFSP2 deficiency show excessive amounts of UFMylation (hyper-UFMylation) and defects in mitochondrial respiration and nucleotide metabolism, indicating as-yet undescribed roles of UFMylation in cellular metabolism. These findings suggest UFMylation as a novel regulator of mitochondrial function and nucleotide metabolism. The long-term goal is to understand how UFMylation regulates cellular metabolism. The overall objective is to understand the molecular mechanism by which UFMylation regulates mitochondrial respiration and nucleotide metabolism, and the molecular mechanism of action of UFSP2-mediated de-UFMylation. The central hypothesis is that UFSP2 deficiency causes (i) hyper-UFMylation of mitochondrial ribosomes (mitoribosomes) and the electron transport chain (ETC) complex I which change protein localization and/or function, leading to decreased protein abundance and/or loss-of-function of ETC complexes; and (ii) hyper-UFMylation of enzymes in nucleotide metabolic pathways, leading to changes in enzyme activity and perturbation of nucleotide metabolism. The rationale is that probing localization and measuring activity of the hyper-UFMylated mitoribosomes, the ETC Complex I and serine hydroxymethyltransferase-2 (SHMT2), will reveal how UFMylation regulates mitochondrial respiration and nucleotide metabolism. In addition, the structure of UFSP2 in complex with UFMylated targets will reveal the structural basis for substrate recognition and de-UFMylation reaction. The central hypothesis will be tested by pursuing three specific aims: 1) Determine how hyper-UFMylation of mitoribosomes and Complex I impairs mitochondrial respiration; 2) Investigate the effect of hyper-UFMylation of SHMT2 on nucleotide metabolism; and 3) Determine the structural basis for substrate recognition and deconjugation reaction of human UFSP2. For the first aim, protein abundance, localization and enzyme activity of mitochondrial ribosomes and the ETC Complex I will be measured in patient-derived UFSP2-depleted versus WT UFSP2 cells. For the second aim, isotope tracing will be used to evaluate metabolic rates of serine and nucleotide synthesis, while the localization and enzyme activity of SHMT2 will be assessed in patient-derived UFSP2-depleted versus WT UFSP2 cells. For the third aim, the UFSP2 in complex with its UFM1-conjugated substrate will be trapped, purified and structure-determined. The research proposed is innovative because it will elucidate the roles of de-UFMylation and the effects of UFMylation on cellular metabolism for the first time. The proposed research is significant because it (1) contributes to the identity of the UFMylome, (2) reveals UFMylation as a novel regulator of mitochondrial respiration and nucleotide metabolism, and (3) reveals the structural basis for substrate recognition and deconjugation reaction of UFSP2.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Extracorporeal Membrane Oxygenation (ECMO) is a form of cardiopulmonary bypass used in critically ill children and adults to support the heart and lungs when conventional therapies fail. More than 79,762 children worldwide have been supported to date, and global use of this tool is expanding. Advances in ECMO and critical care have improved survival of otherwise fatal illnesses, thereby unmasking neurologic injury which itself reduces survival by 50-60% and leads to significant long-term neurologic morbidity. The mechanisms of ECMO-related cerebral injuries are poorly understood. Existing research focuses on evaluating discrete elements, such as underlying illness, coagulation abnormalities, anticoagulation management, or markers of end- organ perfusion as factors associated with brain injury. Prior studies have not considered the temporal and dynamic element of clinical events that may play a large role in the genesis of brain injury, and few have explored which variables could predict significant neurologic injury without the bias of pre-selecting variables of interest. Machine learning is a form of artificial intelligence that employs algorithms to discover patterns in an iterative manner directly from input data: in the context of ECMO, it can identify dynamic patterns and relationships between variables prior to neurologic injury. The long-term goal of this research is to identify modifiable bedside predictors of neurologic impairment and thereby drive the development of early interventions to improve neurologic outcomes of children undergoing ECMO. Towards this goal, we have assembled a multi- disciplinary team with clinical and computational expertise. Our central hypothesis is that a robust risk predictive model for SNI in ECMO patients can be developed based on the physiological and laboratory data routinely collected in real-world clinical settings and that this model can be used to identify parameters of SNI for potential intervention. This proposal will utilize advanced machine learning algorithms to build this prediction model in a large multicenter cohort (0-18 years, n=750). In Aim 1, we will use novel probabilistic machine learning algorithms to train and develop a model to predict SNI by neuroimaging. In Aim 2, we will validate and refine the model from Aim 1 using neuroimaging scores and explore a personalized anytime query algorithm that predicts the timing and type of SNI. This collaborative proposal between clinical and computational scientists will lay the groundwork for a neuroprotective interventional study by identifying modifiable risk factors to improve the tragically high neurologic morbidity and mortality in ECMO survivors.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Chronic kidney disease is a leading cause of death in the United States and places an immense burden on the healthcare system. Many cases of chronic kidney disease eventually result in renal fibrosis, which is characterized by deposition of excess extracellular matrix (ECM) by myofibroblasts and contributes to eventual organ failure. Recent studies have identified renal pericytes, a type of perivascular cell that surrounds endothelial cells of small vessels such as capillaries, as the source of myofibroblasts in fibrosis. Despite this characterized role in a major disease process, little is known about the developmental origin or role of pericytes. We have characterized a novel population of pericytes that surrounds arteries only during development, which we call periarterial pericytes. We have characterized the origin of these pericytes using murine genetic reporters. Furthermore, we have identified a potential regulator of pericyte migration, which is present in the kidney during development. This temporary guidance of periarterial pericytes implies an important role in arteriogenesis. Our preliminary data suggest that pericytes may be required for collagen production and vascular smooth muscle differentiation during nephrogenesis, mirroring their pathogenic role in fibrosis. Thus, the overall goal of this project is to define the role of periarterial pericytes during nephrogenesis in remodeling the ECM of the periarterial niche to support vascular smooth muscle maturation, as well as to elucidate the mechanism by their localization is regulated during development. We will utilize murine genetic lineage tracing tools, in addition to immunofluorescence staining and cutting edge in situ hybridization techniques, to trace the origin, fate, and gene expression of periarterial pericytes throughout development. We will selectively ablate pericytes in vivo to assess their requirement in arteriogenesis and smooth muscle coverage. Finally, we will utilize in vitro models to clarify intracellular interactions that are required for pericyte secretion of ECM and smooth muscle cell maturation, as well as to elucidate the mechanism by which pericyte migration is regulated. Collectively, these studies will characterize a novel role of pericytes during development, as well as elucidate molecular mechanisms that may be dysregulated in disease processes such as renal fibrosis. This information may inform future therapies for chronic kidney disease or regenerative medicine efforts, both which will be badly needed as the prevalence of kidney disease grows in the United States and abroad.

NIH Research Projects · FY 2025 · 2023-07

Mechanistic Investigation of Gut Mycobiota in the Regulation of Lung Immunity and Disease (PA-20-188) Project summary/Abstract Asthma is a common, chronic airway inflammation that affects around 25 million Americans and 334 million people worldwide. In the mammalian gut, a diverse fungal community, known as the mycobiota shapes local and systemic host immune responses. Alterations of the gut fungal community are strongly associated with inflammatory disorders such as asthma, chronic obstructive pulmonary disease (COPD), and inflammatory bowel diseases (IBD). Our recent findings suggest that intestinal specialized phagocytes could mediate the activation of fungal-primed T cells during allergic airway disease. Despite these, the precise mechanisms of gut- lung immune crosstalk are unknown. Based on these preliminary findings, we hypothesize that gut commensal fungi initiate antifungal immunity that affects lung immunity and modulates the progression of airway inflammation. The objectives of this project are to understand the molecular and cellular interplay between gut fungi and host lung immunity with specific focus on T cells. While the current proposal is to illuminate the basic effects of gut fungi on lung immunity and disease and to uncover mechanistic insights about gut-primed antifungal T cell activation and migration on the development of immune-mediated airway inflammation. My long-term goal is to investigate how the gut commensal mycobiota modulates the gut-systemic axis and impacts other pulmonary diseases, such as COPD and cystic fibrosis (CF). During my K99 training, I will be supervised by a team of mentors and scientific advisors with expertise in mycobiota, mucosal immunology, T cell biology and pulmonary diseases. They will provide strong support and mentorship in both research and career transition to independence, and help me to develop necessary technical skills and conceptual knowledge on lung immunity and antifungal T cells. This K99/R00 award will enable me to acquire the skills necessary for a deep analysis and understanding of the gut-systemic axis with the goal of creating novel therapies to treat asthma, COPD, CF, and other immune-related pulmonary diseases.

NIH Research Projects · FY 2025 · 2023-07

Background: Peripheral artery disease (PAD) is a common and highly morbid condition. Nearly 25% of patients die within 3 years of diagnosis, likely due to a high incidence of cardiovascular (CV) events: myocardial infarction (MI) or stroke. A significantly larger proportion experience disability due to leg pain, poor mobility and amputation. The cost of PAD-related hospital care alone exceeds $21 billion. However, research regarding long-term survival, CV, and limb outcomes in PAD and the impact of existing treatments remain limited in large part due to the poor accuracy of PAD diagnosis codes. Our team has developed a novel approach using natural language processing (NLP) to identify PAD patients with a high degree of accuracy within the Veterans Health Administration (VHA). Significance: The Peripheral Artery Disease: Long-term Survival & Outcomes Study (PEARLS) study will advance scientific knowledge for PAD in several ways. Using our novel NLP tool to identify Veterans with PAD, we will examine the trajectory of long-term survival and clinical outcomes, evaluate utilization of recommended treatments (medications, risk factor control and revascularization) and the association of above treatments with the above outcomes. We will also examine disparities in PAD care and outcomes by race and ethnicity and determine the extent to which these disparities our due to access in high quality care. Collectively, our work will address important gaps in PAD research and yield insights for improving care delivery in this high-risk population. Innovation: The use of an informatics-based method to assemble a cohort of newly diagnosed PAD patients in a large integrated health system is highly innovative. We believe that our approach for cohort identification will be transformational and promote big data analytics for research, improving care delivery, and future clinical trials. Specific Aims: A1. Examine the trajectory of long-term outcomes of PAD and assess racial and ethnic disparities. A2. Examine patterns of medical and invasive management of PAD in the Veterans Health Administration A3. Determine the association of medical and invasive management with long-term outcomes Methodology: We will implement our NLP algorithm to identify patients with new PAD diagnosis in VHA during 2015-2020 and obtain data on clinical and treatment related variables. We will follow our cohort longitudinally for mortality, CV events (MI, stroke) and limb events (amputation). We will examine utilization of PAD treatments and risk factor control, identify patient-level and hospital-level predictors of treatment using multi-level models. We will use marginal structural models to evaluate the association of PAD treatments with long-term outcomes. Implementation/Next Steps: Key deliverables will include a) an assessment of long-term outcomes in PAD and identifying racial disparities in care and outcomes; (b) determining the relative impact of PAD treatments on long- term outcomes which can be useful for decision-making and c) an assessment of site-level variation in treatment patterns. We envision that our findings will help us develop comprehensive disease management program to improve quality of care and reduce disparities in use of effective treatments.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract: Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels responsible for excitatory signaling throughout the nervous system. The α7 nicotinic receptor subtype is a widely expressed subtype that is unique among nicotinic receptors in that it forms a functional homopentamer, desensitizes rapidly, and is exceptionally permeable to Ca2+. It plays a prominent role in human disease with dysregulation of α7 linked to Alzheimer’s disease, schizophrenia, and inflammation. α7’s unique characteristics and involvement in disease have made it an attractive therapeutic target leading to the development of numerous compounds aimed at enhancing α7 activity. Among the most promising strategies are positive allosteric modulators (PAMs). PAMs increase receptor activity while maintaining the spatial and temporal characteristics of endogenous neurotransmission. Despite strong functional characterization, precise modulator binding sites and structural mechanisms of potentiation remain unknown, representing a major roadblock towards rational drug design. Moreover, while α7 can uniquely assemble as a functional homopentamer, it can also associate with other subtypes forming a heteromer, yielding increased diversity and altered channel properties. The degree of α7 channel diversity in native tissue is unclear. Thus, the major goal of this application is to resolve key questions related to the α7 subtype by elucidating receptor modulation mechanisms and determining the molecular structure of native α7. Building on previous work defining major gating cycle conformations, I aim to determine high resolution structures of α7 complexed with numerous allosteric modulators. In parallel, I will develop a method to purify native α7 from brain tissue and determine the structure of α7-containing channels using single particle cryo-EM. Taken together, this work will define mechanisms of receptor modulation and native α7 structure and subunit stoichiometry, enhancing our understanding of α7 biology and pharmacology. Furthermore, this work will provide a foundation for future native receptor purification and aid drug development towards the entire nicotinic receptor family.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract: Melanoma is the leading cause of skin cancer deaths because it metastasizes efficiently to distant organs. Metastasis requires significant plasticity for cancer cells to survive the metabolic barriers distinct from the original primary tumor site including higher oxidative stress, limited nutrient availability, and changing microenvironments. The specific metabolic changes required for melanoma to undergo this process are poorly understood. We recently identified a novel suppressor of melanoma metastasis, the enzyme glyceraldehyde 3- phosphate dehydrogenase, spermatogenic (GAPDHS). We characterized the functional role of GAPDHS in a patient-derived xenograft (PDX) model and demonstrated its impact on glycolysis and pyruvate carboxylase activity. In this proposal, we will investigate the metabolic changes associated with GAPDHS that drive metastasis and determine whether its expression can be used to predict tumor progression in melanoma patients. Specifically, we will determine whether the enzymatic activity of GAPDHS in central glycolysis is required for suppressing metastasis (Aim 1), if its downstream suppression of pyruvate carboxylase activity influences metastasis (Aim 2), and whether GAPDHS expression serves as a biomarker of melanoma metabolism and metastasis in patients (Aim 3). As an Assistant Professor of Dermatology at the University of Texas Southwestern Medical Center, I devote 80% of my time to my research interests under the mentorship of Dr. Ralph DeBerardinis, with the remaining time dedicated to clinical practice. My goal is to transition to a career as a successful independent investigator overseeing my own laboratory and research program. To achieve this, I am seeking a K08 award to provide support for an additional period of mentored research to gain experience with cancer metabolism, biomarker development, and advanced biostatistics, which are all necessary to achieve my research goals. With the guidance of a distinguished mentorship committee, I will have access to the training, resources, and support necessary to establish a successful independent research program focused on identifying metabolic vulnerabilities in melanoma metastasis.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY/ABSTRACT This P01 proposal offers a fresh approach to understanding the genetics of an extremely important polygenic autoimmune disease: type 1 diabetes (T1D), which affects nearly three children out of every thousand in North America, and many more around the world. T1D occurs with variable penetrance in Non-Obese Diabetic (NOD) mice, which exhibit a phenotype remarkably similar to that of human patients. Both environmental and genetic factors determine penetrance, but most of the influential mutations and the genes they affect remain unknown. We have developed a powerful technology platform that permits instantaneous identification of point mutations that cause phenotype. Using this platform, we have already identified two spontaneous mutations that cause high and low frequency of disease development in the NOD/NckH and NOD/NckL sublines, respectively. Noting that these sublines, isolated by selective breeding over a period of only seven years, had approximately the same mutational distance from one another as one finds in a pedigree of ENU mutagenized mice as compared to the parental reference strain, we performed a pilot study in which mice were mutagenized on the NOD/NckH background. In a sample of 14 pedigrees, we unambiguously identified twelve ENU-induced mutations with modifying effects on T1D: some accelerating the disease and others suppressing it. Stressing the precision of these studies, which do not merely identify intervals or candidates, but resolve the exact nucleotide change responsible for T1D modification, we propose to expand our efforts, analyzing 21,000 coding/splicing mutations for modifier effects over a period of five years. Our preliminary work suggests that T1D is “balanced on a knife’s edge” from a genetic point of view. Mutations in many genes are clearly capable of influencing T1D development, since randomly induced coding/splicing mutations affecting approximately 1% of the mouse genome caused unambiguous modifier phenotypes. We expect to identify scores if not hundreds of individual modifier mutations during the period of funding. Some of these will have important new facts to tell us about what it takes to develop T1D. Concentrating on those modifier mutations that show large effect sizes, may be amenable to targeting with therapeutic drugs, and/or are particularly surprising in light of what we presently know about T1D pathogenesis, we will rigorously verify causation by re-creating the mutations and/or deleting the causative genes on clean backgrounds (NOD/NckH or NOD/NckL devoid of ENU-induced mutations). We will then systematically examine the mechanism of phenotype modification, both at the level of cellular immuno-pathogenesis, and at the level of molecular pathogenesis. Ultimately, we hope to understand how T1D can be prevented or driven into remission, and we expect many new insights to emerge from the studies planned. A close collaboration between the Bach/Chatenoud group, with its great expertise in the study of T1D pathogenesis in NOD mice, and the Beutler group, with its strength in forward genetics, will contribute to the success of this P01. The Core laboratories and Projects are exceptionally synergistic, assuring that this P01 will dramatically exceed the sum of its parts.

NIH Research Projects · FY 2025 · 2023-06

Lung cancer is the second most common malignancy and the leading cause of cancer-related death in both men and women in the United States, and non-small cell lung cancer (NSCLC) accounts for ~85% of all cases. Localized and regional NSCLC are frequently treated with external beam radiation therapy alone or in combination with surgery and chemotherapy using platinum-based drugs and taxanes. Metastatic NSCLC is typically treated with systemic therapies such as chemotherapy, targeted molecular therapies, immune checkpoint inhibitors alone or in combination. Chemotherapy and targeted molecular therapies do not offer durable complete responses for metastatic disease, which can be achieved only in the minority of patients with immune checkpoint inhibitors. Also, recurrence is common for localized and regional NSCLC treated with radiation therapy (RT). RT has cytotoxic activity by causing DNA damage in NSCLC cells but is limited by intrinsic cellular mechanisms that repair DNA damage and confer resistance to RT. Thus, there is a need for novel agents to overcome DNA repair mechanisms and enhance the therapeutic efficacy of RT in NSCLC and increase the response rates for immune checkpoint inhibitors. We propose that the inhibition of the ATPases RUVBL1 and RUVBL2 with an orally available inhibitor is an effective and cancer-selective strategy for radiosensitization that efficiently blocks DNA repair pathways by reducing protein levels of three key DNA damage repair factors, DNA- PKcs and ATM/AR, in NSCLC cells but not in normal cells. Because of that unique activity, we expect that the RUVBL1/2 inhibitor will be more effectively enhance the antitumor effects of IR than specific DNA-PKcs/ATM/AR in vitro and in vivo and elicit less radiotoxicity. Also, we propose that the RUVBL1/2 inhibition elicits immune stimulation, and therefore will therapeutically synergize with IR and immune checkpoint inhibitors. This project will (1) determine the efficacy, specificity and determinants of radiosensitization by RUVBL1/2 inhibition, (2) characterize immune stimulatory effects of RUVBL1/2 inhibition alone and in combination with radiation, and (3) study the therapeutic potential of RUVBL1/2 inhibition in combination with IR and immune checkpoint inhibition. If our project is successfully completed, it may provide the framework for a new therapeutic strategy for NSCLC patients, which could improve clinical outcomes for this hard-to-treat disease.

NIH Research Projects · FY 2026 · 2023-06

ABSTRACT The mammalian target of rapamycin (mTOR) is an evolutionary conserved Ser/Thr kinase that senses multiple upstream stimuli to regulate cell growth, metabolism, and autophagy. mTOR is the catalytic component of a multi-protein complex called mTOR complex 1 (mTORC1). Elevated mTORC1 activation is common in multiple human disease including cancer, type 2 diabetes, metabolic disorders, and neurodegeneration. Small molecules like rapamycin that target and inhibit mTORC1 are used in the clinic with limited success. Thus, deciphering the molecular mechanisms by which mTORC1 is regulated is crucial to treat mTORC1-mediated disease. The overall objective of this proposal is to decipher the molecular mechanisms by which mTORC1 is regulated by upstream stimuli. Specifically, this proposal is focused on mTORC1 regulation by amino acids and G-protein coupled receptors (GPCRs). We anticipate that the studies in this proposal will yield new insights into mTORC1 regulation by upstream stimuli and will uncover novel therapeutic targets to perturb mTORC1-mediated disease.

NIH Research Projects · FY 2026 · 2023-06

Project Summary/Abstract Age-related neurodegenerative disorders, such as Alzheimer’s disease and related tauopathies, are a major therapeutic challenge. There are critical brain regions implicated in disease pathogenesis and overtime the whole or a large portion of the brain is affected. Therefore, both a focal and broad brain treatment approach is needed to achieve long-term therapeutic benefit. Gene therapy is the use of genetic material to target disease etiology. Viral vectors are used to deliver therapeutic genes to cells to provide long-lasting intervention from a single treatment. Recombinant adeno-associated virus (rAAV) vectors are highly used due to their efficient gene transfer, broad serotype-dependent tropism, low risk of insertional mutagenesis, and long-term transgene expression in non-dividing cells, such as neurons. AAV9 is currently the most frequently used serotype for treating neurological disorders as it can be delivered in blood or cerebral spinal fluid to broadly transduce the brain in a dose-dependent manner. Direct brain injections are effective for focal brain treatments, but are an invasive procedure. With non-invasive systemic delivery, high vector doses are needed to achieve sufficient brain transduction in adults. The greater the number of viral particles received increases the risk of severe immune responses and inhibits use in adults due to manufacturing considerations. We and others have demonstrated that use of FUS to open the BBB can significantly reduce systemic vector doses and target rAAV vectors to select brain regions. While promising for focal treatments, this then limits the ability of AAV9 to efficiently transduce non-targeted brain regions. CSF delivery of AAV9 significantly reduces the amount of vector needed to achieve equivalent or greater brain transduction compared to systemic delivery and achieves high CNS transduction relative to the site of injection. Therefore, we predict that IT delivery of AAV9 combined with transcranial FUS will allow for both focal and broad targeting of gene therapies to the brain and be an effective delivery approach for age-related neurodegenerative diseases. This grant will develop the use of combined FUS and IT injection as an efficient delivery method of AAV vectors to the brain, define mechanisms for this enhancement, determine vector capsid and promoter usage, and test this application in a mouse model of tauopathy. If we are successful, this approach can be readily translated for treating neurodegenerative diseases.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY A growing body of evidence indicates that long noncoding RNAs (lncRNAs), a diverse class of non-protein- coding transcripts >200 nucleotides in length, play important roles in the initiation and progression of cancer. LncRNAs have been proposed to regulate all cancer hallmarks, but, in the vast majority of cases, their molecular mechanisms of action remain poorly understood. This knowledge gap is a major impediment towards realizing the potential of lncRNAs as therapeutic targets in cancer and other diseases. As in many human malignancies, lncRNAs are frequently dysregulated in renal cell carcinoma (RCC). RCC is the most common type of kidney cancer and the most lethal malignant urological tumor, with approximately 70,000 new cases diagnosed annually in the United States. To date, most genomic studies of RCC (and other cancers) have focused on identifying disease-associated alterations of protein-coding genes. Our understanding of the molecular pathways regulated by lncRNAs in RCC, and the functional roles of these transcripts in this malignancy, remains limited. We mined RNA-seq data from RCC patients to identify a set of 805 lncRNAs that are commonly overexpressed in this tumor type. We generated a custom CRISPR interference (CRISPRi) library targeting these lncRNAs and performed screens in multiple RCC cell lines to identify lncRNAs that are essential for RCC cell proliferation. These experiments revealed that the lncRNA Colorectal neoplasia differentially expressed (CRNDE) is required for growth of all tested RCC cell lines. Although this lncRNA has been shown to be overexpressed and is associated with poor patient survival in RCC and other types of cancer, its molecular function remains unclear. We identified a critical region of CRNDE that is necessary for RCC cell proliferation and we identified proteins that interact with this sequence. We also generated novel genetically-engineered alleles in mice that enable constitutional or conditional deletion of critical Crnde sequences. In this proposal, we will leverage our new understanding of this lncRNA, and the novel resources we have generated, in order to dissect the molecular function of CRNDE and define its role in normal physiology and in RCC pathogenesis in vivo. These experiments will take advantage of our extensive experience, and that of our collaborators, in evaluating noncoding RNA functions and RCC biology using cellular and animal models. Successful completion of the proposed research will address two major knowledge gaps in the fields of RNA biology and cancer biology: 1) our limited understanding of the molecular mechanisms-of-action of lncRNAs; and 2) how these mechanisms are co-opted by cancer cells to promote tumor growth, particularly in RCC. We anticipate that the principles revealed by these studies will be broadly applicable to our understanding of the roles of other lncRNAs in cancer cells and may set the stage for developing therapeutics that target CRNDE or the pathways it controls.

NIH Research Projects · FY 2025 · 2023-06

Abstract Type I hyperlipoproteinemia (T1HLP, also known as familial chylomicronemia syndrome or FCS) is a rare, autosomal recessive metabolic disorder characterized by extreme hypertriglyceridemia due to a deficiency of lipoprotein lipase or related proteins. Treatment of these patients is challenging as conventional triglyceride- lowering medications, such as fibrates and fish oil, are ineffective. An extremely low fat diet is helpful, however, despite good dietary compliance, some patients continue to have severe hypertriglyceridemia and recurrent pancreatitis which can be life threatening. There is a pressing need for developing novel therapeutic options for these patients, as currently, there is no FDA approved medication. Our recent preliminary data from a randomized, open-label, clinical trial of orlistat (an inhibitor of intestinal lipase) with a four-period, two- sequence (“orlistat” and “off orlistat” for 3 months), crossover study design in two young males (11 and 9 years old) with T1HLP revealed more than 50% reduction in fasting serum triglycerides with only minimal side effects. However, the long-term efficacy and safety of orlistat therapy for children and adults with T1HLP remains unknown. Potential complications of long-term orlistat use include deficiencies in fat soluble vitamins, steatorrhea, hyperoxaluric nephrolithiasis, and alteration in fecal microbiota. Therefore, we wish to study the long-term efficacy and safety of orlistat for reducing serum triglyceride levels in patients with T1HLP. We plan to enroll 28 patients with T1HLP (fasting serum triglycerides ≥ 1,000 mg/dL) in a randomized, double-blind, placebo-controlled, cross-over trial with an open-label extension. After a screening evaluation, the subjects will be advised to consume an extremely low fat diet (≤15% of total energy from fat) for the entire duration of the study. After the baseline period of 8 weeks, they will be randomly assigned to placebo or orlistat for the duration of 24 weeks (Phase 1). After Phase 1, all patients will enter an open-label extension (Phase 2) and receive orlistat for a period of 24 weeks for a total duration of 48 weeks. During the last week of Baseline Period, Phase 1, and at 24 weeks of Phase 2, patients will be admitted to the in-patient Clinical Research Unit for 4 days to measure serum lipoproteins and chemistry panel for 3 consecutive days, fat-soluble vitamin levels, 24 hour urine oxalate and stone risk profile, mineral balance, 72 hour fecal fat, fecal microbiota, hepatic triglyceride, liver and spleen volume, and will complete gastrointestinal and quality of life questionnaires. The primary endpoint will be fasting serum triglycerides. The secondary endpoint variables will be apolipoprotein B-48 levels, liver fat content and volume. Safety will be assessed by measuring fat soluble vitamins levels, body weight, quality of life, gastrointestinal symptoms, oxalic aciduria, fecal fat excretion and fecal microbiota. Generalized linear mixed models will be used for statistical comparisons. Our data will determine long-term safety and efficacy of orlistat therapy for patients with T1HLP and orlistat may become the first line therapy as an adjunct to extremely low fat diet in these patients.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY/ABSTRACT In species that learn vocalizations, social experience acts on genetically encoded forebrain circuits to enable vocal imitation. Critical periods limit when this social experience can influence the development and function of neural circuits required for vocal learning. In songbirds, HVC is a necessary song neurocircuit component to form a song memory from the social experience of hearing an adult vocal model (“tutoring”). Yet, HVC is also necessary as a premotor region after the critical period. It is not well understood how the tutoring experience acts on HVC to allow this dual sensory and motor function. Our single-nucleus RNA-sequencing results demonstrate that the proportion of HVC’s projection neurons change throughout development. During the sensory critical period, HVC neurons that project to the auditory region and basal ganglia pathway are proportionally more than those that project to the motor pathway. I hypothesize that HVC uses these distinct cell types to function as a sensory region early in development prior to the maturation of its motor function. I therefore expect that the two former projection neuron types experience the most genetic and electrophysiological changes related to learning from the social tutoring experience compared to the latter projection neuron type. To test this, I will examine single-cell epigenomic and transcriptional changes from tutoring experience to measure changes in immediate early genes and genes related to plasticity and learning. I will also use optogenetic circuit mapping to measure the changes in synaptic strength from tutoring experience.

NIH Research Projects · FY 2026 · 2023-05

Viral tropism, or the range of cells, tissues, or species that a virus can infect, is an important concept to understand viral pathogenesis and the emergence of new viral diseases. Noroviruses are the leading cause of diarrheal diseases worldwide, yet little is known about what contributes to norovirus tropism. Filling in this knowledge gap is crucial to better comprehend norovirus biology, pathogenesis, and therapeutic options. Human noroviruses do not robustly replicate in small animal models and thus murine norovirus (MNV) has emerged as a model system to discover mechanisms of norovirus biology and tropism. Over the past several years, it has been established that MNV strains can infect different cells and tissues in mice despite similar routes of inoculation. The molecular basis for these tropism differences are largely unknown. We recently demonstrated that CD300lf is a universal MNV receptor. This finding demonstrates that the primary mechanism for diversifying MNV strain tropism is not differential receptor utilization. These findings suggest the possibility that unknown host antiviral proteins function as restriction factors that limit the tissue and cellular replication of certain MNV strains. Yet, restriction factors for any norovirus have defied molecular identification. Here, we build upon our initial identification of Trim7 and Trim47 as MNV restriction factors. TRIM proteins are ubiquitin E3 ligases, although the substrates and antiviral mechanisms of Trim7 and Trim47 are currently unknown. While Trim7 broadly inhibits MNV strains from replicating, Trim47 selectively inhibits certain MNV strains. In other viral systems, TRIM proteins are responsible for shaping the cellular and species tropism of viruses. Therefore, we hypothesize that Trim7 and Trim47 constitute a family of TRIM restriction factors that regulate the tropism of noroviruses, including human norovirus. In Specific Aim 1, we will determine the molecular mechanism of Trim7 and Trim47 inhibition of MNV. We will also determine the breadth of the antiviral activity of Trim7 and Trim47 on related viruses, including human norovirus. In Specific Aim 2, we will define the physiological role of Trim7 and Trim47 in shaping the cells and tissues that are permissive for MNV replication in vivo. We will also determine the in vivo fitness tradeoffs for viruses that acquire resistance to Trim7 and Trim47 .This project leverages the MNV model system to uncover new mechanisms of viral restriction by a pair of poorly understood TRIM proteins. It will provide insight into drivers of norovirus tropism and evolution.

NIH Research Projects · FY 2025 · 2023-05

PROJECT SUMMARY/ABSTRACT Skin has a remarkable ability to heal wounds through re-epithelialization, a repair process fueled by adult stem cells residing in the epidermis and hair follicles. Following injury, wound-edge keratinocytes proliferate and migrate to initiate wound closure, which is accompanied by activation and infiltration of immune cells. My long-term goal is to elucidate the cellular and molecular basis underlying wound re-epithelialization, how the immune system regulates this process, and how it affects tissue regeneration. Previously we found activation of the transcription factor Stat3 in keratinocytes controls many important aspects of wound re-epithelialization, including basal keratinocyte proliferation, migration and crosstalk with epidermal dendritic T cells (DETCs). However, the molecular mechanism by which wounding triggers Stat3-mediated re-epithelialization and activates the immune system remains unclear and is the subject of this study. Cellular injury is known to produce damage associated molecular patterns (DAMPs) that are sensed by the innate immune system for host protection. We hypothesize that DAMPs produced by skin wounds are sensed by innate immune pattern recognition receptors (PRRs), which then signal to produce cytokines, and further activate Stat3 for wound re-epithelialization. Using a candidate approach and Stat3 activation as a readout, we will first identify, characterize, and verify wound-edge cytokines that influence wound re-epithelialization through epidermal-specific genetic knockouts, gene-expression analysis, and genetic modulation of immune signaling (Aim 1). Next, we describe strategies to identify the immune signaling pathway, upstream PRR, and the cells responsible for the PRR signaling through genetic and biochemical approaches (Aim 2). Finally, we describe an inducible genetic model of wound injury, characterize its similarity to physical wounding, and identify wound- induced ligands using biochemical purification and an in vitro assay (Aim 3). These lines of investigation will 1) offer novel insights into the molecular mechanism of wound initiation and innate immune contribution to skin re-epithelialization, 2) contribute new tools and models to the study of immune regulation and skin repair, and 3) improve our understanding and therapeutic options for autoimmune/autoinflammatory skin conditions and diseases associated with poor wound repair. With an exceptional mentoring team led by Dr. Elaine Fuchs (with Drs. Jean-Laurent Casanova and Daniel Mucida) and a supportive, stimulating training environment at the Rockefeller University, I am ideally positioned to fully develop my technical skills and knowledge in skin biology and immunology. My research, training, and career development will allow me to establish a unique niche in the field of wound-repair and tissue regeneration as an independent investigator.

NIH Research Projects · FY 2026 · 2023-05

Project Summary Classification of protein domains have historically served to contextualize the 3D structural data collectively generated by experimental structure determination methods such as X-ray crystallography, nuclear magnetic resonance spectroscopy, and electron microscopy. Our database, Evolutionary Classification of protein Domains (ECOD), has served the biological community for seven years cataloguing evolutionary relationships between domains from experimental structures. The recent advent of high-accuracy structure prediction methods, such as AlphaFold (AF) and RoseTTAFold (RF), and the consequent release of 1 million predicted structures in AlphaFold Database (AFDB) heralds a paradigm shift in structural biology and domain classification. The rate of structure deposition is expected to jump between a hundred to a thousand- fold. We propose to take advantage of this revolution and transform ECOD into a comprehensive classification of the entire protein university using sequence, structure, and functional evidence. By simultaneously classifying experimental and predicted structures of proteins from model organisms and human pathogens, our classification will help the scientific community to critically evaluate structure models and utilize the evolutionary information to discover and experimentally characterize protein function. Classifying AF models challenges the ECOD pipeline by a 50-fold increase in the workload and by the significant fraction of non-globular and low-quality regions in the models. Thus, our first Aim is to upgrade ECOD’s infrastructure and develop methods to identify single domains from AF models and to integrate sequence, structure, and functional site similarities into our automatic classification. Compared to the current ECOD workflow that relies on human experts for structure-and- function-based classification, these improvements will drastically decrease the need for manual curation and will allow us to achieve our second Aim, i.e., classifying domains of over 1 million released AF models into ECOD via a combination of computational pipelines and minimal manual efforts (0.25%  1% cases). Utilizing the deluge of AF models, the new automatic pipeline, and expertise of human curators, we expect both to significantly improve ECOD and to evaluate the quality of AF models by (1) covering all known protein families in Pfam, (2) confirming remote homology via evolutionary intermediates, (3) comparing evolutionarily related experimental and predicted structures, and (4) resolving errors and inconsistency through periodic quality checks. Finally, we will take the lead in making functional discoveries for biomedically important proteins classified by ECOD in our third Aim, studying virulence factors (VFs) in bacterial pathogens modelled by AFDB or studied by our experimental collaborators, the Orth lab. Fast evolving VFs were a challenge for structure prediction or functional inference by sequence. We will identify candidate VFs in two dozen bacterial pathogens, obtain their structure models, and infer their function using similarities to known proteins in structure and functional sites. Promising hypotheses will be tested experimentally in the Orth lab through biochemical and genetic assays.