Vanderbilt University Medical Center

NIH Research Projects · FY 2025 · 2022-08

Hemodialysis treatment non-adherence is a public health issue because of its association with excessive hospitalizations, high morbidity, and mortality, and increased financial costs. Compared to whites, African Americans have a four-fold higher prevalence of end-stage kidney disease (ESKD), higher non-adherence rates to hemodialysis, and higher odds of hospitalizations. Motivational interviewing, an evidence-based intervention that creates a bond between patients and providers, targets improvement in motivation-related psychosocial factors associated with adherence behaviors. Interventions for such factors are typically developed based on the dominant population and may not be valid and generalizable. Tailored interventions lead to more durable change in African Americans yet there is a lack of studies testing the efficacy of such approaches to improve hemodialysis treatment adherence in African Americans. Use of tailored motivational interviewing in African Americans with ESKD will promote population health by improving dialysis treatment adherence, reducing hospitalizations, and enhancing other critical outcomes in kidney disease, to curb the chronic disease crises. Our long-term goal is to establish tailored strategies and multi-level interventions to improve outcomes in kidney disease. The overall objective of this project is to evaluate the efficacy of a tailored motivational interviewing intervention developed using a rigorous theoretical framework on improving hemodialysis treatment adherence in African Americans with ESKD. The central hypothesis is that tailored motivational interviewing will lead to improved hemodialysis treatment adherence. We will test this hypothesis in the following Specific Aims in a randomized clinical trial (RCT) in African American patients with ESKD. Compared to usual dialysis care, we aim to: Evaluate the efficacy of 8 weeks of tailored motivational interviewing (MOVE) on improving hemodialysis treatment adherence at (1) 3 months, and (2) 6 months post- randomization. At the successful completion of the proposed research, the expected outcomes will include evidence of the efficacy of tailored motivational interviewing on improving hemodialysis treatment adherence in African American patients with ESKD. The proposed research is innovative because of the novel application of a tailored, evidence-based behavioral intervention developed using a rigorous theoretical framework (PEN-3); the use of specifically-trained health coaches to optimize intervention delivery; and the focus on African American patients who are overrepresented in the ESKD patient population, to address the public health issue of hemodialysis treatment non-adherence. Study results will provide a strong basis for conducting an effectiveness and implementation trial, which is expected to have a significant impact on hemodialysis adherence, hospitalizations, morbidity, and mortality. This research strongly aligns with NIDDK’s mission to promote population health by using innovative strategies to optimize key outcomes in kidney disease and curb the chronic disease crises.

NIH Research Projects · FY 2025 · 2022-08

Developmental stuttering commonly emerges between 24-60 months of age with the majority of these children recovering from stuttering. For the remaining children, persistent stuttering into school-age years and adulthood confers significant risk for adverse impact on social-emotional, educational, and vocational outcomes. Although over the past years a variety of risk factors for stuttering persistence have been identified (e.g., stuttering severity, sex, age at onset, time since onset, articulation, language ability), there is still a critical need to optimize the accuracy with which stuttering persistence can be predicted. To date, predictive models have rarely considered the role of emotion; however, our preliminary data suggest that it plays a major role in stuttering persistence. Specifically, our cross-sectional work has demonstrated that cortical and autonomic markers of emotional reactivity and emotion-related cognitive control vulnerabilities in children who stutter (CWS) contribute to stuttering and are associated with persistence (pilot data). We recently extended this work and developed a novel methodology to test the effects of emotional reactivity on speech preparation and production in young children at risk for persistence. Based on our findings to date, the central hypothesis of the proposed project is that emotional reactivity plays a major role in stuttering persistence by interfering with both non-speech cognitive control (e.g. inhibition and execution) and speech preparation and production processes necessary for the early development of speech fluency and thereby confers heightened risk for stuttering persistence. To test this hypothesis, we will conduct a longitudinal study of young (3- to 4-year old) CWS. Annual lab visits will occur for 3 years from study enrollment and will involve a comprehensive stuttering assessment, a speech-language, cognitive, and temperament diagnostic battery as well as the systematic assessment of emotional reactivity, cognitive control, and speech preparation and production processes. The specific aims of the project are to: (1) determine if cortical and autonomic biomarkers of emotional reactivity predict outcome (persist versus recover) for CWS, (2) determine if emotion- related performance during a non-speech cognitive control task and a speaking task predicts outcome (persist versus recover) for CWS, and (3) determine whether markers of emotional contributions to stuttering provide additive predictive value when combined with other established variables associated with stuttering persistence. If successful, the proposed project addresses the continued clinical need to identify markers of risk for stuttering persistence and improve the accuracy of predictive models. These advances will allow clinicians to better pinpoint targets for assessment, set the stage for novel therapeutic approaches, and allow researchers to better evaluate the effects of early intervention due to an improved ability to distinguish persistent from transient cases. Thus, the proposed research supports the mission of NIDCD by discovering new knowledge that has the potential to improve outcomes of young children who stutter.

NIH Research Projects · FY 2024 · 2022-08

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Over the past decade, the federal government has spent more than $34 billion on the meaningful use of electronic health records (EHRs). However, the acceptance rate for clinical decision support (CDS) alerts, a critical component of EHRs, is less than 10%. The large number of low relevance alerts (e.g. a weight loss alert during a cardiac resuscitation) not only increases the burden on clinicians, but can lead to the onset of alert fatigue, resulting in the neglect of important alerts and posing a serious threat to patient safety. Currently, alerts are improved primarily through manual review and by collecting user feedback. However, these methods are labor intensive and do not allow for a timely analysis of user responses to alerts from a comprehensive aspect. The amount of alert log data is large; Vanderbilt University Medical Center generated over 3 million alert firings in 2020. There is an urgent need to utilize the data from the alert log and EHR to develop a data-driven process to generate suggestions for refining alert logic or improving clinical processes. To address this gap, I propose to use explainable artificial intelligence (XAI) combined with bias mitigation techniques to build predictive models that comprehensively learn user responses to alerts and in turn automatically generate responsible suggestions to improve the original logic of alerts. In the K99 Phase, I developed a standards-based taxonomy of features that affect user response to CDS alerts (Aim 1) and a data-driven process to generate suggestions for improving alert criteria using XAI approaches (Aim 2). In this R00 phase, I will evaluate generated suggestions using a mixed-methods design (Aim 3). I will use XAI approaches developed in Aim 2 to generate suggestions. I will ask CDS experts to review the AI-generated suggestions as well as human-generated suggestions for improving the same CDS alerts and rate the suggestions for their usefulness, acceptance, relevance, understanding, work- flow, bias, inversion, and redundancy. Throughout this research, I expect to produce a set of expert-validated suggestions based on the XAI approaches. This study could significantly contribute to the improvement of CDS management and clinical processes. My career development plan and the proposed research are aligned with my current skills and experiences in CDS and machine learning. Overall, this project can help me launch an independent research career in developing explainable, intelligent CDS tools to improve patient safety, provide standardized care, and promote an equitable and efficient healthcare system.

NIH Research Projects · FY 2025 · 2022-08

Primary graft dysfunction, a severe form of acute lung injury, occurs in 20-30% of lung transplant recipients and is a major determinant of both short- and long-term outcomes. Risk of PGD is affected by clinical features of both the donor and recipient as well as by operative management. However, the cellular mechanisms that underlie risk of PGD are incompletely understood and new studies of mechanisms contributing to PGD are essential to development and testing of specific therapies to mitigate PGD risk. Our published and preliminary data suggest that cell-free hemoglobin (CFH) is a major causal factor in the alveolar-capillary disruption that leads to the characteristic development of pulmonary edema in PGD. In a pilot case-control study, we showed that elevated recipient pre-operative plasma CFH is independently associated with increased PGD risk. In a human ex vivo lung perfusion model, CFH in the perfusate caused increased microvascular permeability by oxidative injury to the lung endothelium. Similarly, elevated levels of intra-alveolar CFH are associated with severe lung injury in critically ill patients and intra-bronchial instillation of CFH into ex vivo human lungs injures the lung epithelial barrier and impairs alveolar fluid clearance. New preliminary data show increased CFH in the airspace of donor lung allografts. In this proposal, we will determine how peri-operative management may magnify the impact of CFH on PGD. Cardiopulmonary bypass (CPB) and extracorporeal membrane oxygenation (ECMO) increase hemolysis and release of CFH. Although ECMO has been associated with lower PGD risk than CPB, it is unclear whether this is explained by alterations in CFH. Higher driving pressure during mechanical ventilation may also increase CFH and alveolar-capillary barrier dysfunction. Furthermore, increased FiO2 at reperfusion augments the association between CFH and PGD and hyperoxia exacerbates CFH-induced lung injury in ex vivo human lungs. This strong preliminary data supports the concept that peri- operative management affects PGD by modulating accumulation and oxidation of CFH. In this proposal, we will establish a three-site consortium to test the hypothesis that CFH causes PGD via oxidative injury to the lung endothelial and epithelial barriers. We will also determine how modifiable risk factors including mechanical support and hyperoxia at reperfusion increase accumulation and oxidation of CFH, thereby increasing risk of PGD. There are three specific aims: 1) test the independent effects of intravascular and intra-alveolar CFH on risk of PGD and injury to the endothelial and epithelial barriers, 2) determine how peri-operative factors affect intravascular and intra-alveolar CFH accumulation, and 3) test how CFH oxidation by intra-operative hyperoxia increases risk of PGD. Completion of this large multicenter cohort study of lung transplant recipients will provide novel insight into the relative contributions of intravascular and intra-alveolar CFH to PGD and identify modifiable factors that alter CFH accumulation and oxidation, providing the necessary foundation for development of clinical trials to mitigate PGD with CFH-targeted interventions.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Human and murine lung development requires the coordinated efforts of the lung epithelium with the surrounding extracellular matrix (ECM), but the ECM-directed mechanisms that govern epithelial cell behavior in the lung remain undefined. In epithelial tissues, integrins serve as receptors for the basement membrane components collagen and laminins (LMs), with LMs being the most important ECM protein for lung organogenesis. Lung epithelial cells bind to LMs through integrins α3β1, α6β1, and α6β4. Recent studies reported that mutations in these integrins cause pulmonary hypoplasia or neonatal emphysema complicated by abnormal airways, suggesting they play a major role in human developmental lung diseases. To investigate the role of LM-binding integrins, we generated lung epithelial specific integrin deletions. Deletion of both the β1 and α6 subunits resulted in marked branching defects and early death. Deletion of α3 caused only minor airway branching disruption. β4 deficient mice exhibited normal branching but, surprisingly, were also perinatal lethal. The β4 histological examination was notable for proteinaceous material filling the airways and lack of cilia, similar to α6 deficient mice, suggesting their demise resulted from airway dysfunction. Pathway analysis of α6 deficient lung sequencing data revealed disruptions in BMP signaling, a critical pathway for airway branching. BMP receptor expression was increased in α6-null epithelial cells, but BMP target gene expression remained markedly reduced, implicating α6-containing integrins in regulation of the BMP pathway in the fetal lung. Consistent with loss of cilia in β4 deficient mice, β4-null epithelial cells exhibited reduced expression of transcription factors linked to MCC terminal differentiation. As a critical component of hemidesmosomes, α6β4 controls tight adhesion to the basement membrane and connects with the intracellular keratin intermediate filaments. Keratin also forms a support network apically for cilia, suggesting that α6β4 regulates keratin organization critical for terminal differentiation of MCCs in the lung. Taken together, these findings indicate that: 1) α6β1 is the principal integrin required for airway branching likely through BMP signaling and 2) α6β4 regulates terminal differentiation of MCCs. Based on preliminary data, we propose the hypothesis that α6-containing integrins are critical integrins for fetal lung development through regulation of BMP signaling during airway branching and terminal differentiation of multi-ciliated epithelial cells. AIM 1: Determine the mechanisms whereby α6-containing integrins regulate lung branching morphogenesis. AIM 2: Define the mechanisms whereby α6-containing integrins regulate BMP signaling during fetal lung development. AIM 3: Identify the role of α6β4 integrin in airway epithelial cell differentiation.

NIH Research Projects · FY 2025 · 2022-08

Project summary Postural Tachycardia Syndrome (POTS) affects ~3 million adults in the United States. These patients have a poor quality of life due to chronic presyncopal symptoms and tachycardia that occur upon standing. Our research has shown that meals rich in carbohydrates significantly exacerbate presyncopal symptoms in POTS, however, the underlying mechanism that explains this clinical observation remains unknown. Accordingly, our group conducted a preliminary study to evaluate the pathophysiology of POTS’ excessive orthostatic tachycardia after glucose intake; we surveyed the hemodynamic and neurohormonal changes that occurred after a 75-gr oral glucose challenge for up to 2-hrs (postprandial period) in POTS patients and healthy controls. Compared with fasting conditions, the ingestion of glucose worsened upright tachycardia in POTS patients, which was associated with a more robust reduction in upright stroke volume compared with healthy controls. With regards to the disproportionate decrease in upright stroke volume in POTS patients, this could, in part, be explained by a significant blood pooling in the splanchnic circulation. The splanchnic circulation is the largest blood volume reservoir of the human body, storing ~25% of the total blood volume. Upon standing, there is a significant blood pooling, which occurs mostly in the splanchnic veins. Finally, our study has also shown that 30-min after the ingestion of 75-gr of glucose, POTS patients had a selectively increased secretion of the glucose-dependent insulinotropic polypeptide (GIP) hormone compared with healthy controls. This hormone has vasodilatory properties in the splanchnic circulation. Importantly, the increase in GIP secretion was time-dependently associated with a fall in upright stroke volume after glucose intake in POTS. Consequently, these findings point to the potential contribution of GIP in the pathophysiology of the increased postprandial orthostatic tachycardia and presyncopal symptoms in POTS patients. As such, the overall goal of this proposal is to investigate the mechanisms underlying the exacerbation of orthostatic tachycardia and POTS presyncopal symptoms in response to glucose ingestion. Specifically, we will evaluate the contribution of GIP on the changes in the splanchnic venous capacitance after oral glucose and during upright posture in POTS patients.

NIH Research Projects · FY 2025 · 2022-08

Abstract Despite advances in operative technology, intraoperative methods for primary tumor and lymph node detection have not changed in the past 30 years. To this end, we propose to use a fluorescent and nuclear labeled anti-EGFR antibody (panitumumab) for targeted dual modality imaging (TDMI) of the primary tumor and lymph nodes in head and neck squamous cell carcinoma (HNSCC) In this application, we propose to use labeled panitumumab since it has been successful in several early-stage studies, we introduce two novel concepts for intraoperative detection of very small (1 mm3) tumor deposits. First, we combine the high-resolution/depth-limited imaging properties of optical imaging agents with the low-resolution/not depth-limited properties of nuclear agents. Second, we show that these two agents can be systemically administered for the detection of tumor-positive lymph nodes. We provide extensive clinical data in HNSCC to demonstrate that tumor fragments less than 1 mm3 in size can be detected using anti-EGFR antibody (panitumumab) as the targeting molecular to fulfill the needs of PAR-20-295 entitled, ‘Optical fluorescent methods and technologies for sensitive cancer detection in vivo’. While use of anti-EGFR antibodies for imaging alone is not innovative, innovation should be considered in the context of PAR- 20-295 which requires applications use “…probes with previously demonstrated capabilities for the detection of small tumors”. To this end, we introduce dual modality imaging and leveraging this for molecular imaging lymph nodes. This study is designed to meet FDA guidelines for a surgical imaging trials. Patients eligible for head and neck cancer resection will receive a systemic administration of an anti- EGFR antibody (panitumumab) labeled with either IRDye800 (pan800) or 111Indium (111In-pan) for fluorescent and nuclear signal, respectively. After systemic administration of both agents, a SPECT/CT will be performed to identify the location of tumor-positive margins and tumor-positive lymph nodes. The patient will then undergo resection of the primary tumor and neck dissection using fluorescent and gamma probe guidance (while maintaining the standard of care). We hypothesize that introduction of targeted dual modality imaging (TDMI) will improve intraoperative decision making.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Candidate: Dr. Erin Wilfong, M.D., Ph.D. is a Clinical Instructor at Vanderbilt University Medical Center in the Divisions of Allergy, Pulmonary, & Critical Care Medicine and Rheumatology & Immunology. She has a strong clinical background in both rheumatology and pulmonology with an excellent scientific foundation in chemical biology and translational immunology from both her Ph.D. studies and her post-doctoral research studies following clinical training. Her long-term career plan is to become a leading physician-scientist in inflammatory myositis focused on the use of cutting-edge technologies to identify disease pathogenesis and novel therapeutic targets. To achieve this, her immediate goal is to (1) learn the requisite bioinformatic techniques to independently analyze high-dimensional immunophenotypic and transcriptional profiles and integrate these profiles with clinical outcomes, and (2) apply molecular immunology techniques to determine dysregulated pathways in IIM-specific cell populations. Research Project: This proposal will study the underlying immunologic heterogeneity of IIM and identify associations between immune/transcriptional signatures and clinical characteristics. Dr. Wilfong will (1) identify immune signatures and transcriptional aberrancies in IIM using a cross-sectional IIM cohort and (2) correlate immune features with ILD progression in a longitudinal IIM cohort. She will also take a prior single-cell RNA- Seq finding and (3) perform mechanistic studies investigate how upregulation of the redox sensor TXNIP alters immunometabolism in Jo1+ anti-tRNA synthetase syndrome. Career Development: Dr. Wilfong’s career development plan integrates formal coursework with personalized training from her mentors and collaborators to: (1) master R programming to facilitate analysis of immunophenotypic and transcriptional datasets using cutting-edge analytic tools, (2) learn biostatistical principles necessary to appropriately correlate immunology findings to clinical phenotypes, (3) characterize cellular function using in vitro assays, and (4) develop the leadership and communication skills necessary to become a principal investigator who applies the scientific findings and methodologies from this K08 award towards a future translational immunology R01. Environment: VUMC is the ideal environment to foster Dr. Wilfong’s development as a leader in translational immunology and inflammatory myositis. Dr. Wilfong’s mentoring team includes experts in inflammatory myositis (Crofford/Aggarwal), immunometabolism (Rathmell), single-cell RNA-Seq (Kropski), and computational immunology (Georgiev). In addition to having the necessary mentors and equipment, 71.3% of Vanderbilt University Medical Center career development award recipients (n=236) have successfully received R level funding since 1999. Drs. Crofford and Rathmell have successfully trained many physician-scientists and will ensure that Dr. Wilfong becomes an independently funded physician scientist.

NIH Research Projects · FY 2025 · 2022-07

SUMMARY: Eastern equine encephalitis virus (EEEV) is a re-emerging mosquito-borne alphavirus that causes a debilitating encephalitic illness in humans. About a third of human cases of EEEV infection die and many survivors have long-term, debilitating neurologic problems. The virus is maintained in an enzootic cycle between Culiseta melanura mosquitoes and avian hosts but can be transmitted to humans and horses by some Aedes, Coquillettidia, and Culex species. The infection is unusual in humans but increasing in frequency in recent years, likely secondary to climate changes, vector expansion, and other uncharacterized factors. EEEV also is regarded as a potential bioterrorism threat due to spread via aerosol route. Despite the highly pathogenic nature of the virus, no specific treatment or vaccine for EEEV is available. A primary goal of this project is to define the molecular, genetic, immunologic, and structural characteristics of ultra-potent neutralizing human mAbs with broad activity in vivo against EEEV. Additional goals include defining the mechanistic correlates of protection by these ultra-potent neutralizing mAbs and determining ways to optimize function and deliver to the brain. In these studies, we will elucidate how antiviral Abs with exceptional inhibitory activity exert their action in cell culture and in vivo. The approach will include high efficiency isolation of human mAbs, coupled with innovative antibody gene repertoire studies based on next-gen sequencing. Several hypotheses will be tested, including the concept that ultra-potent neutralizing activity results from features of both the antibodies (selection of optimal V-D-J clonotypes and accumulation of critical somatic mutations) and the antigen (binding to quaternary epitopes on multiple adjacent envelope proteins and blockade of structural transitions critical for virus entry or release). We also will apply new technologies for receptor-mediated transfer of molecules across the blood-brain barrier using engineered sequence changes in the Fc region. Although our focus is to understand how and why ultra-potent human mAbs inhibit EEEV, the studies likely will be relevant to general principles of antibody neutralization of many different encephalitic viruses. In addition to defining the molecular and structural basis of Ab neutralization of EEEV and deploying new strategies for delivery of biologics to the brain, these studies will generate a group of fully human mAbs that can prevent and treat EEEV infection, which could be developed in the near future as a possible therapeutic for humans. Studies in this project, while targeted against EEEV, likely will inform future Ab-based and/or vaccine efforts against other arboviruses that cause human brain infections. We have assembled a unique group of investigators, including a human Ab expert, a molecular virologist with experience in Ab-virus interactions, an animal model and pathogenesis expert with specific expertise in encephalitic alphaviruses, including EEEV, and brain-targeting scientists to pursue these studies.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT Despite significant advances in the care of pediatric heart transplant (PHTx) patients, acute rejection (AR) remains one of the leading causes of death. Cardiac catheterization with endomyocardial biopsy (biopsy) is the standard of care for diagnosing AR and is performed when there is a clinical suspicion for AR or during routine surveillance. Unfortunately, biopsy is invasive and associated with potential risks, including: complications from anesthesia or sedation, valve damage, injury to the conduction system, vascular damage or occlusion, and cardiac perforation. These potential complications are magnified in the pediatric population. Non-invasive methods of detecting AR, such as blood biomarkers and cardiac magnetic resonance imaging (CMR), could decrease the frequency of biopsy. Blood biomarkers, such has donor fraction cell-free DNA and microRNA, have shown potential for diagnosis of AR but have not yet gained widespread adoption in PHTx. Advanced CMR parametric mapping sequences quantify myocardial fibrosis and edema, and our preliminary data suggest a potential for these sequences to diagnose AR. While CMR parametric mapping has significant promise, focusing simply on the average properties across an entire left ventricular plane or region ignores the spatial patterns of disease, resulting in a loss of information and an impaired ability to use the imaging data to direct care. Here we propose advanced image analysis methods that are more granular than plane analysis, including texture analysis, as a means for objectively analyzing different patterns of myocardial disease and developing predictive models that would allow improved clinical decision making. The central hypothesis of this grant is that non-invasive cardiac magnetic resonance and blood biomarkers can detect myocardial abnormalities consistent with acute rejection in pediatric heart transplant recipients and can predict the need for endomyocardial biopsy. To address this hypothesis, Aim 1 will develop and validate a comprehensive predictive model for identifying PHTx recipients having suspected AR and requiring cardiac catheterization. Aim 2 will evaluate whether blood biomarkers improve the CMR model developed in Aim 1. SubAims will include assessment of cost to determine the most cost-efficient screening protocol. Aim 3 will expand modeling to determine severity of AR as defined histologically. This multi-PI proposal is a prospective, multicenter study to perform CMR in PHTx with and without AR who are also undergoing clinical biopsy. The innovation of this study is the use of advanced CMR, texture analysis, and blood biomarkers for the non-invasive detection of AR. This proposal leverages the support of the Congenital/Pediatric Research Committee within the Society of Cardiovascular Magnetic Resonance (SCMR). Application of these data to clinical practice could improve quality of life and decrease associated morbidity by ensuring that only patients with a high probability of rejection undergo biopsy.

NIH Research Projects · FY 2026 · 2022-07

PROJECT SUMMARY The human gut microbiota is increasingly recognized as having essential functions in human health. However, the microbiota is constantly subjected to challenges such as intestinal inflammation, which drives the microbiota into a perturbed state that can exacerbate diseases. Therefore, microbial resilience, which maintains the structural and functional stabilities of the gut microbiome in the face of perturbations, is critical to host health. The overarching goal of our research program is to elucidate the molecular mechanisms that govern commensal resilience in the inflamed intestine. During intestinal inflammation, host processes known as nutritional immunity starve gut microbes from essential micronutrients such as iron. In contrast to the well-studied strategies that pathogens employ to overcome host nutritional immunity, little is known about how gut commensals survive iron starvation in the inflamed gut. The primary goal of our research program for the next five years is to define the resilience mechanisms that maintain commensal iron homeostasis during gut inflammation. Enteric pathogens overcome nutritional immunity by producing iron-chelating molecules termed siderophores. Here, we show that the model gut commensal Bacteroides thetaiotaomicron (B. theta) acquires iron in the inflamed gut by pirating siderophores from an enteric pathogen that causes intestinal iron limitation. Notably, B. theta captures siderophores using a unique system absent in other Gram-negative bacteria. However, such a capture mechanism can be exploited by enteric pathogens to “re-pirate” siderophores from gut commensals to evade nutritional immunity. In addition to increasing iron uptake, we show that B. theta employs small, non-coding RNAs to orchestrate iron conservation and maintain intracellular iron homeostasis in the inflamed intestine. With this MIRA award, we will define commensal resilience mechanisms by addressing two related but independent questions in fundamental bacterial physiology: 1) How does xenosiderophore acquisition mediate B. theta resilience during gut inflammation? 2) How does B. theta manage intracellular iron homeostasis in the inflamed intestine? We will approach these questions using an interdisciplinary pipeline consisting of cutting-edge omics experiments, bacterial & host genetics, and a mechanistic understanding of bacterial physiology in vivo. The completion of these research projects will reveal the mechanisms by which gut commensals adapt to iron limitation in the inflamed gut and how such adaptation shapes the structural and functional stability of the gut microbiome. The proposed work is innovative because it adds commensal iron metabolism as a previously unappreciated dimension to the intricate interactions between pathogen and nutritional immunity. This work is impactful because it will provide much-needed insights into how interphylum iron metabolism contributes to gut microbiota resilience in the inflamed gut.

NIH Research Projects · FY 2025 · 2022-07

The severity of lung injury that develops within the first several days after lung transplantation (LT) is a key indicator of which LT recipients are at greatest risk of death or early development of chronic lung allograft dysfunction. The fact that 30% of lungs deemed suitable for LT rapidly develop severe lung injury suggests that there may be unrecognized subclinical injury already present in donor lungs that renders the lung allograft susceptible to further injury at the time of LT. A critical unmet need is improved ability to detect and interpret the consequences of subclinical donor lung injury that may drive poor clinical outcomes after LT. In this Katz R01 proposal, an accomplished physician scientist will develop a new area of research investigating how subclinical donor lung injury triggers a cascade of events that ultimately results in CLAD. Recent studies have identified donor-derived cell-free DNA (cfDNA) as a biomarker of lung allograft injury, with increased cfDNA detected prior to clinical recognition of acute rejection. However, it is unknown which specific cells and injury mechanisms cause cfDNA release from the donor lung. cfDNA can also mechanistically exacerbate lung inflammation. In models of non-LT lung injury, cfDNA detection in bronchoalveolar lavage or plasma activates the stimulator of interferon genes (STING). STING then promotes ongoing dysregulated inflammation by activating inflammatory pathways previously implicated in lung injury after LT, including NF-B, NLRP3, and MKLK. In this Katz R01 proposal, an ESI physician scientist with clinical expertise in LT and scientific expertise in animal models of ARDS proposes an integrated approach to define mechanisms of donor lung injury that drive cfDNA release and poor outcomes after LT. Using single-cell genomics and CyTOF-based immune cell profiling of serial samples from human donor lungs coupled with a new multi-hit murine model of subclinical donor lung injury, we will determine the specific cellular and molecular mechanisms through which donor lung injury affects early allograft dysfunction. We hypothesize that subclinical donor lung injury drives severe allograft injury through release of donor-derived cfDNA into the allograft airspace, triggering a feed-forward cycle of inflammation and ongoing cellular injury that results in poor clinical outcomes. The Specific Aims are: (1) to test whether subclinical donor lung injury is associated with release of donor-derived cfDNA and poor clinical outcomes in humans, using single-cell RNA sequencing and mass cytometry on donor lung biopsies collected before and after LT and (2) to use a novel animal model of sequential subclinical lung injury prior to ischemia reperfusion to determine how subclinical donor lung injury releases cfDNA to prime the donor lung to develop excessive STING-dependent inflammation after ischemia-reperfusion injury. Together, the combination of longitudinal human observational data and mechanistic murine experimentation fulfills a major gap in LT research and will provide a broad foundational knowledge to understand how subclinical donor lung injury affects clinical outcomes at the cellular and molecular level.

NIH Research Projects · FY 2025 · 2022-07

Project Summary Vascular adaptation after birth is dependent on closure of the ductus arteriosus (DA), a fetal vascular shunt connecting the pulmonary artery and aorta. Failure of DA closure results in persistent patency of the DA (PDA), a common disorder associated with increased morbidity and mortality in the most vulnerable infants. Current pharmacological treatments for PDA are limited and only focus on a single therapeutic pathway – cyclooxygenase-mediated prostaglandin (PG) synthesis. However, recent data reveal complex networks of genes and druggable pathways involved in the vasodilatory-to-vasoconstrictive shift that drives postnatal DA closure. Efforts to identify new DA-selective vasoconstrictors overlook the possibility that ongoing vasodilatory stimuli perpetuate DA relaxation and inhibit its closure. Because critically ill preterm newborns are exposed to multiple medications during the time that DA closure takes place, we postulate that pharmacologic agents used in the neonatal ICU prevent DA closure and contribute to PDA. The DA of prematurely-born infants is developmentally primed to respond to vasodilatory signals. Our prior studies using mouse models and human data show that drugs frequently used in preterm infants have unexpected vasodilatory effects on the DA, including specific antibiotics, antacids, and diuretics. These data suggest that drug-induced DA relaxation is a modifiable contributor to PDA, but this has not been systematically evaluated. We therefore hypothesize that drugs commonly used in the NICU have an adverse effect on closure of the premature DA and that specific drug combinations act synergistically to impair postnatal DA closure. Mouse and human tissues will be used to test this hypothesis in three Aims: 1) Determine whether drugs in the neonatal pharmacopeia prevent the initial phase of DA closure - smooth muscle constriction - that leads to physiologic closure of the DA lumen; 2) Determine whether drugs in the neonatal pharmacopeia impair the second phase of DA closure - fibromuscular remodeling - that leads to permanent sealing of the constricted DA; 3) Identify drug combinations that interact to adversely affect either phase of DA closure. Drug effects will be examined using primary (in vitro) high throughput screening (HTS) of preterm mouse DA smooth muscle cells. A series of secondary screening assays will prioritize single- and synergistic combinations of hits based on potency/efficacy, DA-selectivity, and toxicity for further study of their ex vivo and in vivo vasoactive effects on the DA. A novel ex vivo mouse DA-reopening assay will be used to screen for drugs of interest. The effect of hit DA-vasodilatory compounds will be examined on ex vivo human neonatal DA segments and in a large national database of preterm infants. These studies have high translational potential and will definitively identify which drugs or drug combinations pose increased risks for PDA in preterm infants, providing an innovative approach to enhance conservative PDA management efforts in the NICU.

NIH Research Projects · FY 2025 · 2022-07

SUMMARY This proposal will test the over-arching hypothesis that extra-intestinal pathogenic Escherichia coli (ExPEC) overcomes inhibition by urogenital Lactobacilli via sequential activation of inter-connected acid resistance (AR) mechanisms. We further postulate that transient internalization of ExPEC into vaginal epithelial cells increases fitness by enhancing induction of acid resistance and other mechanisms of persistence that enable bacteria to gain access and survive in otherwise harsh host environments. The hypothesis to be tested has been formulated based on the following strong preliminary data: (1) We discovered a novel AR mechanism in ExPEC that is controlled via a non-canonical signaling system, BtsS-YpdB and it uses L-serine deamination to neutralize bacterial cytosolic pH. We call this new AR mechanism, AR6. (2) BtsS-YpdB signaling is induced during infection and in response to several Lactobacillus species. (3) Deletion of btsS-ypdB significantly decreases ExPEC acid tolerance and vaginal colonization. (4) Strains lacking L-serine deaminases, or BtsS- YpdB differentially react to the inhibitory actions of representative urogenital L. gasseri and L. delbrueckii isolates. (5) Deletion of btsS-ypdB alters the induction and function of the known acid-sensing system EvgSA that controls the most prominent, known AR mechanism, AR2. We will test these hypotheses using the most prevalent ExPEC pathotype, uropathogenic E. coli. Uropathogenic E. coli is the main cause of urinary tract infections (UTIs), an infection that disproportionately afflicts women. Similarly, ExPEC strains are the leading cause of infection-related stillbirths. Despite the dominant paradigm that the low pH of vagina is protective against pathogens, we and others have shown that colonization of the vagina by ExPEC can serve as a nidus for infection of the urinary tract, the cervix, uterine horns and the gravid uterus. Vaginal colonization is therefore a key step in pathogenesis. While several acid resistance (AR) mechanisms have been identified that are active in the gut, the relative contribution of each AR mechanism during ExPEC infection remains undefined. With our aims, we will: Interrogate the significance of transient bacterial expansion in the host as a priming niche for the amplification of acid resistance and other persistence mechanisms (Aim 1). We will evaluate the individual and combined contributions of AR mechanisms to the colonization potential of ExPEC in the vagina, bladder and gut and will elucidate the connection of the novel AR6 pathway we discovered to the induction and function of AR2 (Aim 2). Finally, building on exciting preliminary data we will investigate the potential of urogenital Lactobacilli strains in their ability to override ExPEC acid resistance, aiming to identify effective probiotic strategies to prevent ExPEC reservoir formation in the vagina (Aim 3). Completion these aims will uncover how ExPEC leverage their multiple AR systems to transit the host and evade elimination and will inform our efforts towards developing effective probiotic strategies to prevent, vaginal colonization and combat genitourinary tract infections.

NIH Research Projects · FY 2025 · 2022-07

Clear cell renal cell carcinoma (ccRCC) is characterized by two major chromosome abnormalities, loss of 3p and gain of 5q, which occur nearly universally in this disease. Here we focus on the unique insights these defects provide into activities at the nexus of chromatin and cytoskeletal biology: coordinated activity of chromatin remodelers on spindle microtubules needed for the integrity of mitosis. Colocalized on chromosome 3p are the chromatin remodelers SETD2, PBRM1, and BAP1 and the E3 ligase VHL. Our groups together have pioneered the concept that SETD2 and PBRM1 have active roles on the cytoskeleton that regulate mitotic fidelity, with impacts on genomic integrity that are only now being revealed. SETD2 is a methyltransferase for both histones and spindle microtubules. Using mutant alleles, we isolated loss of microtubule methylation as underlying the genomic instability tied to SETD2 loss. We further discovered that PBRM1, a substrate recognition member of the PBAF chromatin remodeler, specifically recognizes the SETD2 methyl mark on microtubules, and like the mark it “reads”, is required for genomic stability. Finally, we identified the mitotic kinase AURKA as a new target for VHL-mediated degradation, linking this canonical protein (VHL) to complex regulation of mitotic spindle assembly in ccRCC. In Preliminary Data, we find in addition to VHL loss stabilizing AURKA, AURKA regulates SETD2 via phosphorylation on S2080, connecting VHL and SETD2 for the first time in a common oncogenic pathway. We have also discovered another chromatin remodeler, the chromosome 5q histone methyltransferase NSD1, is also acting at the spindle, and excitingly scores in a CRISPR synthetic lethality screen with SETD2 loss. Our Overarching Hypothesis for this application is that VHL and RCC-associated 3p and 5q chromatin remodelers coordinately regulate methylation of spindle microtubules to maintain genomic stability, which when defective, offers unique opportunities for therapeutic intervention. To address our Overarching Hypothesis, we offer three Specific Aims: We will 1) dissect the convergence on the mitotic spindle of 3p and 5q ccRCC chromatin remodelers, 2) define the features of their interactions at the molecular and biochemical levels that promote mitotic integrity or failure, and 3) mechanistically evaluate points of intervention that lend insight into the controls governing mitotic spindle integrity. To accomplish these aims, we will use innovative tools and strategies that precisely evaluate combinatorial mono- and bi-allelic loss of 3p and 5q genes that occur during progression of ccRCC, as well as a rich pipeline of primary ccRCC organoids. The classic genomic features of ccRCC provide new insights on how cytoskeletal activities of chromatin remodelers converge to maintain genomic stability, the principles of study are broadly applicable to many other cancers and have important ramifications for the development of targeted therapies for this disease.

NIH Research Projects · FY 2026 · 2022-07

PROJECT ABSTRACT As of 2018, 30.3 million Americans have been diagnosed with diabetes (10% of the U.S. population with a male sex bias). Its close associations with many chronic diseases, such as heart attacks, strokes, and cancers, make diabetes a leading risk factor for morbidity and mortality. In all forms of diabetes, the inability to maintain normal glucose levels results from progressive dysfunction and eventual loss of insulin-producing b-cells in the pancreas. With high rates of treatment failure on standard therapy, developing new therapeutic approaches to preserve or even enhance b-cell function is a priority. Furthermore, differences in metabolism between men and women during healthy aging and disease are appreciated but poorly understood. Pancreatic b-cells require several key factors to appropriately secrete insulin. One such factor is MafA, a transcription factor fundamental to mature b-cell function. The early loss of human MafA (MAFA) in b-cells in patients with type 2 diabetes highlights its importance to human b-cell health. In addition, a naturally occurring, genetic mutation in MAFA (S64F MAFA) was recently identified to predispose carriers to either familial, adult- onset diabetes or hypoglycemia (low blood glucose). Curiously, S64F MAFA-associated diabetes is much more prevalent in men while women tend to present with hypoglycemia. To better understand the sex- dependent effects of this variant, we generated a mouse model harboring this mutation. This model shows the expected sex-dependent effects seen in humans, suggesting similar mechanisms between mice and humans. Male S64F MAFA mice were hyperglycemic due to widespread, premature b-cell aging and senescence, while female S64F MAFA mice were hypoglycemic by a mechanism which is not yet clearly defined. However, our preliminary studies suggest that S64F MAFA creates different b-cell subtypes in females, one of which is hyperfunctional. Taken together, these results suggest that S64F MAFA can incur diverse b-cell responses to produce sex-dependent diseases: diabetes (b-cell hypofunction) and hypoglycemia (b-cell hyperfunction). This investigation will identify and compare the diverse molecular responses to S64F MAFA in male and female b-cells across mice and humans to understand the sex-dependent, b-cell responses unique to human b-cells. We will first use the penetrant, proof-of-principle S64F MAFA mouse model which mimics several aspects of human disease to identify the diverse b-cell populations by single cell transcriptomics. For example, diversity in premature aging signatures will be related to the dysfunction seen in senescent, male S64F MAFA b-cells. We will then investigate the molecular and functional responses to the S64F MAFA protein in genetically modified, male and female human b-cells using novel pseudoislet technology to identify targets unique to human b-cell function. In sum, our work will advance fundamental understanding of sex-dependent b-cell responses in humans. Mechanisms underlying a relative male vulnerability and female resistance to diabetes in this model can be harnessed to develop therapies tailored to the individual.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Growing evidence indicates that disrupted mitochondrial function in alveolar epithelial cells and in fibroblasts can disrupt lung repair. Mitochondrial bioenergetics and metabolism play central roles in stem and progenitor cell functions in other organs, shifting between mitochondrial respiration and glycolysis to meet the needs of repair, but have received limited attention in the lung. This proposal will use a rare lung disease as model to investigate how bioenergetics and metabolism regulate the dynamic events of alveolar epithelial repair. Hermansky Pudlak syndrome type 1 (HPS-1) patients with mutations in HPS1 exhibit highly penetrant, early onset, fibrosing interstitial lung disease. We hypothesize that by disrupting mitochondrial networking, loss of HPS1 impairs mitochondrial respiration, fostering metabolic reprogramming that drives AT2 progenitor cell proliferation at the expense of differentiation, and stimulates pro-fibrotic epithelial-to- fibroblast signaling. Aim 1 will establish the role of HPS1 in alveolar type 2 (AT2) cell bioenergetics and metabolism, Aim 2 will determine the impact of HPS1 loss on alveolar epithelial repair, and Aim 3 will identify how metabolic reprograming in AT2 cells drives fibrotic repair. We will accomplish these studies using robust MLE15 cell models, primary lung cells from pale ear mice with global inactivation of Hps1, a novel Hps1flox/flox mouse to examine selective contributions of AT2 cells and fibroblasts to repair in the setting of HPS1 loss, patient-derived iPS cells with the common HPS1 mutation to provide translational relevance to humans, and the repetitive bleomycin model to generalize our findings beyond a rare lung disease. The expertise of our laboratory group coupled with the strength of the pulmonary and mitochondrial communities at Vanderbilt uniquely position us to successfully execute these experiments. We expect that proposed studies will establish a role for HPS1 in HPS type 1 lung disease, integrate bioenergetics and metabolism mechanistically into alveolar repair, and provide insight into AT2 cell bioenergetic failure in a growing number of fibrosing interstitial lung diseases.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Childhood asthma is a common, burdensome disease for which there are currently no effective prevention strategies. This is due in part to our limited understanding of the molecular determinants of asthma pathogene- sis. As the development of asthma is highly influenced by genetic and environmental factors, assessment of both genetic and metabolomic profiles can improve our understanding of disease pathogenesis and identify potential targets for treatment and prevention. The overall research objective of this proposal is to elucidate metabolic and upstream genetic pathways underlying childhood asthma pathogenesis. Our central hypotheses are that metabolic profiles at birth are associated with subsequent development of childhood asthma and that this association is due in part to the effect of genetic variants on intermediate metabolic profiles and childhood asthma. To test these central hypotheses, we will pursue the following specific aims: 1) identify metabolic pro- files at birth associated with the development of childhood asthma, and 2) determine the genetic contribution to variation in metabolite concentrations at birth and identify genetic pathways linking metabolic profiles at birth with childhood asthma. To achieve Aim 1, we will capitalize upon a unique resource of longitudinal birth co- horts with newborn metabolic data and rich phenotypic data (n=6209 children). To achieve Aim 2, we will utilize genotypes for children within the largest of these cohorts to perform a metabolite genome-wide association study. We will then leverage these findings, along with summary statistics from the largest GWAS of childhood asthma performed to date, to perform a genetic co-localization analysis. The purpose of this K01 proposal is for Dr. Snyder to build on her prior training in maternal-child health epide- miology and experience with utilizing metabolomics data in large epidemiologic studies by taking on leadership roles in the conduct of epidemiologic studies, applying modern statistical methods for high-dimensional data, and receiving advanced training in genetic epidemiology so that she can more effectively address fundamental questions about upstream pathways of disease development. Through the comprehensive career development plan applied directly to the research, the complementary expertise of the mentorship team, and an outstanding institutional environment, Dr. Snyder will acquire the knowledge, skills, and resources necessary to advance her toward her long-term career goal of becoming a recognized leader in maternal-child health epidemiology, with a focus on integrating genetics and metabolomics in large epidemiologic studies to understand and pre- vent childhood respiratory diseases. The experience, training, and findings generated through this proposal will result in Dr. Snyder developing critical research skills to move toward independence and the submission of a highly competitive R01 application.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY Hypertension (HTN) is a chronic inflammatory disease and is a primary risk factor for ischemic heart disease and stroke, the two leading causes of death worldwide. HTN is associated with vascular “oxidative stress”, yet antioxidant therapy has not proven effective. This may be because reactive oxygen species (ROS) also participate in normal physiological signaling by molecules like Angiotensin II (AngII) and tumor necrosis factor α (TNFα). Pathology may result from excess activation, loss of spatiotemporal constraints, or dysregulation of the feedback mechanisms that control these signals. AngII and TNFα both activate NADPH Oxidase 1 (Nox1), producing extracellular superoxide (O2-•). By an unknown mechanism, this generates an intracellular signal, and disruption of this protects against vascular inflammation and AngII-induced HTN. We previously found that Nox1 physically associates with Volume-Regulated Anion Channels (VRACs) that are encoded by Leucine-Rich Repeat-Containing 8 family proteins. LRRC8A associates with one of four related isoforms (LRRC8B-E) to produce channels with unique properties. ROS production by Nox1 requires functional VRACs, potentially for change compensation, and the oxidized environment created by Nox1 regulates VRACs. Thus, VRACs and Nox1 are functionally interdependent. We now provide new evidence that O2-• also enters cells via these closely associated anion channels. This may allow tight regulation of O2-• delivery to the cytoplasm, providing spatial control of redox signaling which limits off-target oxidation. Blood vessels from mice lacking LRRC8A only in vascular smooth muscle cells (VSMCs) exhibit normal contractility but enhanced vasodilation and these mice are protected from AngII-induced HTN. We hypothesize that by regulating Nox1 activity and O2-• entry into VSMCs, LRRC8 anion channels control cytoplasmic redox signaling pathways that promote inflammation and impair vasodilation. Aim #1 will determine how LRRC8 channels facilitate O2-• influx into VSMCs and determine how this is regulated by local redox conditions. We will use two novel O2-• flux assays that we have developed combined with patch-clamp recording to achieve these goals. Aim #2 will determine how LRRC8A channels and O2-• modulate inflammation and contractility via two cytoplasmic targets: 1) RhoA, a small GTPase that controls vasomotor function, and 2) TRIM21, an E3 ubiquitin ligase that we identified by mass spectrometry as a novel binding partner of LRRC8A. TRIM21 modulates both NF-κB-dependent inflammation and the Nrf2-dependent antioxidant response. Aim #3 will define the contribution of specific LRRC8 isoforms to AngII-induced hypertension in mice. Blood pressure recording, vascular reactivity and molecular biologic studies will define the LRRC8 channel subtype that controls Nox1 and vascular function in HTN . Relevance: Links between inflammation, oxidative stress and cardiovascular disease are clear, but methods to control oxidant-dependent signaling are lacking. This project will identify novel therapeutic strategies that are applicable to the treatment of HTN and vascular inflammation.

NIH Research Projects · FY 2025 · 2022-06

Sepsis with acute organ dysfunction is a common condition with high morbidity and mortality and no specific therapies other than antimicrobials. The NHLBI PETAL Network Phase 2B Acetaminophen and Ascorbate in Sepsis: Targeted Therapy to Enhance Recovery (ASTER trial) is a randomized double blind platform trial that will test the effect of two potential therapies, acetaminophen or vitamin C versus a common placebo to improve lung, cardiovascular and kidney dysfunction in 900 patients with sepsis and pulmonary or cardiovascular dysfunction including patients with sepsis due to COVID-19. The rationale for this clinical trial rests, in part, on novel findings from our group and others that (1) circulating cell-free hemoglobin (CFH) is elevated in patients with sepsis, including those with COVID-19; (2) higher plasma CFH in sepsis is associated with death and organ dysfunction including ARDS and acute kidney injury; (3) both acetaminophen and vitamin C are hemoprotein reductants that reduce the capacity of CFH to cause lipid peroxidation and other oxidant injury and (4) acetaminophen and vitamin C can reduce the injurious effects of CFH on the microvascular endothelium both in vitro and in the isolated perfused human lung. Although ASTER is well designed to test the clinical efficacy of acetaminophen and vitamin C, key information will be needed to understand trial results and plan for potential phase 3 studies. The proposed studies in this R01 will define the mechanisms by which acetaminophen and vitamin C affect organ dysfunction in sepsis (Aim 1) and determine whether there are subgroups that can be identified within the trial for whom a differential treatment effect exists (Aim 2). Specific Aim 1 will determine the mechanisms by which acetaminophen and vitamin C improve lung and kidney dysfunction in sepsis by testing the hypothesis that acetaminophen and vitamin C reduce levels of oxidized ferryl (4+) hemoglobin resulting in decreased oxidative injury, inflammation, and endothelial injury as measured by plasma, distal airspace fluid, and urinary biomarkers of hemoglobin oxidation (ferryl hemoglobin) lipid peroxidation (F2-Isoprostanes, Isofurans), inflammation and endothelial injury. Distal airspace fluid will be sampled at ten participating PETAL Network sites by collecting fluid that condenses on heat moisture exchanger filters placed in the mechanical ventilator circuit, a method that has been developed and validated by Dr. Ware's research group. Specific Aim 2 will identify whether previously described and validated hyperinflammatory or hypoinflammatory subgroups of sepsis patients benefit more from treatment with acetaminophen or vitamin C. A finding of heterogeneity of treatment effect in Aim 2 would be of great value for predictive enrichment in a future phase 3 clinical trial. In summary, the proposed studies will greatly enhance the value of the ASTER clinical trial by determining the biologic mechanisms of the therapeutic effects of acetaminophen and Vitamin C and assessing for heterogeneity of treatment effect in this NHLBI-funded Phase 2B clinical trial.

NIH Research Projects · FY 2025 · 2022-06

SUMMARY Clostridioides difficile (formerly named Clostridium difficile) is a Gram-positive, spore-forming pathogen, and the leading cause of nosocomial and antibiotic-associated intestinal infections. Susceptibility to C. difficile infection (CDI) often follows antibiotic treatment and subsequent disruption of the resident intestinal microbiota, however the rise of infections in healthy young adults suggests that there are additional factors that contribute to CDI. To colonize the gastrointestinal tract, C. difficile must compete with both the host and members of the gut microbiota for critical nutrients. Access to nutrient metals can profoundly impact the outcome of CDI as metals are required cofactors for approximately 30% of all proteins. This fact is exploited by host metal binding proteins which sequester nutrient metals to restrict microbial growth in a process termed nutritional immunity. A hallmark of CDI is the secretion of potent toxins that cause severe damage to the gastrointestinal epithelium and trigger the production of pro-inflammatory cytokines and chemokines. These events initiate the immune-mediated recruitment of inflammatory factors to the site of infection. One of the most abundant inflammatory proteins that accumulates at the site of CDI is calprotectin. Calprotectin is the most abundant protein in neutrophils and is a component of nutritional immunity that directly inhibits microbial growth through nutrient metal sequestration. Calprotectin is also a potent immunomodulatory protein, and a common clinical inflammatory biomarker whose abundance correlates with CDI severity. It is unknown how the massive infiltration of calprotectin affects metal availability in the gastrointestinal tract and shapes competition between C. difficile and members of the gut microbiota. In addition, how C. difficile adapts to calprotectin-dependent metal limitation and resists nutritional immunity during CDI remains unclear. We propose a model whereby nutrient metals make a critical contribution to the outcome of CDI. Toxin driven inflammation drives the recruitment of immune cells into the gut which leads to the accumulation of large amounts of CP. CP chelates available nutrient metals and exerts potent pro- inflammatory activities. This massive inflammatory response and redistribution of nutrients alters C. difficile gene expression and affects the interaction between C. difficile and members of the microbiota. Finally, we hypothesize that C. difficile encodes multiple gene products that compete with both CP and the microbiota for nutrient metals and this competition has a profound effect on the outcome of CDI. Experiments described in this proposal will test this model and define the contribution of nutritional immunity to the pathogenesis of C. difficile, determine the role of metal binding and immune cell recruitment in the protective properties of calprotectin, and identify C. difficile genes required to compete with the microbiome for nutrient metals during inflammation. Collectively, the findings from this proposal will inform the development of effective therapeutic or prevention strategies for the treatment of CDI.

NIH Research Projects · FY 2026 · 2022-06

Project Summary/Abstract More than 20% of patients undergoing major surgery experience acute kidney, brain, and heart injury, and these perioperative complications lead to persistent organ dysfunction, long-term morbidity, and death. My research program is investigating and manipulating mechanisms of perioperative organ injury in order to identify therapeutic targets and develop novel therapies. We are currently focused on the critical impact of oxygen tension on organ injury, because perioperative oxygen administration is inconsistent, unguided, often excessive, and potentially harmful. Both hypoxia and hyperoxia can be harmful to surgical patients, yet both occur frequently, despite the ease with which the fraction of inspired oxygen (FiO2) can be manipulated in the perioperative period. Our laboratory is focused on identifying and investigating molecular pathways and therapeutic targets that a) impact oxygen tension in tissues during surgery and b) impact hypoxia- and hyperoxia-mediated organ injury. We target these molecular pathways to reduce organ injury. We have recently demonstrated that: 1) perioperative oxidative damage increases acute kidney, brain, and heart injury; 2) intraoperative normoxia improves vascular reactivity compared to hyperoxia possibly by reducing intraoperative oxidation of the heme moiety of vascular smooth muscle soluble guanylyl cyclase; 3) normoxia upregulates hypoxia inducible factor (HIF)-regulated transcription and reduces circulating markers of oxidative damage; and 4) increased circulating cell-free hemoglobin (Hb) oxidizes lipids and is independently associated with postoperative kidney, lung, and brain injury. In the next 5 years we will investigate the effects of oxygen tension on mechanisms of organ injury, including oxidative damage, vascular function, HIF signaling, and cell free Hb-mediated organ injury, using a multifaceted translational approach. Our program combines laboratory experiments in human tissues and preclinical models with prospective cohort studies and mechanistic trials in patients having major surgery. We perform experiments on arterioles and arteries isolated from patients during surgery to study the effects of hypoxic, normoxic, and hyperoxic treatments on vascular function. We investigate the impact of oxygen treatments during preclinical models of acute kidney injury in genetically engineered mice in collaboration with oxygen biologist nephrologist Volker Haase, and we are measuring the effect of intraoperative hyperoxia vs. normoxia treatment in samples biobanked from the NIGMS-supported ROCS clinical trial. Examples of these experiments include the measurement of HIF- regulated transcripts in atrial myocardium and the oxidation state of the heme group in plasma cell-free Hb. We will complement these hypothesis-driven experiments with unbiased approaches to measure the transcriptome and protein responses in vascular and murine tissues to identify and support new paths of investigation. This rigorous multimodal strategy provides the framework to advance the understanding of perioperative organ injury and guide the development of therapies for hundreds of thousands of surgical patients.

NIH Research Projects · FY 2026 · 2022-06

Project Summary This administrative supplement is requested to support research continuity and retention of the scientific aims outlined in a K25 NIAAward #1K25AG076864, titled "Development and Application of T1rho Dispersion Imaging of Aging Muscle." The principal investigator is currently managing a high-risk pregnancy and childbirth-related life events that may temporarily affect her research productivity and the success of her K25 grant. The aims of the parent grant are (1) to develop R1p (R1p = 1/T1p - the spin-lattice relaxation rates in the rotating frame) dispersion imaging at weak locking field frequency, a novel approach that can quantify degenerative changes in microvasculature and abnormal macromolecule accumulation in muscle fiber membranes, (2) to validate extracted-MRI indices using gold-standard ex vivo methods through a longitudinal study of skeletal muscle in F344 rats to establish the pathophysiological relevance of these novel MRI biomarkers with aging, and (3) to translate R1p dispersion imaging to human research, utilizing a clinical 3T MRI scanner to assess skeletal muscle changes with healthy aging. The parent grant has enabled successful completion of Aim 1 and Aim 2, and partial progress toward Aim 3. To fully accomplish the objectives outlined in Aim 2, additional computational analysis of histological data is necessary. To support this activity, I have established a collaboration with experts at the Digital Histology Shared Resource (DHSR), in partnership with the Translational Pathology Shared Resource (TPSR) at Vanderbilt University Medical Center. This collaboration will enable cost-effective computational quantification of ex vivo biomarkers, which are essential for interpreting MRI-derived indices from Aim 2. This supplement is also vital for advancing Aim 3 by ensuring continuity of human research activities during a temporary reduction in the principal investigator's availability and productivity. A research assistant (RA) will be hired through this administrative supplement to support subject recruitment, scheduling volunteer visits, and conducting clinical assessments. The RA has received or will receive appropriate training to perform basic clinical evaluation and analyze demographic data, helping to maintain momentum in the human imaging component of the study. Overall, this support is crucial for maintaining the integrity and progress of the parent grant during a period of limited Pl availability.

NIH Research Projects · FY 2026 · 2022-06

Among older adults, heart valve disease is common and often requires treatment with valve repair or replacement. What used to require open heart surgery can now be done with less invasive transcatheter heart valve interventions (THVIs), allowing for less morbidity and quicker recovery. However, despite the safety and procedural success of THVIs, over one-half of patients are dead, have poor quality of life (QoL), and/or are hospitalized 1 year after a THVI. Cardiac rehabilitation (CR) effectively mitigates these poor outcomes in patients with cardiovascular disease, including those undergoing heart valve procedures. However, among Medicare beneficiaries, only 25% of those undergoing a THVI participate in center-based CR (CBCR), which highlights a significant unmet clinical need. To address this need, our long-term goal is to develop a home- based CR (HBCR) program that extends the benefits of CR to more individuals after THVIs. Our over-arching hypothesis is that a HBCR mobile health intervention that innovatively addresses key target health behaviors will reduce clinical events and improve physical activity, functional capacity, and QoL after THVIs and that an interactive delivery and longer duration of the active intervention will produce greater benefits. We propose a multicenter randomized controlled trial enrolling 375 patients undergoing aortic or mitral THVIs who do not intend to participate in CBCR; those who intend to pursue CBCR will be followed in a registry. Participants will be assigned to 3 groups: control (1) vs. a HBCR mobile health intervention with hands-off (2) vs. interactive (3) delivery. The intervention addresses key target health behaviors and includes Apple Watch (to encourage physical activity and give reminders on healthy living) and an exercise prescription (to build strength/balance). The hands-off and interactive delivery groups receive the same intervention, except for a video call (every other week) with an exercise physiologist (interactive group only). Each intervention group (hands-off, interactive) will be randomized to 12-week vs. 24-week duration of the active intervention. The co-primary endpoints are: (1) clinical events composite (death, rehospitalization, skilled nursing facility visits); and (2) average daily total activity counts. Key secondary endpoints: amount, intensity, consistency of physical activity; 6-minute walk distance; and QoL. We test whether the HBCR mobile health intervention (compared to control) reduces clinical events and improves physical activity, function capacity, and QoL after THVI (Aim 1) and whether interactive vs. hands-ff delivery (Aim 2) or 24-week vs. 12-week active duration (Aim 3) of the intervention yields greater benefits. We also explore mediators of clinical benefit and mechanisms of behavior change. With population aging, prevalence of heart valve disease and number of THVIs are rapidly increasing. Overcoming disablement associated with severe valve disease requires more than simply fixing a valve; the potential of THVIs to improve quantity and quality of life is unfulfilled without integrating CR. If successful, our approach will optimize value of care and patient outcomes with broad implications for other patient populations.

NIH Research Projects · FY 2025 · 2022-06

Diffusion magnetic resonance imaging (MRI) enables the ability to probe both tissue microstructure and structural connectivity of the central nervous system. However, there are no validated methods to model and interrogate the pathways that connect the brain and spinal cord, which inhibits our ability to fully characterize and understand the complete damage that may occur in neurological disorders. For example, disease progression in patients with multiple sclerosis (MS) is known to stem from axonal damage in both the brain and spinal cord, yet, coordinated medical image analysis of both structures simultaneously has not been shown. Thus, the overall goal of the proposed research is to develop and optimize simultaneous tissue microstructural mapping of the brain and spinal cord for clinical assessment of MS using magnetic resonance imaging (MRI), specifically interrogating the microstructure and connectivity of motor pathways of the central nervous system. The critical challenges to this goal are (1) quantifying tissue microstructure of the brain and spinal cord in unison has not been performed, (2) clinical MRI lacks specificity for microstructural tissue integrity, and (3) there are few methods available that allow mapping of MS lesions and pathological abnormalities in relation to critical fiber pathways. To address this, in Aim 1 we will develop a cohesive acquisition and image processing pipeline, minimizing artifacts and maximizing reproducibility, in order to facilitate a unified analysis of the central nervous system. In Aim 2, we will utilize diffusion MRI modeling and fiber tractography to characterize tissue microstructure and connectivity from the cortex to the spinal cord. Modeling will enable quantification of highly specific pathophysiological indices of edema, axonal swelling, demyelination, and axonal loss, whereas tractography will facilitate feature localization to specific white matter pathways and along specific pathways. Finally, evaluate microstructure and connectivity of the motor pathways to interrogate pathology in MS, quantifying radiological biomarkers over space and time that may contribute to impairment in this disease. The overall impact of this proposal will be quantitative biomarkers for disease burden that may improve the value of imaging the brain and spinal cord together as it relates to understanding pathology in vivo.