Yale University

NIH Research Projects · FY 2026 · 2024-02

Project Summary Spontaneous intestinal perforation (SIP) is a gastrointestinal complication affecting 3-8% of extremely low birth weight infants, corresponding to approximately 4,000 prematurely born babies annually in the US. All affected infants require surgery and are at an increased risk of brain injury, infection, and death. Yet, the pathogenesis and etiology of SIP are poorly understood. Consequently, there are currently no biomarkers for early recognition or disease-specific therapy. The foundation for this proposal is preliminary and published data where we showed alterations in T cell and epithelial cell subsets in SIP-affected mucosa that was not present in non-SIP mucosal samples. Intestinal immune dysregulation in SIP was characterized by reduced tissue-resident memory T cells, increased naïve T cells that produce more IFNγ upon stimulation, and a decreased proportion of epithelial cells in SIP compared to fetal and neonatal controls. This suggests that the inability to generate tissue-resident memory T cells in utero and the proinflammatory effects of the naïve T cells that infiltrate the intestine in their place after birth could contribute to the development of SIP. We hypothesize that susceptibility to intestinal perforation in extremely premature infants is exacerbated by defects in memory T cell generation and concomitant epithelial cell damage. We will perform a comparative analysis of T cells’ transcriptome and spatial location in patients with SIP compared to samples from fetal and neonatal mucosa. Using a T cell co-culture organoid model, we will investigate the impact of aberrant T cell activation and IFNγ exposure on epithelial cells in healthy and affected mucosa. Completing these aims will provide data to inform a next-step large-scale study into the role of epithelial-immune dysfunction in SIP. The candidate is committed to a career in studying epithelial-immune cell interactions using spontaneous intestinal perforation as a model and is strongly supported by her mentors and her department at the Yale School of Medicine. The proposal builds upon the candidate’s prior research and clinical experience in immune dysregulation in neonatal intestinal disease. It integrates two new domains of expertise in epithelial-immune interactions using organoids and integrative network analysis in a comprehensive training and didactic plan. This proposal is supported by dedicated and experienced mentors with expertise in mucosal immunology, immunobiology, T-cell signaling in immune disorders, neonatal nutrition, and biostatistics. The proposed experiments and didactics will provide the candidate with interdisciplinary skills that will foster her transition to independence as a physician-scientist in studying epithelial-immune cell interactions in intestinal disorders in children.

NIH Research Projects · FY 2025 · 2024-02

PROJECT SUMMARY The high rate of unplanned pregnancies suggests that currently available contraceptive methods are not effectively meeting the needs of women. In addition, contraceptive options for men are limited to vasectomy and condoms, leaving a significant unmet need for contraception. Our long-term goal is to develop a non-steroidal, effective contraceptive that provides a more comprehensive approach to birth control. We propose that the sperm-specific CatSper calcium (Ca2+) channel is an ideal target for the development of such a new class of contraceptives that has no negative side effects in either men or women, as the CatSper channel is a validated target required for sperm capacitation and male fertility in both mice and humans. Drug inhibition of CatSper at the post-testicular and pre-fertilization stages would work without off-target side effects due to its post-meiotic expression in male germ cells and functional divergence from other calcium channels. The recently solved struc- tures of CatSper highlight its high accessibility in the cell membrane, allowing the study of the mechanism of action and reversible contraceptives. However, the inability to reconstitute the channel in vitro has been a bot- tleneck in the development of drugs that directly target the CatSper channel. We have recently overcome this hurdle by creating chimeric CatSper channels that heterologously express functional channels. Using this new tool, the overall goal here is to ultimately develop CatSper modulators that inhibit human sperm function. To this end, in R61 phase, we will perform in-depth biophysical and functional characterization of these novel chimeric channels and do molecular dynamics studies to gain new insights (Aim 1) and develop the necessary assays for primary and secondary screening that measure CatSper activity in high-throughput modes (Aim 2). In the R33 phase, we will perform high-throughput screening for CatSper inhib- itors as well as virtual screening (Aim 3), profile the identified hits, establish preliminary structure-activity rela- tionships and perform the secondary screening (Aim 4), and test the selected compounds on human sperm function (Aim 5). In the immediate term, successful completion of these aims will provide small molecule hits for human CatSper that can be used for iterative lead generation. In the long term, the leads and knowledge gener- ated will ultimately lead to the development of an innovative class of contraceptives targeting CatSper and sperm capacitation with a mechanisms of action foundation.

NIH Research Projects · FY 2025 · 2024-02

Project Summary Rare cell types have been traditionally difficult to study due to their low abundance and lack of markers to facilitate isolation and enrichment. We lack foundational knowledge about how rare cell types contribute to tissue function and how their unique functional roles are established through differentiation from progenitors. Recently, a rare and distinct population of cells has been identified in human small intestine and colon, marked by Bestrophin 4 (Best4). The small intestinal population of Best4+ cells is striking, as these cells are highly enriched for CFTR (cystic fibrosis transmembrane conductance regulator), a feature that Best4+ colon cells lack. This CFTR High Expresser (CHE) cell population may serve an important role in the localized coordination of pH regulation in the small intestine, and is also a compelling target cell type for the small intestinal phenotypes of cystic fibrosis, for which there is currently no effective treatment. Despite mounting need to understand how CHE cells contribute to intestinal homeostasis and disease, we do not know how CHE cells are made (mechanisms controlling their differentiation) or what they do (their contribution to intestinal physiology). The goal of this proposal is to elucidate the fate specification of CFTR High Expresser (CHE) cells in the intestine, with the long- term goal of understanding the lineage origin of CHE cells as they arise from stem cells, and their role in homeostasis and disease. The overall objectives are (i) to define the transcriptional network driving intestinal CHE cell fate specification (Aim 1) and (ii) to define the role of Notch signaling in CHE cell fate specification (Aim 2). As CHEs are found only in rats and humans and genetic tools are limited in rats, I have developed a novel rat intestinal organoid model that allows for genetic perturbation. Aim 1 will determine the role of key candidate transcription factors identified by single-cell RNA sequencing in promoting CHE cell fate in rat intestinal organoids. Then, chromatin immunoprecipitation sequencing (ChIP-seq) will be performed to identify direct interactions between candidate transcription factors and CHE-enriched genes. Additionally, Notch signaling is critical for determining the initial cell fate decision of whether an intestinal progenitor cell becomes absorptive, "Notch on", or secretory, "Notch off." Surprisingly, our single cell RNA-seq data suggests that active Notch signaling may also be involved in specifying later cell fate decisions within the secretory lineage, particularly CHE cell fate. I will investigate this in Aim 2 using genetic manipulation of Notch in the rat intestinal organoid model. Lastly, I will use our ability to modulate CHE cell numbers by regulating Notch signaling to correlate CHE abundance to the physiological function of CFTR-mediated fluid secretion. Completion of this project will identify factors required for specifying CHE cells in the small intestine, informing insights into the specification of CHEs and other rare secretory intestinal cells, building a foundation to observe how CHEs arise from stem cells, and illuminating a potential novel target cell type in pathological conditions associated with CFTR dysregulation.

NIH Research Projects · FY 2026 · 2024-02

Preeclampsia (PE) is a syndrome of new hypertension (HTN) with organ damage that occurs in 3-8% of pregnancies and is a leading cause of maternal mortality. Women who survive PE have a substantially increased risk of future HTN, heart attack and stroke by unknown mechanisms. These women have enhanced blood pressure and vasoconstriction responses to HTN stress that persists months to years after PE. In male mice, T cells are necessary for hypertension and effector memory T cells contribute to exacerbated responses to repetitive hypertensive stresses. To explore mechanisms driving post-PE HTN, we modified two models of PE; one is induced by overexpression of the anti-angiogenic soluble VEGF receptor 1 (sFlt1) during pregnancy and the other is induced by hypoxia during pregnancy. I confirmed that both models cause increased sFlt1 and other features of PE seen in humans. Preliminary data in the sFlt1 model reveals that despite post-partum sFlt1 levels and blood pressure normalizing: (1) post-partum microvascular structure/function abnormalities persist; (2) post-partum HTN stimuli results in an exacerbated blood pressure response, microvascular vasoconstriction and microvascular expression of the T- cell chemokine, CCL5; and (3) kidney effector memory T cells are significantly increased after HTN stimuli. Thus, I propose to test the hypothesis that experimental PE causes long-term T cell- mediated changes in the microvasculature and kidney that increase sensitivity to post-partum HTN stimuli. Aim 1 will examine if T cells are necessary for persistent vascular remodeling and dysfunction after PE. T cell populations, migration and cytokine expression will be measured during and after PE and in response to hypertensive stimuli. T cells will then be depleted and blood pressure and vascular structure/function analyzed. Aim 2 will determine if adoptive transfer of T cells exposed to PE is sufficient to induce the vascular and kidney changes associated with post-PE HTN. Aim 3 will test the specific role of memory T cells in exacerbating the response to hypertensive stimuli after PE. Completion of the aims will provide new insight into the mechanism driving the substantial increase in HTN risk after PE, thereby supporting the NIH mission to improve maternal health. The proposal will also allow me to gain new expertise in HTN diseases of pregnancy and foundational immunology techniques. The mentoring team assembled on this application, with expertise in cardiovascular immunology, nephrology, pregnancy and vascular biology, the environment at Tufts Medical Center and Tufts University and the training plan proposed will further strengthen my ability to become an independent investigator studying mechanisms driving heart diseases in women.

NIH Research Projects · FY 2025 · 2024-01

PROJECT SUMMARY (ABSTRACT) Humoral immunity forms the basis for vaccine-induced immune protection. Following infection and vaccination, a fraction of activated B cells differentiates into long-lived plasma cells (LLPCs) or memory B cells to provide longitudinal protection against pathogens. Whereas LLPCs constitutively produce circulating antibodies, memory B cells in circulation and tissues are reactivated following antigenic reencounter through rapid expansion and antibody production. Existing forms of vaccination rely upon generation of neutralizing antibodies by LLPCs. However, evolving pathogens harboring frequent mutations at sites of immune recognition might be better targeted by heterogeneous memory B cells, with their broader distribution across tissues and enhanced cross-reactivity. Understanding these aspects of memory B cells is thus crucial for leveraging the additional layer of protection offered by the heterogeneous population in vaccine development. Co-expression of Tbet and CD11c (Tbet+CD11c+) marks a subset of such heterogeneous, antigen-experienced B cells that arises in aging, infection, and autoimmunity, respectively, termed age-associated, atypical, and double-negative (in humans, IgD- CD27-) B cells which mediate protection from pathogens in mice and humans. Their contribution towards protection is likely in part due to their unique localization as a resident memory B cell subset in tissue, most notably the splenic marginal zone which via its exposure to circulation provides front-line defense against bloodborne pathogens upon rechallenge. Tbet+CD11c+ B cells also infiltrate non-lymphoid tissues, including the liver, with residency establishment at non-lymphoid tissue sites positioning them for early detection of invading pathogens. Confounding their investigation, however, is lack of knowledge of the relationship between the non-lymphoid and lymphoid populations. Tbet+CD11c+ memory B cells also contain autoreactive clones and appears to be a major source of autoantibodies, which may be pathogenic. Although the ability of Tbet+CD11c+ B cells to mount robust antibody responses at tissue sites of infection makes them an attractive candidate to target in vaccination, we need to understand the relationship between the lymphoid and non-lymphoid pools, and how the balance between their protective and autoreactive functions is regulated. Are these cellular sites, and protective and autoreactive functions, a consequence of two pools of Tbet+CD11c+ B cells? Do cross-reactive Tbet+CD11c+ B cells, recognizing both self and non-self, rely on environmental signals to mediate protection or pathology? To address these knowledge gaps, in this revised application, we will identify the mechanism by which Tbet+CD11c+ B cells infiltrate non-lymphoid organs and determine if they maintained therein and elucidate the nature of their autoreactivity. Both goals fit the R21 Exploratory/ Developmental Grant mechanism. We are at “early and conceptual stage(s) . . . of the development of novel applications that could have a major impact” on our understanding or tissue protection provided by heterogeneous B cells and how they may be safely targeted for vaccine development.

NIH Research Projects · FY 2026 · 2024-01

Type 2 diabetes (T2D) and nonalcoholic fatty liver disease (NAFLD) are emerging as two of the most critical global health challenges of the 21st century. NAFLD is estimated to affect up to one third of the general population, and NAFLD is nearly universally present in patients with T2D, with 75-100% of participants demonstrating hepatic steatosis, and with 50% and 19% demonstrating nonalcoholic steatohepatitis (NASH) and cirrhosis, respectively. Furthermore, NAFLD represents the most common cause of liver disease in children and adolescents. Studies by our group and others have shown a strong relationship between NAFLD, hepatic insulin resistance and T2D, however the cellular mechanisms that lead to hepatic insulin resistance and increased gluconeogenesis remain to be established. The studies proposed in this grant build on our previous studies that have shown that reduction of hepatic fat content through enhancement of hepatic mitochondrial lipid oxidation can reverse hepatic insulin resistance and diabetes in rodent and nonhuman primate models of NAFLD, NASH and T2D. The Overarching Aims that will be addressed in this grant will be to determine if rates of hepatic mitochondrial oxidation are altered in NAFLD, NASH and T2D and whether promoting chronic increases in rates of hepatic mitochondrial fat oxidation by means of a chronic glucagon infusion will reduce hepatic steatosis and hepatic insulin resistance in individuals with NAFLD. To address these questions we will apply a novel Positional Isotopomer NMR Tracer Analysis (PINTA) method that we have recently developed to: i) Assess rates of hepatic mitochondrial oxidation, pyruvate carboxylase flux and hepatic ketogenesis in participants with NAFLD, NASH and type 2 diabetes, ii) Assess rates of hepatic mitochondrial oxidation, hepatic pyruvate carboxylase flux and hepatic ketogenesis and hepatic steatosis in the elderly and iii) Assess the effects of chronic glucagon treatment on rates of hepatic mitochondrial oxidation, pyruvate carboxylase flux, hepatic ketogenesis, hepatic fat content and hepatic insulin sensitivity in individuals with NAFLD. The results of these studies will provide important new insights regarding the role of altered hepatic mitochondrial function in the pathogenesis of NAFLD, NASH and T2D, which in turn will have important implications for the development of novel liver-targeted mitochondrial uncoupling therapies aimed at increasing hepatic mitochondrial fat oxidation to treat NAFLD, NASH and T2D, which are currently being evaluated in Phase 2b trials. The present study will also provide critical information regarding the chronic effects of glucagon on hepatic mitochondrial oxidation, hepatic gluconeogenesis, hepatic insulin sensitivity and hepatic fat metabolism which has important implications for dual GLP-1/glucagon agonists and triple GLP- 1/GIP/glucagon agonists which are now being evaluated in clinical trials for treatment of obesity, NAFLD, NASH and T2D.

NIH Research Projects · FY 2026 · 2024-01

Project Summary/Abstract Macroautophagy is a degradative cellular process that is upregulated in response to stress. Dysregulation of autophagy is broadly associated with several diseases including cancer, aging, and neurodegeneration. However, since this process is essential for mammalian development, diseases affecting core autophagy genes are very rare. For example, ATG3 is a core autophagy gene that is crucial for the formation of the autophagosome and there are no known disease-causing mutations within this gene. Interestingly, there is a patient with an undiagnosed severe neurodevelopmental disease found to have a point mutation in a single allele of the ATG3 gene. Intriguingly, ATG3 has been implicated in other conjugations thought to be independent of autophagy. These other conjugations regulate mitochondria and endolysosomal trafficking, both of which have significant roles in neurologic disease. My thesis work has largely focused on the patient mutation and its effect on autophagy, but my preliminary data indicates that all ATG3 activities appear to be disrupted in patient cells. Therefore, I propose to develop tools to further understand the cell biological consequences of these non-canonical ATG3 activities and use these tools to understand the effect of the patient mutation beyond autophagy. This project is designed to provide training for a successful future career in independent research. The findings from this project will improve the understanding of complex cellular processes and how they can result in disease. This proposal focuses on two aims: Aim One – Determine the impact of a patient mutation on lipidation- independent activities of ATG3; Aim Two – Determine if lipidation-independent ATG3 activity is dependent on membrane binding. Aim One will use microscopy techniques I developed in patient cells and a knockout- rescue strategy in gene-edited cells to directly evaluate the impact of the mutation on the formation and downstream consequences of these autophagy-independent complexes. Aim Two will capitalize on the techniques established in Aim One and biochemical experiments routinely performed in our lab to investigate potential mechanisms by which these autophagy-independent ATG3 complexes are regulated and function. The work in this proposal detailing the effect of this ATG3 mutation has the potential to describe the first severe disease-producing allele of ATG3. Additionally, this work aims to distinguish and characterize previously inseparable ATG3 functions, leading to a new understanding of both foundational biological processes and their downstream clinical relevance.

NIH Research Projects · FY 2025 · 2024-01

PROJECT SUMMARY Agitation is defined as excessive psychomotor activity leading to aggressive and violent behavior in patients and is often due to exacerbation of underlying serious mental illnesses. Coercive measures like physical restraints are currently used routinely on agitated individuals, but are associated with physical trauma, apnea, and death. At the same time, healthcare workers experience stress and burnout from episodes of workplace violence by agitated patients, leading to reinforcement of negative attitudes towards patients with mental health conditions. Interventions to address agitation during psychiatric crises have been hindered by increased boarding, overcrowding, and other system-based challenges. System dynamics modeling is a rigorous method that uses advanced mathematical equations and simulations to study complex systems and identify causal structures that evolve over time. This approach allows us to measure, predict, and improve health and value during agitation care. Our overall objective is to apply systems dynamics modeling to identify and quantify modifiable targets for improving agitation management and assess benefits and costs of potential interventions addressing those targets across different populations and groups. To achieve this objective, we will use group model building focus groups (Aim 1) to adapt our existing qualitative model of agitation care and merge key insights from clinicians, administrators, security/police, patients, and prehospital services to focus on health outcomes. We will then create a mathematical model and incorporate existing datasets of patient records, staff injuries, and survey responses into the model, calibrating quantitative outcomes of restraint use and staff assault and validating parameters of the relationships established in the qualitative model (Aim 2). Finally, this expanded and validated model will guide participatory design sessions with stakeholders in an iterative process where computational simulations for outcomes can be created and predicted in real-time on proposed interventions across three sites (Aim 3). This will allow us to translate research findings from the model into practice to assist hospital leadership in deciding if implementing potential interventions is warranted. This proposed work will make a positive contribution to mental health research by describing, measuring, and predicting outcomes for individuals with psychiatric emergencies. Our study is highly innovative as it will be the first to address staff safety and patient advocacy as one unified issue and applies simulation modeling and systems science methods to address the understudied topic of agitation management and improve health in psychiatric emergency care.

NIH Research Projects · FY 2026 · 2024-01

Project Summary/Abstract Coxiella burnetii is an obligate intracellular bacterial pathogen that grows within a lysosome-derived vacuole. The formation of a replication-permissive vacuole by C. burnetii requires a type IVB secretion system called Dot/Icm, and the >130 effector proteins delivered by this system into the host cell. While it is established that the collective activities of these effector proteins remodel host signaling networks to create a replication- permissive environment, the molecular targets and biochemical mechanisms of most of these effectors are not known. My studies have established that C. burnetii inhibits the activation of the host RIG-I pathway, a cytosolic double-stranded RNA sensing pathway, and that this process requires two effectors, EmcA and EmcB. Although these proteins do not have predicted sequence or structural homology that would indicate possible biochemical functions, we established that EmcB has cysteine deubiquitinase activity. EmcB directly targets RIG-I and preferentially cleaves long, K63-linked ubiquitin chains that are potent activators of RIG-I signaling. The goal of this study is to determine how EmcB functions to cleave ubiquitin chains from RIG-I during infection and identify how C. burnetii infection is sensed by RIG-I. To achieve this goal, I will use biochemical assays, pull-down experiments, and infection studies using a newly engineered emcA, emcB double mutant to determine how EmcB functions to inhibit the RIG-I pathway during infection (Aim 1). Next, I will identify the specific RNAs sensed by RIG-I during C. burnetii infection and test the relative capacity of these RNAs to activate the RIG-I pathway (Aim 2). Together, these studies will enhance our understanding of how intracellular bacterial pathogens modulate host signaling networks for productive infection. By understanding the molecular details of how an intracellular pathogen modulates host processes, fundamental details of C. burnetii infection will be uncovered which could provide insights that lead to improved treatments. These studies will be complemented by a rigorous and comprehensive program of professional training, clinical training, and scientific skill building to provide preparations necessary to pursue a career as a physician- scientist investigating molecular mechanisms of microbial pathogenesis.

NIH Research Projects · FY 2026 · 2024-01

Modified Project Summary/Abstract Section Despite high rates of need, youth prematurely drop out of mental health services at alarming rates. This is due in part to poor therapeutic alliance and concerns about treatment relevance and acceptability. Existing engagement interventions are limited, with few addressing treatment retention for youth at risk for depression and suicide. Measurement-based care (MBC) is the use of patient-reported progress data throughout mental health treatment to promote collaborative, patient-centered treatment plan adjustments. MBC is an outstanding candidate to improve treatment engagement due to its focus on personalized treatment and is also highly effective when integrated in depression treatment. Yet, MBC could be tailored to better address the unique needs of youth. MBC has not been examined as a treatment engagement strategy for youth with depressive symptoms or suicide risk. Further, no clinical protocols, guidelines or training supports exist to facilitate clinician use of MBC with youth and their caregivers. Based on past research, we have developed a theoretically-driven, individually-tailored MBC approach, Strategic Treatment Assessment for Youth (STAY). STAY targets therapeutic alliance and treatment relevance and acceptability (concerns particularly relevant to youth and their caregivers) to improve treatment retention, depression symptoms and suicide outcomes. Pilot data suggest STAY is acceptable, feasible, and appropriate. In Aim 1, we will refine the preliminary STAY protocol and implementation plan to support delivery in a wide variety of clinical contexts. A user centered design approach including cognitive walkthroughs and lab-based testing with N=12 clinicians, N=6 adolescents and N=6 caregivers will be used to rapidly refine prototype versions for usability. In Aim 1, we will also develop STAY instrumentation for clinician fidelity, knowledge, skills, and attitudes to be piloted in Aim 2. Aim 2 will involve a pilot effectiveness-implementation Hybrid Type 2 trial to examine the feasibility, acceptability, appropriateness, and initial effectiveness of STAY as compared to an active control condition (MBC As Usual). Clinicians (N=20) at three community mental health clinics will be randomly assigned to STAY or MBC As Usual and N=60 adolescent patient/caregiver dyads (Total N=120) who meet inclusion criteria will also be recruited to participate. Initial effectiveness of STAY on treatment engagement mechanisms (treatment alliance, relevance), service outcomes (treatment attendance, engagement, completion) and youth outcomes (depression, suicidality) will be assessed. This pilot will inform optimal study procedures, measures, and sites for a fully-powered Hybrid Type 2 trial. Youth depression and suicide rates are a national public health crisis. This project offers an innovative, individually-tailored approach to retain youth in mental health services and improve patient outcomes. This study contributes to NIMH Strategic Goal 4.3 to develop innovative service delivery models to improve outcomes of mental health services received by youth at risk for depression and/or suicide.

NIH Research Projects · FY 2026 · 2024-01

The overarching goal of this proposal is to develop a paradigm for comprehensive whole genome sequencing (WGS) studies of complex genetic diseases and traits by leveraging new approaches for variant analysis and trait association, and to use these methods for improving disease and trait mapping studies, using coronary artery disease as a prototypical example. We will first build a new WGS variant analysis pipeline based on methods that leverage a population of fully assembled reference genomes (a “pangenome”), rather than a single linear reference genome (e.g. GRCh38). By employing variation-aware approaches to read alignment and genotyping, these next-generation methods promise to greatly improve variant detection performance and to alleviate the bias that plagues the current generation of methods. Our pipeline will be designed to detect all forms of variation, including single nucleotide variants, small indels, structural variation, and tandem repeat length variation, and will be constructed based on systematic benchmarking of candidate methods from our labs and the broader community. We will then use these methods in combination with haplotype-aware trait association approaches to study the complex genetic basis of coronary artery disease and cardiometabolic risk factors in a set of ~55,000 deeply sequenced human genomes generated by the Centers for Common Disease Genomics (CCDG). We will assess the contribution of all variant types to coronary artery disease and complex coronary disease risk factors and quantify the improvements in gene mapping studies of common disease that are possible using new pangenomic analysis methods. We will leverage patterns of haplotypic admixture, using a novel tree-based haplotype association method to fine map novel and known coronary artery disease and risk factor loci, and to identify loci where local haplotypic origin modulates risk of coronary disease and risk factors. Finally, we will extend this work to a much larger set of individuals and traits by applying our methods to WGS data from public biobanks allowing us to more broadly assess the role of complex genome variation in human diseases and complex traits. Taken together, this work will yield valuable new methods and data resources for the community, will help pave the way to improve the next generation of human disease gene mapping studies by using pangenomic approaches, and has the potential to yield new insight into the genetic basis of coronary artery disease.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY/ABSTRACT Microbial infections account for nearly one-sixth of all human cancers. Oxidative stress generated by cancer- causing pathogens can drive tumor formation, yet very little is known about the cellular targets of oxidative stress that contribute to cancer development during infection. At the molecular level, one of the primary consequences of oxidative stress is the site-specific oxidation of proteins containing redox-sensitive cysteines. Cysteine oxidation can regulate important cellular functions such as proliferation, metabolism, and other tumorigenic processes. However, almost nothing is known about the cysteines that are oxidized during infection, in part because they are difficult to study. Unlike DNA oxidation, post-translational oxidative modifications cannot be detected by DNA sequencing, transcriptional profiling, or even conventional proteomic analyses. Consequently, methods traditionally used to study host–microbe interactions overlook signaling events that could potentially be targeted to prevent disease. To address this challenge, we have developed and validated a chemical proteomic strategy that can detect specific sites of oxidative modification on host proteins in infected cells. We use chemical probes to quantify proteome-wide changes in thiol reactivity, which decreases upon oxidation. Using this approach, we identified cysteines in several host proteins that become less reactive when human gastric cells are infected with the cancer-causing pathogen Helicobacter pylori. Notably, we discovered an infection-induced oxidation site on Cys219 of the host protease legumain that accelerates tumor growth. The proposed research program will extend these findings by (1) uncovering the molecular mechanisms by which infection-induced oxidation sites influence host protein function and localization, with the goal of defining cysteine oxidation as a new mechanism of cancer pathogenesis during H. pylori infection; and (2) leveraging our approach to identify specific oxidation sites that promote cancer signaling in vivo. Our long-term goal is to establish a platform for the discovery and functional characterization of oxidized cysteines in the context of infection. We will test three specific aims. In Aim 1, we will define the molecular mechanisms linking Cys219 inactivation to defective legumain processing and intracellular trafficking. In Aim 2, we will use metabolic labeling and nucleoside recoding chemistry to establish whether oxidation of the ribosomal protein uL14 on Cys125 inhibits translation. In Aim 3, we will develop a quantitative, tandem mass tagging-based approach to functionally map novel sites of cysteine oxidation induced by a cancer-promoting bacterial protein in vivo. By providing mechanistic insights into how specific oxidation sites affect tumorigenic processes, and discovering additional candidates for characterization, the overall impact of this proposal will be to build fundamental understanding of how reactive cysteines shape cancer signaling and thereby inform the development of new strategies for perturbing these pathways during infection.

NIH Research Projects · FY 2026 · 2024-01

Project Summary / Abstract: The goal of this project is to define dysregulation in key immune checkpoints that regulate microglial activation, including TREM2-TYROBP-APOE signaling, which is associated with a common pro-inflammatory state, and how this contributes to progression of degeneration in age-related macular degeneration (AMD). To explore the hypothesis that proinflammatory microglia are a key component of the pathobiology of retinal degeneration in AMD, we will use CRISPR-Cas9 knockout of key immune checkpoints in human iPSC-derived microglia and expose them to multiple components of extracellular deposits that are characteristic of this disease (Aim 1). This will validate these pathways as potential targets for novel therapeutics aimed at limiting inflammation in AMD. In Aim 2, we will perform single-nucleus transcriptional profiling of retinal microglia from individuals with early and late stages of AMD, and those of controls. In Aim 3, we will investigate how transcriptional alterations in microglia are influenced by the pathologic tissue microenvironment by performing highly multiplexed spatial analysis of RNA and protein in retinal tissue affected by AMD-associated lesions using spatial co-indexing of transcriptomes and epitopes for multi-omics mapping by highly parallel sequencing (spatial-CITE-seq). Assaying transcriptional states in disease associated microglia in retina and within AMD lesions directly using human samples will provide a new paradigm to understand AMD and probe the largely unaddressed question of the role of microglia in AMD pathogenesis. It will also set the stage for further study of neuroinflammatory biology using AMD as a paradigm to discover principles with application to other neurodegenerative diseases. This mentored clinical scientist research career development award is designed to help Dr. Marcello DiStasio achieve his long-term career goal of an academic career with a focus on neuroinflammation in degenerative diseases of the retina. His Mentor, Dr. David A. Hafler, is a leader in the field of neuroimmunology and is committed to the scientific development and execution of the project. He is the William S. and Lois Stiles Edgerly Professor and Chairman, Department of Neurology and Professor of Immunobiology, Yale School of Medicine, and is the Neurologist-in-Chief of the Yale New Haven Hospital. Co-Mentor Dr. Brian P. Hafler is an Assistant Professor of Ophthalmology and Visual Science and of Pathology whose lab focuses on retinal disease using advanced genomics technologies. The environment at the Yale School of Medicine has a strong and established research program related to immunology and neuroscience, and includes the Center for Neuroinflammation, where the research will be conducted. The experience, knowledge, and skills gained through the research plan and career development activities will form the basis for future studies as Dr. DiStasio transitions to independence as a clinician-scientist.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY Vascular function and development are largely mediated by vascular endothelial cells (VECs) that line the inner wall of blood vessels. Fluid shear stress (FSS) generated by blood flow is a major determinant of their function and phenotype with major roles in development, physiology, and disease. VECs in healthy regions of arteries are under unidirectional laminar flow, where they align in the direction of flow and activate anti-inflammatory pathways which confers resistance to atherosclerosis. By contrast, VECs in curved or branched regions of arteries develop disturbances in flow patterns. These disturbed flow patterns fail to align VECs and activate inflammatory pathways, which correlates with susceptibility to form atherosclerotic plaque. FSS direction with respect to cell alignment also regulates inflammatory signaling outputs, which suggests cell polarity and flow direction sensing is important for the differential atheroprotective and atheroprone responses. Thus, how VECs sense and respond to flow direction is an important basic science question pertinent to human health, but the mechanism is unclear. A junctional flow-dependent complex comprising of VE-Cahderin, PECAM1, and VEGF is critical for integrating endothelial cell flow responses. Our lab recently discovered that the polarity adaptor protein, LGN, which binds directly to VE-Cahderin is important for proper endothelial cell alignment. Since LGN directly interacts with a flow dependent mechanosensitive complex and has an established role regulating cytoskeletal dynamics, I hypothesize LGN is important for flow direction sensing. I plan to address this hypothesis using the following specific aims: Aim 1: Characterize the mechanism by generating mutations in LGN’s functional domains to determine which sites are important for mediating endothelial cell flow dependent signaling. I will similarly examine the effects of known LGN interactors if they are shown to be crucial for flow mediated signaling. Aim 2: Examine the role of cell polarity in flow signaling. I will do this first by tracking the intracellular localization of LGN in response to flow and use patterned substrates to separately constrain cell and cytoskeletal polarity to determine which of these variables is important for LGN polarity and inflammatory vs. anti-inflammatory signaling. Aim 3: Determine the role of LGN in vivo by analyzing mice with endothelial deletion of LGN which will address the role of LGN in VEC alignment, inflammation, and resulting atherosclerosis in vivo. Together, these aims will reveal new mechanisms for endothelial flow sensing, vascular inflammation, and atherosclerotic disease.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY Stroke is the second highest cause of death and the leading cause of disability globally. Intracerebral hemorrhage (ICH) is the second most prevalent form of stroke with a high mortality of ~40% and high rates of long-term cognitive decline in surviors.1-4 The B cell response to stroke is well established, and in ischemic stroke models in mice B cells have been linked to slow cognitive decline.5 However, the B cell response to ICH is currently unknown and represents a major gap in knowledge. Our preliminary work has demonstrated an influx of IgA+ B cells into the brain starting 8 weeks post-ICH, similar to the IgA+ B cell aggregates in the brain and spinal cord in multiple sclerosis patients and experimental autoimmune encephalitis (EAE) mouse models.8 While IgA+ B cells are traditionally associated with mucosal tissues like the small intestine, their presence in the CNS is spurring new inquiry into the function of these cells in the context of neuroinflammation. I hypothesize that there is a late influx of IgA+ B cells from the meninges into the brain post-ICH that serves a neuroprotective role in post-stroke recovery. The goal of this project is to utilize a murine collagenase injection model of ICH to elucidate the origin and function of brain-infiltrating IgA+ B cells in post- ICH inflammation and recovery in the following aims. Aim 1. To determine the timing of the IgA+ B cell influx into the post-ICH brain over 16 weeks, investigate whether these cells are locally proliferating or constantly recruited, determine whether tertiary lymphoid structures (TLSs) exist in the post-ICH brain, and identify the brain regions IgA+ B cells localize to. Aim 2. To elucidate the origin of brain-infiltrating IgA+ B cells post-ICH using both meningeal plasma cell depletion and clonal analysis of B cells from blood, gut, meninges, and brain B cells using B cell receptor sequences generated by single-cell RNA-sequencing. Aim 3. To determine the effector functions of brain-infiltrating IgA+ B cells on post-ICH recovery. These experiments will provide much needed insight on the role of B cells in the brain in the weeks and months after ICH, and specifically on IgA in the context of stroke and neuroinflammation more broadly. My results therefore have the potential to shape our understanding of the adaptive immune response to ICH and inform therapeutic approaches to mitigate the long-term effects of this disease on patients and families.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY: Plasticity of neural circuits is a key element underlying the brain's ability to adapt to experience, and harnessing plasticity in the brain may provide new therapeutic avenues to ameliorate the effects of early perturbation or injury. However, our understanding of plasticity in the adult brain remains incomplete. Although adult plasticity in the visual cortex can be selectively induced through loss of sensory input or repeated presentation of single stimuli, the impact of more broadly enriched sensory experience is unclear. In addition, although the role of GABAergic inhibition in developmental visual plasticity has been deeply explored, the role of inhibitory interneurons in adult plasticity remains largely unknown. In particular, cells that express the peptide somatostatin (SST-INs) are thought to play a critical role in shaping the feature selectivity of visual responses in mouse visual cortex, primarily via their robust inhibition of the dendrites of excitatory pyramidal neurons. SST- INs are also critical mediators of visually-evoked activity patterns that facilitate long-range transmission of visual information. However, the functional roles of SST-INs in adult plasticity remain unclear. Our preliminary data suggest the surprising finding that visual experience consisting of repeated presentations of varied stimuli induces a novel form of plasticity in adult mouse primary visual cortex, leading to robust enhancement of visually evoked activity in SST-INs. This experience-dependent plasticity is accompanied by altered visual sensitivity in nearby excitatory neurons. To further explore this observation, we propose to combine a number of methodological approaches, including 2-photon imaging of identified neural populations, optogenetic manipulations, and ex vivo synaptic physiology. We will determine the visual experience required to induce this adult plasticity, identify the underlying cellular and synaptic mechanisms, and examine the functional consequences for cortical visual encoding and transmission. Our results will provide an unprecedented level of insight into a novel form of plasticity in the adult cortex and identify underlying cellular- and circuit-level mechanisms.

NIH Research Projects · FY 2025 · 2024-01

Abstract: Nearly 60% of human conceptions are miscarried surrounding the window of implantation. During this stage within a week after fertilization, the pluripotent epiblast tissue of the embryo transforms into a polarized epithelium with a central lumen. The causes of significant pregnancy loss at this period are still not well understood owing to the substantial challenges associated with human embryo research. Critical gaps in knowledge include mechanistic understanding of the cellular morphogens driving epiblast tissue development at the implantation stages. Further, animal models such as rodents, while highly valuable, have been found to demonstrate distinct processes from humans at the implantation stages. Thus, an in-depth understanding of the levels of conservation or divergence between key mammalian species at this stage remains incomplete, making it difficult to extrapolate findings around the implantation period from model species to human health. The central goal of this proposal is to enhance our comprehension of the human-specific mechanisms that govern embryonic development by examining the regulatory mechanisms through which WNT/β-catenin signaling guides epiblast tissue remodeling during implantation across different species. Based on my preliminary results, the overall hypothesis that WNT/β-catenin signaling will show species-specific differences in its role in epiblast tissue remodeling. I hypothesize that these differences are mechanistically tied to regulation of Ezrin-Radixin-Moesin (ERM) proteins which control cell surface tension and actin architecture. Aim 1 will identify the biomechanical effects of WNT/β-catenin signaling on human epiblast development at implantation using novel human 3D stem cell-based models. Using these highly reproducible models, I will confirm that WNT/β-catenin acts through pERM via Western Blot and loss-of-function experiments. I will then utilize live-cell imaging to analyze changes in actin architecture over development, and then analyze changes in cell surface tension through use a fluorescent membrane tension probe that will be quantified using fluorescence lifetime imaging microscopy (FLIM) to validate previous preliminary data using a secondary 5-dimensional state-of-the-art imaging software-based readout of cell surface tension. Aim 2 will define species-specificity of the mechanism underlying WNT/β-catenin epiblast remodeling control across humans, non-human primates and mice, through performing similar perturbation techniques and characterizations using Western Blot and IF, live-cell imaging with actin reporters, 5D imaging analyses, and FLIM. This proposal directly addresses the NICHDD’s research theme 1: “Understanding the Molecular, Cellular, and Structural Basis of Development” through use of novel models to understand correct processes of early human embryonic development, as well as how abnormal processes lead to undesirable outcomes. A detailed understanding of the morphogen-driven mechanisms at this monumental developmental stage, particularly in the clinically relevant context of human-specific development, can shed light on the pathological alterations that lead to miscarriage at implantation.

NIH Research Projects · FY 2025 · 2024-01

PROJECT SUMMARY Variants of SARS-CoV-2 continue to emerge with mutations in Spike that cause increased resistance to monoclonal antibodies and vaccines. These variants underscore the need for more universal antiviral approaches, such as targeting conserved regions in Spike and utilizing more broadly reactive Fc-mediated immune functions. Spike contains highly conserved regions in the S2 domain which may be an attractive target for the design of inhibitors. These regions are thought to be exposed when S2 undergoes large conformational changes to mediate membrane fusion and viral entry. Molecular dynamics simulations have modelled this process in silico, structural detail is lacking in situ which leaves a gap in our understanding of viral entry and may preclude further inhibitor development. Spike can also be targeted by antibody Fc effector functions such as antibody-dependent cellular cytotoxicity. Antibody Fc effector functions against Spike have been shown to be more broadly reactive and longer lasting in patients than virus neutralization. Eliciting stronger Fc-mediated immunity is therefore an important consideration in the design of immunogens and antibody therapies. However, Spike-IgG-Fc receptor complexes in native membranes have never been described at the molecular level which makes it difficult to define the structural correlates of Fc effector functions. The overarching goal of this proposal is to investigate the conserved Spike S2 domain and its inhibition during membrane fusion and Fc effector functions targeting Spike within native membranes. I hypothesize that Spike-host receptor interactions in native membranes are key vulnerabilities that can be targeted through antibody Fc effector functions and inhibitors to the conserved S2 domain. To test this hypothesis, I have developed a system to observe Spike-host receptor interactions in situ by presenting them on opposing virus-like particles (VLPs) and monitoring their interactions with cryo-electron tomography (cryoET). In Aim 1, I will investigate the conserved S2 domain during membrane fusion by arresting the membrane fusion process at different stages. My preliminary data show how temperature arrests and inhibitors stabilize prefusion Spike and S2 intermediate structures in situ to provide a unique window into viral entry. Further, my data suggests that multivalent inhibitor cross-linking of S2 intermediates may be a key antiviral strategy to disrupt the cooperative arrangements of S2 that orchestrate membrane fusion. In Aim 2, I will identify structural correlates of Fc effector functions by determining how antibody Fc accessibility and the ability to cluster Spikes and Fc receptors on membranes affect Spike-antibody-Fc receptor complex formation. In preliminary data, I have visualized these complexes in situ in unprecedented molecular detail using cryoET. I have also developed a method to directly visualize and quantify antibody-mediated Spike clustering on virion membranes. Collectively, these data will provide detailed molecular insight into Spike-mediated membrane fusion and Fc-mediated effector functions against SARS-CoV-2 to guide the development of pan-coronavirus inhibitors and immunogens.

NIH Research Projects · FY 2026 · 2024-01

Acquired mutations in splicing factors, such as SF3B1, SRSF2, and U2AF1, are prevalent in about half of all patients with the clonal myeloid disorder myelodysplastic syndromes (MDS), a disease associated with aging. Since their initial description in 2011, we have learned that these mutations are non-synonymous, mutually exclusive, and function as disease drivers. Changes in alternative splicing effected by these mutant spliceosomes have been extensively studied but are quite modest, and common patterns of splicing dysregulation that can explain the disease phenotype have not emerged. These studies have focused on the mature transcriptome (RNA-seq of polyadenylated mRNA) and discount the additional roles splicing factors play in RNA processing, such as 5’ end capping, splicing, editing and modification, 3’ end cleavage, polyadenylation, nucleocytoplasmic export, and mRNA decay and stability. Given that RNA transcription and splicing are closely coordinated (co-transcriptional splicing), we determined the changes in transcription kinetics in isogenic cells harboring the K700E mutation of SF3B1, the most mutated splicing factor in MDS. We found that elongation of RNA Polymerase II (RNAPII) leads to increased transcription-replication conflicts (TRC) and increased R-loops (three-stranded structures of newly synthesized RNA within a DNA-helix). Changes in transcription also induce changes in histone marks (H3K4me3) and chromatin organization. Using a short hairpin RNA library, we identified epigenetic regulators (in the Sin3/HDAC complex), which when knocked down reverses the transcription elongation defects and replication stress. SF3B1K700E expression also leads to a paradoxical RNA processing event: transcripts with retained introns are decreased in mutant cells compared to wild-type cells. Taken together, we hypothesize that SF3B1K700E-induced changes in RNA transcription, its subsequent processing, and chromatin organization could have disease-causing effects that are distinct from alternative splicing. The application is organized into three aims that address the following fundamental questions that arise from our preliminary data: (1) How does SF3B1K700E change RNA transcription? (2) How does SF3B1K700E impact co-transcriptional splicing and intron retention? (3) How does chromatin organization change in response to RNAPII, and how can we target them for therapeutic benefit? State-of-the art approaches to be implemented include long read sequencing (LRS) of nascent RNA to define coordination of RNA processing and splicing at the single RNA molecule level (pioneered by the Neugebauer Lab), metabolic labeling and sequencing to determine kinetics of IR-RNA decay, and multiplexed imaging of RNA (MERFISH). The results will be confirmed in primary CD34+ cultures from MDS patient samples and in a mouse model of heterozygous Sf3b1K700E mutation. Our studies will explore a novel paradigm in human disease pathogenesis: disruption of functional coupling between transcription and splicing. It will also inform ways to therapeutically target epigenetic pathways in splicing factor-mutant clonal myeloid disorders.

NIH Research Projects · FY 2026 · 2023-12

Project Summary/Abstract Mechanosensitivity and mechanotransduction are fundamental processes that affect virtually all aspects of human physiology. Sensitivity to mechanical stimuli ranges from light touch to unpleasant or painful and relies on the ability of primary sensory afferents to transduce these stimuli into electrical signaling. In humans, dysfunctions of the somatosensory system have only symptomatic or palliative care, largely because the molecular and cellular aspects of mechanosensitivity and mechanotransduction, and the sequence of events that underlies somatosensory tuning remain enigmatic. Our long-term goal is to understand general principles somatosensory mechanotransduction under normal, adaptive, and pathological conditions. In this collaborative basic scientific proposal, we seek to uncover the mechanism underlying adaptations of mammalian mechanosensory system to cold. We will approach this problem using a novel model organism— hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus). Despite the prolonged severe hypothermia during hibernation, when body temperature drops to less than 10°C, squirrels maintain the ability to detect mechanical force and can be aroused by touch and vibration. The endurance of the sense of touch despite prolonged hypothermia supports the existence of specific molecular and cellular adaptations at the level of peripheral mechanoreceptors. We seek to uncover these adaptations by biophysical analysis of ion channels in peripheral mechanoreceptors that determine baseline excitability, convert touch into excitation and generate and propagate action potential, implementing a mechanosensory tune-up that preserve touch sensitivity despite prolonged exposure to cold. This proposal will elucidate tunable molecular pathways in mammalian mechanosensory neurons to inform clinical practices mitigating somatosensory dysfunctions following hypothermia or nerve damage.

NIH Research Projects · FY 2025 · 2023-12

Project Summary: The brain is an immune-privileged organ; thus, the composition and the nature of the immune response is fundamentally different in the brain than in the periphery, where avoiding immunopathology is prioritized. Prior studies found that human and rodent T cells isolated from brain or CSF have unique transcriptional profiles and increased functional capability to produce cytokines such as IFN-g29. This was also confirmed in my preliminary studies, as I found that steady-state mouse brain is enriched with CD4 T cells that highly express multiple co- inhibitory receptors (PD-1+ LAG3+ TIGIT+) and secrete cytokines robustly upon activation (IFN-g, IL-17A). Interestingly, this steady-state brain T cell population can be modulated by altering in the microbiota composition. Our preliminary results reveal that gnotobiotic mice have ~2-fold fewer brain-resident T cells and significantly fewer IFN-g and IL-17A secreting cells, and mono-colonizing gnotobiotic mice with a single species of bacteria can partially restore this brain T cell population. The microbiome and the gut-brain axis has been demonstrated to mediate the symptoms and progression of a wide variety of neurological disease2-7. While the role of the gut- microbiota-T-cell-brain axis in the context of specific neurological diseases has been studied, its role at steady- state and the mechanism of gut-educated-T cell trafficking in the steady-state brain is not known. It is essential to understand the steady-state gut-brain T cell axis in order to fully understand the molecular mechanisms behind neurological disease and develop better targeted therapeutics. Interestingly in our preliminary studies, we found that the expansion of the brain-resident T cell population correlates with the massive microbiota changes accompanying the developmentally programmed weaning reaction30 and this phenotype is absent in gnotobiotic mice. This weaning period also happens concurrently with large neurodevelopmental changes, including peak myelination of axons, changes in neurotransmitter and receptors, specialization of the prefrontal cortex neural network, and thickening of cortical grey matter31. We thus hypothesize that the gut-microbiota introduced during weaning centrally instruct microglia secrete CXCL10 to recruit CXCR3+ microbiota-educated CD4 T cells from the periphery and establish residence in the brain to “match” brain development with the external environment. To test this hypothesis, we propose the following aims: Aim 1 will focus on investigating how the gut commensal microbiota composition affects brain-resident CD4 T cell plasticity in the steady-state brain. Aim 2 will focus on studying neuroimmune interactions between CXCR3+ microbiota-educated CD4 T cells and microglia at steady- state. Ultimately, results from this study will inform how the microbiota may play a role in optimizing the unique steady-state T cell compartment to regulate homeostatic functions in the brain, using behavioral assays as a readout. The applicant’s multidisciplinary mentoring team will prepare her for research independence and a successful career as a principal investigator in neuroimmunology.

NIH Research Projects · FY 2025 · 2023-12

Alzheimer's disease (AD) is the most common neurodegenerative disease in which neurons and microglia play causal and distinctive roles in its pathogenesis. These cellular mechanisms of AD need to be resolved at the level of individual brain cell types, especially at the protein (proteomic) level to best guide therapeutic and biomarker discovery. Our recent proteomic studies of human post-mortem brains have identified a signaling pathway called the MAPK/ERK pathway, as a strong predictor of AD pathology and cognitive decline. We have also found that activation of the ERK pathway is characteristic of activated microglia in a mouse model of AD pathology, and inhibition of ERK in microglia reduces their pro-inflammatory and detrimental responses. Based on these findings, our overall hypothesis is that excessive ERK activation is a central mechanism of AD pathogenesis that uniquely impacts the proteomic phenotypes of neurons and microglia, leading to neurodegeneration. In order to determine how MAPK/ERK signaling changes with aging and AD pathology in a mouse model, we will apply a novel in-vivo labeling approach called CIBOP-MS that enables us to define the dynamics of proteomic changes occurring specifically in microglia or neurons without the need for cell type isolation (Aim 1). Using a small molecule inhibitor of ERK activity, we will then determine how ERK inhibition impacts neuronal and microglial proteomes and identify novel biofluid biomarkers in an amyloid beta mouse model (Aim 2). Lastly, we will manipulate ERK activity leading to either over-activation or attenuation specifically in either neurons or microglia using genetic approaches (Aim 3). Collectively, our comprehensive studies focusing on ERK signaling in neurons and microglia, will validate CIBOP-MS as a novel approach to resolve brain cell type-specific proteome dynamics in aging and neurodegeneration, with biomarker and therapeutic implications. Our multidisciplinary expertise in in-vivo cell type specific proteomic labeling approaches, systems biology, neuroinflammation, biofluid biomarker discovery and mouse models of AD pathology uniquely positions us to execute this innovative R01 proposal.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY In the United States, colorectal cancer (CRC) mortality is markedly higher in African-American (AA) patients than any other racial or ethnic group. Treatment options, socioeconomic status (SES), comorbidities, and tumor characteristics contribute to survival disparities. Tumor characteristics focused on metabolism and microbiology have been rarely studied in relation to CRC mortality and racial/ethnic group, and thus requires further investigation. Metabolites are substrates and products of metabolism required by tumor cells for gene regulation, growth, and immune responses. They can modulate the metabolism and efficacy of chemotherapeutic drugs, therefore are linked to CRC treatment response and mortality rates. Differences in lifestyle factors (i.e., diet, physical activity), social determinants of health, and environmental exposures exist between racial/ethnic groups and affect metabolite production in CRC. In addition, the colon microbiome is a major modulator of metabolites and has been implicated in CRC. Given that metabolites are susceptible to modulation by lifestyle factors that differ between racial/ethnic groups, and tumor characteristics contribute to disparities in CRC mortality, we hypothesize that differences in CRC metabolites and microbiome between racial/ethnic groups account for disparities in clinical outcomes. Our objective is to address the lack of knowledge in this area by performing a discovery metabolomics approach to identify salient features of CRC metabolism inherent to race. We will also examine how the microbiome and metabolome change during the continuum of CRC care, associate with treatment responses, and are affected by SES. In specific aim 1, we will use untargeted metabolomics, metabolic pathway analysis, and molecular networking approaches to identify CRC tumor tissue metabolites specific to AAs, with stratification by sex, stage, and SES. This will generate the first ever metabolic signatures of CRC in AAs and will include exogenous metabolites derived from environmental exposures and the microbiome. For specific aim 2, we will identify the impact of surgery and chemotherapeutic treatments on the CRC stool microbiome. We will identify microbiota unique to AA patients and determine if factors related to SES including diet, physical activity, and body mass index may confound this association. We will ultimately determine how the response to treatment differs between AA and non-Hispanic white patients to better understand mechanisms of increased mortality in AAs with CRC. The immediate outcomes of this proposal are: 1) generation of the first metabolic signature of CRC by race, 2) identification of novel microbial and environmental metabolites of CRC by race, 3) the first characterization of microbiome and metabolite changes at crucial time points along the continuum of CRC care (e.g., diagnosis, surgery, chemotherapy) and associations to SES. This data will generate new hypotheses regarding tumor characteristics and cancer outcomes. Our long-term goal is to improve patient outcomes by identifying key metabolic pathways and microbiota that differ between race/ethnicity, SES and may be exploited for the development of more effective therapeutic interventions.

NIH Research Projects · FY 2026 · 2023-12

The overall goal of research in my lab is to deepen our understanding of the roles of post-transcriptional gene regulation in the mammalian nervous system and how its dysregulation underlies human disease. In the next five years, we will build upon our previous work on neuronal mRNA metabolism and translation control and focus on three interconnected areas: 1} The impact of microsatellite repeat expansion on mRNA processing and translation. We will conduct targeted and genome-wide analyses to systematically identify the cis-elements and trans-factors that modulate noncanonical processing and translation of repeat-containing RNAs. 2} The functions and regulation of alternative translation initiation. Through our in-depth investigation of a conserved bi-functional mRNA that encodes two proteoforms of the neuronal pentraxin receptor (NPR}, we hope to uncover general principles that will inform our understanding of the regulatory impact of alternative start codon usage. 3} Quantitative measurement of mRNA translation efficiency (TE}. While ribosome footprint profiling, or Ribo-seq, is widely used to measure TE at a transcriptome scale, our recent findings suggest that conventional Ribo-seq procedures may not accurately capture the magnitude of TE regulation. By comparing Ribo-seq to directly measure TE data and optimizing Ribo-seq procedures, we will reexamine fundamental questions about translation control in mammalian cells. Results from these analyses may challenge existing dogmas and open up new lines of investigations.

NIH Research Projects · FY 2026 · 2023-12

Project Summary: Autosomal dominant polycystic kidney disease (ADPKD) affects over 12 million people worldwide resulting in fluid-filled cysts in the kidney and liver and 5-10% of all kidney failure. The one FDA-approved therapy provides only a modest delay in ongoing growth of cysts in the kidney and liver that can progress to kidney failure and devastating abdominal pain. Approximately one third of ADPKD patients have non-truncating mutations in the primary disease gene PKD1/Polycystin-1(PC1), and a significant subset of these likely encode a version of PC1 that is pathogenic because of a quantitative rather than qualitative deficit at its site of action on the cell surface due to inefficient maturation of PC1. PC1 “dosage”—the functional amount of PC1 protein at its site of action—correlates with disease severity. Our laboratory has contributed to the identification of additional disease genes to explain cases of symptomatic polycystic liver disease (PLD) resulting from in inefficient maturation of PC1 in the endoplasmic reticulum (ER). Proof of concept studies using genomic alterations in mice suggest cysts could be avoided in these patients if more PC1 was expressed. Recent studies inhibiting microRNA 17 from repressing Pkd1 mRNA have provided benefit to a Pkd1 missense mouse model. We hypothesize that increasing PC1 protein expression in patients with mutations in these ER genes and in a substantial subset of patients with PKD1 non-truncating mutations in ADPKD will dramatically reduce cyst burden. We have identified that the 5’ untranslated region (5’UTR) of human PKD1 contains alternative sites of translation known as upstream open reading frames (uORFs) whose translation significantly blunts the translation of PC1 from PKD1 mRNA in vitro. Our in vitro data shows that interventions to block PKD1 uORF translation results in 3-4 times more PC1 protein, and that blocking uORF translation is achievable using antisense oligonucleotides (ASO). ASOs are approved therapies for patients with other diseases. For this proposal we will determine the role of PKD1 uORFs on cyst formation in vivo. We have made a novel humanized PKD1 5’UTR mouse model with or without single nucleotide edits to remove uORF initiation codons and we will use this to test effect of uORFs in two models of ADPKD (Dnajb11 model and a Pkd1 p.R2216W model) as well as quantify effect in primary cells in different biological contexts to consider a role for in vivo regulation. Independently, we will test our hypothesis that ASOs targeting PKD1 uORFs are effective treatment in mouse models of ADPKD and would be—due to ability to effect translation efficiency—a unique, specific, and additive approach to any previously proposed therapies.