Vanderbilt University Medical Center

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Neural crest progenitors are essential for development of peripheral ganglia in the autonomic nervous system. Much is known about processes that control differentiation of neural crest progenitors for many autonomic ganglia. However, very little is known about formation of cell lineages within pelvic ganglia. Regulatory processes that control neurogenesis, differentiation, diversification, and maturation of pelvic autonomic neurons are undefined. Pelvic ganglia play essential roles in initiating bladder contraction and mediating efficient emptying. Thus, studies of pelvic neurons will inform understanding of urinary tract conditions that affect a large portion of the population. In humans, pelvic autonomic neurons are scattered primarily in the inferior hypogastric plexus, while in rodents pelvic neurons are aggregated into major pelvic ganglion situated alongside the lower urinary tract (LUT). Our prior LUT studies in mice identified expression of the transcription factor Pax3 in fetal and postnatal pelvic ganglia when pelvic autonomic neurons are differentiating and maturing. Because Pax3 is widely expressed during development, mutations in this gene are typically lethal due to neural tube defects. We generated novel lines of mice that have loss of Pax3 in neural crest lineages. These animals are postnatal viable and exhibit deficits of bladder wall innervation with altered voiding patterns. Neural-crest restricted Pax3 mutants offer an exciting opportunity to identify key regulatory aspects of pelvic ganglia formation and determine how deficits of pelvic autonomic neurons relate to LUT dysfunction. In this study we focus on postnatal stages of pelvic ganglia maturation to test the following hypotheses: 1 – Pax3 is essential for producing the normal allocation of neuron types as the mouse major pelvic ganglion matures postnatally. 2 – neural crest-specific loss of Pax3 reduces total numbers of pelvic autonomic neurons at maturity. Use of single-cell sequencing and high-resolution large-scale microscopy will be applied to assess the final composition of pelvic ganglia in postnatal Pax3 mutants compared to normal littermates. Research outcomes will provide greater understanding of how alterations in pelvic innervation contribute to LUT dysfunction.

NIH Research Projects · FY 2024 · 2023-08

SUMMARY Significance: Urinary tract infection (UTI) is among the most prevalent urologic diseases, and it is caused primarily by uropathogenic Escherichia coli (UPEC). Bladder infection by UPEC is characterized by a transient intracellular stage during which bacteria invade superficial epithelial (facet) cells and divide within the cytosol to form multicellular communities called biofilms. After replicating in the cytosol, bacteria exit the intracellular biofilm – killing the bladder epithelial cell in the process – and disseminate to naïve facet cells or to the upper urinary tract. While in the intracellular biofilm state, bacteria evade innate immune responses and the effects of antibiotics. Similarly, in catheterized individuals, formation of biofilm on the catheter surface creates an additional protective niche for UPEC, from which it can disseminate to the bladder and seed infection. The goal of this proposal is to evaluate the potential of inhibiting biofilm by interfering with UPEC respiration. Rationale and Hypothesis: Although UPEC are facultative anaerobes, they respire oxygen during infection in the hypoxic bladder environment. Aerobic respiration and oxygen sensing have also been linked to the expression of critical UPEC virulence factors. We have previously shown that aerobic respiration is essential for UPEC to establish infection. Of the three respiratory quinol oxidases encoded by UPEC, cytochrome bd has the highest affinity for molecular oxygen, exceeding the affinity of mitochondrial cytochrome c by 1000-fold. Deletion of the cydABX genes that code for cytochrome bd, does not impart a growth defect in vitro, but leads to significant alterations in UPEC biofilm architecture, leading to higher susceptibility to antibiotics in the biofilm state. Furthermore, cydABX deletion mutants are non-motile, exhibit decreased proton motive force (pmf) and are attenuated in a murine UTI model. Finally, deletion of cydABX results in increased expression of the low affinity quinol oxidase cytochrome b0. We hypothesize that cytochrome bd can be chemically targeted to thwart biofilm formation or dissemination from a pre-formed biofilm. We further posit that cytochrome bd has a role in energizing motility. We propose two aims to test the posed hypotheses: Aims: Aim 1 will will evaluate whether targeting of cytochrome bd using known cytochrome bd inhibitors can enhance antibiotic effectiveness. Aim 2 will determine whether the decreased membrane potential of the cytochrome bd mutant imparts generalized or specific effects on pmf-dependent processes and determine how these impaired processes affect motility. Finally, using chemical inhibition of cytochrome bd we will evaluate whether loss of cytochrome bd function impairs dissemination from the biofilm. Impact: These studies will be the first to address the unique contribution of cytochrome bd on UPEC motility and will determine whether targeting bacterial respiration is a viable therapeutic or prevention strategy against UTI.

NIH Research Projects · FY 2026 · 2023-08

SUMMARY Genetic mutations or environmental insults that impair development of neural circuit connectivity can lead to autism spectrum disorder (ASD) and schizophrenia, which together affect the quality of life, independence, and productivity of millions of Americans and cost hundreds of billions of dollars annually. ASD and schizophrenia are associated with an increased ratio of excitatory-to-inhibitory (E/I) synaptic transmission, raising the possibility that drugs that are capable of restoring E/I balance could treat both disorders. GABAergic parvalbumin- expressing fast-spiking interneurons (PV-INs) play critical roles in regulating inhibitory output in striatal networks and coordinating high-frequency oscillations underlying cognition, sensory information processing, motor behavior, and behavior, which are frequently disrupted in ASD and schizophrenia. The ability of PV-INs to fire high-frequency action potentials (APs) is dependent on the expression of the voltage-gated potassium (K+) channel Kv3.1, whose expression is largely restricted to PV-INs. De novo mutations in Kv3.1 are associated with ASD in humans. A growing body of genetic, mathematical modeling, and pharmacological evidence strongly suggests that small molecule potentiators/activators of Kv3.1 channel gating could promote PV-IN firing, inhibitory output, and E/I balance. However, the dearth of potent and specific Kv3.1 channel potentiators with suitable drug metabolism and pharmacokinetic (DMPK) properties has slowed efforts to critically evaluate the therapeutic potential of Kv3.1 in treating ASD and schizophrenia. Here, we propose to employ a molecular target- based drug discovery approach to develop 2-3 state-of-the-art Kv3.1 channel potentiators and then use them in a mouse model of ASD to evaluate their ability to restore PV-IN excitability. In Aim 1, we will employ a fully developed and validated fluorescence-based high-throughput screening (HTS) assay to interrogate approximately 100,000 compounds from the Vanderbilt Institute of Chemical Biology library for novel Kv3.1 potentiators. Fluorescence and automated patch clamp electrophysiology assays will be used to identify potent, selective, and chemically tractable compounds for further development. In Aim 2, an iterative cycle of medicinal chemistry and functional assays will be used to optimize the potency, selectivity, and in vitro DMPK properties of novel Kv3.1 potentiators. The studies outlined in Aim 3 will employ mouse brain slice electrophysiology to evaluate the ability of optimized Kv3.1 potentiators restore PV-IN excitability in the nucleus accumbens and pre- frontal cortex. We will specifically characterize the effects newly developed Kv3.1 potentiators on genetically identified PV-IN current-voltage relationships, AP waveform, and firing frequency. This high-risk/high-reward proposal will create unprecedented opportunities for pharmacologically modulating PV-IN excitability and inhibitory output, critically evaluating the therapeutic value of Kv3.1 for modulating E/I balance, and potentially impacting the clinical treatment of ASD and schizophrenia.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Tumor metastasis requires supportive microenvironment for outgrowth of disseminated tumor cells. The switch of metastatic cells from dormant to proliferative state needs nutrients for energetics and biomass production. Although metabolic regulation of tumor metastasis is not well- established, recent studies demonstrated the importance of fatty acid (FA) uptake and metabolism in tumor cells that drives metastatic tumor outgrowth. However, it is unclear how FA pools in the tumor microenvironment is regulated. The vascular system plays a crucial role in supplying nutrients. This application investigates how vascular endothelial cells regulate FA transportation and utilization in metastatic tumor outgrowth. We discovered that loss of Raptor/mTORC1, but not Rictor/mTORC2, in vascular endothelium inhibits metastatic tumor outgrowth in lung. Raptor/mTORC1 deficiency led to reduced long chain fatty acid (LCFA) and polyunsaturated fatty acid (PUFA) in endothelial cells (EC), decreased expression of membrane proteins that mediate FA transport, reduced FA uptake and transport across endothelium, and decreased lipid droplets in metastatic tumors. Based on these preliminary data, we propose a model in which mTORC1 activities in vascular endothelial cells stimulate transendothelial FA transport, leading to enhanced FA utilization in tumor cells and metastatic outgrowth. To test this hypothesis, we will investigate downstream mechanisms by which vascular mTORC1 regulates transport of fatty acids across the endothelium (Aim 1), upper-stream factors that stimulate the FA transport (Aim 2), and determine if selectively blocking endothelial mTORC1 will suppress metastatic outgrowth and improve chemo- and immunotherapy against metastasis (Aim 3). The proposed work is innovative in its concept that mTORC1 regulates tumor blood vessel transportation of fatty acids. It also utilizes several state-of-the-art technologies, including a metabolomics screen, MALDI-imaging mass spectrometry for lipid/FA detection on tumor section in situ, a breast cancer PDX model, and several new conditional mouse models. These studies would set the stage for future development of strategies selectively targeting endothelial mTORC1 or fatty acid transportation to improve anti-cancer therapy.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT There is a profound need to improve venous thromboembolism [VTE] treatment and prevention, particularly in patients with inflammatory bowel disease [IBD]. This Pathway to Independence Award application is submitted by a pharmacoepidemiologist committed to improving VTE prevention and treatment in populations who are often excluded or vastly under-represented in randomized clinical trials [RCTs]. Of the 3 million Americans affected by IBD, ~1–7% will develop VTE, a significant source of morbidity and mortality, in both the outpatient and inpatient settings. While attention has been paid to the role of anticoagulants for VTE prevention in IBD patients, there are key barriers to prescribing anticoagulants in this population including: (1) absence of data on the effectiveness of anticoagulants for the prevention of VTE in IBD patients; (2) fear of bleeding complications; and (3) lack of evidence on the safety of anticoagulants in high-risk subgroups such as patients with active disease flares or postpartum IBD patients, who are at an increased risk of adverse complications. The overarching goal of this research is to evaluate the use, effectiveness, and safety of anticoagulants in IBD patients in the inpatient and outpatient settings. The applicant will achieve the proposed aims of this K99/R00 award under the guidance of established researchers who span the disciplines of epidemiology, biostatistics, gastroenterology, and hematology at the University of Pennsylvania. First, the applicant will use a combination of machine learning and advanced methodological techniques to examine predictors of use, effectiveness, and safety of anticoagulants in the inpatient setting (K99 phase). In the R00 phase, the applicant will use linked electronic health records/claims database to examine the comparative effectiveness and safety of direct oral anticoagulants, warfarin, and heparins in IBD patients in the outpatient setting. The research proposed in this K99/R00 is critical since it provides timely needed information on the risks and benefits of anticoagulants in IBD patients, data that will not be forthcoming from large RCTs in the foreseeable future. The proposed career development and training goals will provide the applicant with training in predictive modeling, time-varying adjustment, and machine learning. The successful completion of the proposed training, infrastructure, and institutional support at the University of Pennsylvania will guarantee the applicant's success and transition to scientific independence.

NIH Research Projects · FY 2025 · 2023-07

BACKGROUND: Invasive lobular carcinoma (ILC) accounts for approximately 10-15% of all breast cancers and is characterized by hormone receptor positivity (HR+), lack of HER2 amplification, low Ki-67 score, and loss of cell adhesion receptors such as e-cadherin. While these tumors tend to be indolent initially, late recurrences and distant metastasis are common. Effective systemic treatments that can improve both surgical and long-term outcomes for these patients are needed. ILC has very low rates of pathologic complete response (pCR) to both neoadjuvant chemotherapy (3%) and neoadjuvant endocrine therapies (ET, 0%). HER2 mutations are enriched in about 5-10% of unselected ILC, with some reports showing the prevalence as high as 27% in pleomorphic and higher grade ILCs. HER2 mutations in ILC are associated with antiestrogen resistance and a worse prognosis. Neratinib, an FDA-approved adjuvant treatment for HER2-amplified (HER2+) early-stage breast cancer, has shown safety and efficacy in combination with ET in metastatic HER2-mutant HR+ breast cancer. Our hypothesis is that the combination of neratinib and ET will improve clinical and molecular activity in HER2-mutant HR+ ILC, representing a novel and rational neoadjuvant therapy option. TRIAL DESIGN: We propose an unblinded, multi-institutional Phase II clinical trial for 30 patients with Stage I- III ILCs with HER2 mutations. Patients will be initially randomized to either 4 weeks of neratinib + ET OR ET alone; a biopsy will be done at the end of the lead-in phase. All patients then will receive a combination of 20 weeks of ET and neratinib prior to proceeding to surgery. KEY ENDPOINTS: The primary objective of this study is to determine the pre-operative endocrine prognostic index (PEPI) score on the surgical sample in these patients. The secondary clinical objectives will be to evaluate the pCR rate, residual cancer burden (RCB) index, and rates of breast conserving surgery. In addition, we propose correlative studies that will promote understanding of how neratinib synergizes with antiestrogen therapies by evaluating tumor cell cycle arrest and apoptosis markers in the pre-treatment and on-treatment (4 week) biopsy in both the ET ± neratinib arms. Finally, we will study the longitudinal evolution of therapy tolerance and resistance in HER2-mutated breast cancer. We will develop patient-derived organoids and perform single- cell RNA and ATAC sequencing and DNA sequencing to elucidate transcriptional and epigenetic programs that permit breast cancer survival upon continuous HER2-directed therapy and somatic alterations that drive resistance. We hypothesize that the combination of neratinib and ET will improve clinical and molecular activity in HER2- mutant HR+ ILC, addressing an unmet need for this challenging to treat breast cancer subtype.

NIH Research Projects · FY 2024 · 2023-07

Project Summary Several studies have found that infection with SARS-CoV-2 and COVID-19 diagnosis are associated with the development and progression of both Type 1 (T1D) and Type 2 Diabetes (T2D), possibly through infection of beta cells, increased insulin resistance, increased inflammation and fibrosis, and other biological processes. The proposed study will take advantage of robust existing infrastructure to rapidly identify, recruit, and retain diverse cohorts of English and Spanish speaking pediatric and adult patients with recently diagnosed T1D or T2D. The study will include 1600 study participants diagnosed with diabetes in the last 3 months, who have had a known COVID-19 infection in the past 90 days and those with recent diagnosis of diabetes and no known COVID-19 infection in the past year. The study will leverage PCORnet, a unique national network of over 60 health systems with electronic health record (EHR) data on over 80 million patients and a track record for successful study recruitment. We will query EHR records to swiftly identify potential study subjects with recent diagnosis of diabetes and contact them via patient portals, telephone, face-to-face encounters, and other approaches. We will also leverage the T1D Exchange (T1DX), a national network of 54 diabetes centers and an online patient registry of 17,000 individuals with T1D. Consented participants will partake in regular web/mobile or telephone surveys leveraging a previously developed REDCap/Twilio platform. Participants will also come to sites for regular serological testing, and a subsample will participate in more robust testing of glucose tolerance, biomarkers, and vascular function. This data will be supplemented by longitudinal EHR data from participating sites and across PCORnet. Participants will be followed for 2 years. Aim 1 will examine if patients with recent T2D who have recent COVID-19 are more likely to have worse glycemic control, increased inflammation and increased insulin resistance than patients without recent COVID-19. Aim 2 will examine if patients with recent T1D who have recent COVID-19 are more likely to have worse glycemic control, increased inflammation and more rapid reduction in beta cell function than patients without recent COVID-19. Aim 3 will evaluate a subset of patients with diabetes to examine if COVID-19 is associated with worse vascular function, increased inflammation and hypercoagulability. Aim 4 will explore the role of genomic/social/environmental factors on inflammation and metabolic function. Aim 5 will leverage EHR data to explore the role of COVID-19 and COVID-19 treatments on diabetes development and diabetes-related outcomes across the pandemic. The study will be led by a team with significant experience related to COVID-19, post-acute sequelae of COVID-19 (PASC), obesity and diabetes in children and adults, epidemiological research, informatics, health services research, genomics, metabolomics, physiology, patient and family engagement and other areas. The proposed work will provide a deeper understanding of the relationship between COVID-19 and diabetes, that can support future interventions and public health approaches to improve health.

NIH Research Projects · FY 2025 · 2023-07

Project Summary / Abstract HIV remains a major public health concern in the United States. The proportions of people with HIV (PWH) in 2020 who knew their HIV status, were linked to care, retained in clinical care (50%), and had suppressed viral loads (57%), were lackluster. Retention in clinical care is a core quality-of-care indicator and the central stage of the HIV care continuum. Suboptimal clinical retention is strongly associated with virologic failure while on ART, high-risk behavior, and poorer survival. Furthermore, it is estimated that 43% of new HIV transmissions are from PWH who are out of care (the largest proportion from any one care continuum stage). The implication, echoed in multiple public health policies over the past decade, is that high retention and engagement in clinical care are critical for blunting the HIV-related morbidity and mortality and reducing the number of new HIV infections. However, despite consensus that “retention” in care is critical and “engagement” in care must be increased, we do not fully understand how best to measure retention and engagement among patients in high-income settings, particularly in the modern ART era and given changes in care delivery that were introduced during the COVID-19 pandemic. Earlier research indicated that sicker patients (i.e., those with lower CD4) were more likely to miss clinic visits. More recent work, though, found that patients attending clinic visits less frequently may continue to receive laboratory monitoring services; this could well be an indication that healthier patients (i.e., those with higher CD4) who are stably virally suppressed are compliant with newer monitoring guidelines which demand less frequent clinic visits. However, the field has yet to delineate the optimal frequency of clinic visits for these healthier, virally suppressed patients as opposed to individuals in multiple other risk strata, though applying a single metric regardless of sub-population could produce spurious findings of poor retention among clinically stable individuals. In this respect, our proposal is truly novel. The proposed research will therefore extract, harmonize, and analyze readily available data on clinical care patterns within the largest HIV cohort in North America: the North American AIDS Cohort Collaboration on Research and Design. We will use these data to describe patterns of retention and engagement (Aim 1), isolate multiple measures of care receipt (Aims 1 and 2) that predict improved survival and viral suppression, and assess multiple methods for stratifying populations while quantifying the expected causal impact of improved retention on HIV outcomes under existing and novel, optimized metrics (Aim 3). The public health impact of improved retention metrics, based on population-specific HIV clinical care engagement, would be profound, particularly in light of changes in HIV disease and comorbidity clinical management and laboratory monitoring under a primary care model, as well as changing care modalities during the COVID-19 pandemic. This proposal therefore holds unique promise, enabling improved HIV care continuum measurement in the US.

NIH Research Projects · FY 2024 · 2023-07

ABSTRACT Children tend to experience more frequent asymptomatic infections and milder illnesses associated with SARS-CoV-2 infections than adults, but the reasons for these age-associated differences are unclear. While typical patterns of respiratory syncytial virus, influenza, and other viruses were markedly disrupted during the early pandemic, human rhinoviruses (HRVs) remained prevalent. Unlike other respiratory viruses, HRVs, consisting of species A, B, and C, are detected frequently year-round, and HRV infection is often asymptomatic, especially in children, who frequently undergo frequent re-infections with new HRV strains. The immunological consequences of these frequent HRV reinfections are unclear. Studies have suggested a role for HRV infection in rendering individuals less susceptible to infection with heterologous respiratory viruses, including SARS-CoV-2, with recent studies reporting that infection with HRV may trigger interferon responses that block SARS-CoV-2 replication and reduce SARS-CoV-2 transmission. While insightful, many prior observations of HRV interference with SARS-CoV-2 or other viruses are derived from ecological studies or animal models of infection, in which the experimental conditions are closely controlled. Observations from individuals in real-world conditions with detailed information on the sequence of infections, specimen collection prior to development of symptoms, and clinical features of illness are scarce. We aim to overcome these limitations by leveraging an intensive case-based longitudinal SARS-CoV-2 household surveillance platform, with daily nasal sampling and symptom assessment for 14 days following a positive test of the index SARS-CoV-2 case, to test the innovative hypothesis that interactions between HRV and SARS-CoV-2 may reduce the risk of symptomatic SARS-CoV-2 infection, reduce clinical symptoms among symptomatic infected individuals, and reduce transmission of SARS-CoV-2 in humans. We further hypothesize that these interactions may be HRV species-specific. We propose to build upon supportive experimental and in vitro studies to evaluate the clinical relevance of HRV and SARS-CoV-2 interactions in real-world settings by studying two Specific Aims: 1) To test the hypothesis that prevalent HRV infections reduce susceptibility to SARS-CoV-2 infection among exposed household members, and 2) To test the hypothesis that prevalent HRV infection reduces the risk of symptomatic infection or reduces symptom intensity upon SARS-CoV-2 infection. By assessing the impact of HRV co-infection on the individual variability observed in SARS-CoV-2 susceptibility to infection and disease severity, findings from the proposed study may yield new insights into ARI pathogenesis and development of enhanced prevention strategies.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY / ABSTRACT Cardiovascular disease (CVD) kills 1 in 3 individuals and affects >2 in 3 individuals diagnosed with type 2 diabetes (T2D). Despite optimization of available therapies, CVD remains the leading cause of mortality in T2D, highlighting the considerable burden of residual risk. Achieving further reduction in CVD morbidity and mortality in people with T2D requires advancing promising candidate mediators of residual risk. The metabolite α-aminoadipic acid (2-AAA) predicts the development of both T2D and atherosclerosis, independent of other known risk factors. This may represent a novel independent risk mechanism for the development of CVD, particularly among individuals with T2D. Our overarching hypothesis is that 2-AAA is an independent mediator of CVD risk among individuals with T2D. In the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial, both intensive glucose-lowering therapy, and intensive lipid management failed to attenuate CVD risk in individuals with T2D, and indeed showed evidence of increased risk. We hypothesize this was, in part, due to residual risk factors, including 2-AAA. We propose an analysis of 2-AAA in existing plasma samples from N=1,757 participants of the ACCORD study lipid treatment arms, with the following aims: 1) Define the effects of lipid- and glucose-lowering therapies on plasma 2-AAA, and address whether plasma 2-AAA changes in response to lipid-targeted therapy or intensive glycemic management. 2) Address the hypothesis that plasma 2-AAA is a CVD risk mechanism among individuals who experienced events despite optimal therapy. Successful completion of the aims will determine whether 2-AAA levels are impacted by lipid and glycemic management in T2D and establish whether elevated 2-AAA associates with CVD risk. This will provide important information on the utility of 2-AAA as a biomarker of risk and plausibility as a novel therapeutic target, allowing us to refine specific hypotheses to be probed in future studies. These aims represent novel and important questions and use existing NHLBI-supported sample and data resources to add considerable scientific value and address a key knowledge gap.

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY Ion channels pass charged ions through lipid membranes in a regulated manner. Ion channels play major roles in regulating electrically excitable tissues, sensing and responding to the environment, and maintaining cell homeostasis. Over 70 ion channels have been linked to Mendelian “channelopathy” disorders, affecting a diverse set of organ systems. As genetic testing and genomic medicine become prominent, an important challenge is to understand the spectrum of which mutations in ion channel genes cause disease. Unfortunately, a large fraction of variants are currently annotated as “Variants of Uncertain Significance,” which limits the effectiveness and potential of genomic medicine. This proposal seeks to decipher which variants in channelopathy genes cause disease. We will first use large biobank datasets with linked genome sequencing and phenotype data. We will examine associations between genetic variants in 76 channelopathy genes and relevant disease phenotypes. Using control pathogenic variants, we will first determine which gene-phenotype pairs are associated in biobank datasets, then discover novel candidate disease-associated variants that are present in carriers with relevant disease phenotypes. Next, we will use high-throughput automated patch clamping to study hundreds of variants in ion channel genes. Our initial focus will be 5 key ion channel genes that span a range of ion types and organ systems, as well as selected variants from the biobank genetic analyses. Next, we will perform deep mutational scans (a comprehensive mutational study) of every mutation in selected ion channel genes, starting with GABRA1, a ligand- gated ion channel gene (receptor) involved in GABA sensing and linked to seizure disorders. We will generate all possible mutations with degenerate mutagenesis reactions, integrate the mutation library into cells, then measure each mutation's impact on cell surface trafficking and channel function using high-throughput sequencing. Finally, we will integrate these patient and in vitro functional datasets to learn fundamental features of ion channel biology and disease. Through an analysis of the 2D and 3D protein structures, we will decipher protein mutational hotspots. From an analysis of mutational impacts from homologous genes I will determine whether mutation information can be ported to homologous genes. Finally, we will integrate variant data into the American College of Medical Genetics classification framework to clinically reclassify variants. Overall, these experiments have great potential to help resolve the VUS problem for ion channels and decipher novel ion channel biology.

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY. Alzheimer’s disease and related dementias (ADRD) has become a major public health crisis. To date, therapeutic strategies for Alzheimer’s disease have been largely ineffective resulting in an increased focus on identifying effective prevention strategies. Daily 24-hour activity behaviors (sleep, physical activity, and sedentary time) may be modifiable risk factors for ADRD. A better understanding of these risk factors may provide an opportunity for early prevention, particularly in the asymptomatic phase prior to the development of ADRD-related pathology. The availability of longitudinal actigraphy data to objectively-measure 24-hour activity in a strongly ADRD-phenotyped cohort has created the ideal opportunity to investigate potential pathways linking 24-hour activity behaviors with ADRD. The objective of this K01 is to longitudinally investigate changes in 24-hour activity with changes in cognition, structural neuroimaging, and fluid biomarkers of Alzheimer’s disease and concomitant pathological pathways prior to the onset of clinical dementia. I will leverage data from the Vanderbilt Memory and Aging Project (VMAP) cohort with repeated measures of actigraphy, brain MRI, and fluid biomarkers of Alzheimer’s disease neuropathology and concomitant injury. The central hypothesis of this proposal is changes in 24-hour activity will be associated with cognitive decline, structural neuroimaging changes, and Alzheimer’s disease and concomitant pathway changes, preceding symptom onset. Based on this hypothesis, the proposal aims to 1) characterize associations between changes in 24-hour activity and cognitive decline, 2) examine changes in 24-hour activity in relation to MRI markers of neurodegeneration and small vessel disease, and 3) evaluate changes in 24-hour activity behaviors with fluid biomarkers of Alzheimer’s disease neuropathology and concomitant pathways. Taken together, these aims will combine objective measurement of 24-hour activity with cognitive, neuroimaging, and state of the art fluid biomarker data to fill existing gaps in the research. Simultaneously, throughout the award, the candidate will gain advanced training in 1) Alzheimer’s disease pathophysiology and epidemiology; 2) measurement and modeling of structural imaging and fluid biomarkers of Alzheimer’s neuropathology and concomitant pathways of injury; and 3) advanced statistical methods, positioning the candidate to become a leader in the fields of aging and Alzheimer’s disease epidemiology. Vanderbilt University Medical Center and the Vanderbilt Memory and Alzheimer’s Center provide the ideal environment to complete the proposed research and training activities. The overall goal of this K01 is to inform a series of R01 awards and independent research program focused on identifying and characterizing behavioral risk factors for ADRD. With the support of this K01 award and expert mentorship, the candidate will achieve this goal and successfully transition to independent investigator status by the conclusion of the training period.

NIH Research Projects · FY 2026 · 2023-07

While recruitment into clinical research has been a longstanding challenge for NIH funded multi-site studies, it has become clear that there is substantial variability in recruitment success across the national portfolio caused by many factors: eligibility parameters, required procedures, compensation levels, engagement practices, recruitment resources, skill and awareness of recruitment teams, geographic constraints, trust, and perceived benefit/risk. Studies that do not integrate these factors into recruitment and retention often close for poor accrual. In addition, there are now more equitable definitions of what constitutes “recruitment success” beyond an absolute target, including: diversity, representativeness of the actual population, costs of enrollment, retention, and time required. These complex dynamics suggest there is no ‘one size fits all’ solution, and careful attention and consideration must be a part of the recruitment plan. Our team has been forming effective recruitment collaborations for the past 6 years -- considering the study specifics, capabilities of the study team, and needs and values of the participant population -- to together craft feasible, effective plans. In the next cycle, we will Catalyze and Harmonize Operational Innovation for Recruitment (CHOIR) and will continue to be led by a long-standing synergistic partnership between Paul Harris, PhD, as PI responsible for informatics development, and Consuelo Wilkins, MD, MSCI, as PI of community and stakeholder engagement. Formal partnerships with 10 other CTSAs provide broad understanding of Hub needs, along with key areas of expertise. We will extend and build upon existing recruitment-related assets and data tools and resources already in use by our team and others (FasterTogether, ResearchMatch, REDCap TrialsToday, FHIR clinical data-based recruitment infrastructure). These innovations acknowledge recruitment is not a one-time activity but is a continuum. We will provide a national, disease agnostic home for sharing recruitment tools, training, materials, and best practices for diverse populations. Specific Aims are: 1) Partner with study teams to create study-specific recruitment plans, and support ongoing skills development. 2) Evaluate clinical trial recruitment and retention methods and make continual improvements. 3) Enhance national clinical trial awareness through engagement and education; facilitate participant identification of studies with online tools. And, 4) Develop and disseminate technical and procedural approaches to catalyze enrollment in clinical trials across all CTSAs.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Candidate: Paras Karmacharya, MD MS is a Clinical Instructor at Vanderbilt in the Divisions of Rheumatology & Immunology. He has a strong clinical background in rheumatology and an excellent scientific foundation in epidemiological studies from his Master’s in Clinical and Translational Science and post-doctoral research studies following clinical training. His long-term career plan is to become an independent clinical investigator and a leader in pursuing patient-centered, pragmatic research in real-world psoriatic arthritis (PsA) patients and advancing personalized medicine in PsA. To achieve this goal, he will utilize his proposed research project for hands-on training in advanced statistical methods, bioinformatics, and genetics. Research Project: This proposal will study the association of clinical and genetic heterogeneity in PsA with treatment response. Using two unique real-world PsA cohorts, Dr. Karmacharya will (1) Identify PsA phenotypes and their association with treatment response in PsA, and (2) Identify PsA patients with a high probability of responding to therapy and those with a low probability of responding to therapy in the EHR. These aims are foundational to designing future pragmatic trials leveraging the EHR to more efficiently and robustly develop personalized approaches to treatment. Career Development: Dr. Karmacharya’s career development plan integrates formal coursework with individual training from his mentors to (1) Enhance skills in advanced statistical methods, (2) Expand proficiency in applying biomedical informatics techniques to large databases through mentored hands-on training in Vanderbilt’s EHR, (3) Build foundational knowledge in genetics and through experiential learning to apply analytic techniques to a genetic biobank, and (4) Develop expertise in building and managing a prospective PsA cohort. Environment: VUMC is the ideal environment to foster Dr. Karmacharya’s development as a leader in clinical and translational research in PsA. His mentoring team includes experts in PsA (Ogdie/Ritchlin), advanced statistical methods (Byrne/Chen/Ogdie), biomedical informatics (Byrne/Barnado), and genetics (Cox). Additional institutional resources include the Synthetic Derivative (SD), a de-identified EHR with over 3.5 million subjects, and BioVU, a genetic biobank linked to the SD, and departmental support for his multicenter PsA cohort. His mentors, Drs. Crofford and Ogdie, are internationally recognized in rheumatology and PsA with successful track records of mentoring. With Vanderbilt’s institutional commitment to young investigators and expertise in biomedical informatics and genetics, Dr. Karmacharya will successfully leverage his innovative proposal for independent R01 funding.

NIH Research Projects · FY 2024 · 2023-07

Project Abstract Hypertension is a risk factor for stroke, heart attack, kidney disease and death. Hypertension prevalence is high in sub-Saharan Africa, specifically in persons living with HIV. Apart from traditional risk factors such as high body mass index, age, immune activation and lifestyle, dietary salt is one of the driving factors contributing to the development of hypertension directly by promoting pathological changes in the vasculature and indirectly through immune activation and inflammation. Salt intake is driven mainly by salt taste sensitivity and specific genetic variations in the taste receptor genes. High salt consumption is an independent predictor of hypertension, arterial stiffness and cardiovascular disease. Salt consumption is generally high in Low- and Middle-Income Countries including Zambia. The effects of salt on blood pressure (BP) are more pronounced in individuals with salt sensitivity of blood pressure (SSBP). SSBP is when changes in BP mirror changes in dietary salt intake/depletion. It is not clear if salt taste sensitivity correlates with SSBP. Furthermore, genetic variations in the epithelial sodium channel (ENaC) in the tongue associated with salt taste sensitivity are unknown. Therefore, the aims of this project are to: 1. To determine if salt taste sensitivity is associated with salt intake, SSBP and inflammation in persons with HIV. We hypothesize that salt taste sensitivity correlates with SSBP and inflammation. To achieve this, an existing cohort with known SSBP will be utilized and inflammatory biomarkers measured using ELISA and flow cytometry, 24-hr food recall and 24-hour urine will be measured to assess dietary salt intake. Salt taste sensitivity will be analysed using serial diluted salt solutions, and compared with salt intake, SSBP and inflammation between people with and without HIV. Aim 2. To determine if genetic variations in ENaC are associated with salt taste perception, SSBP and hypertension in HIV. We hypothesize that specific genetic variations in the taste receptor genes particularly for ENaC are associated with salt taste, SSBP and hypertension. To achieve this, genetic sequencing of taste receptor genes will be performed to determine linkage with salt taste sensitivity and SSBP in persons with and without HIV. These studies will generate hypotheses for future interventional studies. In addition, the long-term goal is to generate a biobank of saliva and blood samples of persons from Africa for future genomic, proteomic and metabolomic analysis in an R01 grant application.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Primary immunodeficiencies (PIDs) are a group of monogenic diseases caused by inborn errors of immunity (IEI) that confer risk of severe infection, autoimmunity, and cancer. The genetic and immunological study of IEI is instrumental to understand mechanisms of human immunity and to manage and treat patients with PIDs. Their study is also important to understand more common diseases associated with genetic variants in the same genes. However, despite the relevance and importance of studying PIDs from the basic science and clinical points of view, half of the PID patients lack a genetic diagnosis. By studying a patient with a PID of unknown genetic etiology that confers susceptibility to severe mycobacterial disease, we identified a nonsense mutation in SERPINB1. Using a combination of population genetics, biochemistry, and molecular biology, we showed that this is the first case of SERPINB1 complete deficiency ever described. SERPINB1 is an intracellular serine protease suicide inhibitor. Its absence in mice causes uncontrolled intracellular protease activity, leading to neutrophil death and subsequent neutropenia. Interestingly, our patient does not display any neutrophil abnormality, but instead, our in-depth immunological characterization showed a reduction in the frequency of activated T cells and CD4+ memory T cells. Our findings suggest that SERPINB1 has a previously unknown function in T cells and antimycobacterial immunity. Our data also indicates that SERPINB1 orchestrates an intracellular protease-mediated pathway essential for T cell activation and survival. Given our findings, this application will test the hypothesis that SERPINB1 is critical for human T cell activation and, by controlling intracellular protease activity, to human diseases. Capitalizing on the unique opportunity that this patient offers to understand the function of human SERPINB1, we propose three complementary approaches. In Aim 1, we will characterize the immunological consequences of SERPINB1 deficiency on T cell activation and CD4+ T cell differentiation. We will also study the transcriptional consequences of SERPINB1 deficiency in T cells. With this, we will uncover a previously unknown function of SERPINB1 in human T cells. In Aim 2, we will identify and characterize the pathways orchestrated by SERPINB1 in T cells. The absence of SERPINB1 unleashes the action of intracellular proteases. This rare mechanism of PID will allow for chemical inhibition of these proteases for therapeutic purposes. Furthermore, characterizing the SERPINB1-dependent antimycobacterial mechanisms will lead to the identification of additional IEI in its pathway in patients lacking a genetic diagnosis. In Aim 3, we will perform phenome-wide association studies (pheWAS) to identify associations between genetic variants in the SERPINB1 pathway and additional human diseases. This reverse genetic approach will expand what we will learn from the study of a rare disease to benefit a broader group of patients. Our proposed research has far- reaching clinical and biological implications for understanding SERPINB1 mediated immunity, PIDs, anti- mycobacterial immunity, and the contribution of the SERPINB1 pathway to human disease.

NIH Research Projects · FY 2025 · 2023-07

SUMMARY Zinc binding metalloproteins and metalloenzymes constitute approximately 10% of the vertebrate proteome and are essential for many cellular processes. Due to the critical importance of zinc to vertebrate physiology, zinc deficiency leads to growth defects, aberrant immune function, neurological disorders, cancers, and increased risk of infection. This is a pervasive public health problem considering that approximately half of the world's population suffers from dietary zinc deficiency, now the fifth most important risk factor for mortality in developing countries. Although the clinical link between Zn deficiency and infection is established, the molecular mechanisms are not well understood. Despite the known importance of zinc cofactors to cellular function, the mechanism by which zinc is inserted into metalloproteins is poorly understood. In this application we report our discovery of zinc-regulated GTPase metalloprotein activator 1 (Zng1) as the first reported zinc metallochaperone that inserts zinc into cognate client metalloproteins. We identify the protein processing enzyme methionine aminopeptidase 1 (Metap1) and the transcription factor zinc finger homeobox 3 (Zfhx3) as Zng1 clients, and we describe a conserved domain that mediates an interaction between Zng1 and these metalloproteins. This finding forms the basis of proposed structural, biochemical, and physiological experiments to define the mechanism of Zng1-dependent metal delivery to client proteins. Single nucleotide polymorphisms in Zng1 are associated with increased incidence of severe infections in humans, establishing an important link between Zng1 and infection in the zinc starved host. Zng1-dependent metal transfer increases the functional activities of Metap1 and Zfhx3, and loss of Zng1 activity in mice and zebrafish negatively impacts organismal development and increases susceptibility to multiple infectious diseases. Based on these findings, we propose a model whereby the Zng1 family of enzymes are evolutionarily conserved metallochaperones that transfer zinc to Metap1 and Zfhx3 during conditions of extreme zinc deficiency. We predict that this process of co-factor delivery leads to regulated protein processing through Metap1 and coordinated gene expression changes through Zfhx3. Combined, these activities help orchestrate the cellular response to infection during zinc starvation. Experiments described in this proposal will test this model and determine the molecular mechanism by which Zng1 transfers metal to Metap1 and Zfhx3, define the role of Zng1-client interactions in proteostasis and transcription, and reveal the in vivo contribution of Zng1 metal delivery to vertebrate defense against infection. Collectively, findings from this proposal will uncover the biological and pathophysiological relevance of this newly identified family of zinc metallochaperones and make important contributions to nutrition and infection biology.

NIH Research Projects · FY 2024 · 2023-07

In this Research Opportunity Announcement for Area of Interest 4, we describe the seminal work of establishing an infrastructure that operationalizes engagement of diverse research participants, distinct from recruitment/enrollment, and building on the foundational work of integrating research participants within the governance of research programs. Our approach and best practices are embedded into the Engagement Core’s five aims 1)Serve as the central hub for participant engagement – providing resources, expertise, and tools – to the All of Us Consortium for engaging participants as partners in the governance, priority setting, implementation, and evaluation of All of Us; 2) Effectively integrate participants, who reflect the broad diversity of All of Us, as partners in the governance, oversight, and evaluation of All of Us; 3) Capture and integrate the voices of diverse participants to enhance the design, implementation, and use of All of Us data, resources, and results; 4) Continuously assess the impact of participant engagement on all aspects and phases of All of Us including governance, design, implementation, and evaluation; and 5) Advance the science of engagement with an emphasis on engaging historically marginalized and minoritized groups. To date, our Engagement Core has established the infrastructure for participant engagement, by successfully integrating 81 participants (including 58 [72%] from minoritized and historically excluded communities into various All of Us governance roles including 12 members on the steering committee and 6 on the executive committee. Our Engagement Core has responded to 55 requests for engagement reflecting a range of activities including review of a mental health and well-being PPI survey and generating potential solutions to safely extend data access to non-traditional researchers in the All of Us Researcher Workbench. In the next phase, we will continue the multi-faceted strategies above and will expand our approaches to include voices currently missing from existing engagement such as young adults, and parents of children aged 18 and younger (critical given upcoming pediatric enrollment). We will also create new mechanisms such as a rapid feedback panels - a diverse group of participants who will provide time-sensitive feedback in critical areas. Ultimately, we will document successful strategies to contribute evidence-based recommendations for future large-scale studies with a substantial participant engagement component.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Duchenne muscular dystrophy (DMD) is characterized by membrane instability, calcium influx, and necrosis of myocytes. In the heart, progressive breakdown of cardiomyocytes causes fibrosis and an insidious dilated cardiomyopathy. Heart failure is the primary cause of death in patients with DMD, which occurs around the third decade despite traditional cardiosupportive therapeutics. Our preliminary research suggests that signaling from the thromboxane-prostanoid receptor (TPr) in the heart is a driving force leading to cardiomyocyte death and fibrosis, and preventing this activity may preserve cardiac function in muscular dystrophy patients. Our group has found that blocking TPr activity with the antagonist ifetroban improves survival, cardiac function, and cardiac fibrosis in two mouse models of severe DMD and a model of limb-girdle muscular dystrophy. Based on these studies, a Phase 2 clinical study of ifetroban in DMD patients is currently recruiting. However, key knowledge gaps exist. We know that TPr activation leads to fibrosis with enhanced tumor growth factor (TGF)- β activity, but not how it activates TGFβ. Here we will test the hypothesis that TPr activation mediates TGF-β release from the large latent complex, leading to cardiac fibrosis. We will test this in the context of TPr blockade or deletion, using mdx/utrn(+/-) mice or mdx mice containing latent TGF-β binding protein-4 (LTBP4) polymorphism, and confirm with isolated fibroblasts. Our early data also suggests that the mechanism by which TPr regulates cardiomyocyte membrane stability, arrhythmia, and cardiac function may be distinct from this. We hypothesize this occurs via regulation of calcium influx and calcium-activated signaling, and that antagonism improves this in mdx/utrn(+/-) mice in a manner additive with standard-of-care therapies. A DMD mouse model containing a cardiomyocyte-specific deletion of TPr will separate cardiomyocyte-initiated from fibroblast effects. Finally, our ongoing trial presents a unique opportunity to assess molecular outcomes longitudinally in human patients, and identify biomarkers to reflect the cardiac response to TPr antagonism. For this aim, we will do expression profiling of peripheral blood mononuclear cells from DMD patient participants in the ifetroban clinical trial, compared with response to treatment. Resolving these questions will illuminate the role of the TPr in DMD cardiomyopathy, anticipate human response and mechanism of TPr antagonism in DMD patients, and could provide valuable surrogate endpoints for drug response.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY The goal of this work is to refine neuroimaging methods to enable quantitation of choroid plexus (ChP) anatomy and function non-invasively in vivo, and subsequently to use these methods to test fundamental hypotheses regarding ChP activity, cerebrospinal fluid (CSF) flow, and anatomical and protein markers of molecular clearance dysfunction in patients with Alzheimer’s Disease Related Dementias (ADRDs). The premise for this work is based on the known role of the ChP complexes for CSF production, and the recent link between bulk and perivascular CSF flow dysfunction in neurodegenerative disorders, yet a lack of robust methods for quantifying these pathways in humans. We have shown that arterial spin labeling (ASL) magnetic resonance imaging (MRI) methods and deep learning algorithms can be re-parameterized to enable reproducible estimates of ChP perfusion (ml/100g/min) at high spatial resolution and accurate automated localization, respectively: in preliminary data from 139 volunteers, we have (i) demonstrated abilities to obtain reproducible ChP perfusion estimates in healthy adults (n=10); (ii) observed that improvements in vascular health reduce ChP activity (n=23) and progressive intracranial vasculopathy increases ChP perfusion (n=75); and (iii) report here that in older adults with ADRDs, ChP is elevated relative to age- matched adults without dementia (n=31). These data highlight the possibility of evaluating ChP function in vivo in neurodegenerative and cerebrovascular disease. However, extant methods require refinement to improve ChP localization and quantitative accuracy, including an expanded knowledge of ChP physiology and how ChP activity relates to anatomical markers of molecular clearance and symptomatology. Here, we propose to address these gaps in our understanding. In Aim (1), we will perform systematic measurements of ChP MRI relaxation times and circulatory dynamics; findings will improve ChP perfusion accuracy beyond current approaches that utilize convenience calibration values from other tissues. In Aim (2), we will extend prior studies demonstrating circadian variation in CSF production to quantify diurnal variation in ChP perfusion during sleep and wakefulness; results will serve as a necessary prerequisite for future studies that utilize ChP function as a surrogate or complement to glymphatic or CSF flow dysfunction. In Aim (3), we will quantify ChP perfusion in participants with ADRD in sequence with bulk CSF flow velocity through the cerebral aqueduct, parasagittal dural volume, and proteinopathy. Data will be used to test fundamental hypotheses regarding the relevance of ChP activity and impaired trans-molecular passage in the setting of normal and heightened amyloid burden and clinical dementia. Findings will provide the first data on how ChP activity, quantified non-invasively in vivo from high spatial resolution perfusion MRI, reflects variation in traditional or novel fluid efflux. Successful completion will provide new acquisition and post-processing resources, which will provide a foundation for using these methods in the growing number of applications of CSF clearance dysfunction.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract While there is abundant evidence that certain musical behaviors (e.g., regularly listening to loud music) are risk factors for age-related hearing loss (ARHL), human musicality also has the potential to support hearing health across the lifespan. For example, studies in small samples of younger adults show that individual differences in musical aptitude are positively associated with hearing outcomes such as speech recognition in noisy conditions, and that musicians show enhanced hearing outcomes and auditory processing compared to non- musicians. Further, advances in the genetics of musicality show that genes involved in cochlear development are associated with phenotypic variation in musical aptitude. Preliminary studies also show a positive association between music engagement frequency and hearing sensitivity. Given this evidence, could higher degrees of musicality – both aptitude for and engagement with music – be a protective factor against hearing loss as we age? This project systematically tests this novel hypothesis using experimental, epidemiological, and genomic approaches. Aim 1 uses robust experimental approaches to characterize associations between musical aptitude and hearing outcomes in older adults, over and above music engagement (e.g. practice, formal training, listening) and neurocognitive skills (e.g. executive function). Aim 2 uses epidemiological approaches to characterize associations between music engagement frequency and hearing outcomes in large cohorts of middle-aged and older adults. Aim 3 uses computational genomics approaches to investigate shared genetic architecture between human musicality and ARHL, in large cohorts of middle-aged and older adults for whom available health, phenotypic, and genotypic information is known. Evidence for and against our hypotheses will allow us to disentangle three competing theories about the links between human musicality and hearing health, namely that links are either driven by (a) shared genetic and neural architecture underlying both traits, (b) auditory neurocognitive affordances and preferences shaping musicality, (c) or “wear and tear” of sensorineural auditory biology due to cumulative loud music exposure. Taken together, findings from this project will help evaluate musicality as a protective factor against hearing loss as we age, and lay the groundwork for understanding longitudinal and causal relationships between music engagement and hearing health. Further, findings will lay the groundwork for examining specific biological functions (e.g., expression and regulation) of genes linking musicality, sensorineural auditory mechanisms, and hearing outcomes. This work addresses a critical health need: one in three adults aged 70 or older in the United States suffers from hearing loss, with cascading consequences on social isolation, depression, and cognitive decline and dementia. Innovations in personalized prevention and care are sorely needed. Further the work is timely and leverages recent advances in computational genomics and health biobank approaches, and in the genetics of human musicality traits.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY The goal of this R34 proposal and the future R61/R33-funded RCT is to decrease the severity of moderate and severe acute asthma exacerbations in children, sufficiently and quickly enough to decrease hospitalizations. These hospitalizations disproportionately affect Black and low-income children. They often occur because leu- kotriene (LT) induced airway inflammation and bronchoconstriction are incompletely responsive to systemic corticosteroid (CCS) and inhaled albuterol. LT synthesis is induced by viral respiratory infections and aeroaller- gens, the most common exacerbation triggers in children. We have a critical clinical need for a medication that will rapidly decrease LT-mediated airway inflammation and bronchoconstriction. Montelukast (MK), a potent LT-receptor antagonist, may address this need. IV MK caused rapid, sustained improvement at peak plasma levels (Cmax) of ≈1,700 ng/ml in adults with moderate and severe exacerbations. IV MK is not available, and our preliminary pharmacokinetic (PK) study in children with exacerbations found that high-dose oral MK (mean 1.0 mg/kg) achieves Cmax of 1,700 ng/ml in 40% of participants. The R34 Aim is to perform an adaptive, PK- guided, double-masked RCT of standard treatment plus high-dose oral MK or identical placebo, with 3 escalat- ing mg/kg MK dose-levels determined by PK-guided dose modeling, in children with exacerbations that are moderate or severe after initial treatment with albuterol. We will test three Hypotheses (1) High-dose oral montelukast achieves Cmax >1,700 ng/ml in >86% of at least one of three sequential participant groups with escalating weight-based (mg/kg) doses between groups; (2) Participants randomized to high-dose oral monte- lukast have a 2 point or greater improvement of the validated Acute Asthma Intensity Research Score (AAIRS) 4 hours post-treatment in comparison with control group participants; and (3) Among montelukast recipients, Cmax correlates with change of the AAIRS at 4 hours. This R34 research will yield essential and sufficient knowledge to make definitive design decisions for a Phase II RCT (R61-R33 funded), adequately powered for important clinical outcomes. The future RCT will test the hypothesis that the optimal mg/kg MK dose identified in this R34 research improves outcomes as an additional anti-inflammatory and bronchodilator medication in children with moderate and severe exacerbations. The overall Contribution of this research will be to identify an optimal mg/kg dose of oral MK for the future RCT. The Significance of this R34 research and of the future RCT is that high-dose oral montelukast will provide a critically needed medication for exacerbations to decrease the morbidity of this common illness. This research is Innovative by (1) Identifying an optimal mg/kg dose for the future RCT; (2) Providing preliminary efficacy and dose-response data; and (3) Repurposing an inexpensive drug in a novel way to address an unmet need in children with asthma exacerbations. Completion of this re- search will yield knowledge to decrease the morbidity and health burden of asthma exacerbations in children.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract The prevalence of obesity is increasing globally, and chronic systemic and adipose tissue (AT) inflammation in obesity contribute to increased risk of cardiovascular disease (CVD). While several anti-inflammatory agents have shown benefit in reducing adverse cardiovascular (CV) outcomes, none are standard of care in obesity. Thus, there remains a residual risk of CV events and a significant need for new therapeutics to target the inflammation in obesity and prevent or reverse these outcomes. Furthermore, our understanding of AT inflammation in humans is limited due to difficulty obtaining samples in clinical trials, and discoveries in animals do not consistently translate. SLGT2 inhibitors reduce major adverse CV events through unknown mechanism(s) and beyond what is expected from their anti-hyperglycemic or weight loss benefits. An anti- inflammatory effect of SGLT2 inhibition has been demonstrated in animals, and if present in humans could point to a potential mechanism for the CV benefit. Our central hypothesis is that SGLT2 inhibitors decrease systemic and AT inflammation and result in improvements in endothelial function as a surrogate of CVD. With a mentoring team combining expertise in clinical study design, AT immunology and endothelial measures, we can address this scientific gap and advance our understanding of the CV benefits of SGLT2 inhibition. Our team will enroll obese individuals with pre-diabetes and treat them with 12 weeks of an SGLT2 inhibitor or placebo in a randomized double-blind trial. In Aim 1, we will test that SGLT2 inhibition reduces AT and systemic inflammation by quantifying immune cell populations and transcripts. In Aim 2, we will test that SGLT2 inhibition reduces endothelial inflammation and improves endothelial vasodilatory function. We will assess whether changes in endothelial function are associated with changes in AT and systemic inflammation. In Aim 3, we will interrogate the molecular effects of SGLT2 inhibition on immune and endothelial cell interactions in vitro. Through the activities in this proposal, the candidate will (Objective 1) design and implement a mechanistic human trial from inception to completion; (Objective 2) establish and lead a multidisciplinary program at the intersection of immunology, AT biology and CVD; (Objective 3) develop expertise in analyzing immune and endothelial cell interactions in vitro; and (Objective 4) gain investigative skills in surrogate measures of CVD. These objectives will assist the candidate in achieving her long-term career goal to lead a translational research program at the interface of immune function and vascular disease in obesity and define new pathways for targeted interventions to prevent and treat cardiometabolic diseases. She will accomplish these goals within an institution with a tremendous track record of supporting early career physician-scientists. She also has the support of an exceptional mentoring team that has jointly mentored many physician-scientists to autonomy, and that has a diverse and multidisciplinary set of interests that are distinct from, yet mirror and enhance those of the candidate.

NIH Research Projects · FY 2026 · 2023-07

Polarized epithelial tubes are critical for an intact kidney. They control water, electrolyte, and nutrient homeostasis, all of which are deranged in chronic kidney disease. These specialized epithelia require a highly organized actin cytoskeleton that determines cellular shape and function. During renal epithelial repair, the actin cytoskeleton of proliferating cells is rapidly re-organized to form the complex polarized architecture of an epithelial tube. The small Rho GTPase Rac1 is a multifunctional molecular switch and a master regulator of the actin cytoskeleton. We recently demonstrated that Rac1 is required to maintain actin cytoskeletal integrity, epithelial polarity, and cell shape of the mature collecting duct (CD) epithelium. It is still unknown how the actin cytoskeleton is regulated in epithelial cell repair and what role Rac1 plays in this process. For coordinated epithelial tube regeneration, epithelial cells need to rapidly progress through the cell cycle and divide in the correct direction along the tissue plane by undergoing oriented mitosis. A critical step in renal epithelial repair is the activation of the master mitotic kinase cyclin B–CDK1 complex which drives the G2/M transition and prepares the actin cytoskeleton for mitosis. Undergoing mitosis correctly in a confined tight epithelial space is a challenging process, which requires the cells to round up against their neighbors. This so-called mitotic rounding depends on the actin cytoskeleton forming a dense contractile actomyosin cortex, which is tethered to the cell membrane by ERM Proteins (Ezrin), a known target of Rac1 and its main effector p21-activated kinase (Pak1). Defects in mitotic morphology lead to cell cycle delays, mechanical defects, and cell death and abnormal repair. How actin cytoskeletal dynamics are molecularly coordinated and by what means cell shape is connected to cell cycle control during these critical biological processes in kidney repair is not known. We deleted Rac1 in the collecting duct (AQP2Cre) or proximal tubule (γGTCre) and performed reversible unilateral ureteric obstruction (R-UUO) or ischemia-reperfusion induced acute kidney injury (AKI-IRI). Functional and histological assessment indicated that Rac1 is required for repair in both models. Optical clearing and 3D high-resolution imaging revealed that the mutant epithelium was unable to restore normal morphology and actin organization post-injury. Repair in Rac1 mutants showed defects in actin-dependent mitotic morphology, misplaced mitotic figures, and the inability to proliferate due to a G2/M cell cycle defect. 3D high-resolution live imaging of cell division in vitro revealed that Rac1 is required for normal mechanical progression of mitosis and Ezrin-associated mitotic rounding in renal epithelial cells. This forms the basis for our hypothesis that Rac1 controlled actin-dependent mitotic rounding mediated via its effector Pak1 and the linker protein Ezrin is required to promote cell cycling during tissue repair, which will be tested in the following aims. Aim 1: Define the mechanisms whereby Rac1 regulates renal epithelial repair in vivo. Aim 2: Define the mechanism whereby Rac1 controls renal epithelial mitotic morphology in vitro.

NIH Research Projects · FY 2025 · 2023-07

SUMMARY The ocular surface is constantly exposed to the environment and is inhabited by several microbes. The most commonly used methods for ocular surface microbiome characterization consist of traditional microbiological culturing and, more recently, next-generation sequencing. Culture-based methods provide information on absolute abundances but are limited in identifying only fast-growing microorganisms on culture media and a large number of nonculturable microbes. Recent 16S rRNA marker gene sequencing studies provide the most extensive and diverse characterization of the ocular microbiome. This data suggests the existence of the resident ocular microbiome; however, little is known about the core microbiome constituents of the anterior segment in a healthy population. Furthermore, the optimal method of sample collection that will yield the most representative results remains unclear. This study aims to implement whole-metagenome sequencing (WMS) and metatranscriptomics sequencing methods optimized for low-biomass samples to characterize the ocular microbiome inhabiting the anterior segment of the eye and their interaction with the immune system in healthy adults. In this project, we will establish a large human cohort (n=500) and will prospectively collect serial samples from inferior and superior conjunctival fornix from healthy adults for a period of 1 year. In addition to characterizing the core ocular microbiome in healthy adults, we will examine seasonal changes in the diversity and enrichment of specific microbial taxon (bacteria, fungi, and viruses); ocular microbiome differences between sexes and different age groups (young adults, middle age, and older adults). The metatranscriptomics data will enable us to profile active microbial pathways (bacteria, fungi, and virus) and host epithelial (conjunctival) immune response for underpinning associations between the microbiome and ocular immune modulation. The outcomes of this study will contribute towards understanding the core microbial constituents in the anterior segment and their role in immune interactions to maintain homeostasis.