Vanderbilt University

NIH Research Projects · FY 2025 · 2023-05

PROJECT SUMMARY Helicobacter pylori colonizes the stomach of about 50% of the world’s population and is the strongest known risk factor for developing gastric cancer, the fourth most common cause of cancer related deaths. Failure of the host response to control the infection leads to persistent inflammation, which initiates disease progression from chronic gastritis through a histological “Correa Cascade” that results in gastric carcinoma in 1-3% of all those infected. Due to antibiotic resistance, and the fact that antibiotic treatment may not be effective in reducing cancer risk once precancerous lesions are present, we need to develop new therapeutic strategies to limit progression to dysplasia and carcinoma. Our lab investigates the role of the polyamines, putrescine, spermidine, and spermine in gastric inflammation and carcinogenesis. Putrescine is sequentially converted to spermidine and spermine, which is back-converted to spermidine by spermine oxidase (SMOX). We have shown that SMOX expression is elevated in human and mouse gastric tissues infected with H. pylori. Furthermore, infected C57BL/6 Smox–/– mice exhibit depleted spermidine levels, and a decrease in gastritis and carcinogenic signaling compared to wild-type mice. Using FVB/N INS-GAS mice prone to developing gastric dysplasia and intramucosal carcinoma with H. pylori infection, we have seen that Smox–/– mice infected with H. pylori exhibit a significant reduction in gastric intramucosal carcinoma and extent of dysplasia. Spermine catabolism by SMOX generates 3-aminopropanol, which can spontaneously form acrolein, a reactive electrophilic aldehyde that has the potential to damage DNA and proteins. Our preliminary findings demonstrate that acrolein is produced in gastric tissues of H. pylori-infected FVB/N INS-GAS mice and is significantly reduced in Smox–/– FVB/N INS-GAS mice. Additionally, spermidine is an essential substrate for the synthesis of hypusine, a unique amino acid that is only found in the protein eukaryotic translation initiation factor 5A (EIF5A) by the action of the enzyme deoxyhypusine synthase (DHPS). Our recent work with human gastric organoids has revealed induction of hypusinated EIF5A levels with H. pylori infection, which was ablated with the chemical inhibitor of the pathway. Proteomic analysis on these organoids implicated hypusination as a critical pathway for oncogenesis. Taken together, we hypothesize that polyamine dysregulation due to SMOX activity in H. pylori-infected gastric epithelial cells leads to the generation of spermidine and acrolein, and upregulation of the hypusination pathway resulting in increased risk for gastric cancer development. Our specific aims are to determine: 1) the role of SMOX activity in gastric carcinogenesis, including effects of spermidine, spermine and acrolein in FVB/N INS-GAS mice. 2) if spermidine generated by SMOX contributes to gastric cancer development through hypusination using studies in human gastric organoids and mice with an epithelial-specific deletion of Dhps. This proposal seeks to elucidate the mechanisms by which SMOX induces gastric disease progression, thus identifying novel pathways to be targeted for therapeutic benefit, while providing the ideal training for my future career as a principal investigator.

NIH Research Projects · FY 2025 · 2023-04

Small molecule ligands that activate the orphan nuclear receptor Nurr1 (NR4A2) hold promise as neuroprotective therapeutic agents or adjuvants to aging-associated neurodegenerative and dementia disorders characterized by a loss of neuron function including Parkinson's disease (PD) and Alzheimer's disease (AD). Nurr1 activating ligands show functional efficacy in animal models of AD and PD. However, although nuclear receptors are considered to be ligand-dependent transcription factors, Nurr1 is thought to function independent of binding an endogenous ligand that is produced and present in cells. Several synthetic ligands that activate Nurr1 transcription have been reported, but most have not been validated to directly bind Nurr1 and their mechanism of action remains unknown, which has stunted efforts to optimize Nurr1 ligands for AD and PD. Furthermore, Nurr1 regulates transcription as a monomer and as a Nurr1-RXR heterodimer. Synthetic RXR ligands that activate transcription of Nurr1-RXR heterodimers also display functional efficacy in animal models of AD and PD. However, it remains poorly understood how RXR and RXR-binding ligands impact the function of Nurr1-RXR on the structural level. In this project, we will address these knowledge gaps using mechanistic studies to define how small molecule ligands impact Nurr1 and Nurr1-RXR activation on the molecular, structural, and cellular levels using NMR spectroscopy, X-ray crystallography, mass spectrometry coupled to hydrogen/deuterium exchange (HDX-MS) and chemical crosslinking (XL-MS) and small angle X-ray scattering along with biochemical and cellular functional assays. These data will inform the design of new and improved Nurr1 activating ligands to determine if direct targeting of Nurr1 or indirect targeting via RXR is a viable option for AD and PD treatment

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY Suicide is a leading cause of death for adolescents worldwide, and rates have increased over the past decade. Yet, we lack reliable predictors of suicidal behavior (SB) due in part to a need to consider a broader range of risk processes and methods of assessment. There is a particularly critical and urgent need for research on objective, prospective predictors of SB to identify youth at greatest risk and elucidate novel targets for intervention. The goal of this study is to characterize alterations in RDoC’s Positive Valence Systems, particularly neural processes involve in reward valuation, in a large, high-risk sample of adolescents in acute psychiatric treatment for self-injurious thoughts and behaviors (SITBs) who will be followed for 6 months following discharge—a period of high risk for SB. Our preliminary data and theories of SB highlight a key role of reward valuation in that SB partly reflects a choice between immediate relief from emotional pain rather than waiting for potential future rewards that come from continuing to live through difficult times. Consistent with this, rigorous cross-sectional research has linked SB to greater discounting of the value of delayed monetary rewards, and our preliminary data indicate enhanced neural responsiveness to immediate relative to delayed rewards in youth with recent SB. Importantly, preferences for immediate rather than delayed reinforcers are known to increase in times of high stress, and acute interpersonal stress is associated with elevated risk for SB in adolescents, while social support tends to be protective. This multi-method, prospective study will test an innovative conceptual model of SB by which youth with enhanced neural reactivity to immediate reinforcers and blunted reactivity to delayed rewards are more likely to engage in SB, particularly in times of acute interpersonal stress, and social support buffers effects of reward valuation on SB. We will recruit a transdiagnostic sample of adolescents (ages 13-17 years; N=300) in acute psychiatric treatment for SITBs. Participants will complete clinical assessments and behavioral and neural measures of reward valuation in the hospital to capture a high-risk state and minimize participant burden. Following discharge, participants will complete a brief daily survey of interpersonal stress, social support, and SITBs for 90 days. Finally, participants will complete follow-up interviews of SITBs 6 months after discharge to test prospective predictors of SB. This study will characterize patterns of reward valuation associated with SB histories in high-risk youth (Aim 1), examine neural measures of reward valuation as prospective predictors of SB following hospital discharge (Aim 2), and examine the interplay between neural reward valuation and interpersonal factors in the proximal prediction of SB (Aim 3). This study directly addresses NIMH priorities and will be the first to examine neural predictors of future SB in a large, high-risk sample of youth, extending RDoC-informed research on suicide risk to the Positive Valence Systems domain. This research will lead to the identification of objective predictors of SB risk and the development of more personalized, targeted interventions to prevent SB and suicide deaths.

NIH Research Projects · FY 2026 · 2023-04

Project Summary The liver and kidney are the major organs where glucose biosynthesis is coupled to mitochondrial metabolism. Previous studies demonstrate that overnutrition accelerates whole-body gluconeogenesis (GNG) and citric acid cycle (CAC) activity in vivo. A limitation of prior research is that the unique contributions of the liver and kidney to whole-body GNG and CAC fluxes have been difficult to disentangle in vivo. The inability to discern hepatic from renal metabolic fluxes represents a significant gap in knowledge, as obesity may not only cause an ectopic accumulation of lipid but also an “ectopic redistribution” of gluconeogenic function that disproportionately stresses the kidney. We hypothesize that renal GNG and CAC activity are disproportionately elevated in obesity, which contributes to the dysregulation of whole-body glucose metabolism and promotes mitochondrial dysfunction and oxidative tissue damage in the kidney. The scientific aims of this proposal are to (i) determine whether the progressive development of obesity disproportionately impacts renal gluconeogenic and oxidative metabolism, (ii) assess whether gluconeogenic overload on the kidney accelerates oxidative metabolism and stress during obesity, and (iii) identify metabolic mechanism(s) by which SGLT2 inhibitor treatment reduces hepato-renal lipotoxicity in vivo. The aims of our project will be accomplished using a novel, metabolic flux modeling system that simultaneously determines gluconeogenic and oxidative metabolic fluxes in the liver and kidney in vivo. This work is innovative because it examines the etiology and treatment of metabolic disease through the lens of multi-organ fluxomics while focusing on an understudied aspect of gluco(dys)regulation. It is significant because it will identify organ-specific metabolic nodes that may be better targeted to improve glycemic control and reduce damage in the kidney and liver. Results from this K01 project, the unique expertise of each member of my mentoring committee, and the diabetes research infrastructure at Vanderbilt University will be leveraged to achieve my career objective of an independent career studying metabolic regulation in diabetic kidney disease and hypoglycemic counter-regulation. As such, this project integrates a career development plan with the training needed to bolster my disease-state expertise, grantsmanship, and expand my analytical capabilities to ensure a smooth transition toward research independence.

NIH Research Projects · FY 2026 · 2023-04

Project Summary/Abstract Our proposed research seeks to understand the evolutionary origin and capacity to adapt of complex molecular machines. It is challenging to comprehend how protein function, which depends on finely tuned cooperativity between many components, is adapted and altered as an organism evolves. That is, how does evolution satisfy the demands of high performance while maintaining the capacity for diversification and adaptive specialization? To gain insight into these processes we plan to carry out high-throughput mutagenesis and functional studies of a set of proteins involved in DNA replication. High-speed DNA replication relies on proteins known as sliding clamps, which are proteins that encircle DNA and can diffuse rapidly along the double-helix without dissociating from it. Because the sliding clamps form closed circles, they do not readily associate with DNA on their own. Sliding clamps are opened and loaded onto the start sites of DNA replication by ATP-driven molecular machines called clamp loaders. Clamp loaders are members of an evolutionarily ancient family of ATP-dependent molecular machines called AAA+ ATPases, which are a diverse set of proteins that transduce ATP binding and hydrolysis into mechanical action on proteins. Because the DNA polymerase clamp-loaders are very well understood in terms of their three- dimensional structures, they are excellent models for understanding intramolecular force transmission as well as, more generally, the evolution of complex protein machines. The central goal of this proposal is to develop an understanding of the mechanisms and evolutionary divergence of such complex protein machines, leading to advances in our ability to predict and control the behaviors of cellular systems in both normal and disease states. The T4 bacteriophage (T4) is a small virus that infects the E. coli bacterium. The T4 genome encodes its own DNA replication proteins, including a sliding clamp and clamp loader, proteins that are closely related to their counterparts in eukaryotic cells, including human cells. We have developed and validated a powerful high- throughput functional assay for the T4 bacteriophage (T4) clamp loader system. This platform opens up many avenues to investigate mechanism and design principles in a proper biological context. We will use high- throughput mutagenesis to map mutational sensitivity and allosteric coupling in the clamp loader and examine the conservation of these properties in a very divergent AAA+ ATPase, a protein that controls transcription in bacteria. We will use statistical models trained on genome sequences to infer the essential constraints on and between amino acids in clamp loaders and test these inferences in a biological context. The aims of this project represent a unified body of work to use new assay systems to understanding clamp loader and AAA+ mechanism, and to test the potential of emerging sequence-based models for understanding and engineering complex macromolecular machines.

NIH Research Projects · FY 2026 · 2023-03

Fluoroquinolones, such as ciprofloxacin, are among the most efficacious and broad-spectrum oral antibacterials in clinical use. The World Health Organization lists them in their five “Highest Priority Critically Important Antimicrobials,” and these drugs are the most heavily prescribed antibacterials worldwide. The cellular targets of fluoroquinolones are the bacterial type II topoisomerases, gyrase and topoisomerase IV. These essential enzymes regulate DNA under- and overwinding and remove knots and tangles from the genome by generating transient double-stranded breaks in the genetic material. Fluoroquinolones act by increasing levels of double-stranded DNA breaks generated by gyrase and topoisomerase IV, which converts these enzymes into cellular toxins that fragment the genome. Although gyrase and topoisomerase IV are both physiological targets for fluoroquinolones, their relative importance to drug action appears to be species- and drug-dependent. There is a growing crisis in antibacterial resistance and fluoroquinolone resistance is becoming prevalent. This resistance is threatening the clinical efficacy of fluoroquinolones. Initial fluoroquinolone resistance is most often associated with specific mutations in gyrase and/or topoisomerase IV that occur at a serine residue (originally described as Ser83 in the GyrA subunit of Escherichia coli gyrase) and a glutamic/aspartic acid residue 4 amino acids downstream. Based on a published structure and functional studies from the Osheroff laboratory, these residues are proposed to anchor a water-metal ion bridge that serves as the primary conduit between fluoro- quinolones and gyrase/topoisomerase IV. The identification and characterization of novel agents that act against these well-validated enzyme targets and overcome fluoroquinolone resistance could have important health ramifications. Recently, two new classes of gyrase/topoisomerase IV-targeted agents have been described that appear to overcome this resistance, Novel Bacterial Topoisomerase Inhibitors (NBTIs) and Spiropyrimidinetriones (SPTs). Members of these classes, gepotidacin (NBTI) and zoliflodacin (SPT), have advanced to Phase 3 clinical trials. NBTIs are unique, as they induce single- rather than double-stranded enzyme-generated DNA breaks. However, little is known about the actions of NBTIs and SPTs against gyrase/topoisomerase IV or the mechanism of drug resistance. There is an urgent need to identify drugs that display activity against fluoroquinolone-resistant bacteria. Thus, the goals of this project are to further define the mechanism of action of fluoroquinolones, NBTIs, and SPTs against gyrase and topoisomerase IV in vivo and in cells, to characterize the basis of target-mediated drug resistance, and to identify novel compounds that overcome resistance. Research will benefit from the broad library of wild- type and drug-resistant gyrase/topoisomerase IV available in the Osheroff laboratory, which includes enzymes from Bacillus anthracis, E. coli, Staphylococcus aureus, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Francisella tularensis, and Acinetobacter baumannii. These pathogens have substantial effects on human health.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY Our overarching goal is to enable the AI-empowered 3D histopathological interpretation on routine digitized renal tissue biopsies, so as to (1) allow renal pathologists to perform a reproducible 3D phenotyping on serial 2D whole slide images (WSI), (2) advance the characterization of kidney-allograft rejection phenotypes on kidney transplant patients with cutting-edge 3D computer vision, and (3) equip clinical scientists with an advanced 3D spatial transcriptomics analytics tool to investigate the anatomical-molecular associated causes of chronic kidney disease (CKD). Our novel 3D histopathological interpretation, with 3D computer vision (Map3D toolkit) and 3D spatial transcriptomics, will open a new door for performing reproducible clinical phenotyping (Pheno3D toolkit), identifying and validating new 3D imaging and molecular biomarkers (GPS3D toolkit), and ultimately advancing the patient care with personalized diagnosis and prognosis options for a wide range of CKD. Despite more than 25 years of exploitation of digital pathology, the presence, significance and characteristics of 3D contextual information in renal histopathological assessment have been largely overlooked. The current 2D interpretation on renal histopathology is error-prone and less reproducible due to the heterogeneity of tissue morphologies (e.g., glomeruli, tubules, vessels) across 3D serial sections. For example, our previous study on segmental glomerulosclerosis (GS) in patients with nephrotic syndrome and idiopathic FSGS, the percent of GS increased from 31.5 +/- 6.8% to 48.0 +/- 6.6% (P < 0.025) in adults by replacing a 2D single section analysis with 3D serial section analysis. Moreover, 2D based phenotyping can also hinder the discovery of new biomarkers via state-of-the-art spatial transcriptomic techniques. As an example, a glomerulus with focal segmental glomerulosclerosis (FSGS) can have a normal appearance on a specific 2D section, which might lead to an opposite molecular finding using 2D spatial transcriptomics The core tenant of this proposal is NOT developing a new 3D imaging modality, but rather, to develop technologies that enable reproducible 3D characterization on routine 2D renal histopathological biopsies (with trivial added cost), so as to advance the care of future patients with renal diseases. To this end, we will: Aim 1. Develop novel 3D computer vision tools (Map3D) to facilitate renal pathologists in modeling, quantifying, and visualizing 3D renal histopathological tissues from routine 2D digital histopathology. Impact: Allow renal pathologists to perform a reproducible 3D phenotyping on serial 2D whole slide images (WSI). Aim 2. Develop 3D phenotyping tools (Pheno3D) to advance the characterization of kidney-allograft rejection for kidney transplant patients via 3D computer vision and self-supervised deep learning. Impact: Advance the characterization of kidney-allograft rejection phenotypes for kidney transplant patients. Aim 3. Develop 3D computer vision algorithms for 2D and 3D spatial transcriptomics (GPS3D toolkit). Impact: Equip clinical scientists an 3D spatial transcriptomics analytics tool to investigate image-omics interaction.

NIH Research Projects · FY 2025 · 2023-03

PROJECT SUMMARY/ABSTRACT: Degenerative rotator cuff tear (DCT) is among the most common causes of shoulder pain, yet little is known about the genetic and physical risk factors for these tears. Contrary to prior belief that DCTs were fully attributable to repetitive microtrauma, new evidence has emerged on intrinsic tendinous abnormalities that could predispose DCT risk. Obesity and diabetes are common health conditions associated with intrinsic tendinous changes (such as tendon fat and aberrant microstructural fiber composition, as well as increased tendon cell death and abnormal nutrient vessel anatomy) and may contribute to predisposing conditions that promote rotator cuff injury. Several epidemiologic studies link obesity and diabetes with cuff disease. However, most are limited in their scope to establish causal links in part due to inconsistent definitions of cuff disease, lack of temporality between exposure and outcome, and biases inherent to these studies. The objective of the proposed work is to leverage several large international DNA and patient health databases, as well as an ongoing prospective cohort, to evaluate causal roles of diabetes and obesity on DTCs, by incorporating methods rooted in instrumental variable analysis (Mendelian Randomization [MR]) which can overcome traditional challenges faced by previous studies. For my first aim, I will build, validate and compare two algorithms to classify cases and non-cases of DCT. These algorithms will be appropriately matched and applied to a variety of international biorepositories with genetic data that linked to electronic health records (EHR). Using genome-wide association study (GWAS) data for DCT generated from these sources, and GWAS data on obesity and diabetes traits from published studies, I will evaluate evidence for causal relationships between diabetes and DCT (Aim 2), and obesity and DCT (Aim 3) using MR techniques. Additionally, I will determine the potential mediating role of diabetes on the association between obesity and rotator cuff tear using two-step MR methods. DCT is a debilitating condition with great long-term morbidity. With the ever-increasing rates of diabetes and obesity in our population, conclusions drawn from this work (null or otherwise) will be timely and impactful. Together, this work taps into unknown and understudied musculoskeletal consequences of diabetes and obesity, and will inform approaches to mitigating risk of injury, opening the door for future studies on treatment and prevention of DCT in these populations.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY Schools face rising mental health needs among children. U.S. public schools are the most common institutional entry point to mental health services for children, and school-based health centers (SBHCs) increasingly serve as a “medical home” for vulnerable children. Yet there are significant gaps in our understanding of SBHCs’ effectiveness and the extent to which they are reaching underserved subgroups of children. Research on SBHCs has largely neglected the study of children’s mental health outcomes; few studies have sought to study in-depth the mechanisms by which SBHC have the potential to improve children’s outcomes; and we lack longitudinal research on SBHC effectiveness. We will fill these critical gaps in research and generate new, generalizable knowledge on program and policy levers that SBHCs can deploy to increase their effectiveness and improve children’s health and education outcomes. A key innovation of our proposed research is our use of a high-quality, linked health and education dataset that encompasses the population of children in Tennessee who have a Medicaid record at any point in time between 2006 and 2025. Our longitudinal data begin well before the Affordable Care Act-funded expansion of SBHCs in Tennessee, allowing for a long baseline period before the large majority of SBHCs opened. We will employ rigorous quasi-experimental methods with the linked longitudinal data to compare children in schools that gained access to a SBHC with those in schools without access to services provided by SBHCs and examine SBHC impacts on children’s mental health and educational outcomes. We will also advance our understanding of the mechanisms by which SBHCs may improve children’s mental health and education outcomes by: 1) elaborating and testing a child-centered conceptual framework for examining in-depth the organization and implementation of SBHCs and factors that constrain or enable their effectiveness; 2) undertaking a comprehensive documentation of the operations and services of SBHCs and filling gaps in our understanding of how they are operating in rural areas and through mobile/telehealth options, and 3) generating timely new information on how SBHCs adapted their service delivery approaches over time and the extent to which disruptions in children’s access to mental and behavioral health services disproportionately affected underserved subgroups of children. We will actively disseminate the study findings to ensure that they inform program strategies, policies, and evaluation tools that state and local agencies can use to improve the efficacy of SBHCs in serving children most in need of mental health services.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY Late talking is associated with negative academic and socio-emotional outcomes. Despite this, behavioral measures are relatively poor at predicting who will end up having developmental language disorder. Moreover, not much is known about either the typical neurocognitive changes associated with language development or the brain basis of delayed language during preschool, as there have been only three retrospective studies. This project uses well-controlled functional neuroimaging paradigms tapping into receptive language skills and structural imaging of white matter connectivity, as well as an extensive behavioral battery that measures well- established deficits in phonology, semantics and morphology. Four-year-olds, oversampled for late talking, are longitudinally followed when they are 6- and 8-years-old. The first aim determines the sensitivity of the dorsal and ventral pathways to phonological and semantic skills, respectively, motivated by the predictions of the Dual Stream Theory. The second and third aim determine the strength of the directional effects of these pathways on each other, and whether these effects differ depending on age. The fourth aim determines the effect of these pathways on the behavioral development of morphology. Our finding that phonological processing drives the development of semantic and morphological processing would be consistent with Phonological Theory, that semantic processing drives the acquisition of phonological and morphological processing would be consistent with Semantic Theory, or that there are interactive effects would be consistent with Bidirectional Theory. Using a state-of-the-art analytical approach justified by tailored power analyses, we expect to find evidence supporting a model in which early phonological processing drives semantic change and later semantic processing drives morphological change. Although we have theoretically motivated planned comparisons, exploratory analyses will also be conducted to support future work. The scientific rigor of the project is supported by our extensive published research examining phonological and semantic specialization of the brain in kindergarteners into elementary school. The feasibility of the project is supported by our encouraging pilot behavioral and neuroimaging data in 4-year-olds on the exact paradigms to be used in this project. Although we take a dimensional approach to test our model, as research shows that language ability is on a continuum, we also perform exploratory categorical analyses comparing late talkers to typical children. Research is inconclusive with regards to the role of screening of language delay to inform decisions of early intervention. We hope that a more basic mechanistic understanding of the dynamics of language development will allow for the formulation of predictive biomarkers to be used in screening for intervention. We are committed to open science, and plan to pre-register our work, and share our analytical code and data.

NIH Research Projects · FY 2026 · 2023-03

Retinopathy of prematurity (ROP) is a proliferative retinal vascular disease that affects preterm infants and a major cause of childhood blindness. ROP is diagnosed and staged under indirect ophthalmoscopy at the junction between the vascularized and avascular retina (i.e., periphery) using features such as retinal detachment (RD) and increased vascular dilatation and tortuosity. While surgical and pharmacologic therapies exist, poor structural and visual outcomes (<20/200 vision or blindness) occur in >50% of severe cases because RD, schisis, and vascular abnormalities are often missed on clinical examination. Conventional ophthalmoscopic examination in infants is performed using fundoscopy. However, retinal microvasculature is poorly visualized even when combined with exogenous fluorescein contrast. Optical coherence tomography (OCT) is currently the gold- standard for ophthalmic diagnostic imaging in adults and developments in OCT angiography (OCT-A) have enabled in vivo imaging of retinal vasculature without the need for exogenous contrast. While several research groups have developed handheld OCT/OCTA imaging systems and demonstrated imaging in ROP patients, longitudinal quantitative imaging of retinal vasculature to track ROP progression remains limited by several key challenges: 1) infants cannot fixate, making repeated imaging of regions-of-interest (ROIs) impossible; 2) handheld imaging coupled with infant motion results in significant artifacts and poor OCTA contrast; 3) OCT/OCTA quality is severely degraded by vitreous/anterior chamber haze, which is common in ROP; 4) peripheral retinal vascular changes are important for ROP staging but aiming of OCT/OCTA at these ROIs is difficult; 5) vascular volumes provide clinically relevant features such as dilatation and tortuosity, but OCTA is conventionally assessed using only en face projections. To overcome these limitations, we have developed a combination of hardware and image-processing technologies for handheld OCT/OCTA built around a multimodal spectrally encoded coherence tomography and reflectometry (SECTR) ophthalmic imaging platform. SECTR simultaneously acquires orthogonal en face reflectance and cross-sectional OCT images that uniquely benefits volumetric registration for motion-correction and multi-volumetric mosaicking. We hypothesize that the translation of these technologies into a point-of-care ophthalmic imaging system will allow for robust, reproducible, and quantitative longitudinal tracking of retinal vascular changes, which will improve the diagnostic and staging accuracy of ROP in preterm infants. We aim to develop custom hardware (AIM 1) and image analysis pipelines (AIM 2) optimized for handheld SECTR imaging in infant eyes. These technologies will be validated in longitudinal imaging of structural and functional changes in ROP (AIM 3) and provide quantitative insight on the viability of SECTR imaging to benefit treatment decisions. The proposed device and quantitative analysis pipeline are not limited to ROP cases and can be broadly applied in any area of ophthalmology where a robust point-of- care OCT/OCTA may improve current clinical standard-of-care.

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY DNA replication is constantly challenged by a variety of genotoxins that arise from the environment. These genotoxins can be directly produced by the environment (e.g. UV and ionizing radiation) or can arise indirectly in response to environmental agents (e.g. polycyclic aromatic hydrocarbons, reactive oxygen species). Nascent strand degradation (NSD) and fork reversal promote genome stability in response to genotoxins by facilitating replication fork restart. Despite the importance of nascent strand degradation and fork reversal, there are many open questions about this pathway. For example, too much or too little degradation results in genome stability. It is therefore important to understand how nascent strand degradation is efficiently triggered when needed but with enough specificity that spurious degradation is avoided. However, we do not currently understand how nascent strand degradation is triggered. Additionally, current models for nascent strand degradation are too limited to explain the dozens of proteins currently implicated. Inherited defects in several of these proteins are directly implicated in human diseases (e.g. SMARCAL1, BRCA1, BRCA2) suggesting that defects in this pathway may alter individuals’ susceptibility to environmental genotoxins. Thus, it is crucial to develop a robust paradigm for nascent strand degradation and fork reversal to establish exactly how this pathway leads to replication restart and genome stability. Current approaches to study nascent strand degradation and fork reversal lack the specificity and sensitivity to address these questions. To overcome these limitations, we have developed a new site-specific, highly sensitive, and synchronous approach to study nascent strand degradation and fork reversal in vitro using Xenopus egg extracts. This system contains the full set of cellular proteins involved in DNA replication and DNA repair and provides unparalleled opportunities to observe and manipulate these processes. Our new approach has already revealed key insights into the requirements for nascent strand degradation and the mechanism by which it takes place. The proposed work will combine biochemical and single molecule approaches, both in Xenopus egg extracts and human cells. We will leverage our existing insights and exploit the power of our new system to determine how nascent strand degradation and fork reversal are triggered and the underlying molecular mechanisms involved in these processes. This work will enhance our understanding of one of the major cellular pathways that responds to environmentally sourced genotoxins and allow us to better understand how defects in this pathway may alter individuals’ susceptibility to environmental genotoxins.

NIH Research Projects · FY 2025 · 2023-02

Project Summary: Glucose-6-phosphatase catalytic subunits (G6PCs) hydrolyze glucose-6-phosphate (G6P) to glucose and inorganic phosphate. G6PC2 is predominantly expressed in pancreatic islet b cells. Genetic and molecular studies have linked increased G6PC2 activity to elevated FBG. Higher FBG in the non-diabetic and pre-diabetic ranges strongly influence the risk of cardiovascular-associated mortality (CAM) and developing type 2 diabetes (T2D) whereas, in individuals with T2D, elevated FBG increases the risk for diabetic complications and further increases the risk of CAM. These observations strongly suggest that G6PC2 inhibitors may represent a potential novel therapy to reduce FBG. We hypothesize that the proposed genetic, metabolic, physiological and biochemical studies will define the functions of G6PC2 in b cells, demonstrate that G6PC2 represents a viable target for lowering FBG and generate data that fosters the development of G6PC2 inhibitors. Our data suggest a new paradigm in which a glucokinase/G6PC2 futile substrate cycle, rather than glucokinase alone, is the key determinant of glycolytic flux in b cells. Consequently, deletion of G6pc2 increases the sensitivity of glucose- stimulated insulin secretion (GSIS) to glucose, which results in reduced FBG. We have developed novel assays for measuring G6PC2 activity in vitro and in intact cells. In Aim 1 we propose using these assays to identify non-synonymous G6PC2 single nucleotide polymorphisms (SNPs) that impair G6PC2 activity and then explore their effects on human health using Vanderbilt’s BioVU biobank. We will also explore the concept, arising from preliminary data, that G6PC2 regulates metabolic fluxes and pulsatile insulin secretion in b cells through a mechanism independent of the known action of G6PC2 on glycolysis, that involves altered ER calcium oscillations. We hypothesize the results of Aim 1 will define the functions of G6PC2 in b cells and highlight the positive as well as potential negative effects of G6PC2 inhibition in humans. The conspicuous absence of structure- function studies on G6PC2 originated from the absence of a high-resolution molecular model, the low inherent activity of the enzyme and an inability to purify active G6PC2. The groundbreaking publication of the AlphaFold2 algorithm for protein structure prediction, and our development of heterologous expression and purification protocols for isolation of stable and catalytically active G6PC2, have overcome these hurdles. In Aim 2, we will use mutagenesis to probe the functional importance of amino acids within various putative domains in G6PC2 and then leverage a toolkit of biochemical/biophysical assays to define their effects on G6PC2 folding, stability and catalysis. We will also perform studies to characterize the specificity of a human G6PC2 inhibitor and explore its ability to regulate GSIS in human islets. We hypothesize the results of Aim 2 will establish a new paradigm for investigation of G6PC2 by providing a molecular blueprint for understanding the structural basis of catalysis as well as generating data that will provide insight into the mode of action of a known G6PC2 inhibitor, which will inform the rational design of optimized compounds.

NIH Research Projects · FY 2026 · 2023-02

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Overall Center The Vanderbilt Center for Metabolic Phenotyping in Live Models of Obesity and Diabetes (VMPMOD) is built on the strong Vanderbilt history of serving as a resource to study conscious, unstressed mice. During its 20-year lifespan the Vanderbilt Mouse Metabolic Phenotyping Center advanced research in diabetes, obesity, and metabolism by providing the scientific community with innovative and high-quality phenotyping services to study mouse models. Quality services and access to experienced faculty has created a large demand from outside investigators to use the Center. VMPMOD consists of three cores dedicated to serving outside investigators. The Administrative Core provides scientific, financial, and administrative leadership and oversees service requests and data management. The Animal Health and Welfare Core evaluate mice submitted to VMPMOD, oversees mouse health and welfare, and ensures compliance with regulatory bodies and VMPMOD guidelines. The Mouse Metabolic Physiology Core provides a range of surgical services for mice and flexible experimental services performed under customizable environment-controlled conditions. These experimental services are conducted by the Metabolic Regulation Subcore (MRSC) and the Body Weight Regulation Subcore (BWRSC). MRSC uses flexible platforms to study insulin action, hormone secretion, hypoglycemic regulation, exercise metabolism, and metabolic flux analysis with and without simultaneous measurements of ⩒O2, ⩒CO2, respiratory exchange ratio, carbohydrate oxidation, and fat oxidation. BWRSC utilizes techniques that allow for granular measurements of the components of energy balance under a wide range of conditions. Chemogenetics, optogenetics, and fiber photometry are applied in the MRSC and BWRSC to study neural control mechanisms in concurrence with studies of metabolism and energy balance. The proposed VMPMOD has made significant technical developments that create new opportunities for advances in our understanding of diabetes, obesity, and metabolism. The Vanderbilt mouse program has been successful because it is comprised of a faculty willing to develop and make technology that is part of their research lifeline available to the scientific community and because of staff that are so skilled and committed that scientists are willing to entrust their mice, their research lifelines, with them. The same faculty and staff that has made the program successful in the past will continue to lead VMPMOD. Modified Project Summary/Abstract Section Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Administrative Core The Administrative Core provides oversight of Vanderbilt Center for Metabolic Phenotyping in Live Models of Obesity and Diabetes (VMPMOD) activities. This Core ensures that the Center operates efficiently, has a sustainable financial model, and is responsive to the needs of the national scientific community and the recommendations of standing committees. The Administrative Core supports all functions of VMPMOD which include (a) evaluation of the suitability of mice submitted for study to the Center, (b) consultation with investigators in conjunction with core staff for testing procedures on mice submitted to the Center, (c) oversight of research and development, (d) oversight of the outreach/educational component, (e) data management in conjunction with the National Program Coordinating Unit, and (f) interaction and coordinating services with other members of the consortium. This Core is also responsible for ensuring VMPMOD functions within guidelines established by Vanderbilt University School of Medicine and the Program National Steering Committee. The Administrative Core consists of the Center Director, Associate Director, Resource Statistician, and Program Manager. In addition, the Administrative Core receives recommendations from three standing committees. The Center Executive Committee consists of the Center Director and faculty with different perspectives necessary for the success of the VMPMOD. The Executive Committee receives input from other committees and provides recommendations to improve center operations. It consists of the Center Director, directors of other NIDDK-funded center programs, chair of the department home of VMPMOD, and the Vice President of Animal Care. The Center Research Advisory Committee (RAC) consists of Core and Subcore Directors and Vanderbilt faculty with specific areas of expertise in studies of the mouse that are used by the VMPMOD. RAC meets to evaluate whether tests are performed with rigor and are reproducible and to provide recommendations on services that warrant expansion or expiration. VMPMOD provides unique services and expertise exclusively to investigators without direct affiliation to Vanderbilt. The External Advisory Committee (EAC) is comprised of external investigators at different career stages. EAC meets virtually to provide input that assists the VMPMOD to serve investigators most effectively. Center Director, Core Directors, Subcore Directors, and the Program Manager are ex officio members of the EAC. The EAC provides vital input to the RAC and the Center Executive Committee that informs VMPMOD actions. The Administrative Core participates in the National Consortium and directly interact with the National Consortium Coordinating Unit. Modified Project Summary/Abstract Section Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Animal Health and Wellness Core The Animal Health and Welfare Core (AHWC) is responsible for receipt, certification, and husbandry of mice that are sent to Vanderbilt for the purpose of metabolic phenotyping. The AHWC is the interface between the Division of Animal Care and the Vanderbilt Center for Metabolic Phenotyping in Live Models of Obesity and Diabetes (VMPMOD). The responsibilities and services of this Core are critical for the VMPMOD to perform well-controlled experiments in non-stressed, healthy mice. The overall objective of the core is to facilitate the use of mice in diabetes, obesity, and related research, ensure compliance and maintain the health and colony numbers appropriate to the rate of center usage. Specifically, the Core is responsible for 1) receipt and documentation of incoming mice, 2) assignment and oversight of quarantine procedures, 3) provision of day-to-day husbandry, 4) provision of veterinary care and support, 5) performance of pathological assessments, and 6) implementation and maintenance of any specific dietary requirements. Modified Project Summary/Abstract Section Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Mouse Metabolic Physiology Core The Mouse Metabolic Physiology Core and its two subcores, the Metabolic Regulation Subcore and the Body Weight Regulation Subcore, provide investigators both guidance on experimental design and use of state-of-the-art techniques to assess genetic and environmental determinants of insulin action, substrate metabolism, and energy balance in vivo. The experienced faculty and highly skilled staff of this Core have a 20-year history of performing complex procedures to study metabolism in healthy, unstressed mice and have been at the forefront of development, standardization, implementation, and dissemination of new concepts and techniques to study mouse models of metabolic diseases. In the present application, the Mouse Metabolic Physiology Core delivers critically needed mouse-related services to investigators outside of Vanderbilt. Skilled mouse surgeons perform difficult surgeries such as catheter, brain cannula, and brain probe implantations, as well as bariatric surgery and islet transplantation. Experienced staff perform complex experiments such as metabolic flux analyses under conditions such as those created by a glucose clamp or exercise. Measurements of energy balance components and assessment of reward/motivated behavior allow for the physiological and behavioral determinants of body weight to be determined in mouse models of metabolic disease. This Core merges techniques so that oxygen uptake, carbon dioxide output, energy expenditure, activity and feeding behavior are measured simultaneously with studies of metabolism using indwelling catheters or neural regulation using implanted cannula and fiber optic probes. Experience at Vanderbilt over the past 5 years predicts robust use of this Core. The Mouse Metabolic Physiology Core participates in important educational programs for the diabetes community, including a weeklong course that has been given 20 times over 19 years focusing on surgical and experimental techniques necessary to perform glucose clamps in mice. In summary, the Core facilitates diabetes, obesity, and metabolism research by providing novel services that are feasible at few other institutions to investigators outside of Vanderbilt.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY Significance: Urinary tract infections (UTIs) are the most common adult bacterial infection annually affecting over 150 million individuals worldwide. As a common reason for antibiotic prescriptions, UTIs are considered a substantial source of resistance to antimicrobial agents. Thus, improved understanding of the interactions between host and bacterial pathogen during UTI is essential for facilitating the development of new therapeutic agents. The concentration of copper increases 5-fold in the urine during acute infection as a protective strategy against the pathogen. Most copper is excreted in the intestines where uropathogens colonize following UTI forming the basis for recurrent infections. Uropathogenic Escherichia coli (UPEC), the most common cause of UTIs, uses copper efflux and sequestration mechanisms to prevent copper toxicity. Additionally, UPEC inhabits multiple niches (intestines, vagina, and bladder) throughout the host that vary in copper content and oxygen concentration. While copper response mechanisms have been characterized in laboratory strains of E. coli, the relative contribution of each copper response system to UPEC persistence in the host has not been elucidated. This proposal will determine the spatio-temporal induction and regulation of the UPEC response to copper and will define the role of each response system in mediating UPEC persistence in the host. Rationale and Hypothesis: In order to successfully infect the urinary tract, UPEC must tolerate increased copper levels. Similarly, UPEC must tolerate intestinal copper excretion to colonize the host gut and potentially cause re-infections. Based on prior studies, the copper responsive UPEC locus cus is among the most upregulated of all genes in samples from acute human UTIs. The cus locus encodes the two-component signaling system CusSR, a copper sensing and response signaling system, and copper efflux apparatus CusCFBA. Additionally, the copper response systems CueR/CopA facilitate copper export in aerobic conditions. Oxygen availability is a key determinant of which copper response system is preferentially active in E. coli due to the oxidation state of copper and energetic requirements for copper efflux. Oxygen levels vary widely within UPEC’s niches. I hypothesize that oxygen availability and copper levels dictate the copper response systems that are active in the anatomic niches that UPEC colonizes and infects. I propose two aims to test this hypothesis: Aims: Aim 1 will determine which UPEC copper response systems are active in the anatomic niches (vagina, intestines, bladder) that UPEC encounters. Aim 2 will investigate the role of CusSR in mediating copper sensing and tolerance in the anaerobic intestinal niche. Impact: These studies will be the first to address the hierarchy and localization of copper response systems uropathogenesis. Clarification of how UPEC senses and responds to copper will provide insight into whether targeting copper detoxification methods of UPEC is a viable therapeutic option for UTI treatment and prevention.

NIH Research Projects · FY 2026 · 2023-01

Summary Our previous studies have revealed a complex organization of receptive fields (RFs) of neurons in primary somatosensory cortex of monkeys (area 3b). The neurons have small excitatory centers that create the somatotopic map of the hand, a suppressive surround that includes all of the rest of the glabrous hand, and a weaker suppressive surround that includes all of the skin on the other hand (the “extra hand” surround). We propose to determine if neurons in different layers have similar or different surrounds, what surround features are relayed from the thalamus to area 3b neurons, and the connections that mediate different parts of the surround by inactivating cortical areas that directly or indirectly provide feedback to area 3b hand neurons. In addition, we will determine how a major loss of sensory inputs from the hand, due to selective spinal cord lesions, alters the center-surround relationships of neurons in the deprived and non-deprived 3b hand cortex, even after extensive post lesion recovery of 3b reactivation and hand use. The expected results will greatly expand our understanding of how tactile information from the hands is processed in the somatosensory system, and how processing is impaired and recovered following sensory loss. These results will have a clear impact on therapeutic intervention following spinal cord injury.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY/ABSTRACT Schizophrenia is a debilitating neuropsychiatric condition with a strong genetic component but poorly understood biological mechanisms. The strongest signal from genome-wide association studies (GWAS) of schizophrenia lies in the major histocompatibility complex locus and is in part driven by copy number variation in the complement component 4 (C4) gene, an important mediator in the complement cascade. Furthermore, cross-disorder studies implicate shared genetic risk in schizophrenia and autoimmune disorders, suggesting a potential immunogenetic role in pathogenesis. Schizophrenia has been linked to increased levels of peripheral inflammation, and immune biomarkers predict worse outcomes in patients with the disorder. However, it is unclear whether inflammation is a causal factor or a consequence of the disease process, and to what extent immune-regulating genes play a role. This work will investigate the joint role of immune biomarkers and genetics in schizophrenia in a large biobank linked to electronic health records (EHRs). First, a repository of EHRs with longitudinal clinical laboratory data will be used to examine the temporal relationship between immune biomarker levels and schizophrenia diagnoses. Within schizophrenia spectrum patients, these biomarker levels will be assessed for their ability to predict specific clinical outcomes. Second, longitudinal cohorts paralleling those proposed in Aim 1 will be constructed but incorporating whole-genome genotyping data from a subset of ~90,000 individuals that are a part of the BioVU, VUMC’s biorepository resource. Genetic predictors of immune biomarkers will be included in these models and evaluated for their relative contributions to schizophrenia risk and their ability to predict clinical outcomes. Mediation analyses will then be conducted to investigate causal pathways linking C4 CNV and immune biomarker alterations in schizophrenia in BioVU. These analyses will be replicated and meta- analyzed in additional well-powered EHR-linked biobanks from the Million Veteran Program and All of Us research program. The proposed studies will elucidate the poorly understood relationships between immune-regulating genetic risk factors, immune biomarkers, and clinical heterogeneity in schizophrenia spectrum disorders. Understanding these relationships will yield mechanistic insights and uncover potentially clinically useful biomarkers of schizophrenia disease course.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY Although the ability of glucagon-like peptide-1 receptor (Glp1r) agonists to stimulate insulin secretion and reduce caloric intake has been recognized for over two decades, surprisingly little is known about the molecular mechanisms behind these effects. We have previously shown that activation of the hypothalamic Glp1r reduces food intake by engaging key nutrient sensing mechanisms such as mechanistic Target of Rapamycin Complex-1 (mTORC1). Since mTORC1 is also an important regulatory component of -cell function, this suggests that elucidating how Glp1r agonists regulate mTORC1 and its downstream targets will address a key knowledge gap about the mechanism of action of an important class of diabetes and obesity drugs. We have identified a novel interaction stimulated by the clinically relevant Glp1r agonist liraglutide (Lira) whereby the canonical target of Glp1r signaling, cAMP-dependent protein kinase A (PKA), phosphorylates the mTORC1 regulatory protein Raptor resulting in increased mTORC1 signaling. We have also identified the transcription factor Hypoxia- Inducible Factor (HIF) as a target of Glp1r signaling. This is relevant since HIF stimulates glycolysis, a mechanism necessary for the anorectic and insulinotropic effects of Glp1r agonists, and increased HIF expression in the hypothalamus and -cells reduces food intake and stimulates insulin secretion, respectively. We also provide preliminary data showing that Lira no longer reduces body weight or glucose levels in novel knockin mice replacing endogenous Raptor with a PKA-resistant Raptor. Our preliminary data, therefore, lead us to hypothesize that a Glp1r-PKA-mTORC1-HIF-glycolysis axis in the hypothalamus and -cells mediates the ability of Lira to reduce body weight (Aim 1) and stimulate insulin secretion (Aim 2), respectively. The clinical relevance of this is further emphasized by our preliminary data showing that two variants of the Glp1r found in humans that are associated with improved cardiometabolic outcomes and improved responsiveness to Lira also stimulate mTORC1 signaling to a greater degree than wild-type Glp1r. We will, therefore, use mice expressing these human Glp1r variants to test the hypothesis that Lira promotes greater weight loss and improved glucose tolerance in these mice via enhanced mTORC1 signaling (Aim 3). We will complete these Aims by leveraging our extensive expertise in assessing metabolic phenotypes in mice, including real-time measurements of energy balance parameters as well as pancreatic function in isolated islets and in vivo using hyperglycemic clamps. We will apply these approaches to a suite of novel mouse models that allow us to modulate or measure the expression and activity of target proteins in specific cell types. Accomplishing these Aims will delineate specific molecular mechanisms that can be leveraged towards either the improvement of the effectiveness of Lira or the design of more efficient weight-lowering drugs.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY. Loss of pancreatic β cell function and/or mass is central to the development of type 2 diabetes (T2D). Understanding how β cell function is normally regulated in adult human islets will help elucidate mechanisms of dysfunction in T2D, which are not well understood. A large body of work in mouse models suggests that the islet-enriched transcription factor (TF) NKX2.2 is a critical regulator of β cell development and plays a role in the maintenance of adult β cell function. Further, patients with loss-of-function NKX2-2 mutations have neonatal diabetes, highlighting an important role of NKX2.2 in human islet development. However, the role of NKX2.2 in adult human β cells remain undefined. Interestingly, we found increased insulin secretion from primary human pseudoislets following global NKX2-2 knockdown, suggesting different roles of NKX2.2 across species and developmental stages. We hypothesize that, in adult human islets, NKX2.2 regulates insulin secretion via transcriptional repression of β cell-intrinsic pathways. To test this hypothesis, we will first determine the role of NKX2.2 in adult human islet function in a β cell-specific manner. Using florescence-activated cell sorting and CRISPR/Cas9 technology, we will perform targeted knockout of NKX2-2 in adult β cells in primary human pseudoislets. We will assess β cell intracellular Ca2+ signaling events and function in vitro using an integrated live cell imaging and microfluidic platform. To evaluate the impact of chronic loss of NKX2.2, we will examine pseudoislet function in vivo following transplantation into immunodeficient mice. Results of this aim will determine the impact of NKX2.2 on β cell-intrinsic pathways that lead to insulin secretion. Secondly, we will define molecular mechanisms of NKX2.2 function in adult human β cells using a single nucleus (sn)RNA-seq+ ATAC-seq multiome approach on the same nucleus. snRNA-seq will determine if NKX2.2 functions as a transcriptional repressor of insulin secretory machinery in β cells. In combination, snATAC-seq will reveal how NKX2.2 alters chromatin accessibility to regulate the β cell transcriptome. To study the impact of chronic NKX2-2 knockout on β cell phenotype and function, we will analyze harvested pseudoislet transplants for changes in proteins corresponding to top differentially expressed genes of interest. This aim will provide mechanistic insight into how NKX2.2 regulates adult human β cell gene transcription and function at the chromatin and transcript level. Overall, these studies will reveal molecular mechanisms of NKX2.2 function in adult human β cells, with implications for new therapeutic approaches to improve β cell function in T2D. Training under this fellowship will be enhanced by a rich environment, including a large community of islet biology investigators under the NIH-funded Vanderbilt Diabetes Research and Training Center, collaborations with experts in the field, and a variety of opportunities to promote career development, leadership, and scientific communication. Together, the proposed research, training plan, and environment will provide a strong foundation on which to base a career as a physician-scientist.

NIH Research Projects · FY 2025 · 2022-12

Summary With the advent of exome sequencing, a growing number of children are being identified with de novo loss of function mutations in the large GTPase essential for mitochondrial fission - Dynamin Related Protein 1 (DRP1); these mutations result in severe neurodevelopmental phenotypes such as developmental delay, optic atrophy, and epileptic encephalopathies. Though it is established that mitochondrial fission is an essential precursor to the rapidly changing metabolic needs of the developing cortex, it is not understood how identified mutations in different domains of DRP1 uniquely disrupt this process. F-actin and the endoplasmic reticulum (ER) form a complex to prime the mitochondria for fission by pre-constricting the mitochondrial membrane prior to formation of DRP1 oligomers. The effect of DRP1 mutations on protein interactions with F-actin and the ER has never been studied in cell types of the developing cortex. This proposal focuses on testing the mechanism of DRP1 dysfunction both on protein interactions at sites of fission as well as downstream effects on cortical neuron differentiation and maturation. We aim to approach these gaps by leveraging the power of induced pluripotent stem cells (iPSCs) harboring DRP1 mutations in either the GTPase or stalk domains to model cell-fate changes associated with early cortical development. We will functionally assess the capacity for these iPSCs with mutant DRP1 to adopt a neural progenitor fate and progress to active cortical neurons using quantitative analysis of neurite outgrowth and branching, calcium transient recording, and synchronous synaptic firing. To understand how mutant forms of DRP1 interact with F-actin and the ER during fission, we will use live super-resolution Airyscan microscopy paired with in-cell immunoprecipitation to capture changes in the assembly and disassembly of this fission machinery. Successful completion of these aims will improve our understanding of the role of mitochondrial fission during cortical development and at which stages of this process perturbations become highly pathogenic. Furthermore, these results could help shed light on variable patient symptoms and outcomes based on specific DRP1 mutations, possibly leading to individualized therapeutics for mitochondrial disease.

NIH Research Projects · FY 2026 · 2022-12

PROJECT SUMMARY Alzheimer’s disease (AD) is a progressive neurodegenerative condition with profound impacts on memory and cognition that currently affects more than 6 million adults living in the United States. Treatment options for Alzheimer’s remain limited, with high rates of failure in AD drug trials. Drug repurposing to identify new uses for existing drugs offers several advantages over traditional drug development, including higher success rates, lower costs, shorter timelines, and increased assurance of safety. Furthermore, drugs with genetic support of efficacy have been found to be twice as successful in clinical development. This work leverages new developments in genetics and informatics to propose two independent approaches to identify drug repurposing candidates for AD: (1) a virtual transcriptome approach identifying drugs that can reverse gene expression changes observed in AD, and (2) a Mendelian randomization approach identifying drugs acting on genes found to be causally associated with AD. Identified repurposing candidates will be clinically validated using electronic health record (EHR) data from Vanderbilt University Medical Center, as well as external datasets (clinical data from the NIH All of Us Research Program Database or claims data from the Centers for Medicare & Medicaid Services). In AIM 1, we will develop a virtual transcriptomic signature for AD using GWAS summary statistics, and query this signature against large-scale drug perturbation databases. In AIM 2, we will use conditionally independent genetic variants as instrumental variables to perform Mendelian randomization on AD GWAS, and evaluate for causal relationships between actionable druggable genes and AD. Both aims will involve further validation of repurposing candidates using real-world clinical data, which has seldom been done before. Successful completion of this project will yield novel insights into high- priority drug repurposing candidates for AD that can be further investigated in randomized clinical trials, and will establish a high-throughput AD repurposing pipeline integrating genetic, -omics, and EHR data that may be adapted to other diseases of interest.

NIH Research Projects · FY 2025 · 2022-12

PROJECT ABSTRACT Sepsis is a critical problem around the world causing 20% of all global deaths. The lack of highly effective therapeutics leaves critically ill patients with systemic organ dysfunction often caused damage the vascular endothelium. Our lab has shown that one of the drivers of vascular dysfunction in sepsis is circulating oxidized cell-free hemoglobin (CFH). During septic conditions, red blood cells become increasingly fragile leading to lysis and release of CFH into the vascular circulation allowing for oxidation from ferrous (2+) and oxidized ferric (3+, methemoglobin) forms. Data from our lab demonstrates that only the oxidized 3+ form of CFH induces microvascular barrier dysfunction. However, the intracellular mechanisms underpinning this dysfunction are not well understood. My preliminary studies suggest that oxidized CFH causes mitochondrial dysfunction such as increased superoxide production and loss of total mitochondria. In parallel to circulating CFH being increased in septic patients, there is increased circulating extracellular mitochondrial DNA (mtDNA) during sepsis. Importantly, the mechanism underlying release of mtDNA during sepsis remains a key knowledge gap. In addition, it is unknown whether the mtDNA is released as freely soluble molecules or if it is inside extracellular vesicles (EVs). Understanding if mtDNA is contained inside EVs could inform potential effects, distribution, and stability of the circulating mtDNA. In this project, I will test the hypothesis that CFH-induced oxidative damage causes mtDNA release from the vascular endothelium leading to downstream loss of macrovascular barrier integrity. The first aim of this project focuses on identifying the mechanisms behind CFH-induced oxidative damage and its role in mtDNA release from the pulmonary microvascular endothelium. We will evaluate the impact of CFH on mitochondrial oxidative damage and permeability pore activation. In addition, we will quantify whether the mtDNA is freely soluble or contained inside EVs, and if antioxidants block this secretion. The second aim of the project will determine the effect of mtDNA on endothelial barrier function. We will also use platelet poor plasma from a highly characterized prospective cohort of sepsis patients to quantify circulating mtDNA and correlate levels with mortality, ARDS development, and markers of endothelial damage. At the conclusion of this study, we will have uncovered a novel molecular mechanism of CFH induced vascular dysfunction, and characterized both the effects of mtDNA and how it is released from endothelial cells. This proposed project will provide multidisciplinary experience and growth to establish the foundation for a successful career as a mechanistic and translational scientist.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY The innate immune system can be pharmacologically programed to elicit desired immunological outcomes. Retinoic acid-inducible gene I (RIG-I) is a pattern recognition receptor that has emerged as a promising innate immune target for immunopotentiation. RIG-I is activated upon recognizing 5’-triphosphorylated, double- stranded RNA (3pRNA) in the cytosol, which stimulates an antiviral-like inflammatory program that can be harnessed to treat or prevent a diversity of diseases. However, the potency and efficacy of 3pRNA has been limited by major drug delivery barriers, including nuclease degradation, inefficient cellular uptake and cytosolic delivery, and rapid clearance. To address these challenges, we have developed RIG-I activating nanoparticles (RANs). RANs are polymer nanoparticles that are engineered to promote the cytosolic delivery of synthetic, molecularly-defined, and high-affinity stem-loop RNA (SLR) RIG-I agonists recently developed by our team. The objective of this R01 application is to optimize and advance RANs as a versatile platform for pharmacological activation of RIG-I. We will accomplish this through the following Specific Aims. First, we will engineer next-generation RANs with improved properties for systemic administration through optimization of SLR and polymer charge and hydrophobicity. This approach will leverage combinatorial chemical diversity to access a new design space for 3pRNA delivery, which we expect will yield next-generation RANs with higher SLR loading efficiency, reduced cytotoxicity, protection from nuclease degradation, improved stability, and enhanced immunostimulatory activity. Second, we will establish relationships between RAN properties, innate immune activation, pharmacokinetics, polymer and SLR biodistribution, and toxicity. These studies are essential in the preclinical development of new immunotherapeutic modalities and will also yield new insight into how nanocarriers can be engineered for safe and effective activation of RIG-I. We expect these studies to yield next-generation RANs that are optimized for systemic administration of SLR therapeutics. Third, while RANs have broad potential clinical applications, we will evaluate their efficacy as a systemically administered cancer immunotherapy in poorly immunogenic mouse models of melanoma as a clinically important test case. We expect to demonstrate that systemic administration of lead-candidate RANs will activate RIG-I in the tumor microenvironment, resulting in an immunological reprograming of tumor sites that inhibits tumor growth and synergizes with immune checkpoint inhibitors. Collectively, these studies will position RANs as an enabling platform for immunopotentiation with potential to address the significant need for new cancer immunotherapies, antiviral agents, and vaccine adjuvants.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY The mapping of the human genome and genome wide association studies have provided great insights in our understanding of the genetic etiology of hereditary diseases; however, critical gaps remain. A type of genetic variations that has been difficult to detect in genomic studies has been Structural Variants (SVs), disruptions involving more than 50 base pairs. SVs have been implicated in a lot of inherited diseases and cancers, yet their detection remains challenging with conventional DNA sequencing methods. Developments in third- generation sequencing (linked-read and long-read sequencing) and single-cell RNA sequencing (scRNA-seq) provide an opportunity to greatly improve the detection of SVs and Copy Number Variations (CNVs), one common type of SVs. However, existing computational tools do not fully take advantage of the potential and the opportunities that these technologies offer. In this project, drawing from our unique expertise in this rapidly evolving area, we propose the development of a new generation of tools that will improve greatly the detection and phasing of SVs from a large population of samples. We will develop computational tools to generate a high-quality diploid assembly from each individual and to combine data from large populations of controls and patients to characterize SVs that confer risk for any particular disease. We will further design a haplotype- based linkage disequilibrium (LD) mapping approach at the whole genome scale to identify unique sharing haplotype patterns and provide a new perspective for complex disease studies. Detecting SVs in combination with small variants will further allow us to explain the etiology of complex diseases. We will also develop algorithms to detect CNVs from scRNA-seq datasets, which have application in cancer studies. Successful completion of this project will constitute a major step forward in uncovering the genetic cause of complex diseases and cancers.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Early life environments can have profound and long-lasting effects on human health, yet the mechanistic basis of these effects remain poorly understood as do the factors that explain inter-individual variation. These gaps in knowledge severely limit our ability to both predict susceptible individuals and to develop effective intervention strategies. At the molecular level, early life effects on later life health are thought to be mediated by stable changes in gene regulation. However, many gene regulatory elements are responsive to environmental stimuli throughout life, making it challenging to isolate the effects of early life conditions or to understand their stability, especially when within-lifetime environmental variation is absent or longitudinal data are not available. My research program aims to overcome these challenges by working with two subsistence-level groups: 1) the Turkana of Kenya, who are experiencing rapid lifestyle change such that early life and adult conditions are largely decoupled and 2) the Tsimane of Bolivia, who have been followed longitudinally for decades. In both groups, I will generate genome-wide gene regulatory datasets (DNA methylation, gene expression) paired with information on environmental experiences and health, allowing me to identify the mechanisms that embed early life exposures into lifelong physiology. I will also generate genome-wide genotype data for each individual, in order to ask whether genetic variation predicts sensitivity versus resilience to early life challenges at the gene regulatory level. Finally, I will complement this field-based, observational work with lab-based methods capable of testing for causal connections between 1) DNA methylation and gene expression and 2) genotype and environmentally-induced gene expression, both in high-throughput. In doing so, this project will uncover the gene regulatory mechanisms that mediate early life effects on health, as well as the degree to which these relationships are modified by genetic variation. By working “in the field” and “in the lab”, the proposed work will inform our understanding of developmental and environmental processes in natural human populations as well as causal mechanisms. More broadly, the proposed projects will link a major global phenomenon—increasing urbanization and market-integration—with gene regulatory processes and health in two understudied and underserved populations (Africans and Amerindians). It will also generate new methods and resources that will inform our understanding of the general patterns of genotype x environment interactions in human complex traits.