Washington University

NIH Research Projects · FY 2025 · 2024-03

ABSTRACT Sensory neurons convey information about external and internal environment to the brain. Proper function of sensory neurons is regulated by different types of glial cells, and alterations in glial functions have emerged as a major contributing factor in many neurodevelopmental disorders. While Schwann cells surrounding sensory axons are very well studied, much less is known about satellite glial cells (SGCs), which completely envelop sensory neuron soma in various sensory ganglia, including spiral ganglia mediating transduction of sound. Spiral ganglia neurons in the cochlea receive auditory information from hair cells in the ear and convey it to the brain with high speed and precision. Small alterations in conduction velocity in these neurons have major effects on signal processing in the auditory circuits. Whether this peripheral auditory system is dysregulated by FMRP loss remains largely unknown. Sensory deficits, and particularly hypersensitivity to sound is one of the hallmarks of Fragile X syndrome (FXS) and other autism-spectrum disorders. Increasing evidence suggests that sensory hypersensitivity in FXS leads to behavioral alterations such as anxiety and impaired social interactions. The defects have been thus far largely attributed to sensory processing abnormalities in brain circuits. However, core sensory and cognitive deficits may arise from an earlier abnormality in sensory inputs that drive subsequent abnormal development of cortical circuits. Yet, the mechanisms of sensory deficits in FXS remain poorly understood and no targeted treatments are available. In response to this challenge, we began to define potential deficits in spiral ganglia neurons in Fmr1 KO mice, the FXS mouse model. At the ultrastructural level, we observed altered association of spiral ganglia neurons with their enveloping SGCs. We found that genes related to neural development and myelination are dysregulated in Fmr1 KO spiral ganglia. We also observed abnormal number of immune cells in Fmr1 KO spiral ganglia. Our initial observations point to potential deficits in the peripheral auditory system in FXS. The goal of this proposal is thus to unravel the contribution of SGCs to sensory hypersensitivity caused by FMRP loss. To achieve this goal, we will first determine if and how cell type proportions and the transcriptome of cells in spiral ganglia are affected by loss of FMRP using single-cell RNAseq approaches at two critical developmental stages, postnatal day 5 (P5, pre-hearing), P15 (post-hearing), and P28. We will also perform electron microscopy analyses of spiral ganglia at the same stages to define the developmental timeline by which SGCs envelop and myelinate spiral ganglia neuron soma in normal conditions and to identify when abnormalities caused by FMRP loss arise during development. Finally, we will determine if absence of FMRP causes alterations in the excitability of auditory neurons. These experiments will unravel potential defects in peripheral auditory system in Fmr1 KO mice and provide the foundation for future in depth analyses and hypothesis-driven approaches to ameliorate sensory hypersensitivity in FXS.

NIH Research Projects · FY 2025 · 2024-03

PROJECT SUMMARY While pulse oximetry is a standard of care that guides management of critically ill patients, there is growing evidence that racial bias in pulse oximeter accuracy may overestimate oxygenation in hypoxic patients with pigmented skin and cause harm by impeding escalation of respiratory support. Pulse oximeters noninvasively estimate blood oxygenation (SpO2) and peripheral perfusion (perfusion index, PI) by detecting tissue absorption of red and infrared LEDs. SpO2 is based on calibration via the modulation ratio R, the ratio of pulsatile and non- pulsatile absorption from one LED divided by the ratio from the second LED, regressed against arterial blood oxygenation (SaO2). Oxy- (HbO2) and deoxy-hemoglobin (Hb) have distinct absorption at these wavelengths and because, unlike melanin, they exhibit pulsatile variation, it was assumed that SpO2 estimates based on the pulsatile signal would be pigmentation-independent. Alarmingly this assumption appears to be invalid as recent work has found that oximeters are more likely to provide falsely normoxemic SpO2 in hypoxemic patients if they are darkly pigmented. Differences in melanin-related absorption and scattering, particularly at red wavelengths, are thought to underly pigmentation-related errors in estimates of oxygenation, and likely also impact estimates of perfusion. However, studies demonstrating pigmentation-related device error have not controlled for other confounding physiologic factors that may impact pulse oximetry, and it is therefore not known if health disparities faced disproportionately by people with pigmented skin may independently drive pulse oximeter errors. Overcoming pigmentation-related pulse oximeter bias and decoupling it from other sources of oximeter error is critically important to ensuring that darkly pigmented patients, who already face inequities in the health care system, are not denied life-saving care due to devices that fail to meet the needs of a diverse population. To solve these problems, we propose (i) utilizing novel experimental systems that allow pigmentation to be varied while other physiologic parameters remain constant, and (ii) shift pulse oximeter wavelengths from red (with high melanin absorption and scattering) to short-wave infrared (SWIR, 900-1700 nm) where melanin absorption and scattering is minimized. Successful completion of the proposed studies will ensure that life-saving pulse oximeter technology serves the needs of patients across the full spectrum of pigmentation and no longer fails an already marginalized community.

NIH Research Projects · FY 2025 · 2024-03

Huntington’s disease (HD) is a devastating and invariably fatal neurodegenerative disease caused by an abnormal expansion of polyglutamine repeat in the protein called huntingtin (Htt). HD is characterized by progressive loss of selective neurons in the striatum and cortex, but the precise mechanisms underlying neuronal dysfunction and death are not fully understood, and no disease-modifying treatment is currently available. Thus, there is an urgent need to identify critical therapeutic targets and establish effective neuroprotective treatments for HD. One of the major challenges in developing effective treatments lies in the complexity of the brain and our incomplete mechanistic understanding of the disease, The brain comprises diverse cell types, each with distinct vulnerabilities in HD, which complicates treatment approaches. Understanding the individual responses of these brain cell types to the treatment would be crucial for maximizing the effectiveness of the treatment. Transcriptional dysregulation is one of the early molecular abnormalities found in the course of HD and is thought to play a central role in disease pathogenesis. Accumulating evidence suggests that genome-wide perturbations of DNA methylation may drive alterations in gene expression in HD. The fundamental objective of this proposal is to determine the effect of a DNA demethylating agent on gene expression, behavior, and pathology of HD in vivo using two different mouse models. The molecular impact of the treatment on individual cell types in the brain, including neurons and glial cells, in particular, in the striatum, the most vulnerable brain region in HD, will be determined. The underlying hypothesis is that treatment with the hypomethylating agent protects HD brain by inhibiting specific DNA methylation-mediated transcriptional alterations critical for neuronal function and survival, thereby attenuating disease progression. Importantly, therapeutic manipulation of DNA methylation has not been clinically used for neurodegenerative diseases, including HD. To test our hypothesis, the following specific aims will be pursued: 1) Determine the impact of a DNA methylation inhibitor on altered gene expression in individual cell types in HD mouse brains and 2) Determine the effectiveness of the DNA methylation inhibitor on behavior and pathology of HD mice. Given that both neurons and glia contribute to HD pathogenesis, it is crucial to understand the transcriptional responses of individual brain cell types to the treatment. The impact of the demethylating agent in multiple cell types in the striatum of HD model mice will be determined by innovative single-nucleus RNA-seq analysis. If successful, the proposed study will lay the foundation for the development of a new class of disease-modifying treatment for HD. It will also provide a mechanistic understanding of neuroprotection in vivo by this manipulation.

NIH Research Projects · FY 2026 · 2024-02

A. Project Summary This goal of this project is to investigate neuroinflammation in Parkinson disease (PD) using positron emission tomography (PET) with a sphingosine 1-phosphate receptor 1 (S1PR1) specific radiotracer [11C]CS1P1. Neuroinflammation is a key element in the pathogenesis and progression of PD, thus quantitative measures of neuroinflammation provide key evidence for pathogenesis, identification of therapeutic targets and quantification of target engagement in PD. S1PR1 is the most abundant receptor in the S1P receptor 5 member family. In the central nervous system (CNS), S1PR1 is expressed in neurons and glial cells, including astrocytes, microglia, and oligodendrocytes. Dysregulation of S1PR1 signaling plays a critical pathophysiological role in inflammatory diseases in the CNS. We reported that [11C]CS1P1, previously called [11C]TZ3321, specifically targets S1PR1 receptors with increased uptake at sites of inflammation in three animal models of inflammatory disease. We had received FDA approval of [11C]CS1P1 for human use (IND#:146548) and completed whole body dosimetry PET scans for 5 male and 5 female normal healthy participants, and brain PET scans in 10 MS patients; our results demonstrated that [11C]CS1P1 PET is able to detect neuroinflammation in MS. Macaques with a unilateral nigrostriatal brain injury, caused by intracarotid (ic) infusion of the neurotoxin MPTP, were used to directly compare brain uptake of [11C]CS1P1 with the widely used neuroinflammation tracer [11C]PBR28. Four macaques were scanned with each tracer under baseline conditions and following unilateral injury. Surprisingly, 6-8 weeks after MPTP-induced injury, both radiotracers had increased cortical brain uptake demonstrating a robust neuroinflammatory response that highly correlated with each other (r2 = 0.7-0.8). However, when compared with baseline imaging, the post-MPTP increase in ipsilateral frontal cortex compared to contralateral cortex for [11C]CS1P1 (89%) significantly exceeded that for [11C]PBR28 (12%). Therefore, we propose to validate the PET measures and systematically evaluate the ability of [11C]CS1P1 to quantitatively measure neuroinflammation after MPTP-induced injury in macaques. We hypothesize that PET with the S1PR1 radiotracer [11C]CS1P1 can quantify neuroinflammation in humans with PD. To test our hypothesis, we propose two specific aims. Aim-1# is to investigate regional S1PR1 expression in the brain of MPTP-treated NHPs by longitudinal PET imaging and terminal post-PET autoradiography, immunohistochemical (IHC) staining, immunofluorescence (IF) analysis, and ELISA studies of the brain tissues. Aim-2# is to investigate S1PR1 expression in the brains of PD patients and healthy controls through PET imaging as well as in vitro studies of banked postmortem tissue from PD cases and controls, including autoradiography, IHC staining, IF analysis and ELISA studies. We expect to demonstrate that PET with [11C]S1P1 radiotracer can quantify neuroinflammation in PD and other neurodegenerative diseases.

NIH Research Projects · FY 2025 · 2024-02

PROJECT SUMMARY Within the colon, early life bacterial colonization events favorably influence host health, but simultaneously establish a vast reservoir for antimicrobial resistance genes. Bacteroides fragilis represents up to 2.5% of the human gut microbiota and is often found in neonates within the first month of life. B. fragilis is the leading cause of anaerobic sepsis and deep tissue infections. While long recognized as an antimicrobial resistance threat, an increasing number of B. fragilis clinical isolates now express high-level resistance to b-lactam antibiotics including the carbapenems which are considered agents of last resort. Genetic analysis has revealed two distinct phylogenetic clades of B. fragilis, termed clade I and clade II. Clade II strains exclusively harbor the cfiA locus which encodes for metallo-b-lactamase (MBL) activity - an antibiotic resistance enzyme for which no available inhibitors have yet been defined. We have discovered a novel B. fragilis toxin that is specifically expressed in clade II isolates, termed Bcf1. Bcf1 appears to play a critical role in interbacterial competition between B. fragilis isolates, positioning this toxin to enable dominance of clade II strains within the B. fragilis niche, expanding the reservoir for MBL-encoded resistance. The primary goal of this proposal is to examine the molecular mechanisms by which Bcf1 facilitates interbacterial competition. Our preliminary data suggest that competition may rely on the ability of Bcf1 to limit nutrient access within the niche. We will explore this through a series of in vitro and in vivo studies utilizing engineered bacterial genetic variants to discover the protein(s) targeted by Bcf1 and evaluate the necessity of Bcf1 in inter-clade competition within the B. fragilis niche in a mouse model of colonic colonization. These studies will benefit from the use of a novel model of B. fragilis vertical transmission in which the temporal and genetic determinants of niche colonization and interbacterial competition by B. fragilis can be evaluated in young mice. Through a comprehensive dissection of the mechanism by which Bcf1 ensures the success of clade II B. fragilis strains, this study has the potential to illuminate a novel mechanism by which antibiotic resistance to the potent metallo-b-lactam antibiotics is achieved. By focusing on the ecological niche, these studies may discern critical early events in niche occupancy that are subject to perturbation, or alternatively highlight novel probiotic-based strategies by which to re-shape interbacterial competition events mediated by Bcf1 to selectively reduce the prevalence of clade I MBL-expressing strains within the colonic microbiome.

NIH Research Projects · FY 2026 · 2024-02

SUMMARY Mycobacterium tuberculosis (Mtb) causes one of the world's most deadly infections. How Mtb modulates the host immune response in order to establish infection, persist in the face of adaptive immunity, and elicit tissue pathology to transmit is not well understood. Mtb grows intracellularly in lipid-laden (foamy) macrophages and extracellularly within cholesterol-rich caseum of liquified granulomas. Mtb does not make cholesterol, but Mtb can degrade host cholesterol and use it as a carbon source. The host modifies cholesterol by enzymatically oxidizing it to a variety of derivatives, called oxysterols, which modulate the immune response. We found that two oxidized cholesterol metabolites, cholestenone and 3-oxocholestenoic acid, accumulate in Mtb-infected mouse lung, rabbit granulomas, and human sputum. Mtb has two enzymes that can oxidize cholesterol and oxysterols in this way: 3-hydroxysteroid dehydrogenase (Hsd/Rv1106c) and cholesterol oxidase (ChoD/Rv3409c). These enzymes can convert the hydroxy residue at the third carbon position of cholesterol to a keto moiety. We hypothesized that cholestenone and 3-oxocholestenoic acid might be specific markers of TB infection. Indeed, in two geographically distinct cohorts, the level of these metabolites in sputum distinguished subjects with active TB from TB-negative individuals who presented with TB-like symptoms. In addition, their abundance correlated with the degree of smear positivity, a rough estimate of bacterial burden. Why these metabolites are elevated during TB and the impact that they have on infection are unknown. We hypothesize that Mtb Hsd and ChoD disrupt the repertoire of immune active oxysterols by converting them from 3-hydroxy to 3-oxo-derivatives, thereby interfering with the host immune response. In support of this idea, we found that mice infected with hsd Mtb and choD Mtb have altered inflammatory responses in the lungs compared to mice infected with WT Mtb. Here, using two mouse models of TB and a highly sensitive, well-established metabolomics pipeline that is optimized for oxysterol analysis, we will comprehensively profile oxysterols in the lungs, establish the role of Hsd, ChoD, and host enzymes in the generation of the 3-oxo metabolites, and assess the impact of the Mtb-induced metabolites on the host inflammatory response and disease outcomes. In addition, we propose that the shift in oxysterol metabolites creates a unique signature of TB disease that can be used as a diagnostic and treatment biomarker. We will comprehensively profile oxysterols from TB patients in Asia, Africa, and South America and determine whether 3-oxo-modified oxysterols decline in TB patients when they are on effective therapy. Thus, in both mice and humans, we will establish the profile of immune active oxysterols in the lungs and how they are modified by TB. Our studies will have an important impact on the TB field by elucidating fundamental mechanisms of pathogenesis and by advancing biomarker development, which has the potential to substantially improve clinical care. More broadly, our work will provide insight to the role of oxysterols in lung inflammation and immunity.

NIH Research Projects · FY 2026 · 2024-02

To bring rigor and broad representation to research and practice of cancer screening and early detection, and to effectively participate in the NCI Cancer Screening Research Network (CSRN), we will build a CSRN ACCESS Hub at Siteman Cancer Center. Improving early detection is critical to reducing the growing burden of cancer and has the potential to save lives in all communities. For this to happen, we must perform research that reaches a range of geographic areas and settings, including rural and urban. Research conducted at the Siteman Catchment Hub will prioritize rigor and representation by collaborating with community-based healthcare systems in Missouri and Illinois. Led by an experienced multi-disciplinary team, our time-tested administrative structure engages participating sites, community partners, community-based clinicians, and institutional expertise. We draw on the deep clinical research infrastructure and support at Siteman Cancer Center and Washington University School of Medicine, and organized our Hub into four cores: Administrative, Participant Engagement, Study Protocol, and Data Quality. We are founded on intentional integration, engagement, and transparent communication. The specific aims of our Hub are: (1) Contribute to the scientific development of CSRN research including an initial Multi-Cancer Detection Vanguard study; (2) Recruit and retain participants to CSRN studies; and (3) Implement CSRN protocols as indicated by the Network. Building on our experience, we propose to enroll 2000 participants across our region into the Vanguard study. We will work closely with community partners to support recruitment and ensure protocols are accessible, acceptable, and of the highest scientific merit. The Siteman Catchment Hub has several notable strengths and innovations: 1) we serve and are committed to populations that reside in health professional shortage areas; 2) our multidisciplinary leadership team has deep experience with cancer screening research, clinical trials, and recruiting and retaining research participants; 3) we engage regional organizational partners and community partners in Hub leadership; 3) we have strong institutional support and infrastructure that will support the conduct of CSRN research, access to quality data, and rigor in our approach. We need sound evidence to inform advances in cancer screening and early detection. The Siteman Catchment CSRN Hub will deliver on that need and engage fully in the Network.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY Urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) is a costly clinical problem that affects millions of women worldwide. With a rise in antibiotic resistance among UPEC strains, there is a great need to develop alternative strategies for effective UTI treatment and prevention. This effort relies on understanding what factors modify UTI risk, and how. One of the strongest risk factors for UTI is frequent and/or recent sexual activity. It is likely that vaginal bacteria are commonly introduced to the bladder in sexually active women. Indeed, vaginal genera—most frequently Gardnerella or Lactobacillus—have been frequently isolated from urine collected directly from the bladder. Gardnerella is present at high levels in the vagina in women with the dysbiosis called bacterial vaginosis (BV). Women with BV are at increased risk for UTI and recurrent (r)UTI, but the mechanisms driving this BV-UTI link are unknown. Our new mouse model showed that when Gardnerella gains access to the urinary tract, it triggers apoptosis and exfoliation of the bladder epithelium and enhances UPEC UTI in multiple contexts. Gardnerella increased the level of acute bacteriuria and persistent bladder infection achieved by a subsequent UPEC UTI, and also increased the rate of rUTI by triggering UPEC emergence from quiescent intracellular reservoirs that were formed during a prior infection. Importantly, Gardnerella was rapidly cleared from the host, and was absent by the time UPEC UTI was enhanced. These data and suggest that vaginal bacteria influence UTI outcomes in women in ways that have not yet been appreciated because of the transient nature of their interaction with the bladder – a model we term “covert pathogenesis.” In this proposal we will use our innovative mouse models to specify mechanisms by which Gardnerella enhances UPEC UTI, determine whether this effect extends to other uropathogens (Aim 1), and to utilize genetically diverse Gardnerella strains to define the genetic features of Gardnerella strains that correlate with covert pathogenesis activity in the bladder (Aim 2). Finally, we will specify the association between Gardnerella abundance in the vagina, uroepithelial exfoliation, and rUTI in women, using multiple existing sample banks (Aim 3). This work will fundamentally change the way clinicians and scientists think about, treat, and prevent UTI. Our results could lead to the development of new screening strategies to identify women at risk for rUTI and new approaches to UTI treatment and prevention by targeting vaginal bacteria.

NIH Research Projects · FY 2026 · 2024-02

CANDIDATE: I am an Assistant Professor of Medicine in the Division of Pulmonary and Critical Care Medicine at Washington University in St. Louis School of Medicine (WUSM). My long-term goal is to become an independent, federally funded physician-scientist who applies rigorous implementation science and pragmatic, stakeholder-engaged approaches to improve outcomes for patients with asthma. To date, I have received support from the NIH Loan Repayment Program, institutional T32 and KL2 awards, and the American Lung Association. While this training has been foundational, additional mentored training in implementation science methods and real-world intervention design is essential to achieve independence. In this proposal, I am mentored by established leaders in implementation science and health services research, and together we have developed an integrated research and career development plan designed to position me for R01-level or equivalent funding by the end of the award period. PROPOSED ENVIRONMENT: This project is supported by three complementary environments. First, WUSM provides a strong clinical and research infrastructure, including access to EHR-based data systems, pragmatic trial support, and career development resources. Second, the Brown School at Washington University offers nationally recognized expertise in implementation science, where I will complete advanced coursework in implementation science methods during the K23 award period. Third, the study will be conducted within community health centers in the St. Louis Integrated Health Network, a real-world primary care setting serving patients with a high burden of asthma. RESEARCH ABSTRACT: National asthma guidelines recommend single maintenance and reliever therapy (SMART) for patients with moderate-to-severe asthma, based on evidence demonstrating a ~30% reduction in exacerbations. However, uptake of SMART in U.S. practice remains low, including <10% prescribing within our partnered network of community health centers in St. Louis. To address this evidence-to-practice gap, we have developed a multilevel implementation strategy to increase SMART adoption. This strategy includes: clinician education with practice facilitation, audit and feedback, and patient-centered electronic tools including SMART-specific asthma action plans. In this type 1 hybrid stepped-wedge cluster randomized trial, we will: (1) evaluate the effectiveness of the implementation strategy on SMART prescribing (Aim 1, 1° effectiveness outcome), and (2) assess implementation outcomes using the RE-AIM framework, with a focus on adoption (primary implementation outcome) through integrated quantitative and qualitative methods (Aim 2, 1° implementation outcome).

NIH Research Projects · FY 2026 · 2024-02

Project Summary/Abstract A key step in animal germ cell development is the switch in fate from stem/progenitor cells to entering meiotic prophase en route to gametogenesis. Disruption of this switch results in infertility and/or aneuploidy. Conserved features of the developmental transition include stem cell self-renewal followed by initiation of the meiotic program (e.g., homologous chromosome pairing, synapsis, double strand break formation) and repression of mitotic cell cycling. Multiple pathways function in control of the switch, resulting in genetic redundancy that has impeded the identification of key regulators. Furthermore, the regulatory process is heavily reliant on posttranscriptional control mechanisms. Studies of this switch in animals have progressed farthest in C. elegans. This is in part because the developmental transitions follow a convenient spatial continuum, allowing facile analysis of changes in cellular state (e.g. levels of regulators, changes in chromosome behavior) in wild type and mutant backgrounds. We have identified three parallel redundant pathways that control the switch in C. elegans. Yet, important questions remain. How is the switch spatially controlled? Here we focus on one of the pathways, the SCFPROM-1 pathway, an E3 ubiquitin ligase that functions in protein degradation. SCFPROM-1 induces meiotic entry and inhibits mitotic cell cycling, and its activity is controlled by PROM-1 proteins levels that rise from a base in stem cells to a peak at meiotic entry. Proposed studies of the spatial control will uncover how the activity and function of SCFPROM-1 is regulated and is coordinated with the other two known pathways, how meiotic entry is repressed in stem cells, and how mitotic cell cycling is repressed at meiotic entry. What are the missing regulators that execute the switch? The regulatory network contains significant genetic redundancy and there is evidence for missing regulators. Leveraging a novel in vivo genetic system we developed to synchronously switch stem cells to meiotic entry, we are employing genome-wide integrated transcriptomics, translatomics and proteomics, followed by genetic functional analysis to identify missing regulators. This combined genomics and genetically targeted functional analysis will overcome genetic redundancy in the developmental switch, which is difficult in less tractable organisms. Our overall goal is to complete the regulatory network and delineate processes governing an essential step in germline development, which will inform studies in other animals. Our investigations will uncover new developmental, cellular and molecular principles that are important for human health. This fundamental basic research in a genetic model organism will produce foundational knowledge for understanding cell fate switches, as well as the origin of birth defects and fertility.

NIH Research Projects · FY 2025 · 2024-02

ABSTRACT The body maintains an adequate balance between citrate availability and elimination, depending on physiological needs and determined by diet, renal clearance, cell metabolism and bone remodeling. Citrate is used by all aerobic organisms to produce usable chemical energy and is present in bone at strikingly high concentrations (1-5 wt%). In fact, two independent studies using high resolution NMR to model the citrate molecule within the apatite crystal suggest that the degree of citrate incorporation, as well as its spatial orientation within the mineral structure, is critical for maintaining favorable biomechanical properties. These observations prompt several fundamental questions that form the basis for this proposal: 1) What is the mechanism for citrate delivery to bone?; 2) How does the partitioning of citrate in bone influence global citrate handling?; and 3) how is this partitioning regulated? In preliminary studies, we demonstrate functional expression of a membranous extracellular Na+/citrate cotransporter, Solute Carrier Family 13 Member 5 (Slc13a5), in mineralizing osteoblasts. Interference of SLC13A5-mediated citrate transport, either genetically or pharmacologically, disrupts osteoblast mediated mineral nucleation. Mice lacking Slc13a5 show increased serum and urinary citrate levels, reduced bone volume and quality, and defects in tooth enamel, pathological features similar to those seen in humans with mutations in SLC13A5. Intriguingly, metabolic flux analysis revealed striking elevations in 13C-Glucose-derived 13C-Citrate (m+2) in apatite deposited by Slc13a5 null osteoblasts which was allocated to increased mitochondrial citrate production and export. Moreover, we found that Slc13a5 expression was strongly regulated by the calciotropic parathyroid hormone (PTH). These findings suggest the existence of an osteoblast specific mechanism that controls both the production and delivery of citrate to bone as well as systemic citrate availability. Specifically, we postulate that the membrane citrate transporter SLC13A5 senses extracellular citrate concentrations and enables the osteoblast to adjust its endogenous citrate production when extracellular citrate levels drop or in response to calcium regulating hormones such as PTH. Three aims were developed to assess our new metabolic pathway downstream of SLC13A5 in a human disease model using hiPSCs and primary teeth derived from patients with SLC13A5 disease and to define the role of SLC13A5 in citrate partitioning between blood and bone in physiological conditions or in response to PTH. As a young scientist, my ultimate goal is to conduct productive research that provides scientific insights into skeletal mineralization and the integration of these mechanisms in general physiology. My career development plan has been tailored toward this goal with solid mentorship, collaborations, and training opportunities. In conjunction with institutional support, I am confident that the studies/activities outlined in my application will help build upon my existing skillset and facilitate my transition into an independent investigator.

NIH Research Projects · FY 2026 · 2024-02

Project Summary Cardiovascular disease is the leading cause of death worldwide and despite efforts to preserve cardiac function in heart failure patients, preventing adverse remodeling and fibrosis remains a challenging task. Cardiac fibrosis can negatively impact cardiac compliance and function, and is associated with worsened outcomes in heart failure patients. Recent sequencing technology advancements hav e allowed for the identification of fibroblast activation protein (FAP) as a marker of pathologically activated fibroblasts present in failing hearts but not in healthy controls. This has raised the possibility of selectively depleting FAP+ cells for heart failure therapy. One proposed strategy is to use chimeric antigen receptor (CAR) T cells that are engineered to express a surface receptor directed against FAP. Initial studies utilizing these FAP CAR T cells in a mouse model of hypertensive cardiac injury have been promising, but the effects of CAR T cells on the myocardial environment are not understood. The proposed project aims to investigate the feasibility of T cell engaging therapies as a strategy of targeted cell depletion in cardiac fibrosis, and also investigate the modes of crosstalk between FAP CAR T cells and other cell populations in the heart. These questions have broad implications for this novel cardiac application of cellular immunotherapy. Preliminary data demonstrate that FAP CAR T cells can actually worsen the development of fibrosis in an angiotensin II/phenylephrine mouse model of cardiac injury. Importantly, expression of interferon gamma (IFN) and its targets is elevated in the hearts of FAP CAR T cell treated mice compared to those of control T cell treated mice. IFN has been shown to worsen fibrosis in other models of cardiac injury. We hypothesize that FAP CAR T cells can deplete activated fibroblasts, and that this has an anti-fibrotic effect, but it is offset by pro-inflammatory, pro-fibrotic crosstalk between FAP CAR T cells and other immune cells in the myocardium. Aim 1 of this proposal focuses on delineating the feasibility of T cell engaging therapies in cardiac fibrosis. Specifically, it compares FAP CAR T cells to other T cell-based therapies such as bispecific T cell engagers (BiTE®) in target cell depletion, IFN activation, and fibrosis modulation. Aim 2 studies how blocking IFN signaling in FAP CAR T cell treated mice affects the cellular and transcriptional landscape of the heart. It also investigates which cells are the sources of IFN, which cells are responding to this signal during FAP CAR T cell treatment, and what effects these signaling axes have on myocardial inflammation and fibrosis. Together these aims seek to understand the effects and mechanisms of FAP CAR T cell cross talk with other myocardial immune populations, and can potentially inform the field of strategies to refine T cell engaging therapies to maximize efficacy and improve safety in the cardiac setting.

NIH Research Projects · FY 2025 · 2024-02

PROJECT SUMMARY / ABSTRACT Tic disorders including Tourette syndrome (TS) are very common, and often impair quality of life. Many TS patients find symptoms improve over adolescence, but others do not. Very little information is available to inform individual prognosis in TS, and existing studies cannot dissociate behavioral or brain features preceding tics from those that may be caused by prolonged ticcing. A unique data set allows us now to examine these questions prospectively in a large sample of children not selected for the presence or absence of tics. Over 300 children participating in the Preschool Depression Study (PDS), half with and half without some depressive symptoms in preschool, had clinical assessments and behavioral tasks over a 15-year period, along with 5 waves of MRI of the brain. Video recordings were made during and between various tasks at each of 8 visits covering ages 3-19. Our preliminary data (expert review of video from 52 participants) shows that we can detect tics from these recordings, and we calculate that almost half of the children in the PDS will show tics at some visit in this study. We will examine the video from each study visit to identify the presence and severity of any tics. We will also invite all the study participants, now young adults age 21-25, to complete a study visit to diagnose any current or past tics . We will use these data to identify clinical features prior to the onset of tics in children who show tics on video at later visits (Aim 1a). Such a pre-tic follow-up study has never been performed, and may identify novel tic-related clinical / behavioral features that, because they precede tics, cannot be caused by ticcing. In children with tics, we will also identify clinical or MRI features when children first manifest tics that predict outcome at later visits; we will validate these results in extant data from two independent longitudinal studies of tics (Aim 1b). The results of Aim 1 may prove clinically useful for prognosis, and may provide valuable new insights into etiology, pathophysiology, and potentially secondary prevention of tic disorders. A second aim will identify clinical and brain imaging factors that may relate to tic pathophysiology, either (within subjects) by comparing changes over time in these factors as tic severity changes over time, or (between groups) by comparing children with vs. those without tics, controlling for age. This project will leverage existing data funded by NIMH, along with new identification of tics on video from 2,000 study visits, in order to clarify pathophysiology and work towards scientifically- based novel treatments. The investigators are experienced in TS research and child psychopathology, and are ideally suited to bring this study to a successful conclusion.

NIH Research Projects · FY 2025 · 2024-02

PROJECT SUMMARY/ABSTRACT Rotavirus (RV) is the leading cause of viral gastroenteritis in children under the age of 5 globally. Despite the introduction of several WHO-approved vaccines, RV infections still cause 114 million diarrhea episodes, 24 million outpatient visits, and approximately 215,000 deaths annually, and therefore necessitates further study. Aside from the typical gastroenteritis, around 75% of infected children have viremia and 90% have antigenemia, and RV detection in the sera is often associated with high fever. Furthermore, various reports indicate the presence of RV RNA or antigen in extraintestinal tissues, including the hepatobiliary system and pancreas, which have been linked to clinical manifestations such as biliary atresia and type 1 diabetes mellitus. The molecular mechanisms by which RV disseminates to systemic organs from the small intestine are not clear. Studies with reovirus, a closely related non-pathogenic human enteric virus, show that the virus can bypass the intact epithelial barrier to establish intestinal infection and spread systemically in mouse models by exploiting the transcytotic nature of microfold (M) cells. M cells are a subset of specialized intestinal epithelial cells that sample and transport content from the lumen to antigen-presenting cells at the basolateral side of the epithelium. Various enteric pathogens exploit M cells to gain access to host systemic sites. RV virions have been visualized in vivo within porcine M cells and human RV was recently shown by our lab to be transcytosed in vitro by human M cells in an ileal enteroid system. However, it is not known whether RV utilizes M cells for intra- and/or extra- intestinal infection in vivo. Additionally, the endocytic pathways responsible for M cell uptake of pathogens like RV have not been defined. The overall hypothesis of this proposed work is that RV is transcytosed by M cells in a caveolin- dependent manner and that this process is key to RV intestinal infection, systemic dissemination, and infection of extraintestinal organs. In Aim 1, the extraintestinal tissues and cell types targeted by RV will be determined following oral inoculation. Next, Villin-Cre Tnfrsf11aflox/flox mice that lack intestinal M cells will be utilized to study the role of M cells in intestinal infection and extraintestinal dissemination. In Aim 2, CRISPR- Cas9 mediated knockout will be used to dissect the endocytic pathway(s) involved in RV transport by M cells in human ileal enteroids. Conceptually, this study will define the mechanisms by which RV, a major childhood enteric pathogen, can disseminate systemically and resolve the longstanding question of how M cells transcytose substrates, paving the path for rational design of M cell-targeted mucosal vaccines for improved immunogenicity and antigen delivery.

NIH Research Projects · FY 2025 · 2024-02

PROJECT SUMMARY/ABSTRACT Laboratory mice exhibit underdeveloped immune systems which fail to accurately predict human responses to challenges such as vaccination. Intentional exposure of these mice to environmental microbes (creating “dirty” mice) through various approaches has been shown to confer effective maturation of the immune system, but generally requires specialized facilities which are not widely available to the research community. Rapid sequential infection with six murine viral pathogens, beginning in early life to mimic human viral exposures, is sufficient to drive immune maturation and yields mice available for subsequent challenge at 10 weeks of age. Viral pathogens selected include those frequently identified in and transferred from other “dirty” mouse models. This rapid sequential infection regimen dramatically alters circulating immune cells as well as total serum antibody and cytokine levels, and critically, also diminishes specific serum antibody responses to both intramuscular and intranasal SARS-CoV-2 vaccination. Intriguingly, the regimen also stimulates robust changes in the endogenous bacterial microbiota. Thus, this sequential infection model mimics phenotypes observed in other microbially-experienced mice and in human adults, and it can be readily implemented in standard animal facilities without special containment protocols. In the studies proposed here, this sequential infection regimen will be further refined and simplified, and the role of microbiota changes in immune maturation in this model will be explored. In Aim 1, the sufficiency and necessity of combinations of and individual viral exposures to stimulate immune maturation will be defined. Antibody responses to systemic and mucosal SARS-CoV-2 vaccination, as well as robust immunophenotypes observed in the sequential infection model, will serve as key assays. In Aim 2, the effects of sequential infections on the bacterial, viral and fungal microbiome will be defined, and the functional contributions of the endogenous microbiota to immune maturation will be tested. Microbiome profiling, germ-free mice, and antibiotic treatment with fecal transplants will be used to elucidate microbiota contributions. These studies will use established approaches supported by extensive published and preliminary data to reveal the contributions of individual viral pathogen exposures as well as endogenous microbial communities to immune maturation following the rapid sequential infection regimen. Completion of this proposal will yield a simplified “dirty” mouse model that is tractable and affordable for the majority of investigators studying infectious diseases, vaccine responses, or immune outcomes. Additionally, the proposed experiments will address an important open question about the relative importance of the endogenous microbiota versus specific viral pathogen exposures in immune maturation of “dirty” mice. Finally, they will serve to provide novel insights into immune responses directed against mucosal vaccines, which may have predictive value as this vaccination approach is increasingly implemented in humans.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY / ABSTRACT Ebola virus (EBOV), part of the Filoviridae family, causes periodic outbreaks of severe hemorrhagic fever and Ebola virus disease, with case fatality rates as high as 90%. These are highly virulent and emerging zoonotic pathogens, thus a significant threat to human health, potential agents of bioterrorism, and NIAID category A priority pathogens. While a set of EVD clinical symptoms have been defined, the molecular mechanisms that drive EBOV pathogenesis remain poorly understood. Despite current development of a vaccine, anti-filovirus prophylactics and therapeutics are very limited, in part due to the paucity of information on host and viral interactions. EBOV encodes for multifunctional proteins, including VP35. VP35 is a polymerase co-factor necessary for both viral replication and transcription and serves as the main innate immune antagonist. Currently, there is a major gap in our understanding of the role host factors play at critical stages in the viral replication cycle. Our group recently conducted interactome screens for various EBOV proteins to identify key host-viral interactions, which identified a novel interaction between a RING-type E3 ubiquitin ligase and Ebola VP35. In the work proposed here, I will use biochemical and structural biology tools to define the impact of ubiquitination on VP35 function (Aim 1). In Aim 2, I will assess the impact of VP35 on the host ubiquitination machinery, with a specific focus on type I interferon response. Ubiquitination serves powerful regulatory roles in eukaryotes, and there is increasing evidence that viruses can antagonize the immune response by interfering with host ubiquitin- dependent pathways, or by hijacking the host ubiquitination machinery to promote viral pathogenesis. Prior literature and my preliminary data indicate that EBOV proteins interact with the host ubiquitination machinery. I expect my proposed work on VP35 to determine the molecular mechanism of how this interaction impacts host- viral dynamics. This work will also provide an experimental framework to define mechanisms by which other viral proteins interact with the host ubiquitin machinery. My long-term career goal is to establish myself as a physician scientist with expertise to address questions about mechanisms that drive microbial pathogenesis, and use this knowledge to improve patient care. With the proposed studies, I will work towards this goal by uncovering and characterizing macromolecular interactions at the host-pathogen interface. I will also enhance my experimental skills, advance my scientific communication, and expand my teaching and mentorship skills. To accomplish this, I have designed a training plan with an interdisciplinary team of mentors, including my sponsor Dr. Gaya Amarasinghe with a track record of using biochemical and structural biology approaches to define key mechanisms in viral pathogenesis. On my advising committee I also have a biochemist with expertise in post- translational modifications (Dr. Natalie Neimi), an innate immunologist (Dr. Deborah Lenschow), a virologist and cell biologist (Dr. Siyuan Ding), and the chair of my committee is Dr. Jennifer Philips, an infectious disease physician scientist who will lend her expertise in host-pathogen biology as well as career guidance.

NIH Research Projects · FY 2026 · 2024-02

PROJECT ABSTRACT Advanced studies in cardiovascular research innovation are increasingly requiring multi-disciplinary studies that include mathematical, computation, and engineering principles. Likewise, translation of novel discoveries requires clinical knowledge, and a solid foundation in the understanding of vascular pathophysiology. The future in cardiovascular research innovation will depend on successful partnerships between problem solvers comprised of clinician-investigators and engineers. The fundamental premise of this training program is that early engagement of post-graduate surgical trainees and pre-graduate engineering doctoral students will help accelerate development of technology and solutions for the diagnosis, treatment, and prevention of cardiovascular diseases. Research topics will include cardiac arrhythmia, coronary artery disease, peripheral vascular (arterial/venous) disease, aortic aneurysmal disease, and neurovascular disease. With a highly diverse and accomplished faculty mentorship network, high-level of institutional support, and already well- established multi-disciplinary research between surgery and engineering at Washington University in St. Louis, we propose a training program that will develop common synergistic skillsets in translational research and engineering towards solving common and difficult problems in this field. Graduates of this novel training program will have the knowledge and expertise to pursue either an academic or research-related career in the diagnosis, management, and/or prevention of cardiovascular diseases.

NIH Research Projects · FY 2026 · 2024-02

Project Summary/Abstract Neuroinflammation, where immune cell overactivation can lead to catastrophic destruction of otherwise healthy neuronal tissue, is a critical pathological feature of both Alzheimer’s Disease (AD) and Amyotrophic Lateral Sclerosis (ALS). Dissecting the pathways leading to pathological neuroinflammation and defining precise immunomodulatory targets has the potential to greatly enhance therapeutic options for those suffering from neurodegenerative diseases. 97% of ALS patients and 57% of AD patients present with dysregulation of the RNA-binding protein TDP-43 in neurons, leading to disrupted RNA splicing of a variety of critical genes. Our bioinformatic analysis has identified 23 genes commonly dysregulated by TDP-43 pathology in multiple independent human studies; among these is STMN2, which we have characterized as a regulator of NMJ function and stability, and ATP8A2, which has never been studied in the context of AD or ALS, yet is an extremely attractive target for studying neuroinflammation. ATP8A2 is a phosphatidylserine (PS) flippase that regulates PS exposure onto the extracellular leaflet of the plasma membrane, where it acts as the cellular “eat me” signal for immune cell activation and phagocytosis. Aberrantly exposed PS can occur via flippase dysfunction, which is sufficient to cause neuronal phagocytosis by neuroimmune cells. Humans with ATP8A2 mutations have severe neurological dysfunction and ATP8A2 mutant mice develop axon degeneration, implicating ATP8A2 as a primary regulator of neuroinflammation and axon loss, downstream of impaired TDP-43. Excitingly, we have found that ATP8A2/STMN2 double heterozygote mice display a progressive decrease in motor strength that is significantly different from either single heterozygote, demonstrating the utility of this model for investigating TDP-43 pathology. This project will determine the genetic and cellular regulators of PS exposure and neuroimmune cell activation, and test the degree to which ATP8A2 deficiency exacerbates the neurodegenerative phenotypes (e.g. STMN2-mediated NMJ defects) observed in TDP-43 pathology. If successful, this project will characterize the fundamental mechanisms of neuron/immune cell interactions, and define potential therapeutic targets against neuroinflammation in AD or ALS. This project will occur at Washington University in St. Louis, a premier institute for biomedical research, under the guidance of Aaron DiAntonio, who has become a leader in the field of axon degeneration over the past two decades. The DiAntonio Lab’s record of success accentuates the resources and equipment available to perform outstanding science, including two state-of-the-art tissue culture rooms, multiple automated cell imagers and confocal microscopes, and access to a world-class mouse facility. Additionally, the extraordinary neuroscience and immunology communities at Washington University provide excellent opportunities to facilitate the growth and transition from trainee to independent investigator, which will occur through the classes, lectures, journal clubs, retreats, and symposia offered by the institution.

NIH Research Projects · FY 2026 · 2024-02

Project Summary Kidney inflammation contributes to chronic kidney damage, fibrosis and end-stage kidney diseases. Adaptive immune responses are tightly regulated in kidney to prevent excessive inflammation and to maintain self- tolerance. However, infections and medications can break self-tolerance and trigger autoimmunity. Immunotherapy has become a standard therapy for many cancers, but it can cause autoimmune kidney adverse events, including acute kidney injury and glomerulonephritis. Acute interstitial nephritis is the most common immunotherapy-associated acute kidney injury, characterized by immune cell infiltration in kidney. Our group showed it affects 2-3% of the patients receiving immunotherapy, resulting in significantly increased mortality and risk of progression to chronic kidney disease. While immunotherapy-associated acute kidney injury responds to corticosteroids, non-specific immunosuppression also blunts the tumor immune response. Thus, understanding the mechanisms of immunotherapy-associated acute kidney injury and developing targeted therapies that uncouple tumor- and kidney-immune response is critical to improve patient outcomes. Our overall goal is to identify antigenic and immunological drivers of immunotherapy-associated kidney- immune response. Kidney-associated antigens have been identified as targets of autoimmune kidney diseases, such as in membranous nephropathy and podocytopathies. Our data support that antigen-specific T cells also play a critical role in immunotherapy-associated acute kidney injury. Immune cell phenotype and tissue microenvironment are key determinants of inflammation: abundance of effector T cells among kidney- infiltrating lymphocytes is associated with severe kidney inflammation, whereas regulatory T cells (Treg) are associated with tolerance. Inflammatory tissue microenvironment and chemokines are key drivers of lymphocyte trafficking to kidney. However, detailed mechanisms to control effector T cells, Treg and kidney tissue microenvironment in immunotherapy-associated acute kidney injury is not well understood. This project aims to understand the roles of antigen-specific effector T cells (Aim 1), regulatory T cells (Aim 2) and kidney tissue microenvironment (Aim 3) in immunotherapy-associated acute kidney injury, using our novel mouse model, which expresses a series of model antigens in a kidney tubule-specific manner. This unique model will provide an ideal platform to delineate the phenotype and function of antigen-specific T cells in immunotherapy- associated acute kidney injury. By using novel, innovative approaches to study antigen-specific immune response in immunotherapy-associated acute kidney injury, we will better understand the mechanisms of immunotherapy-associated acute kidney injury, and will identify novel therapeutic targets for immunotherapy- associated acute kidney injury. This project will ultimately advance our fundamental knowledge of kidney immunobiology and bring new insights into the mechanisms of other forms of autoimmune kidney diseases.

NIH Research Projects · FY 2026 · 2024-02

Alzheimer’s disease (AD) and primary tauopathies are disorders in which the aggregation and accumulation of tau plays a key role in pathogenesis. In AD, tau aggregation and accumulation begins in the medial temporal lobe and extends into several neocortical regions just as cognitive decline begins and its progressive accumulation significantly correlates with neuronal/synaptic loss, regional brain atrophy, and neurodegeneration. Importantly, the regional presence and progression of brain atrophy and functional decline in both primary tauopathies and in AD highly correlates with tau accumulation. We have produced mice that develop tau pathology and tau-mediated neurodegeneration that are crossed onto a human APOE knockin (KI) background (P301S tau transgenic mice that express human APOE isoforms). We found that tau-mediated neurodegeneration is dependent on the presence of APOE with APOE4 resulting in greater damage than other APOE isoforms. The tau-linked neurodegeneration also requires microglia. While the tau-associated damage is dependent on the brain’s innate immune response via microglia, little is known about the extent and role of the adaptive immune responses in the presence of amyloid-β (Aβ) or tau pathology. Importantly, the contribution of the adaptive immune response to tau-mediated neurodegeneration has been unclear. We recently compared the immunological milieus in the brain of mice with amyloid deposition or tau aggregation and neurodegeneration. We found that mice with tauopathy but not amyloid, developed a unique innate and adaptive immune response and that depletion of microglia or T cells strongly attenuated tau-mediated neurodegeneration. Both CD4 and CD8 T cells were markedly increased in areas with tau pathology in P301S mice and in the AD brain. T cell numbers correlated with the extent of neuronal loss, and dynamically transformed their cellular characteristics from activated to exhausted states along with unique TCR clonal expansion. Inhibition of molecules either secreted by T cells (IFN-γ) or present on the surface of some T cells (PD-1) both significantly ameliorated brain atrophy. Our results have revealed a tauopathy and neurodegeneration-related immune hub involving activated microglia and T cell responses. Given these data, we hypothesize that understanding the role and interactions of T cells, microglia, and antigen presenting cells in tau-mediated neurodegeneration will provide novel insights into the tau-mediated pathogenesis as well as provide new therapeutic targets for preventing neurodegeneration in AD and primary tauopathies. This hypothesis will be tested in 3 Aims. Aim 1 will investigate the mechanisms underlying T cell activation and role of effector functions in microglia and T cells in tau-dependent neurodegeneration. Aim 2 will investigate potential antigen T cell receptor interactions that are relevant to T cell activation in P301S Tau Tg mice. Aim 3 will test whether different classes of clinically-approved immunosuppressant drugs that affect T cells can reduce tau pathology and neurodegeneration.

NIH Research Projects · FY 2025 · 2024-01

ABSTRACT Gastrointestinal (GI) disease is a significant global problem, causing 3 million hospitalizations in the US alone. Conventional antimicrobial treatment of GI infections present challenges such as selection of antibiotic resistance and off-target interactions with commensal microbes. GI diseases are uniquely challenging to treat with protein- based therapies because they often cannot withstand the harsh and rapidly changing environment of the GI tract, which includes a rich collection of proteases. I hypothesize that an engineered probiotic, Saccharomyces cerevisiae var. boulardii (Sb), can deliver anti-infective, protein-based therapies to the GI tract to treat the key enteric pathogens Clostridioides difficile (Cd) and enterotoxigenic Bacteroides fragilis (ETBF). Next-generation therapies targeting Cd and ETBF, including antimicrobial peptides (AMPs), toxin-neutralizing binding domains, and anti-virulence proteins are in development and are amenable to Sb-based expression and secretion. Sb maintains viability throughout the mammalian GI tract and can be modified with sophisticated genetic engineering tools developed for the model yeast Saccharomyces cerevisiae. Herein, I propose to expand upon a genetic toolbox I have begun establishing to achieve finely tuned expression and secretion capacity from Sb in the con- text of the mammalian GI environment and use this toolbox to modulate the expression of a panel of anti-infective therapeutics targeted to either Cd or ETBF. I will thoroughly characterize a panel of Sb strains carrying these therapies in terms of efficacy, tolerability, and impact on the host microbiome in the context of enteric infection. The rationale for this proposal stems from i) the need for improved and controlled delivery strategies for anti- infective protein therapies to treat GI infections, and ii) the extensive potential for Sb, which has inherent anti- pathogen qualities, to be genetically modified for production and delivery of a large variety of therapeutic pay- loads. My central motivation is to demonstrate the capacity for engineered Sb to eradicate the clinically significant gut pathogens Cd and ETBF through therapeutic delivery systems that are both tunable and combinatorial. This will be achieved through two aims: 1) define and expand the therapeutic capacity of Sb specifically in the mam- malian gut environment by building a genetic toolbox of secretion constructs with varying secretion capacities of a panel of anti-Cd and anti-ETBF therapies with distinct mechanisms of action, and 2) demonstrate that engi- neered Sb is capable of selectively targeting the Cd and ETBF in murine models of infection. These experiments are significant as they generate novel treatment regimens for enteric infections, including against Cd which has significant morbidity and mortality worldwide, and for ETBF which has no clinically approved, targeted therapy available. This proposal is innovative as it will develop Sb as a carrier of anti-Cd and anti-ETBF AMPs for the first time and extensively define the impact of this therapeutic system on the host microbiota as well as the target pathogen. Finally, this proposal is impactful as it strives to develop a safe, non-invasive, and targeted treatment for major GI infections, and can be readily adapted to treat other maladies of the GI tract.

NIH Research Projects · FY 2025 · 2024-01

Many adolescents and young adults (AYA, 14-24 years old) in Nigeria have a substantial HIV risk but do not receive HIV prevention services. While pre-exposure prophylaxis (PrEP), HIV self-testing, sexually transmitted infection (STI) testing, and linkage to AYA-friendly clinical services are essential, they have not been widely implemented or sustained in Nigeria. This underlines the importance of implementation science to increase the adoption and maintenance of HIV prevention services for AYA in diverse settings. Our 4 Youth by Youth (4YBY) program (NICHD UH3HD096929) leveraged participatory implementation strategies like crowdsourcing open calls and designathons and participatory learning communities to develop AYA-led PrEP uptake, HIV self-testing, and STI testing services in Nigeria. The success of this NICHD-funded Prevention and Treatment through a Comprehensive Care Continuum for HIV-affected Adolescents in Resource Constrained Settings Implementation (PATC3H) pilot program facilitated transition to an NICHD U-supported randomized controlled trial underway in 32 local government areas. Interim data demonstrates that the 4YBY program is effective, but how to transition this research study into practice and sustain it overtime for AYA in Nigeria requires further investigation. As a multi-disciplinary research team with proven track record, we propose the following specific aims: (1) To use participatory approaches with Nigerian youth (i.e. crowdsourcing designathons and participatory learning communities) to develop new strategies for sustaining the 4YBY program; (2) To evaluate the effectiveness of a finalist sustainment strategy to sustain core elements of the 4YBY program among at-risk, HIV-negative AYA (14-24 years old) in Nigeria using a cluster randomized control trial in 32 new local areas; (3) To estimate the impact and cost-effectiveness of the overall 4YBY program. We define sustainment based on the continuation of core elements of the 4YBY program at sufficient fidelity, the continuation of intended benefits, and adequate capacity for the continuation of the program. The Youth Participatory Action Research framework and the PEN-3 cultural model will inform our study. Implementation science aspects will be guided by the Proctor’s Implementation Outcomes Framework and the Consolidated Framework for Implementation Research (CFIR). We will also estimate the cost of implementing and sustaining the 4YBY program. We are currently implementing 4YBY in 32 local areas among a general population of AYA for 24 months. Within this cohort, we will integrate the final sustainment strategy and collect data on sustainment over time. In addition, we will recruit 32 new local areas with higher risk (PrEP-eligible) AYA that will either receive the same sustainment strategy alongside 4YBY (16 areas) or a standard 4YBY program (16 areas). Findings from the proposed S-ITEST (Sustaining Innovative Tools to Expand HIV Self- testing) study will be relevant in other settings. S-ITEST responds to high-priority topics identified by the United States National Institutes of Health, NICHD, NIMH, FIC, and OBSSR.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY/ABSTRACT Human astroviruses (HAstVs) are a global cause of pediatric gastroenteritis, and can cause disseminated infection in immunocompromised hosts. Seroprevalence studies indicate almost universal HAstV infection during childhood. Despite their clinical importance, HAstVs are highly understudied. Little is known about the host factors needed for infection or their mechanisms of viral entry into cells, and no proteinaceous viral receptor has been identified to date. Beyond providing critical insights into virus-host interactions, identification of host proteins sufficient and/or necessary for HAstV infection is key for creating small animal models permissive to infection and to revealing antiviral strategies to combat infection. Leveraging both genome-wide knock-out CRISPR and surfaceome-specific CRISPR activation screening, novel proteinaceous host receptors for HAstV were identified. These factors, neonatal Fc receptor (FcRn) and dipeptidyl peptidase 4 (DPP4), have also been implicated in viral infection for echovirus and coronavirus infection respectively. Both were confirmed as necessary for in vitro HAstV infection of Caco2 cells and as sufficient to permit HAstV infection of normally non-permissive human cells when overexpressed. Additionally, use of biolayer interferometry confirmed direct physical interactions between both FcRn and DPP4 with the HAstV capsid. These preliminary data serve as the critical foundation for the extensive structural and functional studies proposed here. In Aim 1, the specific protein domains and amino acid residues of FcRn and DPP4 that functionally interact with the HAstV capsid will be identified, as will the viral capsid residues critical for binding these host factors. Cryogenic electron microscopy will be leveraged to reveal how binding to these factors affects the HAstV virion. These genetic and biophysical analyses will provide much-needed insight into HAstV entry biology and form the basis of future pharmacological screens for identification of entry inhibitors. In Aim 2, permissive human intestinal enteroid cultures will be used to determine the necessity of identified entry factors for ex vivo HAstV infection. Blocking of infection via addition of soluble entry factors, anti-factor antibodies, inhibitors, and CRISPR-mediated disruption of factors in these cultures will be performed. Additionally, existing mouse models expressing human FcRn and DPP4 will be used to develop mice permissive to HAstV infection and to evaluate whether these factors are critical for murine astrovirus infection. These studies will reveal the species-specificity of receptor use and provide powerful new tools for study of HAstV pathogenesis and immune response as well as for preclinical testing of vaccines and antivirals. The use of numerous orthogonal approaches, including mechanistic studies of critical receptor domains, state-of-the-art structural techniques, and physiologically-relevant models, will yield a comprehensive picture of how these novel entry factors promote HAstV infection.

NIH Research Projects · FY 2026 · 2024-01

Project Summary Neurofibromatosis type 1 (NF1) is a common neurogenetic cancer predisposition syndrome, affecting 1 in 3,000 individuals worldwide. While most of the tumors are benign neoplasms (neurofibromas, low-grade gliomas), 10-13% of patients will develop an aggressive sarcoma, termed a malignant peripheral nerve sheath tumor (MPNST). Composed of high-grade neoplastic Schwann cells, MPNST most often arise from a benign precursor lesion, such as plexiform neurofibroma (PN). Unfortunately, even with aggressive multi-modality therapy, these cancers recur in >50% of individuals, and most patients die within five years of diagnosis. For this reason, there is an unmet need for better therapeutic modalities. To identify novel targets, we generated a series of patient-derived xenografts (PDX) lines that more accurately reflect the molecular heterogeneity of human MPNSTs. Using these PDX lines, we showed that MPNST exhibit a high degree of aneuploidy and harbor gains involving the long arm of chromosome 8 (Chr8q). Detailed analysis of Chr8q genes revealed that UBR5 is the most highly upregulated gene in MPNST and that UBR5 genetic knockdown (KD) decreased MPNST proliferation, survival, and migration. Based on these exciting data, we hypothesize that UBR5 is a key driver of MPNST pathogenesis and in part responsible for Chr8 gain-mediated MPNST malignant progression. In this proposal, we will: (1) Define when in MPNST progression Chr8q gain occurs and if UBR5 expression through this molecular event correlates with worse overall survival. (2) Utilize a recently developed human induced pluripotent stem cell plexiform model and a murine Nf1 mutant plexiform neurofibroma-prone strain that develops MPNST following lentiviral manipulation of other genomic drivers to determine the sufficiency of UBR5 to promote MPNST formation. (3) Determine the mechanism of action of UBR5 in MPNST including how UBR5 regulates cell survival and whether its role is dependent on its E3 ubiquitin ligase activity. The experiments outlined in this proposal will provide a deeper understanding of the molecular pathogenesis of MPNST, which is required to uncover new opportunities for the development of novel therapeutic strategies that may improve clinical outcomes.

NIH Research Projects · FY 2023 · 2024-01

Gender dysphoria as a measure of proximal stress: Development and psychometric evaluation of a novel measure of social gender dysphoria. ABSTRACT The proposed research will focus on the development and psychometric evaluation of a new measure of social gender dysphoria and distress. Clinical/scientific understandings of gender dysphoria, and its measurement, have been conceptualized primarily through the incongruence of gender identity and assigned sex. Our developmental work has focused on transgender and nonbinary (TGNB) individuals’ critique of existing scales and their descriptions regarding their lived experience of gender dysphoria. Our findings suggest that scientific definitions are too narrowly defined, do not reflect the heterogeneity of experience within the community (Galupo & Pulice-Farrow, 2020), and fail to capture shifting and nonbinary aspects of gender dysphoria (Galupo, Pulice-Farrow, & Cusack, 2020; Galupo, Pulice-Farrow, & Pehl, 2021). We have also documented the ways that social stressors (e.g. discrimination and misgendering) elicit gender dysphoria, and have theorized that social dysphoria acts as a proximal stressor for physical and mental health disparities (Galupo, Pulice- Farrow, & Lindley, 2019). We have subsequently provided empirical support for gender dysphoria as a proximal stressor (Lindley & Galupo, 2020) and used this model to show that social dysphoria works with other proximal stressors to predict problematic substance use among TGNB individuals (Lindley, Bauerband, & Galupo, 2020). We propose to use this pilot work to develop and evaluate a novel measure of social gender dysphoria and distress. Our research team will work with both expert consultants and community panelists to generate and evaluate initial scale items (Aim 1), test and confirm structure, reliability, validity, and invariance of our novel measure of social gender dysphoria (Aim 2), and investigate the relation between social gender dysphoria and mental health outcomes (Aim 3). Our proposed measure of social gender dysphoria will allow a way to capture the role of gender dysphoria as a unique proximal stressor in models of minority stress for TGNB individuals. Our measure represents a critical theoretical and methodological innovation that will allow assessment of the role of gender dysphoria as it is elicited by discrimination and microaggressions (i.e., distal stressors) and is related to well established mental health disparities for TGNB individuals including depression, anxiety, substance use, and disordered eating. Such a measure will not only provide a critical tool for investigating mental health among TGNB individuals, but provide a clinical assessment tool of dysphoria- related distress.