Ohio State University

NIH Research Projects · FY 2025 · 2022-04

Project Summary/Abstract One Health research is a field of international health research which recognizes the complex interconnectedness of human, animal, plant, microbial and environmental health. It takes an interdisciplinary approach to respond to global health needs in the search for causes, prevention and response. Its importance has been experienced globally with COVID-19, a viral disease devastating humans, involving animals, and linked to changes in agricultural, social, environmental and travel practices. One Health research plays a key role in addressing outbreaks, pandemics and the health implications of disasters, humanitarian crises and other emergencies, often requiring research to be initiated urgently – and raising additional ethical challenges. As One Health research increases, so does the need to address its ethical issues. One Health research often involves human subjects, animals (domestic and wild), agricultural practices, cultural practices and environmental issues, all with distinct ethical components. As reports and our team’s OHEARP survey have found, ethics review committees and regulators are already over-burdened and poorly equipped to review and monitor One Health research. As Kenya has received growing numbers of international research projects, the number of people trained in research ethics, or available to conduct such training, has been inadequate. The goal of our MOHERE program is to provide such training and hence build the research ethics capacity for Kenya’s growing international health research, including One Health and emergency research. We will achieve that goal through two specific aims. Specific Aim 1: To strengthen the international health research ethics training capacity at the University of Nairobi and Kenya from a One Health and emergency perspective Specific Aim 2: To Establish a Master’s Level program in international health research ethics with a focus on One Health research and emergency research. A cadre of health professionals will develop expertise in research ethics for One Health so that health research is conducted ethically and in culturally relevant ways for Kenya. The MOHERE team, all with experience teaching bioethics and research ethics, will develop and deliver a 1- year postgraduate diploma (PGD) and 2-year MSc in research ethics. The programs will equip scientists, health professionals and academics as MOHERE trainees to develop the critical competencies needed to provide research ethics education and training, ethics review leadership, and expert consultation to researchers, their institutions, governments and international research organizations. At the same time, another group of doctorate- level professionals (or equivalent depending on their field) will become MA Fellows to complete the OSU MA in Bioethics. They will become the bioethics and research ethics educators and mentors to will help sustain a vibrant research ethics culture at the University of Nairobi and in Kenya. This group will also engage in research into research ethics so that the distinct ethics issues in international research ethics in Kenya will be investigated. As a result, Kenya will contribute to important research ethics scholarship into One Health research ethics.

NIH Research Projects · FY 2026 · 2022-04

Project Summary Double (DH) and triple-hit (TH) lymphomas (L) are rare high grade B-cell lymphomas with diffuse large B-cell (DLBCL) morphology characterized by the co-occurrence of chromosomal translocations involving MYC, BCL2, and/or BCL6. DLBCLs with dual c-Myc (>40% by immunohistochemistry, IHC) and BCL2 (>50% by IHC) protein overexpression without translocation (double-expressor or DEL) are significantly more common than DH/THL, accounting for 20% to 30% of DLBCL patients. Lymphoma with either DEL, DHL, or THL are here collectively called c-Myc overexpressing LBCL and have a significantly worse prognosis compared to the c- Myc-negative counterpart [3-year overall survival of ~30% versus 70%, respectively]. The poor clinical outcome of this subset of lymphoma patients highlights the need for novel therapeutic strategies. Transducin β-like protein 1 (TBL1X) was initially identified as a specific adaptor protein playing an essential role in canonical Wnt signaling by recruiting β-catenin to the promoter region of Wnt targets such as MYC and CCND1 to activate their transcription. Few published reports indicate that the Wnt/β-catenin signaling is constitutively activated in DLBCL, which prompted our initial investigation in this disease. Preliminary data: Our published work shows that, unlike normal B cells, DLBCL cells express abundant levels of TBL1. Genetic deletion of TBL1 or pharmacologic treatment with tegavivint (Iterion), a first-in-class small molecule targeting TBL1, induces significant DLBCL cell death in vitro and in vivo. While tegavivint was initially developed as an inhibitor of the TBL1/β-catenin interaction, our data show that genetic deletion of TBL1 and treatment with tegavivint reduce c-Myc protein expression in a post-transcriptional/β-catenin independent manner. We further show that in DLBCL, TBL1 interacts with a Skp1/Cul1/F-Box (SCF) supercomplex, which controls the proteasome-mediated degradation of critical pro-survival proteins such as c-Myc and components of mTOR signaling such as Rheb. Collectively, these observations establish the rationale for targeting TBL1 as a novel therapeutic strategy to promote c-Myc turnover and to disrupt the driver events coordinated by its activity in c- Myc overexpressing LBCL. Project hypothesis: TBL1 serves as a critical modulator of c-Myc turnover and represents a novel and attractive candidate for targeted therapy for patients with c-Myc overexpressing LBCL. To test this hypothesis, we propose the following aims: Aim 1: Characterize the TBL1/c-Myc feedforward circuit promoting c-Myc overexpressing LBCL cell survival. Aim 2: Initiate a Phase Ib trial with single agent tegavivint in patients with relapsed/refractory cMyc overexpressing LBCL. Aim 3: Identify combination strategies to maximize the therapeutic potential of tegavivint. At completion of this project, we will have a better understanding of the TBL1-modulated mechanism through which c-Myc turnover is regulated, will have developed a novel therapeutic strategy to treat this incurable disease, and will have begun to characterize resistance mechanisms to tegavivint.

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY A decline in mitochondrial quality and activity has been associated with normal aging and correlated with the development of a wide range of age-related diseases. Therefore, rejuvenating mitochondrial function or improving mitochondrial quality control might be an effective strategy to combat aging. Mitophagy is an essential mitochondrial quality control mechanism that mediates the lysosomal clearance of damaged mitochondria. Increasing lines of evidence have established the longevity-extending effects of enhanced mitophagy in various model organisms. Interestingly, recent studies suggest that augmented mitophagy may counteract aging- associated cardiac dysfunction. Therefore, identifying more efficient and specific agents that can modulate the clearance of defective mitochondria via mitophagy are likely to have significant therapeutic benefits. We conducted high-content image-based assays for mitophagy modulators using the pH-dependent fluorescent mitophagy reporter, mt-Keima. We identified the selective neddylation inhibitor, MLN4924, to be the most effective mitophagy activator. Neddylation is a posttranslational modification that attaches ubiquitin-like protein NEDD8 to protein targets via NEDD8-specific E1-E2-E3 enzymes. Of note, our mechanistic studies suggest MLN4924 effectively blocks neddylation of Cullin 2, a component of the elongins B/C-Cullin 2-Rbx1 (Ring-Box 1)-VHL (Von Hippel-Lindau protein) E3 ubiquitin ligase complex (CRL2VHL). The inhibition leads to an accumulation of the Hypoxia Inducible Factor 1 Subunit Alpha (HIF1α), a CRL2VHL substrate, and subsequent activation of the BCL2-interacting protein 3 (BNIP3), a mitochondrial receptor for mitophagy induction. These results provide a novel connection between neddylation and mitophagy. This project aims to delineate the novel mechanistic link between mitophagy and neddylation, and to determine whether mitophagy represents a novel mechanism and therapeutic target for treating age-related cardiac dysfunction. These studies will be facilitated by our recently described mt-Keima mouse model to monitor in vivo cardiac mitophagy. Additionally, we will utilize a set of innovative reagents to genetically and pharmacologically modulate neddylation. To directly assess the role of neddylation in the heart, we have generated mice with the cardiomyocyte-specific deletion of NAE1, encoding a subunit of the E1 neddylation activating enzyme. In Aim 1 of the proposed studies, our goal is to determine the mechanisms by which inhibiting neddylation regulates mitophagy in cardiomyocytes and the heart. In Aim 2 of the proposed studies, we will genetically and pharmacologically manipulate neddylation in the adult heart using mouse models that lack NAE1 or are treated with MLN4924. We will determine whether restoring mitophagy via inhibiting neddylation ameliorates age-related cardiac dysfunction. Completing the proposed studies will produce critical insights into the role of mitophagy in age-related pathological conditions, and will fundamentally advance our understanding of the mechanisms of mitochondrial quality control in the heart.

NIH Research Projects · FY 2026 · 2022-03

Project Summary: Human immunodeficiency virus (HIV) is associated with persistent immune activation and dysfunction, even during suppressive antiretroviral therapy (ART) that was initiated early post-infection. Perinatally acquired HIV (PHIV) infection and lifelong ART likely alter the development and function of the immune system. The exposure of HIV and ART in utero, the decades of ART therapy, the lasting effects of older toxic ART with mitochondrial toxicity and the long-term sub-optimal adherence known to occur with prolonged ART use, heighten concern that sequelae of HIV and ART in children and adolescents may be more frequent and potentially more devastating than in adults. Better understanding of immune dysfunction in PHIV is crucial, as it is often easier to limit or even reverse comorbidities at an early stage. We are exploring the consequences of PHIV and its treatment with ART in a cohort of adolescents in Uganda and have reported significant differences in the immune profiles of HIV- and HIV+ children. These differences may be driven by trained immunity, a process by which cells of the innate immune system (e.g. monocytes, macrophages and natural killer cells) are reprogrammed to respond differently to subsequent exposure to microbial products and proinflammatory lipids. Recent studies, including our own, demonstrate that ART exposure may also contribute to alterations in immune cell activation, potentially through decreased mitochondrial function and through modulation of intracellular signaling cascades. The knowledge gained from this proposal could be substantial, and from a translational perspective will lay the foundation to identify key pathways with biological, clinical, and prognostic relevance for adolescents who are advancing into adulthood. These results could inform intervention trials to mitigate the development of comorbidities associated with immune activation. We propose: Aim 1: To measure innate immune profiles (i.e monocytes and NK cells) in adolescents with and without HIV in Uganda and the United States. Aim 2: To identify the role of trained immunity in innate immune modulation in adolescents with and without HIV infection in Uganda. Aim 3: To evaluate how mitochondrial function plays a role in innate immune profiles longitudinally in adolescents with and without HIV in Uganda. This proposal combines a well-characterized cohort of children with and without PHIV with ex vivo and in vitro assessments of immune cell phenotype and function. We will use high-dimensional flow cytometry analyses, single cell transcriptional profiling, and an in-depth analytical approach to define the potential mechanisms whereby chronic exposure to ART, microbial products, and clinical and environmental factors may contribute to innate immune cell activation or dysfunction. This is a continuation of a highly successful collaboration among the study team members and we are well positioned to perform this innovative project.

NIH Research Projects · FY 2026 · 2022-03

Electronic cigarettes (e-cig) have been promoted as electronic nicotine (NIC) delivery systems (ENDS) without the adverse effects of tobacco cigarette smoking (CS). However, recent studies have reported a wide range of e-cig-induced toxicities with severe inflammatory lung disease observed in e-cig users. While there is a strong association between CS-induced oxidative stress, inflammation and cancer, this has not been established for e-cig use. E-cig generate toxic species, including free radicals and reactive aldehydes with the levels of these toxicants increased with increasing device power. These toxic chemicals produce reactive oxygen species (ROS), which can lead to uncontrolled inflammation and DNA damage with dysregulated cell proliferation that trigger carcinogenesis. In our chronic mouse e-cig exposure model, we observed that e-cig aerosol, generated from e-cig liquid containing NIC, induced lung tumors in 50% of the mice studied at 50 weeks of exposure, as first detected by micro-CT imaging. Histopathological examination of the tumors showed adenocarcinoma or adenoma with focal mixed broncho-alveolar neoplastic and pre-malignant cells. Superoxide radicals, inflammation, DNA damage and stemness markers were detected in the alveoli and bronchioles, preceding cancer development at earlier exposure times. The free radicals and high levels of aldehydes in e-cig aerosol, as well as the NIC derived iminium metabolite, can trigger processes of cellular ROS generation and this may serve as a central trigger of carcinogenesis. Based on the critical public health implications of this work, it is imperative to expand these observations to measure the exposure intensity/duration relationships, and the role of e-cig aerosol free radical levels and NIC in cancer development. Thus, we will perform longitudinal studies with the requisite group size needed to definitively address the critical questions: 1) What is the exposure intensity and duration required for lung cancer initiation/progression? 2) What is the incidence rate of e-cig-induced lung cancer? 3) Is cancer onset and/or progression NIC-dependent? 4) How is it effected by device power and resultant aerosol free radical levels? 5) Is cancer onset and/or progression sex-dependent? 6) What are the mechanisms by which e-cig exposure triggers ROS generation and inflammation in the lung? Studies with longitudinal micro-CT, MRI, EPR spectroscopy, EPRMRI co-imaging and blood tumor markers followed by biochemical assays and histopathology will determine the role of exposure duration, ENDS power, NIC, and sex on initiation and/or progression of lung cancer. ROS generation, lung inflammation and secondary epithelial mesenchymal transition will be evaluated as triggers or steps leading to carcinogenesis. With knowledge of the role of these mechanisms and evaluation of inhibitors that block them, we will identify therapeutic interventions that could be used to ameliorate e-cig induced lung inflammation and carcinogenesis. Thus, this research will provide important insights defining the carcinogenesis risk of e-cig use, and its underlying exposure-dependent triggers and mechanisms.

NIH Research Projects · FY 2025 · 2022-03

Project Summary Dysphagia (swallowing impairment) is a common complication of cardiac surgical procedures, leading to malnutrition, dehydration, aspiration pneumonia, reintubation, increased health care utilization, length of hospitalization, and economic burden. Although preventable, dysphagia-related aspiration pneumonia is a major cause of mortality. Early detection and accurate monitoring of dysphagia are therefore important to facilitate timely interventions to mitigate developing sequelae. Currently, clinical care of dysphagia is hindered by fundamental gaps in knowledge, including 1) contributing risk factors of dysphagia are unknown, prohibiting the use of triaged personalized care pathways; 2) no validated tools to accurately detect and monitor dysphagia in the cardiac intensive care unit exist; and 3) governing mechanisms of swallowing impairment and recovery of function are unknown, impeding the development of mechanistically guided therapeutics and optimization of salient postoperative evaluation time points. Our three specific aims target these knowledge gaps with the broad goal to shift care toward a proactive, multifaceted, and data-driven perioperative Model of Swallowing Integrated Care (MOSAIC). To this end, we will enroll 360 cardiac surgical patients over a four-year period and identify 1) independent risk factors for dysphagia, 2) sensitive clinical markers of swallowing impairment, and 3) governing physiologic mechanisms of unsafe and inefficient swallowing throughout the acute, sub-acute, and long-term postoperative period. Enrolled participants will undergo a preoperative Fiberoptic Endoscopic Evaluation of Swallowing (FEES) to screen out patients with pre-existing dysphagia. Candidate predictor variables will be systematically collected throughout the perioperative time course. Following surgery and within 48 hours of extubation, a simultaneous videofluoroscopy and FEES (VF-FEES) will be performed as well as a battery of simple bedside clinical tests. Detailed blinded analyses will be performed using validated metrics of swallowing safety, efficiency, timing and kinematics to examine acute-phase swallowing function and associated physiologic mechanisms of unsafe or inefficient deglutition. Patients with acute postoperative phase dysphagia will be re-examined at one- and six-months to determine sub-acute and long-term dysphagia trajectories and governing mechanisms of impairment and recovery. Multivariable modeling of dysphagia risk factors will produce a practical dysphagia risk stratification tool to enable accurate forecasting and personalized triaged postoperative care pathways. An accompanying open-access electronic application will be developed to provide seamless clinical adoption and a data-driven clinical decision making tool. The discriminant ability of clinical markers will be determined, and a practical bedside dysphagia screening tool will be validated to enable accurate detection and monitoring of dysphagia in the cardiac intensive care unit. Outcomes will drive future targeted therapeutic and preventative strategies and enhance personalized health care models to ultimately improve patient outcomes.

NIH Research Projects · FY 2025 · 2022-03

The hippocampus and the parahippocampal region of the medial temporal lobe (MTL) consist of anatomically and functionally distinct subregions that are selectively targeted by different pathological processes in neurodegenerative disorders, such as Alzheimer’s disease (AD) and semantic variant Primary Progressive Aphasia (svPPA). In AD, in vivo MRI studies investigating MTL subregions have often shown mixed findings that are inconsistent with post-mortem pathology studies. These inconsistencies likely result from the lack of validity and standardization in MTL segmentation approaches. In svPPA, MTL subregions are severely affected, and automated tools fail to segment the subregions accurately. Sufficient spatial resolution of imaging voxels is required to visualize the internal MTL structures and landmarks that are critical for valid segmentation of the subregions. While more than 15,000 high-resolution scans have been collected worldwide, the lack of standardization among MTL subregional segmentation protocols is still a significant barrier. Variability in the definition of the MTL subregional boundaries among existing segmentation protocols have contributed to the conflicting findings in the field. This proposal seeks to accelerate progress made by our group, the Hippocampal Subfields Group, and complete our standardized, valid, and reliable harmonized protocol for the segmentation of MTL subregions in high-resolution MRI scans. We will leverage our ongoing, successful efforts, and evaluate the proposed harmonized protocol in scans from multicentric datasets. In Aim 1, we will finalize our harmonized segmentation protocol that is a) based on a large histology dataset and developed in collaboration with neuroanatomists, b) assessed by the larger community to reach consensus, c) tested for reliability, and d) implemented in an existing automated software program. In Aim 2, we will validate the harmonized protocol in early-stage AD through associations with markers of specific pathologies: a) test for anatomical specificity of the associations between atrophy rates across MTL subregions to PET measures of MTL tau-pathology and markers of cardiovascular risk in patients with Mild Cognitive Impairment and cerebral β-amyloid. In Aim 3, we will further evaluate the validity and utility of the harmonized protocol in svPPA by performing manual segmentation of the MTL subregions using the HSG protocol and testing for anatomical specificity of volume atrophy. In Aim 4, we will facilitate wide adoption of our protocol by providing training and education materials, in-person workshops for manual and automated segmentation, and making outcome measures publicly available. Our valid, reliable, and reproducible protocol for MTL subregions segmentation will benefit groups worldwide that have and continue to collect thousands of high-resolution data to study a variety of neurological and psychiatric conditions. It also lays the foundation for future research on how distinct disease processes in the MTL subregions may lead to impairments in specific cognitive functions. Our harmonization process can be adapted for achieving valid, harmonized segmentation for other brain regions.

NIH Research Projects · FY 2025 · 2022-03

PROJECT SUMMARY/ABSTRACT Magnetic resonance imaging-guided high intensity focused ultrasound ablation (MRgFUSA) is a transformative neurosurgical approach that produces a precise and visible lesion, such that ‘target’ engagement is clear, and offers an innovative and mechanistic strategy to correct an underlying neuropathophysiology. Unlike current neuromodulation techniques (DBS, TMS, TDCS), MRgFUSA’s thermal effects can be harnessed to non-invasively and precisely target deep brain structures and circuits using the Exablate 4000 (Insightec). MRgFUSA has most recently been applied to the anterior thalamic nuclei (ATN) which has emerged as promising intervention for medication-refractory partial or focal-onset epilepsy, particularly originating from the limbic temporal lobe (e.g., amygdala). Of key relevance here is that the ATN has extensive functional and structural connectivity to the amygdala and that partial epilepsy is often associated with enhanced fear behaviors and clinical anxiety, which is often mediated by exaggerated amygdala reactivity to threat. Moreover, MPI Phan and others have shown that amygdala reactivity to threat, exaggerated at baseline/pre-treatment in anxiety disorders (ADs), can be modified and “normalized” by conventional treatments. It stands to reason that MRgFUSA to the ATN could attenuate anxiety symptoms and do so by reducing amygdala reactivity to threat. Uniquely, MPI neurosurgeon Krishna has obtained a FDA-approved Investigational Device Exemption (IDE) to ablate the ATN for medically refractory epilepsy, providing an unprecedented opportunity to test this notion. This discovery would have exceptionally high impact because existing treatments for ADs are modestly effective, and relapse rates post-treatment are notoriously high and long-term remission is heavily dependent on voluntary continuation of treatment, particularly pharmacotherapy. There is an ongoing urgent need to develop new treatments for ADs that precisely targets an underlying pathological mechanism quickly and permanently. To set the stage for and de-risk future clinical trials and in accordance with the U01 RFA requirements using small (n<10) studies, we propose a first- in-human, proof-of-concept experiment to an existing participant pool who are already undergoing MRgFUSA- ATN for epilepsy (using the existing FDA-approved IDE) and for the purpose of this proposal include only those with high anxiety (as measured by the Hamilton Anxiety Rating Scale, HAM-A; HAMA score > 17) to track the immediate (intraoperative) effect of MRgFUSA-ANT on amygdala reactivity and short- and long-term reduction in anxiety symptoms over the course of 12 months. This project seeks to answer 3 questions: 1) Is MRgFUSA- ATN safe and tolerable, in a high anxiety cohort; 2) Can MRgFUSA-ATN reduce amygdala reactivity to threat; and 3) Can MRgFUSA-ATN reduce anxiety/fear symptoms by post-operative day 1, and how durable are the anti- anxiety effects? If successful, this proposal will provide a guiding and transformative approach towards developing fast-acting, permanent but non-invasive neurosurgical treatments for patients suffering from anxiety and other mental illnesses and inform, optimize and de-risk future clinical MRgFUSA studies.

NIH Research Projects · FY 2026 · 2022-03

In many neurological diseases, specific subsets of neurons are more sensitive to dysfunction and degeneration than others. In Alzheimer’s disease (AD), excitatory (EX) neurons are preferentially vulnerable to tau pathology which defines the pathogenesis and progression of dysfunction in AD. Understanding the molecular origins of selective neuronal vulnerability is of fundamental importance for all of the neurodegenerative diseases including AD. Using single-nucleus RNA-seq dataset analysis and weighted gene co-expression network analysis of the transcriptomic signatures of different cell types from non-AD cases, we identified novel subproteome gene signatures in EX neurons that may serve as potential master regulators of selective neuronal and regional vulnerability to tau pathology in early AD. The ectodermal-neural cortex 1 (ENC1) is one such potential master regulator. Although the role of ENC1 in AD has not been thoroughly investigated, if it functions as a master regulator as predicted by bioinformatics analysis, it may be possible to regulate ENC1 levels to control tau pathology and thwart the onset or progression of AD. Preliminary analysis of human entorhinal cortex from AD and control brain specimens has revealed that ENC1 is enriched in the nucleus of EX neurons in control brains, while cytoplasmic ENC1 levels are elevated within neurons that show accumulated pathological tau species in AD specimens. The interaction between ENC1 and tau correlates directly with levels of pathological tau. Furthermore, forced overexpression of ENC1 mainly in the cytoplasm increases tau aggregation and seeding activity, whereas knockdown of ENC1 reduces these pathological changes. ENC1 has been shown to increase the neurotoxicity of mutant huntingtin under ER stress through the interaction with p62 and the inhibition of autophagy flux. Our new data also show ENC1 overexpression increases pathological tau accumulation, p62 puncta formation and autophagy dysfunction, implicating impairment of p62-mediated autophagy as a mechanism underlying the cytoplasmic accumulation of ENC1 and pathological tau in neurons. Based on these preliminary data, we hypothesize that cytoplasmic ENC1 contributes to the vulnerability of EX neurons to tau pathology, and that reducing ENC1 in EX neurons will enhance the autophagy pathway thereby protecting these EX neurons from selective neurodegeneration in AD. To test this hypothesis, this proposal will (1) determine the effect of ENC1 on tau aggregation and propagation in human cerebral organoids; (2) investigate the role of ENC1 in autophagy pathway and if this pathway is involved in ENC1-induced tau aggregation and propagation in vitro; and (3) determine if cell-type specific manipulation of ENC1 affects neuronal autophagy, AD pathology and cognition in vivo. The proposed studies will provide mechanistic insight into the role of ENC1 as a master regulator of tau homeostasis and will also provide greater insight for developing novel therapeutics targeting ENC1-dependent pathways to prevent, treat or delay the neurodegeneration in AD. Furthermore, this work will elucidate novel mechanisms underlying selective neuronal vulnerability in AD.

NIH Research Projects · FY 2026 · 2022-02

Project Summary: Autoimmunity is a leading cause of chronic illness that encompasses more than 80 individual diseases. Due to the rising prevalence of these diseases, autoimmunity associated health problems currently affect over 20 million individuals only in the USA, constituting a health crisis that requires immediate attention. Autoimmune diseases stem from disturbances in the tolerance of immune system against self-tissues. Immune tolerance is achieved in part by the elimination of self-reactive T cells during their development in the thymus. The self-reactive clones that escape thymic elimination are actively silenced in the periphery by a subset of T cells called “regulatory” T (Treg) cells. Because Treg defects result in fatal autoimmunity, increasing Treg number and activity in the body appears to be a desirable strategy to prevent and treat autoimmune diseases. However, we have a major gap in our understanding of how Tregs perform their inhibitory roles at the molecular level and this hinders the development of effective therapeutic strategies. Recently, I demonstrated, for the first time, that Tregs can inhibit effector T cells in an antigen-specific manner. I reported that Treg antigen receptor (TCR) can remove class II major histocompatibility complex bound antigenic peptide (pMHCII) from surface of antigen presenting cell (APC), dendritic cell (DC) in particular, thus deplete the antigenic stimulus that effector T cell needs to receive to get activated. I revealed that this happens during Treg-Dendritic cell (DC) contact, whereby cognate pMHCII laden DC membrane is captured by Treg in an elegant way that does not reduce the presentation of non-cognate pMHCII by the same DC. I hypothesize that this highly specific mechanism can be exploited to effectively reduce pathological presentation of self-antigen by APC as a promising strategy to combat autoimmunity. I will test this hypothesis by taking the following steps: 1) Determining the antigen specificity of Treg suppression and pMHCII removal in human Tregs and visualizing their interactions with DCs that present self-antigens. 2) Characterizing the molecular machinery employed by Tregs to perform pMHCII depletion and dissect the molecular switches that can be targeted to tune Treg activity. 3) Determining the functional significance of antigen capture by Tregs to reveal potential mechanisms whereby Tregs present captured pMHCII complexes to prime naive T cells resulting in the spreading of antigen specific tolerance. By uncovering novel pathways of antigen-specific immune suppression, this New Innovator Award will identify new targets for immune system modulation that can be utilized for the treatment of chronic diseases such as autoimmunity and cancer. Findings from this project will be instrumental in generating future antigen-targeted immunotherapies, thus the objectives of this New Innovator Award serve the strategic mission of the National Institute of Allergy and Infectious Diseases.

NIH Research Projects · FY 2026 · 2022-02

Project Summary/Abstract Obesity has reached epidemic proportions in the USA, with more than 40% of the population classified as obese (body mass index (BMI) > 30 kg/m2), and nearly 10% as severely obese (BMI > 40 kg/m2). Cardiovascular disease (CVD), especially heart failure, is highly associated with obesity. Reliable non-invasive cardiovascular imaging tests are needed to provide accurate diagnosis and prognosis in this patient group. Unfortunately, obese patients present a diagnostic challenge to current non-invasive cardiovascular imaging modalities. Computed tomography and single-photon emission computed tomography can require excessive radiation for successful imaging of obese patients, while echocardiography suffers from limited windows and signal attenuation leading to degraded image quality. Magnetic resonance imaging (MRI) of obese patients at conventional field strengths (1.5T and 3.0T) is primarily limited by narrow patient bore and table weight limits. As lower field strength allows greater flexibility in magnet design, a new low-field (0.55T) MRI with a unique, ultra-wide 80 cm bore and 700 lb. table weight limit that can accommodate severely obese patients was recently announced by Siemens. One of the first such systems in the USA will be installed at our institution this summer. In this project, we will develop cardiovascular MRI techniques for this system, and demonstrate their clinical value in severely obese patients. Our group and others have recently shown initial feasibility of cardiovascular imaging at low field. We believe that this new low-field MRI platform, when combined with the advanced pulse sequences and image reconstruction methods developed by our team, can unlock the potential of this novel system as a more reliable means to provide non-invasive cardiovascular imaging to patients with severe obesity. Furthermore, the reduced field strength will make MRI safer and more effective in the growing number of patients with implanted devices. We will prove low-field MRI utility for CVD assessment. Aim 1 will establish the feasibility of low-field cardiovascular MRI regardless of body habitus. We will develop image acquisition and reconstruction techniques specifically designed to compensate for the reduced MR signal at 0.55T compared to higher field. This will offer a comprehensive assessment of cardiovascular structure and function, and myocardial tissue. Aim 2 will validate these techniques in normal weight to severely obese healthy individuals and CVD patients, by head-to-head comparison with standard 70 cm bore 1.5T MRI in those able to safely fit in both machines. Aim 3 will apply the validated techniques to evaluate the efficacy of a low-field cardiovascular MRI exam in heart failure patients who are severely obese (BMI > 40 kg/m2) and cannot be safely imaged on standard MRI systems. We will compare the comprehensive diagnostic value of 0.55T MRI against echocardiography, and will correlate MRI markers to clinical symptoms of heart failure severity determined by a six minute walk test in these patients. The cardiovascular techniques that we develop for this imminent novel 0.55 T MRI system provides a timely solution for effective clinical care and management of the increasing population of severely obese CVD patients.

NIH Research Projects · FY 2026 · 2022-02

PROJECT SUMMARY Cutaneous leishmaniasis is a disease caused by leishmania parasites, and exhibits a wide range of clinical manifestations from self-healing lesions to chronic debilitating infections. Currently there are no vaccines for this disease, and the drugs used to resolve the infections are often ineffective. Although the parasites are important determinants of disease severity, the immune response itself causes a large amount of pathology. CD8 T cells have been shown to play a dual role in disease by being both protective when they produce IFN-γ, but pathogenic when they mediate inflammation-inducing cell death in lesions. We found that IFN-γ-producing protective CD8 T cells are restricted to draining lymph nodes, whereas cytotoxic and therefore pathogenic CD8 T cells are found in leishmanial lesions in both experimental murine models of the disease and in patients. Our preliminary results suggest that this dichotomy in CD8 T cell function is a response to the tissue microenvironment and that protective IFN-γ-producing CD8 T cells become cytolytic once they enter leishmanial lesions. The factors involved in this conversion are unknown, and here we propose to fill this gap in knowledge. In our first aim we will determine how the tissue microenvironment initiates the cytolytic pathway in CD8 T cells. We will test the role of hypoxia, IL-1β and IL-15 in promoting cytolytic T cell development and determine how heterogeneous the CD8 T cells are within the lymph nodes and lesions. In our second aim, we will determine what induces the expression of Blimp-1, a transcription factor that regulates cytolytic T cell function and is required for CD8 T cell-mediated disease. In addition, we will test the ability of Blimp-1 to promote pathology by enhancing CD8 T cell recruitment to lesions. Both of these aims will provide information helpful in designing therapies that might block the development of pathogenic CD8 T cells. Finally, in the third aim we will evaluate the role of neutrophils and the skin microbiota in altering the skin microenvironment. Our preliminary results suggest that neutrophils regulate O2 levels in lesions, that the microbiota amplify neutrophil recruitment and both neutrophils and the microbiota are required for CD8 T cell-mediated disease. Overall, these studies will provide information from murine models that will be foundational in understanding the immune responses mediating and regulating disease in cutaneous leishmaniasis.

NIH Research Projects · FY 2025 · 2022-02

Eukaryotic genomes exist in a complex and dynamic 3-dimensional structure that provides important regulatory information controlling gene expression programs essential for the maintenance of cell identity. Genome structure is also critical for packaging the genome in the nucleus and preserving genome stability. Genome structure is primarily dictated by its' association with the histone proteins to form chromatin. The post- translational modifications on histones play a key role in determining genome structure as they regulate the association of non-histone proteins with genomic DNA. An important challenge for eukaryotes is the epigenetic inheritance of the histone modification patterns that govern genome structure following DNA replication and cell division. This proposal seeks to identify fundamental mechanisms that regulate the epigenetic inheritance of histone modification patterns. Following passage of a replication fork, newly replicated DNA is packaged into chromatin that contains a 1:1 mixture of parental histones and newly synthesized histones. The retention of modification patterns on parental histones provides the spatial memory for the duplication of specific chromatin states. The key step in epigenetic inheritance is the transfer of the parental modification patterns to the neighboring new histones. Our work is based on the hypothesis that the new histones play a critical regulatory role in the epigenetic inheritance of chromatin states. The current proposal is based on my lab's identification of previously unknown links between the dynamic acetylation of newly synthesized histones and the epigenetic inheritance of specific chromatin states and the restoration of 3-dimensional genome architecture following DNA replication. The experiments proposed here will characterize the effect of new histone acetylation on the structure and composition of chromatin and identify factors and pathways that function with new histone acetylation to regulate the epigenetic inheritance of chromatin states. In addition, we will characterize a previously unanticipated role of new histone acetylation in the epigenetic inheritance of 3-dimensional genome architecture through regulation of the interactions between chromatin and the nuclear lamina.

NIH Research Projects · FY 2026 · 2022-02

Project Abstract Treatment of biofilm-associated infections using antibiotics can be limited by pathogen antibiotic resistance, as well as antibiotic tolerance displayed by infections. This application seeks to develop new strategies to eliminate persistent wound infections caused by multidrug resistant pathogens. Bacteriophages are natural, abundant, and diverse with minimal toxicity, particularly when used topically, and assumed to have negligible impact on the microbiome. Due to limited cross-resistance, bacteria displaying antibiotic resistance tend to remain phage susceptible and phage cocktails (polyphages) can be developed to minimize resistance to multiple phages, thereby better assuring continued susceptibility of targeted bacteria. Thus, an aggressive mixture of bacteria- and biofilm-disrupting agents – phages and antibiotics – may be employed towards reducing antibiotic-resisting and otherwise challenging-to-treat experimental wound infections. Here we propose to develop bactericidal bacterial viruses (bacteriophages or phages) as adjuvants to antibiotic application, with an aim towards future clinical testing while retaining current standards of care. Emphasis is placed on the treatment of MDR Pseudomonas aeruginosa-infected and mixed- infected wounds. The five key phage characteristics that we developed for therapy are robust antibacterial activity, ability to function against biofilms, host range breadth including multidrug resistant and colony variant P. aeruginosa, limited cytotoxicity against host cells, and phage stability. Aim 1 will evaluate the phage cocktail therapy in mono- and poly-microbial model systems. Aim 2 utilizes eco-systems biology approaches for in vivo testing of cocktails in a complex polymicrobial infection and will investigate how phage therapy-based targeting of a pathogen may cause changes to the wound microbiota. Overall, such therapy will be used to reduce the burden of devastating infections caused by multidrug resistant P. aeruginosa. This study will also lay the foundation for a program targeting each of the major ESKAPE pathogens.

NIH Research Projects · FY 2026 · 2022-02

Older adults (≥65 years old) constitute about 17% of the US population but account for ~57% of the population who use ≥3 drugs (polypharmacy). Currently, ~30million US adults use ≥3 drugs which increase their exposure to high-order drug-drug interactions (HDDIs), i.e. drug-drug interactions involving ≥3 drugs. HDDI is a major risk factor of serious adverse drug events (ADEs) that result in emergency department (ED) visits or hospitalization. Older adults, compared to younger adults, are 7-times more likely to be hospitalized and 2 to 3-times more likely to visit the ED for ADEs. However, knowledge on how to identify HDDI-ADE associations, data on the comparative safety of different 3-drug combinations and clinical and pharmacokinetic mechanisms for HDDI- ADE associations are all critically limited. The current project is designed to address these major gaps by specifically: (1) applying our state-of-art data mining techniques to discover HDDIs that are associated with higher risk of ADEs as well as those associated with lower ADE risk; (2) performing a comparative safety assessment of 3-drug combinations involving anticoagulants, antidiabetic agents and opioids; and (3) validating and evaluating the clinical validity and pharmacologic mechanisms of HDDI-ADE associations. These aims will be implemented by focusing on older patients who had an ED visit based on real-world data from health insurance claims (MarketScan and Medicare) and Electronic Health Records (EHR) data. We will focus on gastrointestinal (GI) bleeding, hypoglycemia and opioid-induced ADEs as the primary ADEs of interest for all three aims. These three ADEs account for 60% of all ADE-induced ED visits and are a target priority for prevention and surveillance by the US Health and Human Services. Our preliminary data from the MarketScan claims data (2012-2018) alone has confirmed that older adults are at higher risk of ADEs and that the risk of these ADEs increase with the counts of concomitant drugs used by patients. We will apply our mixture drug-count response (MDCR) and graphical models to identify the specific 3- or 4-drug combinations that have the highest risk and those with the lowest risk of ADEs in ED settings among older adults (Aim 1). For Aim 2, we will apply pharmacoepidemiologic study designs and casual inference approaches to assess the comparative safety of specific high and low-risk 3-drug combinations. We will control for the potential confounding effects of several patient-level (demographic, clinical, chronic comorbidities, drug characteristics, healthcare utilization, etc) and provider/healthcare system-level factors to assess the causal associations between high-risk versus low- risk 3-drug combinations. Finally, we will use a clinician expert team to evaluate the HDDI-ADE pairs and associations identified from Aims 1 and 2 for clinical validity and utility. The identification of high- and low-risk 3-drug combinations is vital knowledge that could guide the development of new polypharmacy prescribing recommendations to help address the significant public health challenge created by ADEs.

NIH Research Projects · FY 2025 · 2022-01

PROJECT SUMMARY/ABSTRACT Acute myeloid leukemia (AML) is a common and aggressive hematologic malignancy caused by a pathologic expansion of immature myeloid cells. Despite significant research efforts spanning 50 years, the 5-year survival rate in AML has remain relatively unchanged at <27.4%, underscoring the need for innovative approaches to treatment. Natural killer (NK) cells are a member of the group 1 innate lymphoid cell (ILC) family that play a pivotal role in the detection and elimination of leukemic cells. However, we and others have previously shown that AML is able to inhibit NK cell maturation, promoting disease progression. Mechanistically, we have shown that AML blasts are capable of secreting agonists for the aryl hydrocarbon receptor (AHR) transcription factor, which inhibits NK cell maturation and function. Accumulating evidence suggests that AHR is required for the maintenance of a related group 1 ILC subset termed an “ILC1.” Notably, ILC1s have been shown to be pro- tumorigenic in solid tumor mouse models. Therefore, we hypothesize that AML is able to skew ILC populations to suppress immune-surveillance by NK cells while promoting ILC1s. Preliminary data from our group support this hypothesis by demonstrating that AML is capable of expanding ILC1 populations in a tissue-specific manner in an AML mouse model. Furthermore, we predict that these ILC1s are functionally hyperactive in the setting of AML and promote disease progression. Our long-term goal in this project is to determine how AML leads to dysregulation of group 1 ILCs and to elucidate the downstream consequences on AML progression. Thus, our aims are 1) to identify and characterize the mechanism(s) which promote ILC1 expansion in AML and 2) to determine the functional consequences of expanded ILC1 populations in AML. To address these aims, we will use a combination of ex vivo and in vivo studies. First, we will determine whether AML promotes ILC1 expansion through interconversion from NK cells, skewing of ILC progenitors towards an ILC1 phenotype, and/or from direct proliferation of ILC1s. We will also use transgenic mouse models to assess the contribution of Ahr and ILC1- derived cytokines in promoting AML progression. By identifying these mechanisms, we will be able to better target these pathways to restore group 1 ILC homeostasis and inform the development of future immune-based therapies. This project represents a novel and innovative approach to targeted therapy in AML by focusing on how leukemia targets ILC populations, an area which has been understudied to date. Successful completion of these studies will fill in critical knowledge gaps of how ILCs are dysregulated in AML and how these cells can be targeted therapeutically to improve patient survival.

NIH Research Projects · FY 2026 · 2022-01

PROJECT SUMMARY Gene therapy for Duchenne Muscular Dystrophy (DMD) is currently being tested in clinical trials in young patients. However, limitations of the adeno-associated virus (AAV) delivery system, including its small carrying capacity and its low efficiency transfection of muscle stem cells, will remain a barrier to a cure. The miniaturized transgene being delivered that is based on a Becker muscular dystrophy dystrophin (micro- dystrophin) will still result in some skeletal muscle turnover with subsequent inflammation and cardiomyopathy. Additionally, injury resulting from normal muscle use will be repaired with muscle stem cells that will likely not express dystrophin. Both of these issues will result in at least low-level chronic inflammation, which will likely exacerbate muscle damage and ultimate loss of transgene expression, limiting efficacy. Prednisone, which has served as the standard of care for DMD, but has many severe side effects, continues to be given as an anti- inflammatory to prevent an immune response to the transgene. Published and preliminary data support the scientific premise that mineralocorticoid receptor (MR) antagonists, which have clinical benefit for DMD cardiomyopathy, stabilize muscle membranes, improve skeletal muscle force, and reduce fibrosis, are also anti-inflammatory and represent an ideal drug for combination with gene transfer. However, the anti- inflammatory properties of MR antagonists in muscular dystrophy have not been explored. Since prednisone directly competes with MR antagonist binding to its receptors, these studies are crucial for clinical use of MR antagonists as an adjunct therapy to replace prednisone. In this application, we will test whether prednisone and MR antagonists have the same or different effects on specific immune cell populations in dystrophic muscles, whether cytokine reductions by MR antagonists are dependent on MR signaling mechanisms in muscle fibers or myeloid cells, and whether MR antagonists limit accumulated damage after acute injury in dystrophic mice treated with micro-dystrophin gene therapy. We have developed methods to flow sort immune cell populations from single muscles from the mdx genotypic model of DMD that will allow the first identification of the immune cell populations suppressed by prednisone, despite decades of clinical use, and a direct comparison with MR antagonists. These methods will also allow for the unbiased identification of gene expression changes in inflammatory myeloid cells induced by MR antagonists and prednisone. These studies will inform optimal MR antagonist clinical use as a co-therapy to extend efficacy of emerging genetic therapies for DMD and potentially other forms of muscular dystrophies. The data generated will also identify novel potential anti-inflammatory treatment targets.

NIH Research Projects · FY 2025 · 2022-01

Emotion dysregulation is a transdiagnostic maintenance factor involved in a wide array of costly and debilitating psychiatric disorders. Although numerous full-model behavioral health treatments have been designed to improve patients’ emotion regulation capacities, these treatments consist of multiple components, making it difficult to discern which are active mechanisms leading to reductions in negative emotion intensity. Further, it is unclear whether the delivery of these evidence-based components can be tailored to the individual patient. The proposed Mentored Patient-Oriented Research Career Development Award (K23) is a four-year plan to support the applicant’s long-term career goal of becoming a clinical scientist with expertise in (1) identifying active mechanisms of emotion regulation interventions for psychopathology, (2) tailoring these interventions to individual patients, and (3) developing scalable interventions for wide dissemination. The applicant’s training and career thus far are aligned with these long-term goals. Throughout his graduate work, he conducted studies testing emotion regulation mechanisms in transdiagnostic samples and used this information to explore for whom these mechanisms were most impactful. The immediate goals of the K23 award are for the applicant to become skilled at intensive longitudinal experimental designs to disaggregate between- from within-person mechanisms of change and enhance his proficiency in conducting and analyzing multimethod assessments of emotion regulation. This proposal uses a two-phase approach to address these goals. In line with an experimental therapeutics approach, the goal of Phase 1 is to compare the effects of teaching one or three emotion regulation skills on daily changes in negative emotion intensity among participants with elevated emotion dysregulation. The first goal of Phase 2 is to apply a personalization algorithm based on Phase 1 data to an independent sample to determine which baseline participant characteristics predict greater reductions in negative emotion intensity in each experimental condition. The second goal of Phase 2 is to compare the effects of teaching participants emotion regulation skill(s) according to their optimal or non-optimal delivery condition, based on the personalization algorithm. The training plan closely matches the proposed research and long-term goals, including (a) developing advanced understanding in statistical methods to test between- and within-person mechanisms of emotion regulation interventions, (b) gaining proficiency in applying novel personalization algorithms, and (c) enhancing expertise in the implementation and analysis of multimethod assessments of emotion regulation skills. The broader aim of this research and training is to address the need for more efficient, personalized, and scalable interventions for transdiagnostic psychiatric conditions, in line with the NIMH strategic plan. This study will answer important theoretical and practical questions about the efficacy of different emotion regulation mechanisms on clinical outcomes that may promote the development of more targeted and disseminable interventions.

NIH Research Projects · FY 2025 · 2022-01

PROJECT SUMMARY/ABSTRACT Acute myeloid leukemia (AML) is a disease characterized by clonal expansion of myeloid cells with blocked differentiation. Despite newly approved targeted therapies for AML, there is still a large subset of patients whose disease progresses or does not respond to treatment. One group of patients that have demonstrated resistance to targeted therapy (entospletinib, enasidenib, ivosidenib, and venetoclax) have protein tyrosine- phosphatase non-receptor type 11 (PTPN11) mutations. The PTPN11 gene encodes for the phosphatase Shp2, which regulates multiple signaling pathways including the RAS-MAPK pathway. PTPN11 mutations (PTPN11+) commonly co-occur with mutations in the nucleophosmin (NPM1) gene. Our preliminary data demonstrates that the co-association of PTPN11 and NPM1 mutations results in inferior outcomes for patients treated with intensive chemotherapy. Intriguingly, we have seen that PTPN11+/NPM1+ patients rarely have FLT3-ITD, an alteration that is well-known to worsen outcomes in NPM1+ AML. Because PTPN11 mutations activate many downstream signaling pathways similar to FLT3-ITD, we hypothesize that the addition of PTPN11 mutations to NPM1+ AML can clinically phenocopy NPM1+/FLT3-ITD AML in terms of adverse outcomes and therapy resistance. Therefore, we have generated a novel transgenic Ptpn11E76K/Npm1cA mouse model to study the development of AML and the resulting immunosuppressive phenotype. Using this model, we will interrogate whether expanded myeloid cells from Ptpn11E76K/Npm1cA animals can initiate AML in immunodeficient mice and confer resistance to chemotherapy. In addition, we will use modified AML cell lines to study the mechanism of resistance, particularly the roles signaling pathways, antiapoptotic proteins, and immunosuppression play. The knowledge gained will clarify the biology of PTPN11 mutations in AML and provide the foundation to develop therapies that will be effective in patients with PTPN11+ AML. This research will be completed by an MD/PhD student under the supervision of two leading translational researchers in the field of AML. The MD/PhD program is well established at Ohio State, and cancer research at the Comprehensive Cancer Center is robust and exceptionally strong. The training plan contains opportunities for the development of critical thinking, scientific knowledge, technical expertise, clinical skills, communication capabilities, and rigorous experimental design. Successful completion of the research proposal and training plan will provide the skills necessary to become an independent researcher in the field of hematology.

NIH Research Projects · FY 2025 · 2022-01

Molecular features associated with time-to-event outcomes, such as overall or disease-free survival, may be prognostically relevant or potential therapeutic targets. Therefore, analyzing data from high-throughput genomic assays with clinical follow-up data has been of growing interest. The Cancer Genome Atlas (TCGA) Project has collected baseline demographic, clinical characteristics, and follow-up data for 11,125 patients for 32 different cancer types and corresponding tissue samples were processed for examining SNPs, copy number, methylation, miRNA expression, and mRNA expression. Because the number of variables (P ) exceeds the sample size (N), one strategy frequently employed when associating molecular features to survivorship data is to fit univariable Cox proportional hazards (PH) models followed by adjustment for multiple hypothesis tests using a false discovery rate approach. However, most chronic conditions and diseases, including cancer, are likely caused by multiple dysregulated genes or mutations. It is therefore critical to fit multivariable models in the presence of a high- dimensional covariate space. Traditional statistical methods cannot be used when the number of features exceeds the sample size (e.g., P > N), though penalized methods perform automatic variable selection and accommodate the P > N scenario. Penalized approaches including LASSO, smoothly clipped absolute deviation (SCAD), adaptive LASSO, and Bayesian LASSO have all been extended to Cox's PH model for handling high-dimensional covariate spaces. However, when modeling survival or other time-to-event outcomes, the Cox PH model assumes that all subjects will experience the event of interest, which is violated when a subset of subjects are cured. Instead, when a subset of subjects in the data are cured, mixture cure models should be fit. Although mixture cure models have been described for traditional settings where the number of samples exceeds the number of covariates, limited variable selection methods and no methods for high-dimensional model fitting currently exist for mixture cure models. Therefore, this project will overcome a critical barrier to progress in this field by developing penalized parametric and semi-parametric mixture cure models applicable for high-dimensional datasets. The specific aims of this application are to: (1) Develop penalized parametric mixture cure models for high-dimensional datasets; and (2) Develop a penalized semi-parametric proportional hazards mixture cure model for high-dimensional datasets. For both aims we will characterize the performance of the methods using extensive simulation studies, develop software, and distribute R packages to CRAN. In aim (3) we will identify molecular features associated with cure and survival using our large unique AML dataset from the Alliance for Clinical Trials in Oncology and assess robustness of findings using AML datasets from Gene Expression Omnibus and The Cancer Genome Atlas project. This research will fill a critical gap as there are currently no mixture cure models for high-dimensional data. We anticipate application of our methods to our AML data will enhance existing risk stratification systems used in daily clinical practice that determine treatment intensity and modality.

NIH Research Projects · FY 2026 · 2021-12

Abstract Extracellular vesicles (EVs) have gained significant attention since their discovery in 1983 as important mediators of intercellular communications, potential disease markers, therapeutic targets, and drug delivery vehicles. Though it is widely accepted that EVs get packaged inside the cell, pass through the extracellular environment, and deliver the cargo to the target cells. However, even after 37 yrs it is not determined, 1) how EVs handle the differential ionic environment (cytoplasm vs extracellular), 2) whether EVs possess any functional ion channels, and 3) whether any of these channels play a physiological role. We focused on answering these questions and focused on an ion with the largest gradient, i.e., potassium. Using the in silico approach, we discovered several ion channels, and the most prominent ion channels, we discovered in exosomes is BK. We incorporated a novel electrophysiology approach, near field electrophysiology, as canonical patch-clamp methods are not feasible due to the size of exosomes. We discovered that functional BK channels exist in exosomes, and decide the integrity of exosomes. Our preliminary data also indicate that exosomal BK can protect the heart from ischemia-reperfusion injury. We will now test the hypothesis that exosomes containing BK determine the content of exosomes, facilitate their survival in variable ionic environments, and protect the heart from IR injury. Overall the data supports the above hypothesis which will be tested using multiple approaches and pursuing the following specific aims to, 1. establish a presence, molecular identity, and biophysical properties of BK in exosomes, 2. determine the physiological role of BK in exosomes., and 3. elucidate the mechanistic role of exosomal BK channels in cardioprotection. In our proposal, we have incorporated genetic mice models, and innovative as well as a novel technology to understand a very basic and broad biological question. The outcome of this program will open an opportunity to study exosomal ion channels including BK channels, and advance the exosome field by determining how exosome survive variable osmolarities, establishing the molecular identity of exosomal ion channels, understand how cargo content is regulated by exosomal ion channels, and the role and mechanism of exosomal ion channels in cardioprotection. In the future, our study will set the ground for exploring other ion channels in exosomes from different living beings as well as organ systems.

NIH Research Projects · FY 2026 · 2021-12

A unique neurogenic niche in the adult hippocampus hosts neural-lineage stem cells that can persist throughout the lifespan in a wide range of adult mammals. Uncovering the functional role of these stem cells and how they interact with other cell types in the niche can provide insight in to the mechanisms that mediate hippocampal cognitive-emotional functions, as well as potential mechanisms for regenerating tissue in the adult brain. Recently, stem cell secreted proteins (i.e. the stem cell secretome) have emerged as influential players in tissue homeostasis. However, relatively little is known about either the content or the function of the secretome of endogenous neural stem cells and their progenitors (NSPCs) in the adult hippocampus. Our preliminary data reveal that adult hippocampal NSPCs may regulate their microenvironment through the production of the soluble protein, vascular endothelial growth factor (VEGF). We find that NSPCs synthesize large quantities of VEGF in their hippocampal niche and that NSPC-derived VEGF is necessary for sustaining healthy hippocampal function. We propose to investigate the hypothesis that NSPCs support hippocampal function by direct actions of VEGF that suppress neuronal hyperexcitability, ultimately supporting memory function, as well as protecting it from injury. In Aim 1, we will use RNAsequencing as well as genetic code expansion coupled with biorthogonal non- canonical amino acid protein tagging to determine the specific local contributions of NSPCs to VEGF isoforms in the cell layers of the dentate gyrus subregion where these cells reside. In Aim 2, we will use transgenic knockdown and viral rescue models to investigate how dentate gyrus circuit activity is regulated specifically by NSPC-derived VEGF. In Aim 3, we will use transgenic knockdown and viral rescue models to determine how NSPC-VEGF influences hippocampal behavioral functions and vulnerability to excitotoxic injury. The completion of this work will establish a new functional dimension of endogenous NSPCs via their secretome, and advance understanding of how hippocampal health is actively maintained in a unique niche of the adult brain.

NIH Research Projects · FY 2026 · 2021-12

An estimated 53,260 new oropharyngeal cancer cases and 10,750 deaths will occur in U.S. during 2020. Unfortunately, oral squamous cell carcinoma (OSCC) is one of the most challenging-to-treat human cancers. Even if surgical resections are curative, facial structures vital for function and esthetics are sacrificed. OSCC, however, doesn't occur de novo, but arises from initiated keratinocytes. This pre-transformation interval provides a therapeutic window for secondary OSCC chemoprevention. Specific situations such as tobacco and/or alcohol use or diseases associated with DNA repair deficits e.g. Fanconi anemia (FA) can render the entire oral cavity at risk to develop OSCC. Although systemically-delivered chemopreventives should conceptually provide full mouth field-coverage, bioavailability challenges and drug-related systemic toxicities have generated disappointing outcomes. In contrast, local delivery formulations can deliver therapeutically-relevant levels of chemopreventives-at markedly lower doses relative to systemic administration-to target tissue without drug-related systemic side-effects. Notably, the oral cavity is bathed in a protective, viscoelastic, adhesive coating hydrogel (mucous). While mucous can impede local drug delivery, mucoadhesive and mucopenetrating nanoparticle chemopreventive formulations address this issue. Nanoparticles also function to stabilize drugs, minimize off-target side effects, prolong chemopreventive-oral epithelial contact time and facilitate delivery to the underlying keratinocytes. The chemopreventives for this study were selected based on our results and their complementary mechanisms of action. IL6, produced by oral keratinocytes and other cells, is a pervasive cytokine throughout the mouth including saliva. Via its proinflammatory, pro-proliferative and proangiogenic properties, IL6 can facilitate malignant transformation of oral intraepithelial neoplasia to OSCC. To suppress this autocrine-paracrine loop, the IL6 receptor inhibitor, tocilizumab (TCZ) was selected. In addition, our labs have shown that the synthetic vitamin A derivative, fenretinide (4HPR), not only possesses growth modulatory effects, but it also demonstrates high affinity binding/inactivation of signaling kinases upregulated during carcinogenesis i.e. FAK, Pyk2, STAT3, Wnt, c-Src and c-Abl and perturbs cytoskeletal components necessary for OSCC cell invasion. Our data also show that while single agents are beneficial, TCZ+4HPR combination treatment enhances the agents' chemopreventive efficacy in both in vitro and in vivo models. The Specific Aims of this proposal are: 1) Optimize Janus nanoparticles (JNPs) for targeted co-delivery of 4HPR & TCZ to surface oral epithelium, 2) Identify the lead JNP formulation by using bioassay-based in vitro studies and an in vivo PK model., 3) Conduct a Phase 0 pharmacokinetic/ADME trial in healthy volunteers. Experimental methodology will include: electrohydrodynamic co- jetting in conjunction with dynamic light scattering, zeta sizing and electron microscopy to formulate the JNPs, ex vivo mucoadherence studies, in vitro monolayer and raft culture functional assays, LC-MS, IHC (quantified by image analysis), and in vivo (rabbit model) and human clinical trial PK analyses.

NIH Research Projects · FY 2025 · 2021-12

Project Summary: The proposed research explores mechanisms of dysregulated lipid metabolism in failing hearts within the dynamic processes of long chain fatty acid (LCFA) delivery from the circulation to metabolism in the cardiomyocyte (CM). We will explore the roles of LCFA delivery by endothelial cells (EC) to CMs and rates of LCFA uptake by CMs in adverse metabolic remodeling in a mouse model of heart failure that recapitulates key metabolic defects in failing human hearts. Hearts rely on LCFA oxidation, up to 70% of fuels, to meet ATP demand. LCFA are also esterified into the neutral triglyceride (TG) pool and into physiologically active acyl- derivatives. In failing hearts, LCFA oxidation is reduced and the lipid profile becomes toxic. We have shown a reduction in the central LCFA metabolite, acyl-CoA, in failing hearts is detrimental, and increased acyl CoA production by ACSL1 improves metabolic state and mitigates functional decline. 13C NMR of hearts revealed an exponential component of 13C LCFA entry into TG that reflects LCFA uptake rate and is sensitive to activity of the LCFA transporter, CD36. LCFA uptake is also accelerated by metabolic trapping via esterification of LCFA to acyl-CoA by ACSL1, a process that is sex-dependent. Experiments on mice with cell- specific, CD36 deletion in ECs (EC-CD36 KO) and CMs (CM-CD36 KO) will support the objective to study the separate roles of LCFA delivery by ECs to CMs and uptake by CMs on LCFA metabolism within competing pathways, including deleterious ceramide formation in failing hearts. Potential differences in LCFA uptake and metabolism between two primary physiological sources, albumin-bound LCFA and lipoprotein-bound TG, will be studied. The hypothesis is: a) the contributions of EC CD36-dependent and independent transendothelial transport of LCFAs into CMs of normal and diseased hearts determine the metabolic fate of LCFAs, separate from CM CD36 activity, and depend on the LCFA source; b) there are sex-dependent differences in both CD36 transport of LCFA and trapping of LCFAs into CMs via esterification that contribute to the lipotoxic profile of failing hearts. Specific aims are: 1. Elucidate EC CD36 contributions to transendothelial transport of LCFA uptake kinetics and metabolic fate in normal and failing hearts of male vs. female EC-CD36 KO mice. 2. Distinguish CM-CD36 from EC contributions to LCFA uptake kinetics and metabolic fate in hearts of normal and failing heats of male vs. female CM-CD36 KO mice. 3. Elucidate reciprocal effects of CD36 transport and metabolic trapping by ACSL1 on LCFA use in normal and failing hearts, by silencing of CD36 in hearts having low overexpression of ACSL1 (MHC-ACSL1 J3) and in hearts from crossed, MHC-ASCL1xEC-CD36 KO and MHC-ASCL1xCM-CD36 KO mice. 4. Distinguish contributions of EC-CD36 and CM-CD36 to LCFA uptake rates and metabolism from albumin-bound vs. chylomicron-bound sources in normal and failing hearts of wild type, EC-CD36 KO, and CM-CD36 KO mice. Outcomes will provide unique information on molecular events modulating lipid content as new targets to resolve metabolic imbalances in failing hearts.

NIH Research Projects · FY 2026 · 2021-12

PROJECT SUMMARY Mitochondrial dysfunction and altered mitochondrial metabolism have been implicated in the development of heart failure (HF). Mitophagy is a specialized autophagic pathway that mediates the lysosome-dependent clearance of damaged mitochondria, and is essential for mitochondrial quality control. However, our current knowledge regarding mitophagy in the heart and how it relates to myocardial metabolism is limited. Under normal conditions, the heart relies predominantly on fatty acid β-oxidation (FAO) to fuel ATP production. In contrast, a failing or hypertrophied heart usually shows impaired FAO and increased reliance on glucose utilization. Despite significant advancements in understanding the regulatory programs that attenuate FAO, it is unclear how shifts in myocardial substrate utilization contribute to the regulation of mitophagy. Therefore, this research proposal focuses on the functional significance of cardiac mitophagy in response to altered myocardial substrate utilization that occurs, for instance, in the setting of impaired FAO. The goal of this project is to delineate the novel mechanistic link between mitophagy and myocardial substrate utilization in the heart, as well as to determine whether mitophagy represents a novel mechanism and therapeutic target for the treatment of heart disease. These studies will be facilitated by our recently described mt-Keima mouse model to monitor in vivo cardiac mitophagy, as well as a set of innovative reagents to genetically and pharmacologically modulate mitophagic flux. To directly assess the role of FAO in the heart, we have generated mice with cardiomyocyte-specific deletion of CPT2 (CPT2-cKO), encoding a single gene required for FAO. We have demonstrated a decline in mitophagy precedes the development of impaired cardiac function in FAO-deficient hearts. Our genetic analyses suggest cardiac CPT2 deletion impairs the PTEN-induced putative kinase 1 (PINK1) signaling pathway, which positively regulates mitophagy through mitochondrial ubiquitination. Augmentation of mitophagy by modulating USP30, which mediates the reverse reaction, deubiquitination on mitochondria, mitigates the functional decline in FAO deficient hearts. Therefore, in Aim 1 of the proposed studies, our goal is to define the magnitude and Kinetics of cardiac mitophagy in response to impaired FAO, as well as to dissect the underlying molecular mechanisms connecting mitophagy to myocardial substrate utilization. In Aim 2 of the proposed studies, we will genetically and pharmacologically manipulate USP30 enzymatic function in the heart using mouse models that lack USP30 or are treated with a novel USP30 inhibitor. We will determine whether the detrimental cardiac phenotype, induced by cardiac CPT2 deletion or pressure overload, could be reversed, at least partially, by restoring mitophagy in cardiomyocytes via the inhibition of USP30. Completion of the proposed studies will produce critical insights into the role of mitophagy in normal cardiovascular physiology and in pathological conditions, and will fundamentally advance our understanding of the interaction between mitochondrial metabolism and mitochondrial quality control in the heart.