Baylor College Of Medicine

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY A rapid rise in syphilis cases has caused cases of congenital syphilis, the second leading cause of stillbirth worldwide, to skyrocket. Despite its prevalence, very little is known about the mechanisms through which Treponema pallidum (Tp), the causative agent of syphilis, is able to invade the placenta, impact placental function, and cause adverse pregnancy outcomes. This is largely due to a complete lack of viable in vitro models to study pathogenesis of Tp in the placenta. The overall objective of this proposal is to use novel in vitro models to investigate mechanisms through which Tp invades and impacts the placenta. By combining expertise from the Mysorekar and Edmondson/Norris laboratories, Tp has successfully been cultured in vitro in trophoblast cells and this system is being adapted to work with 3D stem cell-derived trophoblast organoids (SC-TOs). This project investigates the hypothesis that pairing these new in vitro models with an integrative, multi-omic approach will allow identification of key pathways and targets underlying placental invasion and replication of Tp at the maternal-fetal interface. Aim 1 will further define the optimal conditions for co-culturing Tp with Jeg-3 cells and SC-TOs. Advanced microscopy techniques will be used to characterize Tp attachment, growth, and possible internalization and migration of various Tp strains in our models. Aim 2 will elucidate the metabolic alterations and nutrient siphoning mechanisms that occur during Tp infection. Unbiased metabolomics will be conducted on trophoblasts infected with Tp to identify changes in trophoblast nutrient and metabolite levels due to host nutrient siphoning by Tp. Western blotting, immunofluorescence, and confocal imaging will then be used to examine changes in the expression and localization of nutrient transporters which regulate materno-fetal nutrient allocation across the placenta. Aim 3 will use integrated multi-omics to explore mechanisms underlying and driving Tp infection of trophoblasts by identifying changes in chromatin accessibility, gene expression, and protein expression elicited by infection. Bioinformatics will be used to integrate these -omic outputs and identify overarching pathways altered in trophoblast cells by Tp. Together, these studies will begin to identify key mechanisms through which Tp infects the placenta and impacts trophoblast function. This will be integral to increasing basic understanding of Tp pathogenesis in the placenta and for identifying novel biomarkers and treatment targets for syphilis-associated pregnancy outcomes.

NIH Research Projects · FY 2025 · 2025-08

The uncertain etiology of Alzheimer’s disease poses significant obstacles to the development of both therapeutic and prevention strategies. At the root of this quandary is the fact that familial disease accounts for a very small fraction of cases, with the vast majority remaining genetically sporadic. The most widely known risk factor, the ApoE4 allele of the gene coding for apolipoprotein E, has been identified, but mechanistic ties to disease development are still emerging. Neuroinflammation has long been suspected as a contributing factor, and regular exercise documented as a mitigating factor, with theories to explain their effects being posed, but again with little mechanistic proof. A picture of a very complex process of initiation and progression of Alzheimers disease and related dementias (AD/RD) including Pick’s disease, fronto-temporal dementia (FTD) and other tauopathies, is slowly emerging. A key factor in the etiology of AD/RD that repeatedly crops up is viral infection. COVID-19 has brought new attention to this factor, with the observation that a fraction of those infected experience “brain-fog”, a catch-all term that includes cognitive dysfunction, memory dysfunction and mental fatigue, marked by impaired ability to perform mental tasks compared to before the infection. Several factors however, complicate an understanding of the effect of COVID-19 on AD/RD and constitute the driving questions for this proposal: These include (1) What are the relative contributions of genetic risk factors and COVID-19 on initiation and progression of AD/RD? (2) What are the effects of SARS-CoV-2 variants: extinct strains e.g. Wuhan, alpha, delta vs. current strains e.g. omicron and its lineage, and their sequential infection on AD/RD initiation and progression? (3) What are the effects of prior infections with other highly prevalent viruses e.g. HSV-1 on the initiation and progression of AD/RD by SARS-CoV-2? Successful completion of this work will probe the relationship between COVID-19 and neurodegeneration, while yielding deep mechanistic insights into the role of SARS-CoV-2 in combination with HSV-1 in triggering/accelerating an AD phenotype and focus a search for therapeutic targets to counteract AD initiation/acceleration/progression.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Post-transcriptional mechanisms play crucial roles in maintenance of progenitor cell identity and have been implicated in cancer development, including leukemia. However, our understanding of the role of post- transcriptional regulatory processes in leukemogenesis remains limited. To address this gap, we conducted parallel CRISPR dropout screens in both normal and transformed hematopoietic progenitor cells, identifying the RNA helicase DDX6 as a selective vulnerability of leukemia cells. Our findings reveal that DDX6 is overexpressed in leukemia patients and plays a crucial role in initiating and sustaining acute myeloid leukemia (AML). Importantly, depletion of DDX6 in vivo significantly reduced leukemic burden and extended lifespan in patient-derived xenografts and mouse models of AML. Mechanistically, DDX6 coordinates the sequestration and translational suppression of mRNAs encoding chromatin and transcription factors in cytoplasmic condensates termed P-bodies. Overall, our data unveil DDX6-mediated RNA processing as a central pathway dysregulated in leukemia cells, relative to hematopoietic stem and progenitor cells, and required for AML maintenance. While these insights are significant, the roles of DDX6 in promoting malignant transformation remain unclear. Furthermore, the direct gene-regulatory networks of DDX6 in leukemia are yet to be fully understood. To address these questions, this application proposes three complementary aims. In SPECIFIC AIM 1, we will utilize two novel genetic mouse models for deletion and overexpression of DDX6 in vivo in a cell- and time-specific manner to rigorously dissect the roles of DDX6-mediated RNA sequestration during normal and malignant hematopoiesis. Additionally, we will assess the impact of perturbing DDX6's helicase activity on leukemia cells, both in vitro and in vivo. In SPECIFIC AIM 2, we will explore the relationship between DDX6 and RNA sequestration in cytoplasmic condensates of normal and leukemia cells. Specifically, we will characterize the molecular landscape of P-bodies in human AML as well as normal hematopoietic progenitors and investigate how DDX6 cooperates with other RNA-binding proteins to regulate the storage of critical RNAs during leukemogenesis. We will also identify DDX6's direct protein interactors and assess how these interactors facilitate RNA sequestration and contribute to leukemogenesis. In SPECIFIC AIM 3, we will elucidate the direct role of DDX6 in suppressing the translation of target mRNAs during leukemogenesis. By analyzing the translatome and proteome of leukemia cells following acute degradation of DDX6 using our novel FKBP-degron AML lines, we will identify factors regulated by DDX6 and examine their functional and cellular roles in AML cell self-renewal and leukemogenesis. Collectively, our study will advance the understanding of post- transcriptional regulation in AML and pave the way for innovative therapeutic strategies targeting DDX6 and downstream effectors. Furthermore, our work introduces the concept of RNA condensates as potential biomarkers in myeloid leukemia diagnosis and treatment.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Thoracic aortic aneurysms and dissections (TAAD) are life-threatening conditions for which therapeutic options are limited. While genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with sporadic TAAD, the molecular mechanisms through which these SNPs contribute to disease development remain unclear. The long-term objective of this project is to elucidate the role of TAAD-associated SNPs in sporadic TAAD pathogenesis, with a focus on their epigenetic and regulatory functions in aortic cells. Recent research has shown that altered expression of extracellular matrix (ECM) and inflammatory genes, and related epigenetic changes play critical roles in the progression of aortic disease. Integrating GWAS data with single-cell chromatin accessibility (scATAC-seq) data from human aortic tissues has revealed that many TAAD-associated SNPs are located within accessible chromatin regions of aortic cells, suggesting that these SNPs influence regulatory elements activity. This project tests the hypothesis that the risk alleles of TAAD-associated SNPs alter the binding affinities of transcription factors (TFs), reshaping the local epigenetic landscape and driving target gene dysregulation. The proposed research will systematically investigate the epigenetic mechanisms by which sporadic TAAD-associated SNPs influence gene regulation. The first aim is to identify the functional cell types where these SNPs exert their effects by integrating GWAS data with scATAC-seq data to determine the cell types in which disease-associated SNPs are located within accessible chromatin regions. The second aim focuses on exploring the epigenetic modifications and TF binding at SNP-containing chromatin regions using integrative analyses of GWAS and CutRun-seq data, targeting key histone modifications and TFs. The third aim will conduct allele-specific analyses by combining whole-genome sequencing, scATAC-seq, and scRNA-seq data to determine the direct target genes of SNPs in specific aortic cells. By uncovering the functional and regulatory mechanisms of TAAD-associated SNPs, this study will provide critical insights into the genetic and epigenetic basis of aortic disease, potentially leading to new therapeutic strategies.

NIH Research Projects · FY 2025 · 2025-08

Abstract: Neural tube defects (NTDs) are among the most common and severe birth defects, affecting over 300,000 infants worldwide each year. NTDs are often lethal and, if not, lead to devastating health consequences and early childhood mortality due to the improper development of the brain and /or spinal cord. Prevention of NTDs is also challenging because primary neurulation begins in the third to fourth week of pregnancy, often before a woman even knows they are pregnant. During this early pregnancy timepoint, the yolk sac acts as the interface between the maternal and fetal environments. It supplies the growing embryo with the correct balance of metabolites for successful growth and development. MicroRNA-290 is expressed exclusively in extraembryonic tissues, including the yolk sac, and when knocked out, leads to a partially penetrant NTD phenotype. However, with pregestational hyperglycemia, as a model of maternal metabolic dysregulation, loss of miR-290 leads to increased incidences of neural tube closure defects and cellular stress in both the yolk sac and placenta. Additionally, preliminary metabolic analysis of the yolk sac and cranial region of E10.5 embryos reveals decreased folic acid is reaching the embryos from hyperglycemia dams. Folic acid is an essential vitamin supplied from the maternal diet, and deficiency is known to contribute to NTDs; however, precisely how folate prevents NTDs is unclear. We also found decreases in antioxidant glutathione and increased methyl donor SAM in the miR-290 KO embryos, likely exacerbating the metabolic dysregulation caused by maternal stress. Folic acid, SAM, and glutathione are all part of one-carbon metabolism, a series of interlocking, essential metabolic cycles that provide methyl groups for numerous cell functions, including lipid synthesis. DNA and protein methylation, and production of antioxidants. Therefore, we hypothesize that miR-290 protects extraembryonic tissues from metabolic dysregulation by regulating one-carbon metabolism. We aim to build off our preliminary metabolic results to establish a mechanism by which defects in extra-embryonic tissue metabolism drive transcriptional changes in the neuroepithelial cells, impeding neural tube closure. We plan to investigate how maternal metabolic stress impacts the yolk sac function, leading to impaired fetal central nervous system development, and to identify strategies that can be implemented before and during the earliest pregnancy time points.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Chagas disease is a neglected tropical disease endemic to Latin America but is increasingly being detected in the United States. Chagas disease is caused by the Trypanosoma cruzi (T. cruzi) parasite and presents itself in two phases: an acute phase and a chronic phase. The diagnosis of both phases is challenging as patients can be asymptomatic or have nonspecific symptoms. If untreated, the acute infection progresses to a chronic phase where 20 ‒ 40% of patients will develop life-threatening illnesses, including cardiomyopathy, heart failure, or cardiac arrest. Acute infection can be detected by microscopy; however, the level of parasitemia during chronic infection often drops below the limit of detection of these techniques, making it unsuitable for diagnosis. Therefore, the diagnosis of chronic infection relies solely on serological detection of anti-T. cruzi antibodies can persist in the blood throughout the life of the infected individual. However, existing serological tests are prone to inconclusive or false-negative/positive results due to inadequacies of the native T. cruzi capture proteins used in these assays and for differences in the six discrete typing units (DTU) subtypes. For this reason, the WHO and PAHO recommend testing using at least two different serological techniques to diagnose Chagas infection. In the United States, discordant results from these two tests require the sample to be forwarded to the CDC for additional testing to confirm diagnosis. This repetitive testing process imposes a significant resource burden on the healthcare system, and the poor performance of existing Chagas serological tests can lead to increased disease transmission and life-threatening illness due to misdiagnosis. Therefore, this project aims to develop and validate a highly sensitive serological assay for detecting all geographic variances of T. cruzi infection with a single test. This assay will employ a collection of carefully designed recombinant T. cruzi antigens, enabling high-sensitivity detection of all T. cruzi strains. The rationale for the proposed research is supported by the applicants’ preliminary data demonstrating the generation of a recombinant T. cruzi antigen (Tc24) that can accurately detect anti-T. cruzi IgG in patient samples from multiple Latin American countries and to provide a test-of-cure in a prospective longitudinal post-treatment study. To achieve this goal, we will pursue the following specific aims: 1) Optimize a Chagas Tc24 Direct ELISA and high repetitive region quantitative PCR for T. cruzi 2) Use Tc24 ELISA and qPCR to provide a proof-of-principle for a test of cure in chronic Chagas This approach is innovative because it combines the use of a collection of carefully designed recombinant T. cruzi antigens for detecting all geographic strains of T. cruzi while exhibiting no cross-reactivity with other parasites with a single test and it is significant because it will provide a test-of-cure and post-treatment efficacy for people with Chagas disease.

NIH Research Projects · FY 2025 · 2025-08

Abstract/Summary The Fundamentals in Bioinformatics (FunInBio) program develops, deploys, and evaluates a continuing education program to address skills gaps in post-doctoral scientists. The FunInBio curriculum conveys bioinformatics skills while participants contribute to a citizens science research problem about the environmental component of antibiotic resistance. Participants will gain exposure to many facets of computational biology, including genome sequencing/analysis, comparative genomics, and training tailored to their professional needs. FunInBio is directed towards post-doctoral scientists from universities that employ scientists who would benefit from bioinformatics training. In Aim 1, we will develop a three-part curriculum covering programming background, an in-person short course on comparative genomics, and customized online modules tailored to participant needs. In Aim 2, we will deploy the FunInBio curriculum at regional educational institutions in the life sciences. In Aim 3, we will assess the effectiveness and outcomes of the FunInBio continuing education program to iteratively improve our curriculum. Integral to these courses is the program website, developed as a repository for educational material and a database that aggregates course-generated data. The primary outcome of FunInBio is the group of early-career scientists trained in bioinformatics skills. In addition, we will publish our course materials to extend the impact of our curriculum, and we will publish results quantifying the effectiveness of our approach. FunInBio will bring bioinformatics education to the growing population of post-doctoral scientists as they address a meaningful research question through citizen science. Contact PD/PI: Wright, Erik Scott Project

NIH Research Projects · FY 2025 · 2025-08

Baylor Research Education Program in Clinical Neurosciences Abstract Baylor College of Medicine has been the premier medical training program in Houston, TX for over half a century. The primary neurological training programs include Neurosurgery, Neurology, and Pediatric Neurology. These programs have always had a strong academic tradition, and graduates have served as faculty at many leading medical schools, but historically there was a decidedly clinical emphasis. Over a decade ago, a strategic decision was made to focus on research and research education by capitalizing on the extraordinary scientific resources at Baylor College of Medicine and affiliated institutions in the Texas Medical Center. Many residency programs, including these three, were retooled to substantially enhance their ability to train the next generation of academic neurologists and neurosurgeons. In 2009, the BCM Department of Neurosurgery was awarded the predecessor NINDS R25 grant to support resident research. Our successful renewals in 2014 and 2019 evidence the strength of clinical neurosciences research at BCM that is available to support our trainees and propel them towards independent careers as clinician-scientists. In this UE5 renewal, we expand the breadth of the program to offer the same high-caliber training to residents in Neurology and Pediatric Neurology and make these opportunities available to future trainees interested in neuroscience research in related disciplines such Radiology, Anesthesiology, Pathology, and Emergency Medicine. We will select outstanding residents who have the background, talent, and motivation to become successful clinician-scientists, and then carefully integrate additional specialized research education into their residency training and beyond. The UE5 program will mentor these residents through the entire research process, from project conception to experimental design, data analysis and interpretation, to publication of results, and finally to the development of an effective plan for beginning a career as a physician-scientist. The mentorship established by this program will continue beyond residency into fellowship and the early career stages. In addition to carrying out a research project, residents selected for this research education program will be trained in experimental design, scientific writing, oral presentation, and in the responsible conduct of research. Furthermore, they will receive considerable oversight and career counseling from a team of expert mentors with the intent of paving their way to success in obtaining a mentored career development award and becoming productive, independent physician-scientists.

NIH Research Projects · FY 2025 · 2025-08

SUMMARY Understanding brain function requires tools capable of recording membrane voltage with high spatial and temporal resolution, cell-type specificity, and compatibility with animal models. Genetically encoded voltage indicators (GEVIs), protein-based fluorescent biosensors of membrane potential, have the potential to achieve these specifications and enable optical measurements voltage dynamics in the brain. This project focuses on developing next-generation GEVIs optimized for two-photon (2P) microscopy, the gold standard for deep-tissue imaging in scattering brain tissues. Specifically, we aim to improve GEVI sensitivity for detecting subthreshold signals and enhance response kinetics to reliably capture rapid action potentials in awake, behaving animals. A key objective is to tune indicators for maximal performance when using resonant scanning microscopy, the most widely accessible two-photon imaging method. Key innovations include refining our high-throughput screening platform to identify fast-kinetics GEVI variants, engineering ultrasensitive GEVIs tailored for synaptic activity, and designing advanced subcellular targeting strategies to minimize background fluorescence. These efforts will incorporate directed evolution, synthetic dosage compensation circuits, and clustering strategies to enhance signal-to-noise ratios. Preliminary data demonstrate substantial advancements in GEVI sensitivity and voltage response dynamics, including new mutations that improve fluorescence changes across a broad voltage range. The optimized GEVIs will undergo rigorous validation in vitro and in vivo, combining state-of-the-art electrophysiology and 2P microscopy to benchmark their performance against existing tools. By addressing current limitations in GEVI technology, this project aims to expand the utility of voltage imaging, providing neuroscience researchers with powerful tools to explore neural computations, synaptic integration, and their disruption in neurological disorders. These innovations are expected to catalyze widespread adoption of GEVIs, advancing our understanding of brain function in health and disease

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT The major source of early morbidity and mortality resulting from a nuclear incident comes from acute radiation syndrome in the gastrointestinal tract (GI-ARS). Radiation targets the cycling intestinal stem cell (ISC) causing the cells to apoptosis and subsequently affecting the entire intestinal epithelial renewal which originates from the ISC. Although GI-ARS accounts for a significant portion of acute morbidity and mortality after high dose ionizing radiation exposure, there are no therapies or treatments to prevent, mitigate, or treat GI-ARS. We propose to explore the use of the microbiome, which lives in intimate contact with the intestinal epithelium, to deliver growth factors that promote survival and proliferation of radioresistant ISC populations. We have engineered Limosilactobacillus reuteri 6475 (LR6475), a commercially used organism that is inexpensive, easy to store, able to be administered orally in the event of a nuclear disaster, and adept at surviving in the gastrointestinal tract, to deliver key growth factors necessary to promote radioresistant ISC regeneration of the intestinal epithelium. The overarching hypothesis is that LR6475 can serve as a platform to develop mitigating medical countermeasures for GI-ARS. The objective of this proposal is to assess our candidate genetically modified LR6475 in a rodent model of radiation damage for the ability of the medical countermeasure to accelerate recovery from GI-ARS. To achieve this objective, we propose to determine the efficacy of engineered LR6475 as medical countermeasures in humanized microbiome mouse in which the recovery of the ISC population can be tracked. The results obtained at the end of this project will support the continued development of LR6475 as a stockpiled general nuclear countermeasure.

NIH Research Projects · FY 2026 · 2025-08

SUMMARY This proposal aims to fundamentally advance our understanding of astrocyte physiology by using novel genetically encoded voltage indicators (GEVIs) to measure the coordinate of astrocyte membrane potential (Vm) and local neural activity in vivo. Historically considered “electrically uninteresting,” because of the minimal variations in somatic voltage observed in patch recordings, our preliminary data with JEDI3, a sensitive, stable, two-photon compatible GEVI, reveals robust changes in membrane voltage in vivo that track closely with variations in neural activity and behavioral state. In Aim 1, we will characterize the spatiotemporal coordination of astrocyte Vm fluctuations and neural activity across multiple contexts—cortical up/down states under anesthesia, transitions between quiet wakefulness and active locomotion, visual stimulation, and seizures—and test the extent to which these voltage changes occur independently of astrocyte calcium signaling. In Aim 2, we will determine how neuromodulatory inputs (noradrenergic and cholinergic) shape astrocyte Vm and whether manipulating astrocyte voltage can influence state-dependent neuronal dynamics. We will also validate optogenetic tools for reliably controlling astrocyte voltage, a critical advance that will enable causal tests of the effects of manipulating astrocyte membrane voltage. In Aim 3, we will resolve subcellular hotspots of astrocyte depolarization at the scale of individual dendrites and axons, and test whether presynaptic activity alone is sufficient to drive these focal changes. Finally, as part of Aim 3, we will develop and validate improved astrocyte-optimized GEVIs, ensuring that these optical methods are made accessible to the broader neuroscience community. By illuminating the “hidden” electrical dimension of astrocyte function, this work promises to transform our understanding of glia-neuron interactions, reveal novel mechanisms of brain state regulation, and open new therapeutic avenues for neurological diseases where astrocyte dysfunction is implicated.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Necrotizing enterocolitis (NEC) is a devastating intestinal morbidity of preterm infants preceded by microbial dysbiosis and intestinal necrosis. But even after decades of research, the etiopathogenesis is still unclear. The gut epigenome-microbiome axis may determine the phenotype that predisposes to NEC but studying epigenetic variation in the gut is difficult to do non-invasively. Focusing on genomic regions that display systemic interindividual variation in DNA methylation (CoRSIVs) therefore offers great promise. The systemic nature of these stable epigenetic variations means that measuring CoRSIV methylation in blood DNA provides information about epigenetic regulation throughout the body. CoRSIVs are essentially epigenetic polymorphisms, which have been linked to human disease including immune disorders and cancer. The role of CoRSIVs in NEC and preterm pathophysiology has not been investigated. Our overall hypothesis is that interindividual variation in CoRSIV methylation, which is established in the early embryo and maintained in various tissue lineages during differentiation, influences the development of the intestinal microbiome (including microbial dysbiosis) leading to intestinal inflammation and necrosis in NEC. We intend to test our hypothesis by 2 specific aims. Aim 1. Among preterm neonates, determine if CoRSIV methylation in peripheral blood at birth is associated with subsequent diagnosis of NEC. In a pilot nested case-control study design, we have enrolled preterm infants (< 32 weeks or born at < 1500 g) and collected stool and blood samples every week for 4 weeks (11 NEC patients and 22 gestational age matched controls). We will investigate CoRSIVs in peripheral blood leukocytes and perform whole genome sequencing for methylation quantitative loci (mQTL) effects at CoRSIVs. Aim 2: In preterm infants, determine if blood leukocyte CoRSIVs methylation at birth associates with subsequent development of stool microbiome diversity and microbial composition. Animal data supports the hypothesis that individual variation in DNA methylation informs the development of the diversity and composition of the stool microbiome. We will correlate stool microbiome in the first 4 weeks, evaluated by metagenomics with blood leukocyte CoRSIVs methylation at birth. We propose an innovative DNA methylation profiling approach to investigate the role of interindividual epigenetic variation in the pathophysiology of NEC in preterm infants. Our high risk- high reward approach opens the door to a new area of research in preterm infants that has the potential to inform novel preventive strategies, which improve clinical outcomes. The novel data generated in the proposal will seed a future R01 proposal with a large multi-center study and mechanistic studies in organoid models and germ free mice. New knowledge on epigenetic influences on microbiome development is relevant to a broad-spectrum of diseases such as cancer, infection, immunity and growth

NIH Research Projects · FY 2025 · 2025-08

Project Summary Spontaneous cardiomyocyte (CM) regeneration has been demonstrated in embryonic and neonatal mammals; however, adult CMs go into cell cycle arrest as their turnover has been reported as minimal in human hearts and rodents. Several recent discoveries to induce CM proliferation in adult CMs, such as Cyclin A2, mir199, YAP- 5SA, or the combination of CDK1, CDK4, cyclin B1, and cyclin D1 (collectively known as 4F), have provided new mechanistic insights of our understanding of the cell cycle block in adult CMs. However, a pertinent question is what is the major signaling pathway that dictates CMs to spontaneously proliferate in fetal/neonatal stages and be abolished in adulthood. Our recently published temporal single-cell RNA-seq data from 60-day mature hiPS-CMs indicates the presence of a unique subpopulation of primed starting CMs that respond to the 4F. One of the major characteristics of this primed population is the expression of the transmembrane scavenger receptor; CD36. CD36 is a fatty acid internalization receptor with recent indications of regulating several signaling pathways. Our single-cell RNAseq from primary CMs isolated from P1 hearts demonstrated that CD36 is expressed only in the spontaneously proliferating CMs population and the responders to the 4F cell cycle stimulation. Our preliminary data show that loss of CD36 global and CM-specific knockouts are born with smaller hearts, which contain fewer CMs compared to their WT littermates. Furthermore, compared to their WT littermates that completely regenerate the heart apex following apical resection at P1, CD36KO showed minimal regeneration of the apex. Mechanistically, bulk RNAseq data from the P1 CD36KO hearts showed significant downregulation in the expression of the cell cycle genes and the retinoic acid (RA)-cell cycle induction signaling genes, such as FABP5, PPARd, and RXR that coordinately play a critical role in cell decision between proliferation and growth. Spatial metabolomics analysis demonstrated a decline in retinoic acid esters within the CD36CKO hearts, which indicates a deficiency in retinoic acid internalization. To confirm the causality of this proposed mechanism, in vivo, crossing CD36KO with CM-specific PPARd overexpression (PPARdCTG) only in CMs rescued the proliferation deficiency phenotype in CD36KO in the P1 hearts. We hypothesize that CD36 modulates RA signaling through esterification and internalization of the RA to bind to FABP5 to activate the PPARd/RXR transcriptional program, thereby regulating the decision of CMs to progress through the cell cycle when needed. To test this hypothesis, we will address these aims: Aim 1: To address the hypothesis that CD36 internalizes RA to bind to FABP5 to initiate the signaling mechanism. Aim 2: To test the hypothesis that RA/FABP5 binding is the rate-limiting step in the proposed pathway to activate the PPARd/RXR transcriptional activity. Aim 3: To test the hypothesis that FABP5/PPARd overexpression is a therapeutic target for ischemic heart failure.

NIH Research Projects · FY 2025 · 2025-08

Obsessive-compulsive disorder (OCD) has a lifetime prevalence of 2-3% and is a major cause of global disability. OCD is characterized by obsessions (i.e., unwanted intrusive thoughts) and compulsions (i.e., repetitive ritualistic behaviors) and exemplifies a pathologically avoidant phenotype where greater degrees of avoidance are associated with greater symptom severity and treatment resistance. In the 20-40% of OCD patients that are treatment refractory, deep brain stimulation (DBS) of the ventral capsule and ventral striatum (VC/VS) is an effective therapy that achieves significant benefit in 66% of patients. While VC/VS DBS does not acutely relieve OCD symptoms, DBS often produces a “pro-approach” response including an increase in talkativeness, a desire to engage in activities, extroversion, and affiliative behavior. Our central therapeutic hypothesis is that VC/VS DBS achieves eventual benefit in OCD by providing the “boost” in pro-approach behavior that allows individuals to overcome their pathological fear-based avoidance. Thus, we focus on the approach-avoidance axis as one of the critical neurobehavioral axes underlying the pathophysiology of OCD. Our overall goal is to develop a mechanistic understanding of DBS for OCD by rigorously investigating the relationship between clinical symptoms, behaviors (i.e., approachful vs. avoidant), and neurophysiology. Based on our preliminary data from on-device DBS recordings in OCD patients, we hypothesize that greater variability in neural activity in the VS, a structure known to influence motivated goal-directed behavior, allows the agent to shed maladaptive ritualistic behavior in favor of reasoned behavior aligned with long-term goals. In other words, the transition from repetitive, ritualistic actions in the symptomatic state to more adaptive and flexible behaviors after response is accompanied by increased dispersion and decreased predictability of VS activity. In 10 patients with recording-capable DBS devices, we will collect a broad array of neurobehavioral data in the clinic and the home. The first 2 Aims test our hypothesis by studying the pattern of VS neural activity in the controlled environment of the lab/clinic during two complementary paradigms: one based on a psychophysical behavioral task, the other based on exposure and response prevention, a therapeutic behavioral intervention. The third aim tests this hypothesis in an ambulatory, naturalistic setting with chronic neural on-device recordings paired with time resolved behavioral measures from wearables (e.g., sleep, heart rate, activity) and video diaries (e.g., facial affect, speech). We will investigate a possible common neural basis underlying approach and avoidance across these 3 paradigms. These data will allow us to use VS intracranial recordings to inform and validate the approach-avoidance framework and investigate the degree of explanatory power this framework has for OCD. By understanding the therapeutic mechanism driving OCD symptom improvement after VC/VS DBS, we will pave the way for rigorous, data-driven neuromodulatory strategies to regulate behavior and ameliorate symptoms in other disorders characterized by dysregulated approach-avoidance behavior.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Developmental and epileptic encephalopathy 5 (DEE5) is a rare neurodevelopmental disorder caused by monoallelic pathogenic variants (PV) in SPTAN1, which encodes non-erythrocytic αII-spectrin. Individuals with c.6908_6916dup (p.D2303_L2305dup) and the c.6619_6621del (p.E2207del) PV experience infantile epilepsy with refractory seizures, profound developmental delay, hypotonia, and microcephaly. There is no available cure for the affected children, and the current treatment goal aims to improve quality of life. However, there remains a significant need for therapy development as multiple reports of failure to respond to anti-seizure medication have been documented. Typically, children harboring the duplication variant succumb to the disorder before 6 years of age. Previous studies in patient-derived fibroblasts and transfected mouse neurons had demonstrated a dominant-negative consequence of the variants resulting in aggregation of αII with β spectrins and a disrupted axon initiation segment based on Ankyrin G and voltage-gated sodium channel signals. However, there is a lack of connection between the molecular phenotypes and the clinical seizure phenotypes. Hence, there is a critical need to clarify the fundamental molecular factors involved and assess the cellular and biochemical properties using patient-derived cells and a mouse model to develop feasible therapy for the affected individuals. The overall goal of the proposed work is to better the understanding of the molecular consequences of DEE5 (Aim 1) and develop appropriate assays and a generalizable pipeline for screening antisense oligonucleotide (ASO) therapy that addresses the dominant-negative neurodevelopmental disorders (Aim 2). The applicant hypothesizes that the SPTAN1 PV variants aggregate with SPTBN4 at the axon initiation segment to cause aberrant action potential and thus seizures, and the wildtype protein function can be restored by knocking down the variant allele using ASO. The applicant will use lineage-appropriate patient-derived cell lines as well as develop a humanized mouse model to fully assess the disease mechanisms underlying DEE5 and will design and screen RNase-H-dependent ASOs in patient-derived cell lines to determine the effective therapeutic approach for alleviating cellular phenotypes. The proposed work serves instrumental value to the scientific community and patient families by providing an extensive understanding of SPTAN1 dominant-negative disease mechanisms, a valuable mouse model for future research, and supportive preclinical evidence for treatment development. This project is designed to prepare the applicant for a career as an independent scientist in translational research and furthers the applicant’s long-term goals of contributing to the preclinical development of rare genetic disorders affecting the central nervous system.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Over 90% of human genes undergo alternative splicing, generating numerous transcripts or isoforms with distinct functions for each gene. This highly regulated process is often disrupted in cancer, leading to the production of harmful protein isoforms that contribute to tumor growth, survival, metastasis, and immune evasion. Accurately identifying these aberrant isoforms is essential for understanding cancer biology and developing targeted therapies. While RNA sequencing has advanced our understanding of alternative splicing in cancer, the study of protein isoforms at the proteomic level is still in its infancy. Advances in mass spectrometry (MS)-based shotgun proteomics have enabled unbiased identification and quantification of more than 10,000 protein coding genes from biological samples. However, because shotgun proteomics data analysis involves searching MS spectra against a reference protein database to identify peptides, and most identified peptides map to multiple protein isoforms, it remains challenging to accurately identify and quantify known protein isoforms in the reference database. Furthermore, novel isoforms absent from the reference database cannot be identified. Efforts have been made to address these challenges, such as the development of our recently published SEPepQuant algorithm, but integrating known and novel protein isoform identification and quantification into routine cancer research remains elusive. This integration faces two primary hurdles. Firstly, there are software-related obstacles in protein isoform identification and quantification, including the lack of user-friendly tools, insufficient benchmarking to guide software selection, difficulty in result interpretation, and compatibility issues with rapidly evolving MS technologies. Secondly, even with advanced software, its usage is confined to bioinformaticians, limiting direct benefits for most biologists and clinicians to gain insights into protein isoforms from the wealth of public cancer proteomic datasets. The overarching goal of this project is to overcome these hurdles by developing an integrated computational framework for shotgun proteomics- based protein isoform analysis and facilitating the dissemination of protein isoform data through accessible platforms. Leveraging the complementary expertise and unique resources of our investigator team, we will achieve this goal through three specific aims: Aim 1) Further develop SEPepQuant to improve robustness, interpretation, compatibility, and proteogenomic integration; Aim 2) Create a unified framework for protein isoform quantification and conduct systematic benchmarking; and Aim 3) Democratize protein isoform analysis for ordinary biologists and clinicians. Through these aims, we will create new informatics technologies to advance protein isoform analysis in cancer research, enhancing our understanding of protein isoform regulation, and facilitating the identification of dysregulated isoforms as potential biomarkers and therapeutic targets.

NIH Research Projects · FY 2025 · 2025-08

Project Summary/Abstract Clinically credentialed immunomodulatory drugs bind to the substrate receptor cereblon (CRBN) within the CRL4CRBN E3 ubiquitin ligase complex, creating a ‘neo-epitope’ on the CRBN surface that recruits non- endogenous substrates (neosubstrates) with an 8-amino acid G-loop degron motif for subsequent ubiquitination and proteasomal degradation. Such a drug modality is known as molecular glue degraders (MGDs), a subclass of molecular glues (MGs) that are characterized by their ability to induce protein-protein interactions. A significant gap remains in understanding the full scope of CRBN neosubstrates beyond the known G-loop-containing zinc finger proteins in the human proteome, often leading to new CRBN interactors being discovered serendipitously. Using bioinformatic analysis, we have identified 2,458 canonical G-loop targets as potential CRBN interactors and aim to create a more comprehensive atlas of CRBN interactome. The overarching objectives of the proposed work are: 1) to create a comprehensive atlas of CRBN interactome, encompassing canonical G-loop, mutant G- loop, and even the hidden non-G-loop targets, and 2) to further validate these uncharted proteins as bona fide CRBN interactors in the presence of novel MGDs. To achieve objective 1, we will leverage our expertise in bioinformatic analysis, while realizing objective 2 will require the development of efficient target-based MGD discovery methods, building on our knowledge of the CRBN interactome. We plan to employ not only a degradation activity-first, protein-protein docking-guided rational design and screening approach, but also utilize our DNA-encoded chemical library (DEL) platform to develop a ternary complex binding-first, unbiased DEL screening approach. To showcase our strategy, we have prioritized several uncharted G-loop targets, aiming to set compelling examples that will inspire future research into targeting the remaining predicted CRBN interactors with the MGD modality. Upon completion, this proposed work will significantly expand our understanding of the CRBN target landscape, and the molecular mechanisms and cellular pathways influenced by both existing and future CRBN ligand-based MGDs. Given MGD’s unique proximity-inducing mechanism and chemical knockdown pharmacology, our work is also poised to catalyze the development of numerous new CRBN-targeting MGD chemical probes or drugs that address many previously ‘undruggable’ wild-type proteins and a significant number of recalcitrant mutant proteins, thereby significantly advancing biomedical research and ultimately making a difference in public health.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Biliary atresia (BA) is a neonatal cholestatic liver disease and the leading cause of pediatric liver transplants. The primary treatment, known as the Kasai procedure, aims to promote bile flow by creating a direct connection between the liver and the intestine. However, the success of this procedure varies, and many children still require liver transplants within two years. The progression of fibrosis, driven by hepatocyte injury, bile duct obstruction, ductular reaction, and hepatic stellate cell (HSC) activation, is a key factor leading to liver transplant necessity. Identifying approaches to reduce fibrosis progression is a potential mechanism to reduce the need for liver transplant in BA patients. Bile acids are often considered key factors in initiating liver inflammation and promoting fibrosis development. However, in addition to elevated bile acids, infants with BA have elevated blood endotoxin levels, originating from gut bacteria translocation. Endotoxin is known to activate hepatic stellate cells, promoting hepatic collagen deposition, even without elevated bile acids. Our preliminary results in bile duct-ligated neonatal piglets demonstrate that oral antibiotic treatment reduces fibrosis development without preventing increased bile acid concentrations or ductular reaction over a two-week period. This finding suggests that fibrosis progression can be suppressed by reducing endotoxin load from gut bacteria. This study will investigate the use of a clinically relevant, non-absorbable antibiotic to reduce bacterial-derived inflammation and subsequent fibrosis in neonatal cholestasis, potentially offering a safer alternative to high-potency, absorbable antibiotics that pose a risk of drug- induced liver injury during BA. We hypothesize that suppression of bacteria-derived inflammation using non- absorbable antibiotics will reduce fibrosis development during obstructive cholestasis in the neonatal period. To research this hypothesis, we will: 1) determine if a non-absorbable antibiotic suppresses the development of liver fibrosis and injury, and 2) determine whether high concentrations of bile acids enhance fibrosis activation induced by endotoxin in a novel 3D spheroid cell culture model.

NIH Research Projects · FY 2025 · 2025-08

Zika virus (ZIKV) and closely related dengue virus (DENV) are major human pathogens. ZIKV and DENV are transmitted by Aedes mosquitoes in the tropical and subtropical regions, where approximately 3 billion people or 40% of world population live and are at risk of these infections. ZIKV has caused three major outbreaks on Yap Island (2007), in French Polynesia (2013), and in Brazil and other American countries (2015-2016). Several million people in 48 Pan-American countries and territories have been infected, showing symptoms including fever, rashes and conjunctivitis. Although most people recover in a few days, ZIKV infection has been found to cause a 20-fold increased incidence of serious neurological diseases, such as Guillain-Barré syndrome and >4,000 cases of microcephaly (small brain/head) and other neurological defects in newborns. WHO announced ZIKV is a “Public Health Emergency”. It is estimated that DENV infects ~400 million people per year with 100 million developing symptoms including fever, headache, rash, conjunctivitis and muscle and joint pain. ~500,000 cases/year progress to serious and potentially life-threatening dengue hemorrhagic fever or shock syndrome, with approximately 22,000 deaths annually, mostly among children. Except for mosquito control, there have been no available antiviral drugs to prevent or treat ZIKV and DENV infections. There is therefore a pressing need to find effective antiviral agents against ZIKV and DENV. In this project, we propose to perform medicinal chemistry and structure-activity relationship (SAR) studies to develop potent anti-ZIKV/DENV compounds (Specific Aim 1) and use chemical biology, biochemical, and cell biology methods to identify the protein target of these compounds.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Tick-borne diseases are an endemic and emergent public health threat in the US. The historic geographic range of established tick vectors have expanded and new invasive tick species have been introduced over the last decade. Additionally, novel tick-borne pathogens of human importance have been discovered at a rate unparallel by other vector-pathogen complexes over the last 50 years. Unfortunately, our understanding about the pathogenicity and ability to detect human tick-borne disease has been outpaced by the identification of new species of tick-borne pathogens. This is especially true for spotted fever group Rickettsia (SFGR), which has expreicenced a significant rise in disease and the identification of multiple novel pathogenic members of the family since its initial discovery in 1920. We critically need new tools and resources to tackle this growing public health problem. The overarching goal of this proposal is to develop a bank of Rickettsia spp. isolates from ticks collected in the southern US and Central America to be used as a resource for further research into host- pathogen interaction, pathogenicity, diagnostic, and vaccine development for SFGR. Our specific aims are as follows: (1) Develop a Rickettsia isolate library using a cohort of previously collected and processed ticks from the southern US and Central America, and (2) Conduct molecular characterization of Rickettsia spp. isolates obtained from ticks collected in the southern US and Central America. We will utilize a cohort of >3,500 ticks previously collected from Texas and Belize to conduct this research. We will select a stratified sample for culture of 350 ticks from the 1,878 (53%) ticks that screened positive for Rickettsia that are representative of all Rickettsia spp. and locations. We will conduct molecular characterization of the isolated species uing PCR and sequencing of gltA, ompA, ompB, and htrA genes. Ultimately, we will make the isolate libarary available to the broader scientific community for future research to advance the field. This study brings together a well-suited team to tackle a growing public health problem. Our findings will serve as the foundation for many important future studies to determine pathogenicity and develop effective diagnostic tests. Additionally, it will serve as an important resource for the larger scientific community.

NIH Research Projects · FY 2025 · 2025-08

Recombination is essential for repairing DNA double-strand breaks (DSBs) and maintaining genomic stability. DSBs can be repaired through non-homologous end-joining (NHEJ) or homologous recombination (HR). Even minor deficiencies in DNA recombination can result in cancer, immune deficiencies, or other severe diseases. The goal of the proposed research is to understand the mechanisms and regulation of DNA recombination using the model organisms of budding and fission yeast. We will focus on three areas of research: 1. The initial processing of DNA double-strand breaks (DSBs) into single strands, a process termed 'DNA end resection', is the critical first step in homologous recombination. It is necessary for the loading of damage response and repair proteins. Systematic studies of resection have thus far been conducted only in euchromatin. We propose to study this process in constitutive heterochromatin, which constitutes a large fraction of eukaryotic genomes. We have designed multiple assays to study DSB ends resection and repair within heterochromatin using a fission yeast assay. 2. Repair of one-ended double-strand breaks (DSBs) can initiate extensive repair-specific DNA synthesis. This pathway, known as break-induced replication (BIR), is facilitated by Polδ/Pif1 helicase-driven D- loop migration. BIR is crucial for the maintenance of telomeres in telomerase-negative cancers, which constitute approximately 5-10% of all cancers. It is also implicated in many genomic rearrangements and in the mitotic DNA synthesis occurring at under-replicated genomic loci. Two fundamental questions about BIR will be addressed in this application: How does lagging strand synthesis proceed during BIR? Second, what mechanisms restrict efficient BIR to subtelomeric regions? Understanding the constraints that limit BIR to areas near telomeres is vital, as BIR is a highly mutagenic pathway prone to template switching. 3. Repair of DSBs can be associated with templated insertions, a type of genome rearrangement where a DNA segment is inserted into a DSB. These copy number variations (CNVs) are among the most common in cancer genomes, yet their formation mechanism remains unclear. Templated insertions can occur at programmed breaks at VDJ locus or at HO or CRISPR/Cas9 endonuclease-induced breaks and typically range from approximately 50 bp to 1 kb in size. Templated insertions are mediated by Ku-mediated non-homologous end joining (NHEJ). We have developed a simple and cost-effective amplicon sequencing-based assay, termed 'Break-Ins', which allows systematic studies of templated insertions. Using this assay, we screened for mutants with a high level of templated insertions. We propose to study in-depth a group of identified mutants that are closely related to human neurodegenerative diseases and cancer. The primary goal is to understand the cellular conditions that drive the formation of templated insertions. Additionally, using 'Break-Ins', we identified mutants and conditions that lead to elevated release of mitochondrial DNA (mtDNA) and its transfer to the nucleus. Our goal is to understand the mechanisms that restrain the release of mtDNA from mitochondria and its transfer to the nucleus.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY A group of eighteen NIH-funded investigators requests funding for the purchase of a BD FACSDiscoverTM S8 spectral and imaging cell sorter, for installation in the Cytometry and Cell Sorting Core at Baylor College of Medicine. These investigators use samples from a variety of species including humans, rats, and mice for their research in the fields of immunology, cancer biology, cardiovascular disease, cancer immunotherapy, virology, parasitology, and neuroscience. Besides the users from two different institutions listed in this application, the instrument will be accessible, through an existing memorandum of understanding, at internal cost to all investigators at Baylor College of Medicine and the Texas Medical Center. Baylor College of Medicine, Rice University, the University of Houston, the University of Texas Health Science Center at Houston, the University of Texas M.D. Anderson Cancer Center, the University of Texas Medical Branch at Galveston, and the Texas A&M Health Science Center - Institute of Biosciences and Technology.

NIH Research Projects · FY 2025 · 2025-08

PAR: 22-080 S10 Biomedical Research Support Shared Instrumentation Grants CAGT Shared Instrumentation Grant: IncuCyte SX5 Imaging System Project Summary/Abstract The development of cell and gene therapies to target cancer has revolutionized the approach to treat patients with blood cancers. However, this treatment has had little effect on the treatment of solid tumors which comprise about 90% of all cancers. Therefore, novel strategies to develop therapies must be considered and multiparametric evaluation of those products are critical to understand their functionality. Utilizing state-of-the-art technology such as live-cell image analysis in complex models will provide improved accuracy and consistent testing of current and future products that will translate to the treatment of patients. We aim to purchase a new IncuCyte SX5 with funds provided through the Shared Instrument Grant application that can be used in the Center for Cell and Gene Therapy (CAGT). The IncuCyte is a live cell imaging system that allows easy quantification of cell morphology and behavior over time. The SX5 provides up to five color fluorescent imaging with the capability of three-color multiplexing compared to the current two-color fluorescence with the S3 model. This instrument improves productivity by allowing automatic acquisition and analysis for up to six culture plates or vessels in parallel. Analyses include the visualization and quantification of cell proliferation, tumor and immune cell interactions, cytotoxicity, cell type specific metabolic activity, and three-dimensional tumor spheroid assays. The SX5 increases the flexibility of experimental parameters and reagents when evaluating complex therapies developed by investigators at CAGT. Novel evaluations with the IncuCyte SX5 will prove to be invaluable to assess the functionality of cell and gene therapy products for clinical translation.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY RNA helicases play essential roles in post-transcriptional gene regulation and have been implicated in immune regulation. However, the role of RNA helicases in hematopoietic stem cell (HSC) function and immune cell fate specification remains poorly understood. Recently, we discovered that the RNA helicase DDX6 sustains leukemia by orchestrating the translational repression and sequestration of mRNAs in RNA condensates known as P-bodies. Whether DDX6 is also crucial for HSC function and immune homeostasis remains unknown. To address this gap, we generated a novel transgenic mouse model to study the loss of DDX6 in HSCs. Preliminary data suggest that DDX6 regulates HSC quiescence and function. Specifically, the loss of DDX6 leads to extramedullary hematopoiesis and induces HSCs to exit quiescence, increase metabolic activity, and undergo cell division. These changes led to the expansion of HSCs in vivo under steady-state conditions but caused their premature exhaustion during competitive transplantation. Mechanistically, our initial analysis of DDX6-bound transcripts within P-bodies of hematopoietic progenitors revealed enrichment of untranslated mRNAs encoding critical regulators of quiescence exit. While these insights are significant, the mechanisms by which DDX6 enables HSCs to maintain longevity and function under regenerative and infectious stress remain unclear. Additionally, how DDX6 regulates the translational landscape of HSCs is yet to be fully understood. To address these questions, this application proposes two complementary aims. In SPECIFIC AIM 1, we will use novel genetic mouse models to delete DDX6 in vivo in a cell- and time-specific manner to rigorously dissect the roles of DDX6-mediated RNA sequestration in HSCs following regenerative stress and infection. In SPECIFIC AIM 2, we will explore how DDX6-mediated RNA sequestration in cytoplasmic condensates controls HSC function. We will characterize the molecular landscape of P-bodies in HSCs under regenerative and infectious stress and elucidate DDX6's widespread role in suppressing the translation of target mRNAs using Ribo-ITP, a highly sensitive and quantitative method for single-cell and low-input ribosome profiling. Additionally, we will identify factors regulated by DDX6 and examine their functional roles in HSC function. Collectively, our study will advance the understanding of RNA helicases and their underlying mechanisms in immune homeostasis, paving the way for innovative therapeutic strategies targeting DDX6 and its downstream effectors to combat infectious diseases.

NIH Research Projects · FY 2025 · 2025-08

Project Summary The vaginal microbiota during pregnancy is an important reservoir for infant microbial seeding, but factors that impact the composition are complex and largely unknown. Vaginal presence of neonatal pathogens, like group B Streptococcus and extraintestinal pathogenic Escherichia coli, are associated with increased risk for adverse infant outcomes. Current antibiotic-based therapeutics are not optimal given the rise in antibiotic resistant bacterial strains, off-target microbial alterations, and potential long-lasting adverse outcomes for the neonate. Thus, it is critical to identify factors that impact the vaginal microbiota during pregnancy to identify alternative methods of limiting colonization and transmission of pathogens. Human milk oligosaccharides (HMOs) may be one such factor that can impact the vaginal microenvironment during pregnancy. HMOs are the third most abundant solid component in human breast milk, with over 100 unique HMOs identified thus far. HMOs provide health benefits to breastfed infants, like promoting the growth of beneficial microbes and limiting adhesion and invasion of pathogens in the gastrointestinal tract. HMOs are present in maternal circulation as early as gestational week 10 and the presence of specific HMOs are correlated with composition of the vaginal microbiota. This suggests there is a broader impact of HMOs in the mother that has not been explored. Our central hypothesis is that HMOs within the vaginal microenvironment act on both the microbial community and on the host to impact pathogen colonization and immune homeostasis. This hypothesis will be tested in the following specific aims: 1) identify utilization of HMOs by vaginal bacteria and the impact of HMO supplementation on vaginal microbial communities, and 2) investigate the potential of exogenous HMOs to confer a health-associated state on the vaginal microenvironment. We will use a combination of vaginal swabs from pregnant human donors, mini-bioreactor arrays, human vaginal epithelial organoids, and gravid humanized microbiota mice to explore the interplay between HMOs, vaginal bacteria, and the host response during pregnancy. This proposal will provide the applicant with training in organoids as a model system, animal models of pregnancy, and microbiome analyses, while expanding on the applicant’s previous background in immunology. Training will take place at Baylor College of Medicine under the mentorship of leading experts in the human microbiome and host-microbe interactions. Ultimately, this proposal seeks to identify if HMOs impact the vaginal microenvironment during pregnancy and assess their potential as a novel therapeutic alternative to antibiotics to limit pathogen colonization during pregnancy.