Virginia Polytechnic Inst And St Univ

NIH Research Projects · FY 2024 · 2023-09

Project Summary Acute alcohol use and intoxication proximally increase the likelihood of college students’ intimate partner violence (IPV) perpetration underscoring the need to detect and intervene with alcohol-related IPV during critical drinking periods. Problematically, retrospective self-reported alcohol use has been the only method used to assess college students’ alcohol use in relation to IPV perpetration in their natural environment (e.g., ecological momentary assessment [EMA]). College students’ self-reported alcohol use underestimates their breath alcohol content (BrAC) by as much as 54% and is fraught with limitations; investigators’ ability to deliver and evaluate crucially-needed ambulatory alcohol-related IPV interventions (e.g., just-in-time interventions) is hindered by inaccurate alcohol assessment. Recent advancements in mobile breathalyzer technology allow investigators to remotely and objectively assess undergraduates’ alcohol use and intoxication in naturalistic settings. When paired with EMA of IPV, mobile breathalyzers offer a novel approach to strengthening alcohol-related IPV monitoring; no study has used portable breathalyzers to remotely assess alcohol use in relation to IPV. As a critical first step toward enhancing remotely-delivered alcohol-related IPV interventions, the overall objective of the proposed study is to determine the feasibility of using smartphone-linked, portable breathalyzers in conjunction with smartphone-based EMA to capture alcohol-related IPV episodes and contexts among heavy drinking college students with a recent IPV history. We will leverage our team’s established remote alcohol monitoring and EMA procedures to address three aims: The first aim is to examine the feasibility and acceptability of using portable breathalyzers paired with EMA to investigate the association between BrAC and IPV among heavy drinking, previously-aggressive college students. The second aim is to determine if breathalyzer-derived BrAC is a stronger predictor of different forms of IPV perpetration than is self-reported alcohol use, and whether there are gender differences in these associations. Our exploratory aim is to examine contexts (e.g., where, when, and with whom one drinks) in which alcohol-related IPV events occur, which will help optimize EMA delivery in future alcohol-related IPV studies. Across 30 consecutive days, 100 heavy drinking college students with an IPV perpetration history will use smartphones to complete 5 daily (1 morning, 4 evening) self-reports of IPV and drinking context; smartphones will prompt 4 evening BrAC submissions to a smartphone-linked portable breathalyzer (plus event- triggered reports) to provide the most rigorous, accurate, and ecologically-valid assessment of alcohol-related IPV to date. Results will provide EMA+ feasibility data to inform a future R01 using EMA+ with a large sample of couples to identify intra/interpersonal and contextual antecedents of college alcohol-related IPV. Consistent with the NIAAA’s priorities, the proposed study will enhance methods of tracking alcohol-related IPV in college students’ natural settings and is an essential first step toward strengthening alcohol-related IPV interventions.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Dopaminergic signaling is proposed to be critical for gating items into working memory and for maintaining representational content over delays in the presence of distracting information. Working memory deficits due to dysregulated dopaminergic signaling are associated with a broad range of psychiatric illnesses. However, the precise role of dopamine in working memory has yet to be fully understood due to limits on our ability to measure it in humans. Recent advances by PI Montague’s research group allow for tracking neuromodulator release in epilepsy patients using fast-scan cyclic voltammetry augmented with machine learning (elastic net regression). Our team can now measure dopamine responses in awake humans with sub-second temporal resolution, allowing for precise characterization of phasic and tonic dopamine release in cortical areas that have been associated with cognitive processes that constitute working memory. This application merges this technique with measurement of working memory processes to test fundamental predictions about dopamine’s function in cognitive control. We will record dopamine release in the lateral prefrontal cortex, anterior cingulate cortex, and hippocampus during performance of a set of working memory tasks. Medication-resistant epilepsy patients will participate in the study during phase-II monitoring for seizure activity with surgically implanted electrodes. These electrodes will be implanted at Banner Hospital in the Phoenix metropolitan area (Co-I Bina). During recording, patients will complete Delay Match-to-Sample and Delayed Estimation Visual Working Memory tasks. Our research group (Co-I Brewer, Co-I Bae, and Co-I McClure) has used these tasks to characterize individual differences in working memory and to explore the neural basis of working memory for the past 15 years. The overall goal of this project is to collect and model direct dopamine recordings during stimulus presentation, distractor presentation, and delay periods of canonical working memory tasks to better characterize the role of phasic and tonic dopamine release in working memory.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Addiction is a physiological process that involves changes in neural plasticity in response to drugs of abuse. Although genetic factors have been recognized as a strong influence on the susceptibility to addition, environmental stimuli and life experiences also form important risk factors. There has been increasing evidence that epigenetic mechanisms mediate the influence of environmental factors on gene activities in the CNS and therefore are likely involved in brain development and pathology of drug abuse. Furthermore, DNA sequence variation is known to impact epigenetic landscape, chromatin structures and molecular phenotypes via influencing the cis-regulatory elements such as promoters and enhancers. In this project, we are interested in probing how epigenomic mechanisms mediate environmental factors such as adolescent drug exposure during addiction development and how a genetic variant affects such mediation. We will use the state-of-the-art omic and bioinformatic technologies to decipher the links among genetics, epigenetics and environmental factors involved in opioid addiction. The low-input approaches will allow us to characterize epigenomic and transcriptomic dynamics with cell-type and brain-region specificity.

NIH Research Projects · FY 2025 · 2023-09

Transmission is a fundamental component of host-pathogen systems. Despite complex biological processes interacting to determine whether a pathogen successfully infects a host, transmission parameters often assume a constant per-contact transmission probability. Factors that can influence risk of infection include the frequency of exposures, duration of a contact, and dose acquired during an interaction, which are collectively referred to as transmission determinants. These determinants of infection can vary among hosts and between routes of transmission (e.g. direct and indirect) and have profound effects on disease dynamics. Ultimately, a mechanistic understanding of how heterogeneity in transmission probabilities among exposure events contributes to disease dynamics remains an important outstanding question in the ecology and evolution of infectious diseases. Here we seek to elucidate how factors determining transmission success influence the probability of acquiring infection, epidemic dynamics, and pathogen evolution. Specifically, we will investigate how transmission determinants (pathogen dose, contact duration, and contact frequency) vary among exposure routes (direct and indirect) and ultimately contribute to the dynamics of an emerging multi-host disease of snakes. Snake fungal disease (SFD) is caused by, Ophidiomyces ophidiicola, which is an environmentally persistent fungal pathogen that, to date, has been documented in more than 42 species of wild snakes on three continents, and has contributed to the severe declines of several species. O. ophidiicola is transmitted through direct and indirect contacts, and the behavior of snake species affected by SFD naturally vary along an exposure duration and contact intensity gradient, making this an ideal system for understanding the effects of variation in exposure events on disease dynamics. This work will highlight important mechanisms that contribute to variation in snake declines, and more broadly, provide insight into the theoretical underpinnings and profound effects that factors determining successful transmission can have on infectious disease outbreaks. This proposal will leverage our recent advances in this multi-host system and use a combination of field, experimental, and modeling approaches to provide critical insight into how the dynamics of exposure events have cascading effects on infectious disease.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Despite the wide application of commonly used drugs for type 2 diabetes (T2D) treatment, the prevalence of T2D continues to rise in the US. Insulin resistance and progressive decline in functional β-cell mass are two key driving forces for T2D. Obesity is a leading pathogenic factor for developing T2D, which is a significant obstacle for effective glycemic control in many patients with T2D. Thus, identifying novel agents that can simultaneously ameliorate obesity and promote insulin sensitivity and β-cell function would be a more effective strategy for preventing and treating T2D. In searching for agents with both anti-obesity and anti-hyperglycemic activities, we found for the first time that elenolic acid (EA), a small molecule generated from hydrolyzing olive leaf-derived oleuropein, is such a highly promising compound. Excitingly, oral administration of EA reversed hyperglycemia while also promoting weight loss and suppressing food intake in obese diabetic mice, Notably, EA was more effective in managing hyperglycemia and obesity than that of metformin. Interestingly, EA induced peptide YY (PYY) and glucagon like peptide-1 (GLP-1) secretion from intestinal L-cells. In this grant, we propose to test hypothesis that EA is a dual acting agent for simultaneous treatment of obesity and diabetes via triggering PYY and GLP-1 secretion. Aim 1 will characterize the anti-diabetic and anti-obesity effects of EA. In that regard, diet- induced obese mice and obese diabetic db/db mice will receive EA treatment once daily via oral gavage. The effects of EA on metabolic profiles of obese diabetic mice will be examined for determining its anti-obesity and anti-diabetic efficacy. In addition, euglycemic-hyperinsulinemic clamps in combination with ex vivo analyses of peripheral tissues will be performed to examine the effects of EA on insulin action, fat metabolism, and gluconeogenic programs. Immunohistochemistry will be carried out to analyze Islet β-cell mass and function. Further, oral bioavailability, metabolism, and potential toxicity of EA will be studied. Lastly, mouse models with T2D will be used to investigate the synergistic metabolic effects of EA plus metformin combination therapy. Aim 2 will identify the mechanisms by which EA suppresses food intake and protects against obesity. First, the effects of EA on feeding responses and stomach emptying in mice will be evaluated, followed by ex vivo analyses of hypothalamic pathway controlling food intake. Next, pair-feeding in combination with energy expenditure analyses will be performed to examine the extent to which the anti-obesity efficacy of EA is driven by reduced energy intake. Additionally, intracerebroventricular administration of pharmacological inhibitors targeting GLP-1 receptor (GLP-1R) or PYY receptor (Y2R) as well as the receptor null mice will be used to investigate whether EA inhibition of food intake requires the central PYY/Y2R and/or GLP-1/GLP-1R signaling systems. The results of this project are expected to defining the efficacy of a novel compound for treating both diabetes and obesity as well as uncovering the mechanism underpinning these effects, which will potentially lead to developing new, safe, and effective therapy for battling both diabetes and obesity.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT Nearly 30% of all cancer deaths are attributable to tobacco use. Tobacco taxes have positively impacted the prevalence of tobacco use and subsequently the incidence of tobacco-related cancers. The objective of this project is to provide critical insight for tobacco control, by developing and testing a novel integrated tax proposal based on the abuse liability of tobacco and nicotine products in the unique Experimental Tobacco Marketplace (ETM), including how this proposal may interact with tobacco-related socioeconomic cancer disparities. The ETM is an experimental model to forecast, before implementation, the effects of potential public health policies on patterns of tobacco purchasing, including between-product substitution. The ETM places the mix of products, prices, and specific regulations under experimental control to provide estimates of policy impact under conditions that simulate “real-world” circumstances. To achieve these goals, three aims are proposed. Aim 1 will develop an integrated abuse liability ranking of commercial tobacco and nicotine products through a systematic review and a novel application of network meta-analysis. Aim 2 is a proof-of-concept study that will examine the impact of different tax strategies based on the abuse liability of tobacco products in a laboratory-based sample of cigarette smokers. Aim 3 will examine the equity of the most effective abuse liability dependent tax strategy by testing its effects in a nationally representative prospective sample of lower, medium and higher socioeconomic status smokers. These aims support the candidate’s long-term goal to become an independent researcher investigating the differential impact of policies on tobacco initiation, use and cessation among individuals who experience tobacco-related cancer disparities. To further support this goal, a mentored career development plan is proposed, consisting of training in the fields of advanced statistical methods including network meta-analysis, tobacco products abuse liability, government tax policies, and tobacco-related cancer disparities. The advice, guidance and technical expertise provided by the candidate’s mentoring team, collaborators and consultant, and the Virginia Tech’s supportive research environment will provide the resources necessary to successfully complete the research and training plans. Importantly, this K01 award will provide the training, mentoring, and research experiences needed for a highly competitive R01 application to compare the abuse liability dependent taxes to other existing tax proposals. As importantly, this proposal will provide the essential first step in launching an independent cancer-related research program focusing on developing and testing novel policies for the elimination of tobacco use and its unequaled harms.

NIH Research Projects · FY 2024 · 2023-08

|| Project Summary . Impaired endothelial wound healing in blood vessels like the coronary artery and saphenous vein is a primary contributor to vascular stent failure and deadly coronary thrombosis. Identifying the mechanism of delayed endothelial repair is essential for improving vessel patency and patient survival rates. As such, this proposal focuses on identifying regulators of normal endothelial healing and understanding how these factors are dysregulated when healing is impaired. Previous studies have shown connexin 43 (Cx43) gap junctions may regulate wound healing. However, the specific role of connexin 43 in endothelial cell (EC) healing is unknown. Preliminary data in this proposal indicates that ligation-induced vascular injury promotes increases in the expression of EC Cx43 surrounding the damaged area in the aorta and carotid arteries in mice. Cx43-mediated gap junction intracellular communication is controlled by phosphorylation at the Cx43 C-terminus. My preliminary data demonstrate that specific Cx43 phosphorylation at its serine (s) 368, associated with gap junction closure, is present during the final stages of EC wound healing in vivo. Cx43-s368 also reduced the rate of wound closure in cultured human EC. These findings inform the hypothesis that Cx43 expression and channel functions are critical for EC wound healing in large arteries. The aims of this proposal are to define the role of connexin 43 in endothelial wound healing (Aim 1), investigate if a loss of Cx43 limits healing in vivo (Aim 1), and test if channel functions, regulated by posttranslational modifications, improve/delay vascular repair (Aim 2). This investigation will be completed using a novel mouse carotid EC injury survival surgery in mice I developed, which will allow for the assessment of Cx43 expression in carotid EC during the healing process. Both genetic and pharmacological strategies will be used to alter Cx43 expression and phosphorylation in mouse injury models and in cultured human EC, and the impact of these modifications on the rate and quality of EC healing will be quantified. Additionally, RNAseq approaches will be used to identify Cx43 gap junction-dependent signaling in the regenerating endothelium. These aims will be accomplished under the mentorship of researchers with extensive experience in vascular biology, Dr. Scott Johnstone, Dr. Robert Gourdie, and Dr. Brant Isakson, at Virginia Tech’s Fralin Biomedical Research Institute (FBRI). The FBRI is home to research equipment including but not limited to animal facilities, confocal microscopes, flow cytometers, and cell culture equipment. This state- of-the-art research environment will allow for a detailed and mechanistic investigation. Ultimately, this study will characterize a novel role for Cx43 in vascular EC. This project is designed to identify new potential therapeutic targets in vascular disease that will promote the mission of the NIH to enhance patient health and to promote longer life free of illness.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Non-enzymatic protein-protein and protein-DNA cross-links are deleterious post-translational modifications that have been associated with many severe pathologies, including cancer metasta- sis, retinopathy, chronic renal failure, skin and bone disorders, aging, diabetes, Alzheimer’s, Par- kinson’s and cardiovascular diseases. However, the development of therapeutic strategies is hin- dered by our poor understanding of their formation. We propose to address this gap in knowledge using computational methods based on intrinsic electric field calculations. Our goal is to identify the structural and dynamical molecular factors at the origin of the formation of non-enzymatic protein-protein and protein-DNA cross-links. We focus our study on sugar-mediated cross-links, initiated by glycation reactions, as it has been shown to occur in a broad range of systems. We hypothesize that partial depletion of the protein (or DNA) hydration layer exposes side chains (or nucleobases) to surrounding carbohydrates. This facilitates glycation reactions whereby reducing sugars (glucose) react with the free amine groups. Glycated proteins and DNA then have enhanced ability to form adducts, altering their biofunction. Our proposed research seeks to provide a molecular interpretation of sugar-mediated cross-link formation and can be divided into three thrusts ; each of which with the potential to expand into a standalone research direction. First, we propose to characterize the density of the hydration layer of healthy and pathological proteins and DNA strands known to aggregate (collagen, elastin and α-synuclein) at the quan- tum level. Our preliminary data on mineralized collagen systems show that water adsorption is controlled by the nature of the environment rather than the nature of the adsorption site, consistent with experimental observations. This suggests that the density functional theory protocol we de- veloped for this study is suitable for the characterization of macromolecule-water interactions. Second, we propose to model carbohydrate reactivity in dehydrated and hydrated biomole- cules, as a proxy for non-enzymatic glycation reactions. The novelty of our approach is to intro- duce accurate reactivity information in classical molecular dynamics simulations using intrinsic electric fields as a metric for bond formation. Our preliminary data verify the feasibility of such study and include the development of an open-source code that allows this type of calculations. Finally, we propose to integrate our atomistic data into a microscopic kinetic model of protein- protein and protein-DNA cross-linking processes. With this model, we aim to predict the critical density, location, cooperativity and strength of cross-links that are associated with known patho- logies, paving the way towards the identification of therapeutic points of intervention.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY Aerobic life on earth harnesses the oxidizing power of molecular oxygen (O2) through a diverse range of enzyme cofactors and employs that high oxidation potential to mediate numerous oxidative transformations during metabolic functions. Many of these cofactors are coupled binuclear sites consisting of two metal centers such as copper and iron in close proximity. This proposal details fundamental research in the field of bioinorganic chemistry and aims to address several intriguing questions about the potential roles of redox aromatic active amino acids such as tyrosine and tryptophane chains in the enzyme mechanism and function. We identified three different classes of O2-utilizing copper enzymes including cytochrome c oxidase (CcO) which reduces O2 to water, multicopper oxidase (MCO) which couples that reaction to four one-electron oxidations of the substrates, and a new class of copper enzymes called BURP domain cyclases which catalyze 2-electron oxidative macrocyclization of the Tyr/Trp chains in peptide substrates.The use of O2 as either a substrate or terminal electron acceptor has been established in these enzymes except for the BURP domain enzymes which is yet to be confirmed. In all three classes, appropriately tuned and positioned Tyr/Trp chains play different roles either as an integral part of the active site (i.e., CcO) or they may be assigned a mediatory role to provide an alternative path for oxidation of the more challenging substrates (i.e., MCO). They may even act as the direct substrate for the active site (i.e., Cu-dependent BURP domain cyclases). In all cases, despite the diverse use of these chains, their main function is to delicately supply the electron/proton needed for the O2 reduction. How are these residues designed/optimized for a particular function? What are the similarities and differences between these enzymes? Our studies aim to reveal the potential role of these Tyr/Trp chains play in enzymatic function and mechanism and address some of the questions about copper biochemistry and aerobic metabolism.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT Substantial progress in psychiatric genomics has led to the identification of several copy number variants (CNVs) and single genes that are associated with extremely high risk for schizophrenia. A major question facing the field now is whether these discrete genomic loci all act independently or disrupt a common set of neurobiological pathways to produce a similar clinical phenotype. Studies on the effects of the 3q29 deletion, a CNV that confers >40-fold increased risk for schizophrenia, indicate that mitochondrial function may be disrupted in the developing central nervous system. Strikingly, the CNV with the next strongest risk for schizophrenia (22q11.2Del) has also been reported to produce mitochondrial phenotypes. In addition to the 3q29 and 22q11.2 deletion, at least nine other neurodevelopmental disorder-associated CNV loci also contain genes that encode mitochondrial proteins. These data motivate the hypothesis that neural mitochondria may be a site of convergent biology downstream of schizophrenia-risk CNVs. This career development project is designed to address this hypothesis with extensive new training in mitochondrial neurobiology. I have developed a unique set of isogenic human induced pluripotent stem cell lines that, in combination with mouse experimental models of each CNV, will be leveraged to test the hypothesis that 3q29 and 22q11.2 deletion similarly disrupt mitochondrial function in the developing nervous system. The goals of this project are to define the extent of mitochondrial phenotypes produced by these schizophrenia-associated CNVs and to determine the degree of biological convergence at molecular and functional levels. To this end we will assess the effects of each CNV on the proteome of mitochondria isolated from CNV-model mouse brain and isogenic human cortical organoids. Additionally, the transition from glycolysis to oxidative phosphorylation is a critical stage of neuronal development. We will test the capacity of 3q29 deletion and 22q11.2 deletion neural progenitor cells to adapt to metabolic stress by using media formulations to force cultures to either utilize glycolysis or oxidative phosphorylation to meet energy demands. Finally, we will utilize an engineered heterologous cell system to screen for gene drivers of 3q29 mitochondrial phenotypes. These experiments will yield important data related to the concept of convergent biology, a timely and significant question in translational psychiatry which could have profound effects on our understanding of risk alleles and future therapeutic approaches.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY During the 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 mitochondrial health in skeletal muscle. Thus, bioenergetics of peripheral tissues, and skeletal muscle in particular, may have an underappreciated role in AD etiology. Exercise is an effective means to promote mitochondrial, as well as, skeletal muscle 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. We show in 5xFAD mice that muscle dysfunction is present at a young age before observable cognitive decline and that muscle loss and impaired mitochondrial health manifest along by an age associated with cognitive decline. We present evidence that mitochondrial respiration does not improve following 12 weeks exercise training in 22-week-old 5xFAD mice compared to WT littermates. In sum, bioenergetic dysfunction in muscle may underlie a maladaptive response to exercise prior to overt manifestation of AD-related pathology. There is a critical need therefore to define the adaptive mechanisms in muscle in relation to neurophysiological changes over the continuum of AD pathology to identify novel therapeutic targets. Our central hypothesis is that impaired bioenergetics precedes manifestation of overt AD neuropathology resulting in maladaptation in muscle to exercise training. To test our hypothesis, we propose two aims: Aim 1) Determine the adaptive response of muscle mitochondria to endurance exercise training in AD mice before development of AD. 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 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 muscle function in vivo (Aurora), neuromuscular junction integrity (histochemistry) and mitochondrial quality (confocal microscopy) in novel MitoTimer/5xFAD transgenic mice (1b), assess central (hippocampus) and peripheral (plasma NfL) neuropathology (1c), and perform untargeted metabolomics of muscle and hippocampus following exercise training (1d). Aim 2) Determine the tissue-specific and functional roles for AMPK⍺1 in AD etiology in 5xFAD mice. We will assess mitochondrial function, proteostasis, development of neuropathology, and metabolomics in both muscle and hippocampus at 3, 6, and 9 months of age in muscle- and motor neuron-specific AMPK⍺1 knock-out mice, as well as novel gain- and loss- of-function AMPK⍺1(T172A) knock-in mice. Our findings will elucidate the maladaptive response of skeletal muscle mitochondria to exercise training in context with AD neuropathology and the integrated isoform-specific functional role of AMPK⍺ in AD etiology. These studies will provide mechanistic to the integrated pathology along the continuum of AD pathology between skeletal muscle and brain and the role of exercise as a therapeutic.

NIH Research Projects · FY 2024 · 2023-07

Project Summary: Hepatitis E virus (HEV) infects >20 million people worldwide annually leading to 3.3 million clinical cases of hepatitis and >44,000 deaths due to hepatobiliary diseases. As a non-enveloped virus, HEV is surprisingly present as quasi-enveloped exosome-like virions in circulating blood that resist neutralization. Genotype 1 HEV (HEV-1) infection is associated with fulminant hepatitis with high mortality (>25%) in pregnant women. HEV-1 replicates in placental tissues, and HEV-1 vertical transmission is associated with a high neonatal mortality. Due to the lack of an efficient cell culture or animal model for HEV-1, the mechanism of HEV-1-associated severe diseases during pregnancy is unknown. Significantly higher levels of TNF-α were found in HEV-infected pregnant women with fulminant hepatitis, and HEV-infected pigs with detectable HEV RNA in CNS tissues had significantly higher levels of proinflammatory cytokines (TNF-α and IL-18) than in pigs without detectable HEV RNA in CNS tissues. The long-term goal is to delineate the mechanisms contributing to HEV-associated high mortality during pregnancy. Unfortunately, we currently do not have a suitable system, in vitro or in vivo, to study HEV-1 infection at the maternal-fetal interface. In aim 1, we will establish an in vitro placental barrier in Transwell insert to study HEV-1 infection in the maternal-fetal interface. We hypothesize that HEV-1 in circulating blood during peak viremia crosses the placental barrier leading to fetal infection. We will develop a placental barrier culture mimicking the critical maternal blood- and fetal blood-facing layers that constitute the human placental barrier in vivo, by co-culturing BeWo placental trophoblastic cells and human umbilical vein endothelial cells on basolateral and apical sides of an extracellular matrix-coated Transwell insert. The integrity of the barrier will be confirmed by measuring TEER and barrier permeability to small molecules. We will determine whether HEV-1 can cross the barrier by infecting barrier cultures in the maternal chamber with HEV-1 and HEV-3, respectively, and measuring the amount of HEV in the fetal chamber of the barrier. In aim 2, we will determine the mechanisms of HEV-1 infection in the maternal-fetal interface leading to fetal infection. We hypothesize that quasi-enveloped exosome-like HEVs in circulating maternal blood during peak viremia more easily cross the placental barrier when the barrier is inflamed by pro-inflammatory cytokines such as TNF-α and IL- 18 that are consistently produced during HEV infection, and that HEV-1 infection in the barrier produces type III IFNs to limit viral infection. We will inflame the barrier cultures with TNF-α and IL-18, separately or in combination, and then infect them with non-enveloped HEV-1, HEV-3, quasi-enveloped HEV-1, HEV-3, respectively, to determine the amounts of HEV that have crossed the barrier. We will also determine the expression levels of IFN-α, IFN-β and IFN-λs in infected cells, and determine if the antiviral resistance environment induced at the barrier can be transferred to HEV-susceptible liver cells. We anticipate to establish a placental barrier mimicking the critical maternal blood- and fetal blood-facing layers in vivo, and show that quasi-enveloped HEV-1 will more easily cross the barrier especially when the barrier is inflamed with proinflammatory cytokines TNF-α and IL-18. We also expect that HEV-1 induces IFN-λ1/λ2/λ3 in the barrier to limit virus replication, and that the antiviral resistance environment induced at the barrier is transferable to HEV-susceptible liver cells.

NIH Research Projects · FY 2025 · 2023-07

Abstract Sex is critical to the survival and evolution of sexually reproducing organisms including mosquitoes. A dominant male-determining factor (M factor) is the primary signal that controls sex-determination in mosquitoes. Nix, the M factor in the yellow fever mosquito Aedes aegypti, is the first M factor found in mosquitoes. In a simplified model, the expression of Nix, a predicted RNA-binding protein, leads to male- specific splicing of the pre-mRNAs of two conserved transcription factors, doublesex (dsx) and fruitless (fru), which program male sexual differentiation. In Aedes the M factor is located within the male- determining locus (M locus) on one of the “autosomes”. This pair of “autosomes” are so-called homomorphic sex chromosomes that are cytologically indistinguishable except in the region around the sex locus. The Ae. aegypti M locus is a ~1.3 Mbp repeat-rich region that contains Nix and four other protein-coding and 25 long non-coding RNA genes. The Nix transgene alone, in the absence of the M locus, is sufficient to convert females into fertile albeit flightless males, and myo-sex, a myosin heavy chain gene also in the M-locus, is required for male flight. The M- and m-bearing chromosomes in Aedes mosquitoes provide an opportunity to gain insights into the evolution of homomorphic sex chromosomes. In addition to its basic biological importance, Ae. aegypti is a major vector for the dengue, chikungunya, and Zika viruses. No specific treatment for dengue exists and the first dengue vaccine is recommended only for a limited population. Prevention of these vector-borne infectious diseases relies heavily on effective vector control. However, increasing insecticide-resistance poses a significant threat. Therefore, novel control strategies are urgently needed. Only female mosquitoes feed on blood and transmit pathogens, and for the most part females determine the size and distribution of the mosquito population. We are interested in deciphering the mechanism of sex-determination, investigating sex chromosome evolution, and translating such fundamental knowledge into safe, efficient, and diverse methods to control diseases that are transmitted by Ae. aegypti. Building on recent progress, we will pursue the following specific aims: 1) Decipher the sex locus in Ae. aegypti, 2) Identify and characterize the target(s) of Nix, and 3) Develop efficient sex-separation methods through Nix-mediated innovations.

NIH Research Projects · FY 2025 · 2023-07

Environmental exposures affecting human health are traditionally associated with industrial sources in urban environments, while exposures and associated health outcomes unique to rural landscapes have been less emphasized in environmental health sciences training. To address this gap, the Rural Environmental Health (REH) training program will leverage Virginia Tech’s strengths in agriculture, engineering, veterinary, and natural resources research and outreach in rural areas to advance biomedical and public health training and research. The REH program will provide predoctoral fellows with environmental health training in the areas of toxicology, environmental epidemiology, exposure science, and risk assessment. Trainees will focus their dissertation research within one of three focus areas: mechanisms of environmentally-mediated diseases prevalent in rural areas, characterization of exposures in rural landscapes, or community-engaged approaches to address environmental health priorities in rural areas. A total of six REH trainees will be recruited over the 5-year funding period from participating PhD degree granting programs at Virginia Tech including: 1) Translational Biology, Medicine, and Health, 2) Biomedical and Veterinary Sciences, 3) Environmental and Water Resources Engineering, 4) Biological Systems Engineering, 5) Fish and Wildlife Conservation, and 6) Biological Sciences. Over their 4 years of training, they will participate in formal coursework in environmental health sciences, including toxicology, epidemiology, and exposure science, to gain depth in methodological skills. REH trainees will gain team science and science communication skills, experience working at the science-policy interface, and receive career planning guidance through participation in the Interfaces of Global Change (IGC) Interdisciplinary Graduate Education Program. The IGC will expose them to the wide range of global change research on interacting health threats from pollution in rural environments. REH trainees’ will participate in a journal club led by REH leadership to build camaraderie, learn from each other, and keep abreast of the relevant literature on rural environmental health. The REH program is supported by a leadership team spanning four colleges (Medicine, Veterinary Medicine, Engineering, and Natural Resources) and an additional 22 faculty mentors with cutting-edge interdisciplinary research programs funded through grants from NIH and other federal agencies including NSF, USDA, and NASA. As a new program, the leadership team has developed a mentoring and evaluation plan that includes multiple methods of independent feedback from trainees, faculty mentors, and an external advisory board made up of directors of long-standing NIEHS-funded training programs to guide progress and ensure the continued growth and success of the REH trainees

NIH Research Projects · FY 2025 · 2023-06

SUMMARY Synopsis: This is a K01 application by Dr. Luis Escobar, researcher on zoonotic infectious diseases. Escobar’s project Integrative Spatial Epidemiology Study of Wildlife Rabies Spillover will combine spatial modeling with large epidemiological, genomic, and ecological data to investigate pathogen spillover from wildlife to humans and livestock. Candidate: PI Escobar, DVM/MSc/PhD, is a tenure-track Assistant Professor at Virginia Tech, with prior postdoctoral training at SUNY Upstate Medical University and the University of Minnesota and a strong record of scientific achievements. This application has three training objectives to strengthen Escobar’s research as an independent investigator: (1) Training in bioinformatics; (2) Integrate bioinformatics with spatial epidemiology; and (3) Gaining skills in laboratory management and research proposal development to transition to career independence. Mentors: Prof. X.J. Meng, with a solid history of NIH funding, outstanding expertise in bioinformatics, and at the applicant’s institution, will serve as Primary Mentor. Four additional mentors (Drs. Z. Tu, W. Hopkins, S. VandeWoude, A.T. Peterson) will provide complementary expertise and will assess Escobar’s progress towards career independence. Research: Emergent infectious diseases, such as Ebola, Nipah, and COVID-19, have been linked to pathogen spillover from bats to other species. A gap in knowledge exists in the fundamental ecology of pathogen spillover transmission (i.e., Where does it occur and why?), which limits understanding of previous epidemics and capacities to prevent disease emergence. This project will study pathogen spillover from bats to humans and livestock using rabies from the common vampire bat (Desmodus rotundus), a well-documented system of a virus that frequently spills from D. rotundus bats over to other species. While D. rotundus-rabies is restricted to Latin America, it is rapidly expanding towards the United States (US) for unknown reasons. The central hypothesis is that D. rotundus-rabies spillover does not occur randomly in the geography but follows specific biodiversity and habitat patterns, which can be used to predict where pathogen spillover will occur. Aim 1 will determine the role of habitat and virus lineage on rabies-spillover to humans and livestock across Latin America. Aim 2 will identify effect of biodiversity composition on rabies virus spillover transmission. Aim 3 will investigate genomic, geographic, and environmental factors shaping the spread of rabies virus. This hypothesis-driven project has conceptual and technological innovations and combines the expertise of PI Escobar and mentors to illuminate ecological drivers of rabies spillover. Findings will offer new mechanistic insights to explain and anticipate pathogen spillover. As such, this K01 application will facilitate Escobar’ s transition towards research independence and offers exciting discoveries on the ecology of spillover transmission, aligned with the NIH mission to seek fundamental knowledge about the nature of living systems to enhance human health.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY Chemotherapy-induced peripheral neuropathy (CIPN) manifests in nearly 70% of patients treated with anti- neoplastic drugs and persists after treatment discontinuation, thereby impacting patient quality of life. The anti- depressant duloxetine (selective inhibitor of serotonin and norepinephrine uptake) offers limited pain relief, while long-term use of opioids for chronic pain carries safety risks. Thus, novel non-opioid analgesics are needed for pain management in cancer patients. Our findings show that supraspinal (in the brain) delivery of cocaine and amphetamine-regulated transcript peptide II (CART II) reverses pain-like behaviors (tactile) in male and female mice with CIPN. Multiple behavioral outputs would provide clinical face validity and bolster translatability of a given target, thus a comprehensive dose-evaluation of CART II anti-hyperalgesic effects during chronic pain states of CART II in both sexes at multiple endpoints of chronic pain in both sexes is warranted. Previous studies have uncovered broader pharmacological actions of CART II and systemic administration of a neuropeptide likely will impose a challenge, so it is critical to determine the neurochemical signaling mechanisms of CART II in CIPN. While the lack of a known receptor for CART II has prevented such mechanistic studies, we discovered that Lysophosphatidic Acid Receptor 2 (LPAR2) is a high affinity receptor for CART II in the brain. We used cell- based assays and in vivo pharmacology tools to show that supraspinal LPAR2 is necessary CART II-induced acute analgesia. However, the role of LPAR2 in chronic pain states has not been evaluated. Our findings also demonstrate that therapeutic doses of supraspinal CART II increase glutamate release in ventrolateral periaqueductal gray (vlPAG) and nucleus accumbens (NAcc) in naïve mice, and previous work shows excitatory inputs into these brain sites can relieve neuropathic pain states. However, neurotransmission during CIPN in the vlPAG and NAcc has not been fully established. The central hypothesis in this proposal is that CART II produces its anti-hyperalgesic effects via activation of LPAR2 and increased glutamate release. Thus, we propose to fully examine anti-hyperalgesic actions of this signaling pathway in vlPAG and NAcc in males and females via two independent yet interconnected aims. Aim 1 will comprehensively evaluate dose-dependent anti-hyperalgesic effects of supraspinal CART II in 4 different behavioral output measures (tactile, cold, anxiety, depression) to interrogate any sex differences in therapeutic potential for CIPN. Incorporation of a positive allosteric modulator and knockout mice will reveal if LPAR2 is necessary and sufficient for the anti-hyperalgesic actions of CART II. Aim 2 will examine CART II-mediated neurotransmission as a mechanism of action for its anti-hyperalgesic effects in CIPN. Collectively, the expected results will address significant gaps in understanding of the supraspinal mechanisms underlying neuropathic pain states and interrogate the CART II/LPAR2 axis as a novel therapeutic strategy in reversing already established CIPN.

NIH Research Projects · FY 2026 · 2023-06

Project Summary/Abstract One of the core attributes of cells is their ability to grow and divide. This process is driven by a network of proteins, commonly called the cell cycle machinery, which acts like a miniature engine driving cycles of growth and division. How the proteins of the cell cycle machinery work together is relatively well understood. However, the rules of their production from the corresponding genes are much less clear, and yet are equally important to create a functional engine. Genes can differ widely in the strategy they use to produce their corresponding protein. For example, they may produce more or less of the messenger RNA intermediate, the messenger RNA or the protein may have a long or a short lifetime, and production can be directly linked to those of other proteins or not. These features in turn influence the dynamic range of protein levels, the interaction between proteins, and the robustness of the system in the face of perturbations. How expression strategies are encoded in the DNA and how they contribute to proper protein function is poorly understood and therefore limits our understanding of cell cycle regulation. Interestingly, different cell cycle regulators use different expression strategies even when operating in the same pathway and it is likely that each strategy is selected for optimal function of this protein within the cell cycle machinery. Our long-term goal is to understand how these different expression strategies support cell cycle regulation, and how the information for a specific expression strategy is encoded on the DNA. Our immediate goal is to understand how messenger RNA features of select cell cycle regulators support the function of the protein that they encode. We will address this question in fission yeast, a eukaryotic model organism that shares many features with human cells but uses a minimal cell cycle machinery that is experimentally well-tractable. Gaining a deep understanding of how different expression strategies are encoded in the genome will enhance our understanding of cell physiology and will provide insight into the consequences of non-coding mutations that are observed in cancer or other genetic diseases, thus promoting advances in precision medicine.

NIH Research Projects · FY 2026 · 2023-06

(PLEASE KEEP IN WORD, DO NOT PDF) Germline mutations in SAMD9L are a major cause of inherited bone marrow failure and pediatric myelodysplastic syndrome, frequently associated with poor clinical outcomes. Although these mutations are known to impair hematopoietic stem cell function, the cellular mechanisms linking genetic disruption to disease progression remain incompletely understood. This project aims to define the physiological role of SAMD9L in maintaining normal hematopoiesis and to determine how pathogenic variants compromise this process. Using patient-relevant human induced pluripotent stem cells and genetically engineered mice models, the proposed studies will investigate how dysregulated cellular stress responses, metabolic pathways, and inflammatory signaling contribute to stem cell dysfunction and disease evolution. The work integrates genetic, cellular, and functional approaches to establish mechanistic links between SAMD9L disruption and impaired blood formation. Expected outcomes include identification of key regulatory pathways that influence disease severity and therapeutic response. Successful completion of this project will advance understanding of inherited bone marrow failure syndromes, support the development of targeted intervention strategies, and establish a strong foundation for the investigator’s independent research program in translational hematology.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY: Pancreatic cancer accounts for approximately 3% of all cancers in the United States and approximately 7% of all cancer related deaths. New treatment paradigms are direly needed. Emerging tumor ablation techniques have shown significant promise. This proposal will focus on High-Frequency Irreversible Electroporation (H-FIRE), which delivers a series of electric pulses through electrodes inserted directly into the tumor to produce structural defects in the target cell membrane resulting in cancer cell death. The objective of this proposal is to utilize our mouse and novel pig preclinical animal models to expand upon the preliminary data presented in this proposal and generate critical mechanistic, safety, and efficacy data necessary to support future H-FIRE clinical trials in pancreatic cancer patients. Our overarching hypothesis is that H-FIRE will effectively mitigate heterogeneity in physiologically and clinically relevant pancreatic tumors, with treatments leading to contiguous zones of ablation near critical tissue structures. We further postulate that the benefits of H-FIRE will ultimately extend beyond focal tumor ablation and generate a predictable, tunable systemic anti-tumor immune response reducing metastatic burden and preventing recurrence. Specific Aim 1 will characterize the biophysical response of pancreatic cancer cells and tissues to H-FIRE. This Aim will evaluate the hypothesis that H-FIRE pulse parameters can be tuned to achieve different cell death outcomes (apoptosis, pyroptosis, necroptosis, or necrosis) that are highly relevant to tumor ablation, the tumor microenvironment, and anti-tumor immune responses. In concert, we will assess ablation development with real time treatment feedback using Fourier Analysis Spectroscopy (FAST). We expect to determine which parameters (i.e. pulse width, energized time, interphase/interpulse delay) play significant roles in tuning cell death elicited within relevant cancer cell lines and ex vivo tissues. Specific Aim 2 will establish H-FIRE treatment strategies for pancreatic cancer that optimize tumor ablation and systemic anti-tumor immune responses. Using Pan02 mouse models, this Aim will test the hypothesis that H-FIRE is an effective treatment modality for precise and complete pancreatic tumor ablation in vivo. We also postulate that due to the unique features of H-FIRE mediated cell death and resultant changes in the tumor microenvironment, focal tumor ablation will result in predictable and tunable systemic anti-tumor host immune responses reducing metastatic burden and preventing recurrence. Specific Aim 3 will define H-FIRE treatment parameters and determine its safety profile utilizing physiologically and clinically relevant porcine models of pancreatic cancer. This Aim will test the hypothesis that H-FIRE can effectively ablate orthotopic pancreatic tumors under physiologically and clinically relevant in situ conditions. To test this hypothesis, we will utilize novel, orthotopic, porcine pancreatic cancer models featuring a diverse range of clinically relevant physical properties that are predicted to impact H-FIRE efficacy in human patients.

NIH Research Projects · FY 2024 · 2023-05

Abstract Among the many social justice issues that our nation continues to confront, health disparities, especially underrepresentation in the biomedical workforce remains one of the most tractable. The lack of representation in the proportion of minorities, especially black men, who go on to obtain a STEM degree remains alarming. Among the factors responsible for this lack of diversity are the switching out of STEM majors by the end of the sophomore years and the challenging environments many URMs face at research intensive (R1) institutions. Virginia Tech’s ESTEEMED (VT ESTEEMED) program will provide the foundation and tools necessary from disciplines that include biological sciences, chemistry, biochemistry, math and statistics for our scholars to succeed in obtaining their undergraduate degrees and pursuing graduate degrees in biomedical engineering. To accomplish this broad goal of increasing diversity in the biomedical workforce, this revised VT ESTEEMED, which addressed all the “minor weaknesses” in the “01,” will use program faculty and PIs with outstanding history of research productivity, training and mentoring of students from diverse backgrounds. Specific Aims of the VT ESTEEMED research education program are: 1. Recruit 8 entering Freshmen, each year, for a total of 32 to become VT ESTEEMED scholars who will have the foundation necessary to succeed in getting a BS and pursuing a graduate degree in biomedical engineering; 2. Create a bridge program that will provide a smooth transition to college for VT ESTEEMED scholars and to help them develop a familiarity with the fundamentals of biomedical science and the process of “entering research;”3. Provide a mentored research experience that will enhance student learning and engagement that will also translate into academic retention and success; 4. Create learning opportunities for ESTEEMED scholar-led developmental activities including literature review sessions, research progress/status updates, and the cohosting of speakers; and 5. Engage ESTEEMED scholars in summer activities that includes research training and the acquiring of skills that include communication including writing and oral, and mentoring and leadership. With outstanding leadership skills locally and nationally, the ESTEEMED program directors and the potential faculty mentors bring a history of exemplary scholarly achievements in STEM and URM training and development.

NIH Research Projects · FY 2025 · 2023-04

PROJECT SUMMARY Stroke is one of the most common causes of death and disability in the United States and worldwide. The vascular system is meticulously regulated throughout life to adapt to changes in metabolic demand and blood flow under widely variable conditions. Many ischemic stroke patients however fail to fully recover following an acute attack. This impaired recovery is related in part to the limited return of perfusion within the brain microcirculation, even after restoring the patency of occluded vessels – a scenario referred to as the “no-reflow” phenomenon. Blood circulating within the vascular system exerts different types of forces on the surrounding vessels. These forces are sensed and interpreted by the vascular cells to guide their development during embryogenesis and regulate remodeling during postnatal and adult life. It has been also suggested in recent years that there are signals downstream of mechanical changes that are exchanged between vascular cells. Specifically, pericytes and endothelial cells integrate these cues to dynamically regulate blood vessel physiology, capillary permeability, and changes in microvascular tone in health and in disease. Despite recent advances in our knowledge of flow-mediated biomechanical inputs, the underlying molecular processes and their link to hemodynamic forces in vivo are still emerging, in part due to limitations in the tools and models to measure these forces. To help fill this gap in knowledge, the proposed study aims to investigate the impact of abrupt changes in blood flow on two components of the blood-brain barrier -- pericytes and endothelial cells -- and their interaction in mature brain vessels under static conditions following the loss of flow. We will utilize both ex vivo and in vivo models to establish the mechanistic interactions underlying how pericytes and endothelial cells process, interpret, and organize various mechanical signals. Additionally, we will look at corresponding changes in the surrounding extracellular matrix that might accompany this cellular interplay, specifically interactions between endothelial cell integrin α5 and pericyte-derived vitronectin within the capillary wall. Our preliminary data suggests a two-phase response over time following an acute shift towards static conditions. We propose that an early stage marked by a rapid inflammatory response, involving elevated interleukin-1beta expression, is overlaid by a hypoxia-driven response in a subsequent phase, both contributing to cerebrovascular instability and an increased risk for hemorrhagic conversion of ischemic stroke patients after re-establishing cerebral blood flow. Identifying the key mechanistic determinants responsible for blood vessel destabilization in the brain during the hyper-acute phase of stroke will provide targetable signals that could be clinically significant in advancing stroke therapies.

NIH Research Projects · FY 2026 · 2023-04

Abstract Cryptococcal meningitis (CM), caused by the fungal pathogen Cryptococcus neoformans (Cn), is among the most prevalent HIV/AIDS-associated opportunistic infections and causes 15% of AIDS- related mortality globally. In healthy individuals, exposure to Cn in early childhood results in a pulmonary latent infection that is asymptomatic, but leads to the formation of lung granulomas. Following HIV-associated compromise of the immune system, control of latent Cn infection within pulmonary granulomas is lost and the fungus disseminates to cause meningitis. Most studies examining host-pathogen interactions in Cn are observational studies in human cohorts or analyze reference Cn strains in acute disease models of cryptococcosis. We recently showed that the mouse model accurately recapitulates differences in human survival that are observed across Cn clinical isolates and used these data to develop a mouse model of latent Cn infection. Our preliminary data using these mouse models to analyze the immune response to over 50 Cn clinical isolates from individuals with advanced HIV revealed a continuum of disease outcomes that we classified into 3 groups: 1) latent infection resulting in granuloma formation and control; 2) lethal disease similar to that observed with Cn reference strains; and 3) hypervirulence resulting in rapid mortality. Previous studies with reference strains revealed lethal disease is associated with various Cn virulence factors and a detrimental host Th2-mediated type-2 immune response. In contrast, disease prevention is associated with the type-1 cytokine IFNγ. How these Cn-host interactions differ to cause the continuum of disease observed in immunocompromised individuals with HIV is not well defined. We will use analysis of clinical isolates in mouse models of disease to test our central hypothesis that antigenic differences between Cn clinical isolates lead to either protective or detrimental immune responses in the host. We will test this hypothesis by pursuing three specific aims. Our first and second aims will determine the host cellular and effector functions that result in either latency (Aim 1), lethal disease (control infections), or hypervirulence (Aim 2). Our third aim will identify Cn gene alleles for antigens that influence the immune response and ultimately disease outcome. Taken together, these translational studies will define the molecular processes underlying the continuum of Cryptococcus disease with the goal of developing novel immune-modulatory treatment strategies for at-risk patient populations.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY Loss-of-function mutations in the genes encoding the cardiac isoform of the voltage-gated sodium channel (Nav1.5) have been associated with the Brugada Syndrome (BrS), and loss-of-function of plakoglobin has been associated with Arrhythmogenic Cardiomyopathy (ACM). Both diseases are associated with inconsistent experimental findings, can be revealed by basic experimental differences, often affect the right ventricle, are associated with intercalated disc proteinopathies, and for both, there are few treatments to prevent arrhythmias. We have previously demonstrated that another loss-of-function in the intercalated disc protein connexin43 can be concealed by choice of experimental perfusate, potentially explaining why disease-related conduction slowing can be measured in some laboratories but can remain concealed in an intact organism with “normal” electrolyte composition. We also previously demonstrated that induced acute interstitial edema (AIE) is greater in the right relative to the left ventricle. Since AIE can unmask gap junction uncoupling, we hypothesize that AIE can unmask two other intercalated disc diseases: BrS and ACM. Finally, if AIE can unmask intercalated disc diseases, our data suggest that these diseases may be treatable by managing intercalated disc microdomain separation. In this project, we propose an innovative hypothesis that the concealed nature of BrS and ACM in intact tissue is mechanistically tied to a newly discovered form of cell-to-cell communication called ephaptic coupling. Upon successful completion of these aims, we will produce new methods to unmask these diseases in their pre- manifest stage, allowing for early detection. Further, the work will suggest important new therapies for two diseases with few effective therapeutic options and poor patient outcomes.

NIH Research Projects · FY 2026 · 2023-04

Project Summary/Abstract The lack of practical and effective strategies to manage hunger and adhere to a weight management intervention represents a critical barrier to the weight management field. In proof-of-concept efficacy studies, we have demonstrated that premeal water consumption (500 ml) acutely reduced perceived hunger and meal energy intake among middle-aged and older adults, and that premeal water consumption (500 ml, 3 times per day) increased the amount of weight lost (i.e., 2 kg greater loss) after 12 weeks among middle-aged and older adults with overweight or obesity. However, water consumption may be important for weight management regardless of when it is consumed. To address this possibility, we propose a rigorously-designed randomized controlled intervention trial in adults aged 50+ years with overweight or obesity comparing three groups with different diet prescriptions: 1) pre-meal water consumption (500 ml, before each main meal) with a hypocaloric diet; 2) 1500 ml water consumed throughout the day with a hypocaloric diet; 3) hypocaloric diet with no instructions regarding water consumption. Smart water bottles will objectively assess water intake timing and volume. Urine osmolality, urine volume, and serum osmolality will be used as objective indicators of compliance with the water intake prescription. We will investigate changes in perceived hunger and fullness and appetite-regulating hormones as potential mechanisms by which premeal water could improve appetite regulation. We will also investigate the impact of water consumption and hydration on executive function capabilities, which may influence intervention adherence. We hypothesize that weight loss at 12 weeks, and weight loss maintenance at 12 months, will be greater in the premeal water group compared to the daily water volume and control groups. Furthermore, when combined with a hypocaloric diet, premeal water will reduce hunger and increase fullness; these changes will mediate adherence to the hypocaloric diet and weight loss outcomes. Although increasing water intake could be an effective weight management strategy, no evidence-based recommendations exist for the timing of water intake needed for this benefit. Our findings could identify a low-cost, actionable dietary strategy that could be incorporated into clinical practice guidelines for obesity treatment, and improve adherence to a hypocaloric diet prescription through reduced hunger, increased fullness, and improved attention and inhibitory control. This proposal is aligned with the 2020-2030 NIH Strategic Plan for Nutrition Research (Objective 3.5) and the Dietary Guidelines for Americans 2020-2025 due to its focus on older adults, an understudied population at increased risk for obesity and inadequate hydration. If effective, this hydration/weight control intervention approach could be applied to clinical populations such as adults with obesity who are prescribed increased water intake to prevent or treat kidney stone disease or urinary tract infections, which are common in older adults.

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY Existing approaches targeting selected pro- or anti-inflammatory mediators during experimental sepsis mostly ended in failure, due to the un-resolved predicament of sepsis patients experiencing the dichotomy of severe immune-suppression as well as exacerbated pathogenic inflammation, collectively contributing to increased multi-organ injuries and compromised immune defense toward secondary infections. Our integrated experimental and computational studies combined with scRNAseq analyses expanded our understanding of the traditionally defined immature Ly6Chi myeloid derived suppressor cells (MDSCs) seen in human and animal sepsis, in that these less-differentiated Ly6Chi monocytes are not only immune suppressive (with elevated immune suppressor PD-L1 and reduced immune activator CD86), but also highly pathogenic inflammatory (with sustained ROS, and elevated inflammatory mediators S100A8/9, CD38 and CX43), characteristic of septic monocytes from human patients and animals with experimental sepsis. Instead of the traditional narrow definition of MDSC partially emphasizing their immune-suppressive features, we propose the holistic concept of “exhausted memory monocytes” encompassing both immune suppression and pathogenic inflammation. Mechanistically, we recently reported that the less-studied TLR4 adaptor molecule TRAM is critically involved in the generation of exhausted monocytes, and that TRAM deletion can alleviate experimental sepsis. Based on these novel findings, our long-term goal is to define novel therapeutic targets for relieving innate immune exhaustion and preventing/treating sepsis. As a crucial first step, our key objective is to better characterize the generation of “memory” exhausted monocytes and key underlying mechanisms. We plan to test the central hypothesis that the generation of monocyte exhaustion memory during sepsis pathogenesis are mediated by the novel TRAM signaling circuitry, and that targeting TRAM will hold a therapeutic potential in restoring monocyte homeostasis and preventing/treating severe sepsis. To test this hypothesis, we plan to perform the following integrated studies. Aim 1. To test the novel phenotypic hypothesis that TRAM mediates the generation of memory exhausted monocytes during sepsis. Aim 2. To characterize novel mechanisms underlying TRAM-mediated generation of exhausted monocytes. Aim 3. To examine the translational potential of sepsis intervention via reprogramming exhausted monocytes. Completion of this project will holistically reveal important and novel mechanisms responsible for the generation of monocyte exhaustion memory leading to sepsis pathogenesis, and facilitate the development of effective therapeutic strategies in restoring monocyte homeostasis and reducing sepsis mortality/morbidity.