Virginia Polytechnic Inst And St Univ

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY: Despite aggressive definitive treatment of osteosarcoma (OS), survival expectations for human and canine OS have not improved in decades due to refractory metastatic disease. The poor immune responsiveness of osteosarcoma to immunomodulating therapies contributes to the persistence of metastatic disease development. Novel therapeutic paradigms to activate this “cold” tumor microenvironment and induce an effective anti-tumor immune response in OS are direly needed. To fulfill this need, our research team proposes a combination therapy approach using 2 highly novel modalities – histotripsy tumor ablation and N-dihydrogalactochitosan (GC) to treat OS. Histotripsy is a non-thermal, non- invasive and non-ionizing tumor ablation technique that utilizes high intensity focused ultrasound waves to mechanically disintegrate tissue. Histotripsy releases non heat-denatured tumor antigens, which can potentially induce a more robust anti-tumor immune response. GC is a semisynthetic derivative of chitin with demonstrated ability as an immunostimulant that enhances the anti-tumor effects of various ablation modalities across different cancer types. Despite the exciting potential of histotripsy and GC to synergistically induce an effective anti-tumor response, combining both therapies to treat OS has not been reported. Thus, this multidisciplinary proposal will evaluate combining histotripsy tumor ablation with GC as an innovative immunomodulatory strategy for treating osteosarcoma using a unique comparative oncology study that incorporates murine preclinical models and pet dogs with spontaneous appendicular osteosarcoma. The overall objective of our proposed study is to evaluate local and systemic immune responses in OS after combining histotripsy tumor ablation with intratumoral injection of GC. Our overall hypothesis is histotripsy-GC treatment of OS induces effective and durable intratumoral and systemic anti- tumor immune responses. We will test this hypothesis with the following aims: Aim 1: Characterize the immunomodulatory responses to and effects on metastasis of histotripsy-GC treatment of OS in a murine heterotopic syngeneic tumor model. We hypothesize that histotripsy-GC treatment of OS heightens the intratumoral and systemic immune activation profiles, induces an abscopal effect, and reduces metastatic burden. Aim 2: Evaluate the feasibility of histotripsy-GC treatment of OS in dogs with appendicular OS, and the effects of treatment on the immune profile of the OS TME and on progression-free survival. We hypothesize that histotripsy-GC treatment of canine appendicular OS does not result in major adverse events, stimulates increased intratumoral immune cell infiltration, upregulation of intratumoral and lymph node immune activation gene profiles, and achieves increased PFS compared to standard-of-care therapy. Significance: These data will inform subsequent studies that generate clinically relevant data to design future human clinical trials for histotripsy-GC therapy in OS.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Consumption of unsafe drinking water is associated with a substantial burden of disease. For children <5 years old, unsafe drinking water is one of the leading risk factors for enteric pathogen infection and diarrhea incidence. Violations of health-based regulations in public drinking water systems are highest in lower-income rural areas of the United States (US) overall, and particularly in the Central Appalachia region. Appalachia is also impacted by substantial disparities in health outcomes, including higher mortality rates for 7 of the 10 leading causes of death. Estimates indicate ~40 million people in the US live in homes with unregulated private wells. However, our understanding of drinking water contaminants and associated health impacts for individuals living in house- holds with private water supply in rural areas of the US is severely limited by a lack of data – particularly for individuals living in low-income households in rural areas of Appalachia, and Central Appalachia especially. Our preliminary studies in Central Appalachia indicate that many lower-income households with private wells or spring water have relatively higher exposures to waterborne pathogens (and some chemical contaminants). Our overarching goal is to assess the potential of a low-cost, household-level, water treatment intervention for ex- panding safe water access and improving health outcomes and wellbeing for rural households without utility- supplied water. We propose to enroll 480 lower-income households (~1,584 individuals) with well or spring water and conduct a cluster-randomized controlled trial in 10 Central Appalachia counties (in VA and TN) to evaluate the hypothesized benefits of point-of-use water treatment using countertop water filters. We will build on estab- lished relationships with a range of state and local agencies for recruitment, and will address the following aims: 1) Measure the effect of drinking water filtration on the incidence of acute gastrointestinal illness among infants, children, and adults; 2) Measure the effect of drinking water filtration on the presence and quantity of bacterial, protozoal, and viral waterborne pathogens detected in saliva and stool biospecimens; and 3) Quantify fecal indicator organisms, waterborne pathogens, and regulated chemical contaminants in drinking water samples from treatment and control households. This will be the first randomized controlled trial in the US to evaluate point-of-use water filtration for households with well water. This effort is designed to rigorously assess and quan- tify the benefits of a relatively low-cost and easy to use water treatment approach that can also improve the taste of water, and is a less-costly alternative to bottled water reliance. Findings from this study will also substantially improve our understanding of which waterborne and enteric pathogens infants, children, and adults living in households with private water supplies in rural areas are exposed to and infected with, enabling more informed estimates of the associated burden of disease for rural populations without utility-supplied water. Such results will help to increase awareness of the need for improved policies and programs to expand access to safe drinking water for vulnerable and underserved populations in rural Appalachia and other rural areas of the US and beyond.

NIH Research Projects · FY 2025 · 2025-07

Project Summary/Abstract. The current treatment regimen for glioblastoma, surgical resection followed by radiation and chemotherapy, increases median survival from 12.1 to only 14.6 months. Generating effective therapies against glioblastoma is difficult because treatments must: 1) gain access to the brain and target tumor cells without damaging healthy cells, 2) target spread out cells since glioblastoma is not contained within a defined mass like other cancers, and 3) trigger the host’s immune response to overcome immune suppression mediated by glioblastoma cells, including highly invasive and resistant glioblastoma stem cells (GSCs). Neurotropic oncolytic viruses (NOVs) are a promising treatment option since they cross the BBB and preferentially target rapidly dividing cancer cells throughout the brain while stimulating host immune responses to target the tumor. OVs can also be engineered to produce foreign proteins to augment the vector’s killing capacity. Thus, NOVs overcome many of the limitations of other glioblastoma treatment options. Semliki Forest virus (SFV) is a potent and safe NOV, but there are limitations in its effectiveness since some glioblastoma cells are refractory to SFV infection, resulting in incomplete tumor removal. Our long-term objective is to create virus-based therapies that reduce human disease burden. This proposal aims to enhance SFV’s therapeutic efficacy against glioblastoma using directed evolution and virus-mediated delivery of a potent anti-GSC peptide. Our preliminary data show that we can effectively adapt a virus to a new environment using directed evolution, and we have also created highly safe vaccine candidates that express cytokines. Finally, we have developed a highly effective GSC-targeting peptide, generated SFV expressing the peptide, and showed it has more potent killing activity against glioblastoma cells while being safe in healthy cells. We hypothesize that more potent, yet still safe, oncolytic SFV vectors can be generated using directed evolution and virus-driven expression of cancer-targeting peptides. We will test our hypothesis using rigorous molecular virology, ex vivo, and in vivo methods and a multidisciplinary team of experts in virology, immunology, and oncology. In Aim 1, we will use directed evolution in 3D glioblastoma organoids to select for a glioblastoma-adapted virus. The outcome of this aim will be a glioblastoma-adapted virus, which we expect will improve disease outcomes and have minimal adverse effects. In Aim 2, we will test SFV- expressing a potent GSC-targeting peptide in relevant in vivo and ex vivo models. The outcome of this aim will be a virus targeting GSCs and differentiated glioblastoma cells. Impact: These studies will develop two novel strategies to create more potent OVs. Completing these high-risk, high-reward studies will lay the groundwork for studies aimed at clinical testing of NOVs to treat all types of cancer in humans and will launch an exciting research direction in the lab of a productive young investigator with a drive to improve cancer treatment outcomes.

NIH Research Projects · FY 2025 · 2025-07

Structural and biochemical insights into the signaling mechanism of the histidine kinase GacS from Pseudomonas aeruginosa Project Summary The nosocomial pathogen Pseudomonas aeruginosa causes acute and persistent, chronic infections using distinct virulence mechanisms. The sensor signaling kinase GacS is a major player in regulating these virulence mechanisms. GacS itself is regulated through a complex set of non-canonical interactions. In recent work, our structural and biochemical studies have revealed that RetS and GacS form a novel heterodimeric DHp-DHp domain interface but that the overall GacS dimer remains intact. Consequently, GacS is predicted to undergo substantial conformational changes to accommodate RetS. Here, we propose to test a novel hypothesis positing that binding of the extracellular GacS ligand modulates RetS-GacS interactions by controlling the rigidity of the protein. Under the first aim, we propose experimentally validate the GacS kinking motion predicted by molecular dynamics simulations. The second part of the aim will directly measure the impact of ligand binding on shape and flexibility of a CitAGacS chimeric protein to test our hypothesis. Under aim 2 we seek to determine the identity of the elusive GacS ligand and use virtual screening to identify drug-like modulators of GacS. The in vitro screening approach is informed by our recent discovery of an inside-out signaling mechanism, whereby RetS primes GacS for signal recognition. Virtual screening is aided by the creation of a molecular model of the ligand-bound conformation of the sensory domain.

NIH Research Projects · FY 2025 · 2025-07

Project Summary Pulmonary Arterial Hypertension (PAH) is a disease characterized by pulmonary vascular remodeling leading to right heart failure, and death. PAH is defined as a mean pulmonary arterial pressure greater than 20 mmHg at rest, as assessed by right heart catheterization. Despite advances in our understanding of PAH pathology and therapeutic interventions, the survival rate is still considerably low, with poor prognosis. Thus, there is an urgent need to find novel factors that may contribute to PAH pathogenesis and progression. Evidence has implicated sleep disturbance as a risk factor for cardiovascular diseases. However, the effects of sleep disturbance on PAH pathology are limited in the literature. Thus, this project seeks to investigate the impacts of sleep disturbance on PAH in human pulmonary vascular cells and in different mouse models of PAH. Knowledge from this study will better inform clinicians on whether to include poor sleep as an important factor in risk-stratifying PAH patients. Additionally, findings from this project may open new therapeutic windows for pulmonary hypertension by developing small molecules to manipulate the molecular clock and therefore improve the related quality of health of PAH patients. I hypothesize that sleep disturbance impairs pulmonary vascular cell phenotypes, increases the hemodynamic burden, and exacerbates PAH in mouse models of PAH. This hypothesis will be tested under two specific aims. Aim 1 determines the circadian clock oscillations in pulmonary vascular cells from healthy and PAH-diseased patients and examines the effects of clock genes on cell cycle dynamics and pulmonary vascular cells functions. To accomplish this aim, qPCR, immunoblotting, proliferation, migration and Bioluminescence assays will be performed. Aim 2 investigates whether sleep disturbance affects PAH severity and progression by using two models of sleep disturbance (sleep fragmentation and chronic jet lag), genetically predisposed mouse models of PAH (Egln1 KO, IL-6 transgenic, and Bmpr2 mutant mice), as well as Bmal1 KO mice to investigate the role of clock genes in PAH. Magnetic Resonance Imaging, cardiac hemodynamics, morphometric, histological, and molecular profiling will determine whether sleep disturbance exacerbates and/or accelerates PAH. Using integrated but independent approaches, this proposal will dissect the effects and mechanisms of poor sleep on PAH, and thus make advances in understanding and treating PAH. This project is driven by a unique and comprehensive training plan designed to integrate expertise in molecular techniques, circadian biology, sleep medicine and advanced hemodynamic measurements in cardiopulmonary disease. It emphasizes the development of technical, professional and communication skills, as well as grantsmanship and undergraduate mentorship. This proposal is supported by a sponsor (Dr. Sassi) and co-sponsor (Dr. Gourdie) whose labs have expertise in in vitro and in vivo models of cardiovascular diseases. Therefore, the project will be conducted in an ideal environment to study the impact of sleep disturbance on pulmonary arterial hypertension.

NIH Research Projects · FY 2026 · 2025-07

PROJECT SUMMARY During early stages of Alzheimer’s Disease (AD), skeletal muscle mass and function precipitously declines in comparison to those who are cognitively intact, potentially due to poor neuromuscular health. Thus, bioenergetics of peripheral tissues may have an underappreciated role in AD etiology. Exercise is an effective means to promote mitochondrial and neuromuscular health. However, whether regular exercise has therapeutic potential for delaying or preventing AD is an outstanding question. We present evidence of impaired skeletal muscle AMPK-signaling response to exercise in 5xFAD mice, a model of AD and show neuromuscular dysfunction is present at a young age before observable cognitive decline. Also, we present evidence that mitochondrial respiration does not improve following 12 weeks exercise training in 22-week-old 5xFAD mice compared to WT. In sum, impaired neuromuscular function may underlie a maladaptive bioenergetic response to exercise prior to overt manifestation of AD-like pathology. There is a critical need therefore to define adaptive neuromuscular mechanisms in relation to established neuropathological changes over the continuum of AD-like pathology to identify novel therapeutic targets. We hypothesize impaired bioenergetics precedes manifestation of overt AD- like neuropathology resulting in neuromuscular maladaptation to exercise training. We propose two aims: Aim 1) Determine neuromuscular adaptive response to endurance exercise training in AD mice before AD-like pathology. We will assess mitochondrial respiration and reactive oxygen species (ROS production in intact muscle fibers and as well as synthesis (i.e. biogenesis) and breakdown (via D2O labeling - GC/MS) of muscle mitochondria and intact sciatic nerves in 22-week-old 5xFAD and APP/PS1 male and female mice following 12 weeks voluntary wheel running (exercise training) (1a), determine pre- and post-exercise training neve- stimulated muscle function and sciatic nerve conductance in vivo, neuromuscular junction integrity (histochemistry) and mitochondrial quality (1b), assess central (hippocampus) and peripheral (plasma NfL and NMJ) neuropathology (1c), and perform untargeted metabolomics of muscle, sciatic nerve, and hippocampus following exercise training (1d). Aim 2) Determine tissue-specific and functional roles for AMPK⍺1 in AD-like etiology in 5xFAD mice. We will assess mitochondrial function, sciatic nerve conductance, proteostasis, development of neuropathology, and metabolomics in both muscle, sciatic nerve, and hippocampus at 3 and 9 months of age in muscle- and motor neuron-specific AMPK⍺1 knock-out, as well as novel gain- and loss-of- function AMPK⍺1(T172A) knock-in mice. Our findings will elucidate neuromuscular responses to exercise training in context with AD-like neuropathology and integrated isoform-specific functional roles of AMPK⍺ in AD- like etiology. These studies will provide mechanistic and integrated insight into novel roles for neuromuscular dysfunction as a sentinel for AD and provide training, career development opportunities, and protected time to establish a research program focused on mechanisms of age-related disease and healthy aging.

NIH Research Projects · FY 2026 · 2025-07

PROJECT SUMMARY/ABSTRACT Understanding the top-down control of the neurobiological basis for aggression could help us identify ways to reduce uncontrolled rage eliciting numerous social issues and economic burdens. Cognitive control that includes abilities to pay attention is a fundamental ability essential for recognizing social cue and establishing social relationships. Individuals with cognitive impairment like inattention, however, often externalize socially disruptive behaviors, such as impulsive aggression. Notably, early life trauma (ELT), in the form of child abuse/neglect, is a critical risk factor for cognitive decline and pathological aggression in later life, yet the neural circuit mechanisms that transduce ELT into maladaptive neuronal changes resulting in impulsive aggression are not well understood. The thalamic nucleus reuniens (RE), the largest midline thalamic nucleus, is thought to be a key component connecting an extensive network between the ventral hippocampus (vHip) and medial prefrontal cortex (mPFC). The RE highly expresses L-type calcium channels (LTCCs), potential therapeutic targets for psychiatric diseases. Our preliminary data showed that mice received a systemic treatment of Bay K8644 (Bay K; LTCCs agonist) display decline in cognitive performance with poor attention and impulsive responses during the five-choice serial reaction time task (5-CSRTT), while they exhibit escalated aggression in the social context. Likewise, a same set of behavioral phenotypes was observed in mice exposed to ELT (e.g., pup-dam/littermate separation) who exhibit increased LTCC expression in the RE. We also found that optogenetic/chemogenetic activation of the vGlut2 RE projecting to the vHip (vGlut2 RE vHip), but not vGlut2 RE mPFC, is sufficient to induce the aggression in control mice, whereas optogenetic inhibition of vGlut2 RE vHip circuit normalizes aggression of ELT mice. Our long-term goal is to test the hypothesis that vGlut2 RE vHip is the primary circuit that mediates the maladaptive effects of LTCCs on early adversity-induced aggression. To achieve our objective, we will first characterize the in vivo activity of vGlut2 REvHip neurons during aggressive behaviors of ELT mice. We will examine whether ELT disinhibits in vivo vGlut2 RE vHip activity leading to increased neural responses during aggression (Aim 1). Using CRISPR-Cas9 approach, we will then test whether LTCCs in the RE circuitry are necessary for eliciting impulsive aggression associated with cognitive decline. Using molecular profiling methods, we will further examine how ELT induces circuit-specific molecular adaptation in the vHip- projecting RE neurons (Aim 2). Lastly, we will focus on circuit-specific role of the vHip in controlling ELT-induced aggression. Using intersectional optogenetic manipulation, we will determine whether REvHip hypothalamus circuit plays a key role in promoting impulsive aggression following ELT (Aim 3). Completion of these experiments will transform our understanding of the social aggression associated with dysregulated cognitive control and provide new insight into the circuit-specific roles of RE in driving the risk of maladaptive aggression that often follows early adversities.

NIH Research Projects · FY 2026 · 2025-03

SUMMARY The prevalence of obesity in the United States (US) has been consistently rising. For individuals with obesity the lifetime risk of developing type 2 diabetes (T2D) is 3-7 times greater than for those without. Despite substantial advances in obesity treatment, obesity and T2D remain global public health challenges. Recent estimates suggest dietary factors contribute to >300,000 deaths per year in the US, making decisions about what foods to eat a leading and modifiable driver of disease burden. While changes in brain systems involved in energy balance and homeostatic regulation are thought to contribute to obesity, importantly, changes in neural systems associated with attention, reward learning, and affective regulation also occur. These changes may allow reward systems to override satiety signals and promote eating. Consequently, a detailed understanding of the underlying neurochemistry of attention, reward, and valence is key to understanding the neurobiology of eating behavior and obesity. A wealth of literature, largely generated in model organisms, supports the general hypothesis that alterations in brain catecholamine signaling (i.e., dopamine and norepinephrine release) are key drivers of food intake and reward-based decision making. The importance of studies in rodents on mechanisms of food reward, learning, motivation, and by extension the neurobiology of obesity cannot be overstated. However, much of this literature has not been translated to humans. This represents a fundamental gap in our understanding of the essential human health behavior of food choice and food reward. The long-term goal of this research is to provide a framework to translate and extend mechanistic studies of food reward in rodents to humans. To that end, here we will leverage innovations from our group in two domains: 1. the capacity to detect sub-second changes in dopamine (DA) and norepinephrine (NE) and distinguish them from each other, 2. the ability to perform these measurements on electrodes routinely implanted in clinical settings, in this case for phase II Epilepsy Monitoring. Bringing together a team that combines expertise in human neuroscience of ingestion, neuromodulation, and computational modeling, we will test the central hypothesis that DA and NE dynamics will differentially encode aspects of food and non-food reward dependent on body mass index (BMI). In Aim 1, we will measure catecholamine dynamics from the amygdala during a food-based learning task. In Aim 2, we will measure catecholamine dynamics from the amygdala during a task using emotion, food, and neutral words. Completion of this proposal will begin to bridge the translational gap between studies in rodents and humans. It will also provide a needed foundation for more in-depth testing of the relationship between brain mechanisms of food reward and peripheral markers of metabolic health and disease.

NIH Research Projects · FY 2025 · 2025-02

Abstract The biomedical research enterprise is rapidly expanding and evolving, and conventional training programs are often inadequate to create employees that are effectively trained to thrive in many programs. Additionally, underrepresented minorities continue to comprise a very small population of this trained workforce. This application aims to help address these issues, and is seeking to support a previous R25-funded Initiative for Maximizing Student Development (IMSD) training program at Virginia Tech (VT). The training program will partner with 15 departments and several interdisciplinary graduate education programs across VT, and takes advantage of VT’s long history of academic excellence. Historically, the VT-IMSD predoctoral program was very successful, resulting in 40 PhD recipients in disciplines such as biomedical engineering, immunology, neuroscience, infectious diseases, and drug/bio/ pharmaceutical chemistry. We propose to use a training approach that has yielded exemplary outcomes, including a significantly improved PhD degree completion rate for UMRs compared to the national average of 60%. Our proposed training plan involves three structured phases designated as “Moving-in, Moving-through, and Moving-out.” Each phase involves structured and purposeful engagement with faculty and alumni, and a purposeful cohort-based approach to trainee development. This T32 program aims to develop adaptable, highly trained and resilient, diverse, and transdisciplinary PhD students using the following aims: 1. Recruit 5 URM doctoral scholars each year for a total of 25 over five years to successfully complete PhDs. 2. Prepare scholars to effectively compete for postdocs or biomedical research jobs through activities that build confidence in being a scientist and effective skill development. 3. Use the VT IMSD/PREP Alumni Network to advance our training goals. Training program recruits will participate in activities that include lab rotations, foundation courses, seminars, and peer mentoring. These activities will provide opportunities for scholars to develop skill sets that include diverse research experience, oral and written communication, effective grant and scientific writing, diversifying job interest, surviving the challenges of a doctoral program, and, ultimately, a biomedical research career. The scholars will also participate in scientific and career development activities that include a biweekly IMSD forum, a weekly program area seminar, a monthly multicultural assembly, and an annual research symposium. A robust evaluation plan is in place to thoroughly assess implemented initiatives and program outcomes. Illustrious alumni from previous training cohorts, five of whom are Assistant Professors and others who are research scientists at prestigious institutions and industry will form our Advisory Committee. The 37 distinguished faculty with outstanding URM mentoring records and state-of-the-art interdisciplinary research programs funded by NIH and other federal agencies including NSF, DOD, and USDA, are enthusiastic and ready to support this critically needed program. The new IMSD leadership and unwavering institutional support will ensure the success of this T32 program.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY/ABSTRACT Temperature significantly influences physiological processes and behaviors, impacting chemical reactions, biomolecule activity, and species distribution. Mosquitoes, vectors of diseases like malaria and dengue, are responsive to temperature cues, shaping survival, reproduction, and host-seeking behaviors. Fruit flies and mosquitoes utilize the same receptors for innocuous thermosensation, which can be studied in fruit flies due to the abundance of available genetic tools. Unraveling the cellular, molecular, and neural basis can offer insights into controlling vector-borne disease transmission. Drosophila melanogaster (fruit flies) is a model for studying insect thermosensory processes. Its thermosensory systems rely on manipulable sensory neurons and a balance between high- and low-temperature activated neurons. Specific neurons, including aristal heating and cooling cells, guide avoidance responses to temperature shifts. The warm receptor GR28B(D), which is essential for avoiding high temperatures, is not well understood. This proposal will uncover the precise molecular mechanism of GR28B(D) in sensing temperature changes. I will utilize the CRISPR-Cas9 system to determine critical protein/amino acid domains necessary for temperature sensing. Employing a top-down strategy, I will systematically identify the critical domains/amino acids associated with the receptor. The successful execution of this project will reveal significant insight into the molecular intricacies of temperature sensing in fruit flies. Our approach integrates genetic manipulations, electrophysiology, advanced imaging, and behavioral analysis to uncover specific roles of Exon 1 and smaller protein domains in GR28B(D), advancing our understanding of sensory processes in Drosophila melanogaster and related insects. The approaches employed in this proposal offer a breadth of innovative in vitro and in vivo techniques to the PI. Training for the expertise of these techniques will take place in the sponsor’s laboratory. Throughout the fellowship, career professional development training will occur through journal clubs, seminars, and associations at Virginia Tech, as well as at national and regional conferences to prepare the PI for a career as an independent researcher.

NIH Research Projects · FY 2026 · 2025-01

Project Summary/Abstract The proposal presents a four-year research career development program focused on the evaluation of the RELAX (Regulating Emotions Like An eXpert) intervention for families of adolescents with attention- deficit/hyperactivity disorder (ADHD), and the development of ecological momentary intervention (EMI) procedures a cutting-edge approach to support skill rehearsal in real life via smartphone apps. The candidate is currently an Assistant Professor of Psychology at Virginia Tech. The outline proposal builds on her previous research and clinical experience working with families of adolescents with ADHD by providing training and mentorship that will facilitate her growth in four areas: (1) Developing proficiency in managing and responsibly conducting clinical trials research; (2) Acquiring skills in the development and implementation of EMI; (3) Gaining skills in data management and conducting longitudinal, multilevel analytic methods and clinical trial analyses; and (4) Building community partnerships to ensure successful disseminating, implementing, and sustainability of evidence-based interventions. The proposed clinical trial and didactic work will position the candidate with a unique set of cutting-edge skills and statistical abilities that will enable her to transition to independence as a clinical scientist conducting clinical trials research with families. Adolescents with ADHD experience a host of negative outcomes, including risk for substance abuse, suicidality, and mood disorders, partly due to the high levels of emotion dysregulation (ED) they experience. Despite this, no evidence-based intervention exists for adolescents with ADHD that directly targets ED. As such, the proposed research is both significant and innovative. Specifically, RELAX targets ED among families of adolescents with ADHD in a brief, group-based intervention. In-person and telehealth pilot studies of RELAX provide promising findings; however, the effectiveness of RELAX delivered through real world clinical settings is needed. Families also indicated the desire for more opportunities to practice skills learned during RELAX in focus groups following the pilot study; EMI could address this need. Despite EMI being used in other clinical populations, EMI has not been explored among youth with ADHD, nor by simultaneously utilizing it with parents and adolescents. This K01 will implement a small clinical trial to assess the acceptability, feasibility, and preliminary efficacy of RELAX delivered by community clinicians relative to control. Additionally, it will iteratively develop the EMI procedures to use in the future for skill rehearsal during and following RELAX. The aims of this research are: (1) Quantify the acceptability, feasibility, and efficacy of RELAX relative to control at post-intervention and short- and long-term follow-ups; (2) Identify the extent to which changes in RELAX main outcomes during RELAX are associated with improvements in social-emotional and behavioral functioning at follow-up; and (3) Identify stakeholder feedback on the EMI procedures. This study has potential to support future independent research, and holds promise for transforming treatments for adolescents with ADHD.

NIH Research Projects · FY 2026 · 2025-01

PROJECT ABSTRACT The experience of pain involves a combination of brain, autonomic, and behavioral responses, and dysfunction in these neural and physiologic processes underlie several chronic pain conditions. How these systems interact to generate the experience of pain, though, is poorly understood. The posterior insula (PI) and dorsal anterior cingulate cortex (dACC) are core brain regions that serve critical functions in pain and autonomic processing, however their causal role in these functions remain uncertain. The PI and dACC are thus promising therapeutic targets for non-invasive neuromodulation, but unfortunately these regions are deep to the cortex, precluding access with existing non-invasive techniques like transcranial magnetic stimulation (TMS) or transcranial electrical stimulation (TES). Low-intensity focused ultrasound (LIFU) is an emerging non-invasive neuromodulation approach that focuses mechanical energy to selectively target brain regions at any depth to transiently and reversibly alter neural activity. The Legon lab was the first to demonstrate its potential for neuromodulation in humans and has performed several foundational studies to establish LIFU’s potential as a next-generation non-invasive technique. In this F30 proposal, we are mapping the neural and physiologic responses to pain and then causally manipulating the PI and dACC to identify their specific contributions in these processes in healthy participants. To accomplish this, we will leverage electroencephalogram and multimodal physiologic measures including electrocardiogram, electrodermal activity, and continuous blood pressure monitoring during a tonic, pain-inducing stimulus at various intensities. This work will help to determine key brain- body interactions that subserve fundamental roles in the human pain experience.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY Exercise is a powerful strategy to improve skeletal muscle metabolism that can both prevent and reverse disease. However, the complex signaling that drives the immediate and long-term changes in muscle metabolism is incompletely understood. Skeletal muscle consists of multiple cell types, such as myocytes, vascular endothelial cells, pericytes, and immune cells. When subjected to exercise stress, these cells communicate with one another to coordinate the heightened energy production required by the skeletal muscle. Our long-term goal is to understand intercellular skeletal muscle signaling initiated in response to exercise, thus informing strategies to promote health and prevent disease. Reactive oxygen species (ROS) are molecules known for their ability to both initiate signaling cascades and to cause damage. Skeletal muscle produces ROS in response to exercise, but whether ROS harm or protect the muscle has been debated. Initially, ROS were believed to be solely deleterious, contributing to conditions like diabetes and sarcopenia. However, recent findings indicate that ROS play a crucial role in skeletal muscle's metabolic adaptation to exercise. Despite this, we still have limited knowledge about where ROS are produced, the enzymes involved, and the specific processes that rely on ROS for beneficial signaling during exercise. Building on our previous research, our current proposal focuses on NADPH oxidase 4 (NOX4), a ROS-producing enzyme we identified as a critical factor in initiating skeletal muscle's metabolic responses to exercise. Importantly, this enzyme is most highly expressed in the vascular endothelial cells of skeletal muscle. If we remove this enzyme from only the endothelium, we observe a reduced metabolic response to acute exercise. Based on these findings, we will investigate the hypothesis that endothelial signaling, dependent on NOX4, plays a crucial role in determining the mitochondrial metabolic responses to exercise in skeletal muscle. To investigate this, we will utilize advanced tools that allow us to specifically manipulate the expression of Nox4 in endothelial cells. This will enable us to gain new insights into exercise-induced endothelial signaling and examine the impact of endothelial cell-derived ROS on both 1) endothelial signaling and 2) the mitochondrial responses to exercise in skeletal muscle. Ultimately, we hope these findings will contribute to developing targeted exercise interventions and potentially provide a foundation for treatment strategies based on exercise as medicine.

NIH Research Projects · FY 2025 · 2024-09

This study is guided by a long-term goal of optimizing alcohol-related intimate partner violence (IPV; psychological, physical, sexual, and other types of non-heterosexual abuse [e.g., threatening to out a partner]) interventions for bisexual and other multigender-attracted (bi+) young adults and their partners. As a critical first step toward this goal, this R21 will identify modifiable intervention targets for alcohol-related IPV by developing new scientific knowledge of the unique day-to-day processes that potentiate and ameliorate IPV after alcohol use among bi+ young adults and their partners. This study will also explore subpopulations of bi+ young adult couples who should be prioritized in future alcohol-related IPV research. Alcohol use proximally increases the likelihood of IPV perpetration, particularly among young adults ages 18-25. Relative to those with other sexualities, more bi+ young adults endorse heavy alcohol use and IPV, but research has not identified daily experiences that may potentiate or mitigate alcohol-related IPV within this priority population. This critical scientific gap may be attributed to (1) limited inclusion of bi+ populations in alcohol-related IPV research, with no daily diary studies focusing on bi+ young adults, (2) a lack of data on bi+-specific stressors in existing alcohol-related IPV research, and (3) limited couple-level data from bi+ young adults despite IPV being a dyadic process impacted by both partners’ alcohol use and stress. This study will address these gaps by examining potential intervention targets (i.e., bi+ specific stress, partner support) implicated by integrative alcohol-related IPV theories that exacerbate or mitigate IPV after drinking in the naturalistic settings of bi+ young adults and their partners. A rigorous, 60-day, daily diary approach will be used to collect daily reports of alcohol use, stress (e.g., bi+-specific stressors), partner support, and IPV perpetration/victimization from 50 bi+ young adults and their partners (N=100 individuals; 25 men/men or women/women couples, 25 men/women couples). Study aims are: (1) Determine if one’s own and one’s partner’s alcohol use increases IPV perpetration on days when bi+ individuals and their partners report high, but not low, levels of stress. (2) Determine if one’s own and one’s partner’s alcohol use increases IPV perpetration on days when individuals perceive their partners as providing low, rather than high, levels of partner support. (3) Across Aims 1 and 2, explore descriptive differences between (a) men/men, women/women, and men/women dyads, (b) couples in which only one partner is, rather than neither partner being, heterosexual, and (c) IPV types to identify priority populations for future research. Data generated from this study will provide the most comprehensive, theoretically-informed assessment of alcohol-related IPV among bi+ young adults and their partners to date. In doing so, these results will provide the foundation required for future research to develop alcohol-related IPV interventions effectively tailored to meet the needs of bi+ young adults.

NIH Research Projects · FY 2025 · 2024-09

|| Project Summary This R21 is in response to FOA PA-20-195/ NOT-HL-21-024: development of new ideas and first-generation prototypes for therapeutics and nanotechnologies as applied to the cardiovascular mission areas of the NHLBI. Our proposed research aims to design and test novel small antibodies (12-15 kDa) called nanobodies. Nanobodies are unique monoclonal antibodies, found in camelid species, that have the specific advantage of being able to enter the cell. The developed nanobodies will target a specific protein interaction inside smooth muscle cells (SMC) with the aim of disrupting their pathological proliferation. We have identified connexin 43 (Cx43)-cyclin E interactions as a significant contributor to pathological SMC proliferation and cardiovascular disease progression. This interaction occurs inside the cell, so therapeutic approaches to disrupting interactions are very limited. The focus of the research will be to develop Cx43-cyclin E targeting nanobodies as potential therapeutics in the treatment of cardiovascular disease. Acquired injuries are associated with pathological proliferation. For example, stent placement surgery damages the blood vessel wall, leading to rapid SMC proliferation, a hallmark of stent failure. SMC proliferation thickens the blood vessel walls, called neointima formation, eventually blocking the artery. Current therapies target general proliferation pathways using adapted chemotherapeutic agents. However, these non-specific agents kill healthy cells as well, reducing vascular healing and leading to long-term failure rates of 25%. By targeting the mediators of only pathological proliferation in SMC, it may be possible to stop neointima formation and promote the healthy recovery of endothelial cells on the blood vessel wall. In large vessels, Cx43 is expressed only in SMC, and the target site is a post-translational modification that we have shown does not alter Cx43 channel conduction. Preliminary data in this proposal suggest the Cx43-cyclin E interactions occur in models of human SMC proliferation. We have generated novel disruptor peptides replicating the Cx43-cyclin E binding site and provide data that they can limit neointima in mice and humans. As peptides are of limited therapeutic use, due to in vivo stability issues, we have adapted these peptides for injection into llama, generating nanobodies. The project objectives are to define the best binding nanobodies from screening, alter these to enable cell entry, validate functions in vitro, and demonstrate the translatable nature in mouse and human models of neointima. This research is specifically responsive to NHLBI-NOT-HL-21-024. Our study aims to produce prototype nanotechnologies (nanobodies) that significantly improve cellular therapeutics (intracellular targets), and address gaps in the repair and regeneration of vascular disease states. Doing this will achieve more effective therapies for future translation and our data will feed into future NHLBI Catalyze Program applications.

NIH Research Projects · FY 2025 · 2024-09

Project Summary/Abstract Cardiovascular disease (CVD) and type II diabetes (TIID) are intimately linked, as TIID more than doubles a patient’s risk of developing CVD. This pathology is termed ‘diabetic cardiomyopathy’ (DC), and occurs independent of other risk factors. A major but poorly understood component of DC pathogenesis is impaired insulin signaling, or insulin resistance (IR). However, current therapeutics center on blood glucose management and reduction, rather than insulin sensitivity restoration. As such, this proposal centers upon the identification and investigation of putative major molecular mediators of cardiac insulin sensitivity and resistance, in an effort to identify novel therapeutic targets for DC to reduce disease burden. Utilizing unbiased genome-wide studies, my laboratory identified ‘regulated in development and DNA damage 1’ (Redd1, also Ddit4) as the most transcriptionally active, insulin-inducible gene in the murine heart. This increase in transcriptional activity translated to increases in both REDD1 gene and protein expression. REDD1 is known to inhibit mTORC1 signaling, but the exact mechanism by which this occurs is unknown. Notably, REDD1 is shown to be involved in both insulin sensitivity and resistance. Our preliminary data indicate that high fat diet (HFD) and fatty acids increase REDD1 expression, further suggesting a role for REDD1 in IR. Our data also outline a novel mechanism in which insulin increases REDD1 expression, nuclear localization, and chromatin binding. This increase in chromatin binding is associated with alterations in transcriptional activity, enhancing the transcriptional activity of oxidative metabolic genes in the heart. Thus, it is our hypothesis that high fat diet prevents REDD1 nuclear localization, resulting in suppressed oxidative metabolism mainly via the loss of REDD1-dependent transcriptional regulation, as well as enhanced inhibition of mTORC1, ultimately driving IR. We will test this hypothesis with two specific aims. We will, first, determine the role of REDD1 in mediating cardiac insulin resistance and, second, investigate the mechanism of REDD1 nuclear localization and its transcriptional role in mediating cardiac insulin sensitivity. Here, we will utilize a novel murine model with cardiac REDD1 deletion for in vivo studies and isolate cardiomyocytes for in vitro studies. These mice or cardiomyocytes will be subjected to high fat/fatty acids to induce insulin resistance. We will also employ a novel REDD1 mutant to examine the contribution of REDD1 nuclear localization to cardiac insulin sensitivity and resistance. Overall, we expect that high fat will prevent insulin-inducible REDD1 nuclear localization, chromatin binding, and activation of oxidative metabolic genes. We hypothesize that this is a major mechanism by which high fat mediates IR. These studies are critical, as we predict that restoration of nuclear REDD1 will restore cardiac sensitivity to insulin, prevent subsequent heart disease. Investigation of these pathways is essential for the development of new therapeutics for TIID and DC. This novel research will be conducted under the mentorship of Drs. Jessica Pfleger and Robert Gourdie, with numerous opportunities for technical and professional training as described in the training plan.

NIH Research Projects · FY 2026 · 2024-09

PROJECT ABSTRACT Chronic kidney disease (CKD), characterized by chronic inflammation and progressive fibrosis, is prevalent and can eventually lead to end-stage renal disease (ESRD), necessitating renal replacement therapy (hemodialysis or transplant). CKD is also a strong risk factor for cardiovascular disease. Further, the costs incurred by the Nation’s health system in caring for CKD patients are enormous. Current treatments to slow CKD are limited and non-specific. An ideal medicine would reverse tissue scarring and restore kidney function, but even the more modest goal of slowing fibrotic progression is beyond therapeutic reach currently. Thus, new experimental therapy strategies are needed. CKD involves endothelial dysfunction resulting in vascular rarefication and subsequent hypoxia. Inhibition of either sphingosine 1-phosphate (S1P) synthesis at the level of sphingosine kinase 2 (SphK2) or transport (via Spns2) opposes these pathologies. By virtue of inhibiting S1P clearance from blood, SphK2 inhibitors drive an increase in blood and plasma S1P. The steepening of the plasma: tissue S1P gradient maintains endothelial barrier function while the rise in erythrocyte S1P drives an increase in 2,3- bisphosphoglycerate, which results in increased oxygen delivery. SphK2 inhibition is also anti-inflammatory, although the mechanism remains uncertain. SphK2 deficiency, whether accomplished via genetic manipulation of Sphk2 alleles or administration of SphK2 inhibitors, consistently results in substantially reduced renal fibrosis in multiple animal models. Spns2, which transports S1P to the extracellular environment, generates a local extracellular S1P gradient at the kidney pericyte, where SphK2 is the major source of S1P. Like SphK2 inhibition, Spns2 inhibition suppresses inflammatory signaling in pericytes in vitro and ameliorates renal fibrosis in mice. These results, although encouraging, are preliminary in that the inhibitors used are suboptimal. We will rectify the deficiencies by making our SphK2 inhibitors orally available (Aim 1). Likewise, we will improve our Spns2 inhibitors regarding potency, selectivity, and oral bioavailability (Aim 2). When drug-like compounds are realized, we will validate their efficacy in multiple models of renal fibrosis (Aim 3). These studies will determine whether there is synergism in blocking both SphK2 and Spns2 in vivo. In sum, our studies will serve to increase the fundamental understanding as to the effects of systemic SphK2 and Spns2 inhibition in vivo and to validate inhibitors of S1P signaling as potential therapies for retarding progression of CKD to ESRD. Further, if successful, we will have facilitated translation of SphK2 and/or Spns2 inhibitors into clinical trials for a CKD indication.

NIH Research Projects · FY 2025 · 2024-09

Summary The nervous system interacts with the immune system in ways that have been identified to contribute to a number of diseases, including Alzheimer’s disease, infection, brain cancer, and multiple sclerosis. The field of neuroimmunology is a quickly growing research area that involves complex models and manipulations, most often using in vivo models in mice. These models, though useful, lack human components, and have the added difficulties of complex manipulations, difficulty in modulating individual organs without affecting others, and difficulty in the acquisition of dynamic data. In contast, organ-on-chip systems offer benefits in these areas as well as flexibility of cellular components and experimental conditions, but have rarely been applied to neuroimmunology beyond models of the blood-brain barrier. Here we propose to develop a microphysiological system that recapitulates the brain-meninges-lymph node axis in both healthy conditions and in a model Alzheimer’s disease. To do so, we will integrate three tissue engineered models recently established in our laboratory – of the human brain, lymph node, and meninges – into a user-friendly microfluidic device for media recirculation, and validate the function of each compartment separately and together. We will deliberately retain modularity of the system, so that we can examine the interactions of these components while easily manipulating single organ compartments with fluid flow, drugs, mutations, or disease states. We will characterize the response of each component to fluid flow, enable T cell circulation between organs, and test the response to inflammation of each component separately and together. Next, we will convert the baseline model into an Alzheimer’s specific model by incorporating a suite of tissue engineered models of Alzheimer’s brain, derived from neural stem cells from individual Alzheimer’s patients. After confirming lymphatic drainage of amyloid similar to what has been shown in vivo, we will quantify the impact of the Alzheimer’s brain on the inflammatory state of the meninges and draining lymph node compartments, and determine the physical and chemical requirements for biomimetic T cell migration into and within the brain. If successful, we will have both a baseline “normal” system linked through multiple compartments and created from all-human components, poised as a foundation for future use across neuroimmunological research, and a model of interactions between the brain, meninges, and cervical lymph nodes in Alzheimer’s Disease. Ultimately, we envision using these systems for mechanistic tests of disease onset and progression, as well as to test variations in drug responses between individuals of varied ancestry, sex, and age, by sourcing cells from patients representative of different cohorts.

NIH Research Projects · FY 2026 · 2024-08

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. PROJECT SUMMARY Young adulthood is a developmental period during which health risk behaviors (HRBs) (e.g., substance use) peak, but the factors that lead to sustained addiction are not well understood. Current neurobiological models of risk taking focus on a developmental imbalance between the brain’s control and reward systems to explain the typical heightened risk taking seen in adolescence. We have shown that the two systems involved in value-based decision making—i.e., the valuation system (risk and reward processing) and the control system (cognitive control)—predict HRBs in adolescence. However, patterns of brain development during adolescence that predict continuity vs. discontinuity in HRBs in young adulthood are unknown. To fill these significant gaps, we will conduct longitudinal analyses to prospectively measure developmental trajectories of neurobehavioral mechanisms underlying HRBs throughout adolescence and young adulthood, and to characterize psychosocial risk and resilience factors that influence these developmental trajectories. We propose that poverty, abuse, and neglect are risk factors that are linked to HRBs through their impact on neurobehavioral mechanisms underlying HRBs, and that social integration is a resilience factor that deters HRB progression during young adulthood. We address the following aims: (1) we will assess longitudinal and bidirectional associations between valuation and control systems and HRBs from adolescence to young adulthood; (2) we will examine whether poverty, abuse, and neglect (experienced in childhood and adolescence) are associated with young adult HRBs through altered developmental trajectories of valuation and control systems in adolescence (i.e., neurocognitive vulnerability); and (3) we will examine whether social integration mediates the link between valuation and control decision-making systems and HRBs, and moderates the effects of psychosocial stress on HRBs. This application addresses these aims by extending a prospective longitudinal study to follow up 167 adolescents through young adulthood (23-27 yrs) who were previously assessed during adolescence (13-22 yrs). Our sample is from communities with elevated substance use rates, and thus is well-poised to provide critical insights about HRBs. Our intensive longitudinal multiple-level data (11 prospective measurement occasions over 15 years) will provide an unprecedented opportunity to understand both individual differences and within-person developmental changes in neurobehavioral mechanisms underlying HRBs from adolescence to young adulthood. The proposed study will (i) advance developmental theory regarding dynamic processes of brain-HRB associations; (ii) generate new knowledge regarding how psychosocial risk factors alter developmental trajectories of neural mechanisms underlying HRBs; and (iii) uncover neurocognitive and social resilience factors that can be targeted for strategic intervention to prevent HRB progression and addiction.

NIH Research Projects · FY 2024 · 2024-08

PROJECT SUMMARY/ABSTRACT Rift Valley fever virus (RVFV) is a viral zoonosis that causes severe disease in ruminants and humans [1]. Concerns are heightened regarding the introduction of RVFV to the U.S. as competent mosquito vectors have been identified and increased global commerce makes it probable that RVFV will spread to new areas [12]. Despite its pathogenic potential, there are no FDA-approved therapeutics or vaccines to challenge the global spread of this infectious organism. The nonstructural small (NSs) protein is the main virulence factor of RVFV, making it an attractive antiviral target [15]. This proposal aims to close the gap in knowledge regarding the role that NSs plays in the host autophagy pathway, by identifying the impact of the NSs-LC3 interaction on autophagy, viral replication, and pathogenesis. Bioinformatic analysis identified four putative LC3-interacting region (LIR) motifs in the RVFV NSs protein (LIR1-4), suggesting that NSs interacts with LC3 family members, the host key autophagy proteins. Autophagy is a homeostatic process in which cellular material is degraded and recycled and can be exploited by viruses to facilitate replication or can be antiviral. [31]. Preliminary studies confirmed that NSs interacts with all six LC3-family members in vitro and in RVFV-infected cells. Isothermal titration calorimetry (ITC) demonstrated favorable binding between LIR4 and all six-human LC3 family members, however little to no binding was observed between NSs LIR1-3 and LC3, indicating that LIR4 is mediating interaction between NSs and LC3. Additionally, ITC data and co-immunoprecipitation data in RVFV infected cells demonstrated the importance of F261 within LIR4 for the NSs-LC3 interaction. Crystal structures showed that the predominant NSs-LC3 interactions at the binding interface involve the side chain of LIR4 F261, furthering the importance of LIR4 F261 for NSs-LC3 interaction. Aim 1 will define the autophagy molecular mechanism of the NSs-LC3 interaction and the effect of loss of NSs-LC3 interaction through F261S substitution on autophagy. Aim 2 will determine the impact of NSs-LC3 interaction and regulation of autophagy on viral pathogenesis and survival through mouse studies using RVFV ZH548 wild type vs. F261S viruses. This fellowship will prepare the trainee for a career as a Professional Investigator in Academia through an intensive individualized training program facilitated by my strong sponsor Dr. Kehn-Hall, collaborators, and opportunities provided by Virginia Tech. Taken together, this fellowship will define the mechanism by which NSs and LC3 interact to modulate autophagy and the impact of the NSs-LC3 interaction on RVFV pathogenesis in a mouse model, providing a potential future therapeutic target.

NIH Research Projects · FY 2024 · 2024-08

We seek to establish a summer experiential learning (EL) program for undergraduate biomedical engineering (BME) students that focuses on identifying unmet needs in healthcare through Biodesign immersion. The Biodesign process is a systematic approach for the identification of unmet medical needs, the generation of novel technologies to address them, and the development of commercialization plans to bring them into fruition (i.e., identify, invent, and implement). We plan to capture key elements of these three phases through a unique EL framework that combines needs identification with parallel research and development (R&D) activities on distinct projects. The Specific Aims include: (1) rural and urban clinical immersion and needs identification; (2) parallel R&D component to support a later-stage technology; (3) assessment of healthcare advancement metrics and professional development by exploring the growth of the students’ Engineering Habits of Mind (EHoM). The proposed program is a strategic partnership between Virginia Tech (VT) and Carilion Clinic, an integrated healthcare system in Southwest Virginia with a variety of rural and urban healthcare environments. The improvement of health outcomes among rural patients is more intractable and provides fertile ground for innovation. In the clinical immersion, teams will apply contextual inquiry methods with mentors in the departments of surgery and nursing to identify design opportunities and develop EHoM in systems thinking and problem finding. Preparation will include training on user-centered research methods, such as task analyses for identifying stakeholder “pain points.” The teams will plan and document their design research at the clinical sites to create a database of >25 needs with supporting information related to stakeholders, existing solutions, regulation, reimbursement, market, and public health. Students will prepare a product development plan on a design opportunity well-suited to improve patient care and promote health equity. They will then recruit and lead a future senior design team to solve the associated challenge. The concurrent R&D component of the program captures a majority of the EHoM through the signature design pedagogy. Teams will be assigned to existing R&D projects at Carilion Orthopedics (CO), a regional hub for cutting-edge research and healthcare innovations. Students will carry projects through reduction-to-practice and proof-of-concept testing at the Carilion Clinic Center for Simulation, Research, and Patient Safety (CSRPS). Utilizing assigned projects will give students added exposure to tinkering, verification and validation, fabrication technologies, and manufacturing. Building this knowledge base will feed back into the clinical immersion component as an additional set of lenses for filtering needs. Further, multitasking on needs identification and parallel R&D will prepare students to manage different sets of deliverables and timelines in their future careers. Taken together, this program will offer a deep dive into best practices associated with identifying medical needs and serve as a career bridge for students planning to enter the medical device industry.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Exercise is a powerful strategy to improve skeletal muscle metabolism that can both prevent and reverse disease. However, the complex signaling that drives the immediate and long-term changes in muscle metabolism is incompletely understood. Skeletal muscle is composed of numerous cell types (e.g., myocytes, vascular endothelial cells, pericytes, immune cells, etc.). In response to exercise stress, these cells "talk" to each other to orchestrate the increased energy production needed by the skeletal muscle. Understanding the signaling initiated and the pathways activated with cell type clarity will provide the molecular resolution necessary to unravel the protective effects of exercise. Our long-term goal is to understand intercellular skeletal muscle signaling initiated in response to exercise, thus informing strategies to promote health and prevent disease. Reactive oxygen species (ROS) are fast-acting, transient molecules recognized for their ability to initiate signaling cascades. The current proposal builds upon our K01 research, where we have identified a ROSproducer, NADPH oxidase 4 (NOX4), as a critical initiator of skeletal muscle metabolic responses to exercise. lmportanUy, this enzyme is most highly expressed in the vascular endothelial cells of skeletal muscle. If we remove this enzyme from only the endothelium, we observe a blunted metabolic response to acute exercise. We have previously shown that endothelial NOX4 increases expression and activity of endothelial nitric oxide synthase (eNOS). Based on these observations, the current proposal seeks to understand if eNOS lies downstream of NOX4 in mediating skeletal muscle responses to exercise. Therefore, we propose to uncover the role of eNOS in skeletal muscle mitochondrial metabolism and determine whether eNOS and nitric oxide production activate exercise-induced mitophagy. We will utilize innovative tools to address these questions and expect this project to reveal novel information regarding exercise-endothelial signaling. These studies will generate new hypotheses and will advance the Craige Lab research program by uncovering the underlying pathways influenced by eNOS. Ultimately, we hope these findings will contribute to the development of targeted exercise interventions and potentially even provide a foundation for treatment strategies based on exercise as medicine.

NIH Research Projects · FY 2024 · 2024-08

PROJECT SUMMARY / ABSTRACT The primary objective of this proposal is to develop a localized cancer immunotherapy using particle-anchored cytokines with prolonged intratumoral retention to elicit durable anti-tumor immune responses. It is well known that immunostimulatory cytokines can elicit robust anti-tumor immune responses in preclinical studies but also exhibit severe immune-related adverse events due to systemic exposures. The current drug delivery methodology cannot sustainably supply cytokines for days to weeks, failing to fully address issues of toxicity and limited efficacy. We hypothesize that anchoring cytokines to large-sized particles for intratumoral injection would enhance the local retention of cytokines (e.g., IL-12 and IL-15) to drive tumor inhibition while avoiding the systemic exposures of such cytokines that cause adverse effects. To test this hypothesis, we develop a novel local delivery system that physically anchors the Fc-cytokine to particles having surface-decorated Fc-binding peptides. Our preliminary studies show that the intratumoral administration of our new particle-anchored cytokine significantly increases tumor retention of cytokines and markedly reduces systemic toxicity. Moreover, local treatments of our micron-meter-sized particle-anchored cytokines promote cures in poorly immunogenic tumor models, and elicit anti-tumor immunities with controls over distant untreated lesions. Overall, two specific aims will be pursued in this proposal, including: (i) to develop liposome-anchored cytokines with prolonged tumor retention over one week and minimal systemic leakage of free cytokines; and (ii) to elucidate the mechanisms by which particle-anchored cytokines elicit durable anti-tumor immunity to improve efficacies in poorly immunogenic tumors. Our simple formulation technology would fully address the dose-limiting toxicity problem in the use of cytokines in cancer immunotherapy, and provide a new and effective treatment option for difficult- to-treat solid tumors when combined with other immunotherapeutics. This work will also advance the research on drug delivery technology by elucidating crucial physicochemical characteristics to circumvent systemic leakage for local drug delivery. This work will also significantly advance our understanding of how localized immunostimulatory cytokines impact tumor microenvironments, leading to enhanced systemic anti-tumor immunity and better treatment outcomes.

NIH Research Projects · FY 2025 · 2024-08

The rigor and reproducibility of research relies on the appropriate use of statistical methods, in addition to the careful planning and designing of studies to answer research questions and make meaningful inferences based on data. While statistical methodology is widely used and disseminated in all areas of research, it is often misused due to lack of knowledge and training in statistical concepts and inadequate planning strategies. The field of neuroscience in particular has been criticized for overall poor study planning and misuse of statistical methodology, specifically related to inadequate sample size and power to detect clinically meaningful effects, as well as inattention to fundamental statistical assumptions. Although true for all disciplines, it is particularly important to encourage collaboration and team science in the field of neuroscience to not only offer a larger scope of perspectives and expertise, but also to result in improvements in the rigor and reproducibility of the resulting research. In partnership with CENTER, the team of collaborative biostatisticians, epidemiologists, and data scientists at Virginia Tech’s Center for Biostatistics and Health Data Science (CBHDS) will develop, evaluate, and disseminate a series of educational units in rigorous and reproducible approaches to study planning and design within a team science framework, including: 1) creating a formal plan for experimental design, execution and analysis; 2) exercising a sound process for choosing appropriate descriptive statistics; and 3) identifying assumptions and sources of error. The proposed educational units will be adaptable across various disciplines, formats, levels of learners, learning environments, and diverse backgrounds and perspectives.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with no known cure. Current therapies focus on symptom relief with corticosteroids or immunosuppressants but have significant side effects. The gut microbiome plays an important role in immune regulation, and modulation of the gut microflora could provide a novel avenue for SLE therapy. The Luo Lab has demonstrated that bacterial DNA (bDNA) can stimulate IL-10 production and regulatory B cell (Breg) proliferation, which are both associated with improved lupus symptoms. Toll-like receptor 9 (TLR9) is an endosomal sensor that recognizes bDNA and is associated with less severe lupus symptoms. CpG-B oligonucleotides (ODNs) are short nucleotide sequences that mimic bDNA and agonize TLR9. We have shown that CpG-B ODNs can robustly induce IL-10 production and Breg proliferation in vitro through two distinct, TLR9-dependent mechanisms: a B cell intrinsic mechanism, where TLR9 is agonized in B cells themselves, and a B cell extrinsic mechanism, where TLR9 is agonized in a secondary cell type, creating IL-6 that subsequently expands Bregs. IL-6 can expand Bregs but is also capable of expanding inflammatory cell subsets. We have in vivo data to show that increased IL-6 is associated with increased Bregs but is also associated with increased renal damage in lupus prone (MRL/lpr) mice. Therefore, we aim to enhance the IL-6 independent, B cell intrinsic mechanism, while limiting the IL-6 dependent, B cell extrinsic mechanism. This proposal aims to determine the therapeutic potential of CpG-B ODNs in the management of SLE. We propose to study these TLR9 agonists with MRL/lpr and humanized models, both in vitro and in vivo, to demonstrate the translatability of my work. We hypothesize that CpG-B ODNs can increase the number of Bregs and protect the host against SLE by acting through B cell intrinsic, TLR9 agonization. This application is broken down into two aims: Aim 1 is to delineate the cellular and molecular mechanisms by which CpG-B ODNs stimulate Breg differentiation in vitro. The first objective is to elucidate the mechanism of the B cell extrinsic pathway by using cell-specific Tlr9-/- co-culture experiments. By elucidating its mechanism, we can more effectively limit the B cell extrinsic pathway in the future. We will then analyze the pro- vs. anti-inflammatory profiles of the intrinsic and extrinsic mechanisms. Lastly, we propose to validate these findings in human PBMCs. Aim 2 is to determine the efficacy of using CpG-B ODNs in vivo. We aim to enhance the B cell intrinsic mechanism, while limiting the B cell extrinsic mechanism using Tlr9 conditional knockout mice. After determining that we can confer protection in MRL/lpr mice, we will demonstrate that we can protect mice with a human immune system. These experiments will demonstrate the potential efficacy of CpG-B ODNs in future human trials. We believe that CpG-B ODNs have the potential to be passive, yet effective treatments for lupus. This work will be the focus of the applicant’s Ph.D. candidacy and training plan. The applicant also plans to attend conferences, shadow physicians, and continue to practice the skills necessary to being a successful and productive physician-scientist.