East Tennessee State University

NIH Research Projects · FY 2026 · 2026-06

Abstract Sepsis is a systemic inflammation triggered by infection that leads to organ dysfunction. With 750,000 cases and 250,000 deaths per year, it is the most common cause of in-hospital deaths in the U.S. Immune paralysis is a high risk for the secondary infections and mortality of survived sepsis patients. However, the cellular and molecular mechanisms of immune paralysis have not been elucidated. Therefore, our long-term goal is to determine the mechanisms of sepsis-associated immune paralysis. Lactate level is an independent biomarker for the prognosis, severity, and mortality of sepsis. Recent clinical studies have reported that sepsis patients with high lactate levels exhibit impaired immune responses. In this application, we will define the role of lactate in mediating B cell dysfunction, thereby contributing to immune paralysis in sepsis. B cell immunity plays a pivotal role in the immune response following sepsis. We discovered that lactate induces the numeric, phenotypic and functional changes of B cells following sepsis. Transcription factor enrichment analysis (TFEA) revealed that FoxO1 is the most significantly suppressed transcription factor by lactate in B cells following sepsis. FoxO1 is a master transcription factor governing the expression of genes associated with B cell development, proliferation and immune response. In addition, we made a novel finding that lactate drives the production of γ-amino-butyric acid (GABA) in B cells post-sepsis. GABA, traditionally known as a neurotransmitter in the brain, is reported to be an effectively immunosuppressive molecule. We demonstrated that GABA is a B cell-associated metabolite, which may impair immune responses following sepsis. Mechanistically, we found that lactate promotes the lactylation of FoxO1 and histone 3 in B cells, as demonstrated by immunoprecipitation assay and supported by a machine learning-based prediction tool. Importantly, we observed that the expression of AARS1, a newly identified lactate sensor and lactyl-transferase, is highly enriched in B cells when compared to other immune cells, suggesting that B cells are sensitive to elevated lactate levels during sepsis due to their abundance of lactylation machinery. Based on our preliminary studies, we hypothesize that lactate suppresses B cell immune function via FoxO1 lactylation and promotes B cell-derived GABA release, thus contributing to immune paralysis during sepsis. To test this hypothesis, we will integrate genetic and pharmacologic approaches, utilize FoxO1 mutant mouse models, perform multi-omics analyses, and apply machine learning-based tool. We propose the following specific aims: 1) Define the mechanisms of lactate-induced FoxO1 lactylation in B cell dysfunction in sepsis; 2) Determine the mechanisms of lactate-promoted GABA production in B cells and its contribution to sepsis-associated immune paralysis; 3) Elucidate whether lactate-impaired B cell immune function is mediated by lactate transport (MCTs) and lactate receptor (GPR81).

NIH Research Projects · FY 2026 · 2026-06

This proposal requests funding for a Liquid Chromatography-Mass Spectrometry (LCMS) system to support biomedical research and educational initiatives across East Tennessee State University's Academic Health Science Center. The instrument will primarily serve researchers and students in the Colleges of Medicine and Pharmacy, while remaining accessible to investigators from the Colleges of Public Health and Arts and Sciences. The acquisition of this LCMS system addresses a critical infrastructure gap that emerged in 2022 when the university's previous LCMS instrument became non-functional. From 2009-2022, LCMS capabilities at ETSU facilitated significant research productivity across multiple investigative teams, resulting in over 40 peer-reviewed publications. This research spans an array of biomedical applications including: pharmacokinetic studies of therapeutic drugs and substances of abuse; development of novel drug delivery systems; quantification of endogenous biomarkers in disease states; stability studies of compounded pharmaceuticals; analysis of environmental contaminants; and investigation of lipid mediators in cardiovascular disease. This productivity has been severely hampered by the lack of this essential analytical capability since 2022. Beyond supporting faculty research programs, this instrument will provide exceptional educational opportunities for a broad spectrum of students, including PharmD, MD, PhD, MS, and undergraduate trainees. Hands-on training with sophisticated LCMS technology will equip these learners with specialized analytical skills highly valued in both academic and industrial research settings. This training represents an uncommon opportunity, particularly for undergraduate science students, enhancing their competitiveness for advanced educational programs and future employment. The strategic placement of this instrument within our shared research infrastructure will maximize its impact, supporting ongoing NIH-funded investigations in areas including pharmacokinetics, drug metabolism, natural product chemistry, biomarker discovery, and neonatal abstinence syndrome research. Additionally, the instrument will enable new collaborative research directions that align with institutional priorities in addiction science, infectious disease, and rural health disparities. In summary, this LCMS system will rejuvenate research capabilities that previously flourished at ETSU, while simultaneously enriching the educational experience of our student population in the biomedical sciences.

NIH Research Projects · FY 2026 · 2026-03

PROJECT ABSTRACT Substance use disorder (SUD) and overdose deaths represent a national public health crisis. Recovery is the recommended outcome for individuals with SUD, however, access to treatment and recovery supports varies greatly across the U.S., and many who could benefit from them do not receive services in part due to stigma, difficulty navigating a complex service continuum, wait times, and costs. Peer support specialists (PSS) are individuals in recovery from a SUD who are trained, certified, and employed to assist and provide guidance to patients in various states of recovery. The prima facie practical value of peers in support roles is high, as they can engage individuals outside traditional boundaries and extend the reach of care beyond clinical settings. PSS lived experience may make them more relatable than other medical or social service staff. Moreover, peer support represents a mechanism that could advance long-term recovery at lower expense relative to services delivered by other licensed clinical personnel. Regrettably, the evidence for PSS services is relatively scant in part due to methods that are ill-fitted to measure the dynamic, behavioral, and interactive process of recovery across time and within a complex service continuum. Consistently, the existing literature points to the lack of a uniform taxonomy (job titles, service roles, job-related activities, work settings) as an impediment to advancing the research on PSS provider service. In response, the Developing an Index of Peer Support Specialist Work Settings and Activities study will identify the fundamental components of PSS service delivery providing the foundation for subsequent researchers to perform rigorous effectiveness research on the public health impact of PRSS service provision. The following Aims will be accomplished building upon two preliminary studies characterizing PSS work roles and activities: 1) conduct a psychometric review of Survey 2.0 and align survey constructs within a social ecological systems framework; 2) verify PSS work roles and activities within regional recovery networks; 3) authenticate the Peer Support Specialist (PSS) Work Settings and Activities Index. A dynamic team, jointly led by investigators and consultants within the Consortium on Addiction Recovery Science (CoARS) in collaboration with state and national PSS associations and credentialing bodies, offer assurance of the successful execution of these Aims. This proposed work is innovative as it seeks to shift the standard paradigm of research design and survey construction by engaging working PSS and researchers with lived experience of SUD in the co-creation of survey instruments grounded in social ecological system theory. The proposed work is significant as it addresses a key barrier to the accurate assessment of PSS service. Results will open the door for high-quality scientific exploration into the effectiveness of PSS service provision and inform the effective deployment of PSS interventions across regional service systems.

NIH Research Projects · FY 2026 · 2026-02

PROJECT SUMMARY/ABSTRACT The Central Appalachian region is a rural area that has some of the highest rates of opioid related mortality in the U.S. It is essential to fully understand factors contributing to these challenges and identify solutions as the region is heavily burdened with this major public health crisis. The East Tennessee State University (ETSU) Mentored Substance Use Research (EMSUR) program is a collaborative, transdisciplinary program that aims to mentor and train the next generation of researchers and practitioners focused on substance use in Central Appalachia. The EMSUR summer program will train 10 undergraduate students each year over five years through experiential faculty-mentored substance use research, engagement with the proven digital Substance Use Research Education and Training (SARET) curriculum that will include regionally-focused ETSU-developed modules, and participation in dynamic real-world presentations from guests speakers and experts in rural health, treatment and recovery research, and those with lived experience of substance use disorder (SUD). Every attempt will be made to recruit highly qualified students with an interest in living and working in the Central Appalachia region. The goal of the EMSUR program is to increase undergraduate student interest in SUD-related research and practice, thereby increasing the number of trained SUD researchers and practitioners living and working in the Central Appalachian region, that will ultimately be able to find solutions for these SUD related challenges. The EMSUR summer program will be accomplished through three specific aims: 1) Build a robust recruitment pipeline for undergraduate students through collaborations across ETSU and partnering institutions, leveraging faculty, staff, and direct student outreach to promote program awareness and engagement. 2) Train a cohort of 10 undergraduate students per year in a mentored, transdisciplinary summer research experience, fostering critical thinking skills, scientific rigor in addiction research, and practiced-based experiences. 3) Equip trainees for dissemination of their research through both conventional (e.g. poster, oral presentation) and innovative methods (e.g. infographics, data visualization). Program success will be evaluated across recruitment, program completion, and trainees continued engagement in SUD research in the short and long term.

NIH Research Projects · FY 2025 · 2025-09

Per- and polyfluoroalkyl substances (PFAS) are a large group of highly stable chemicals used in the production of water-, grease-, and stain-repellent properties. PFAS are recognized as persistent organic pollutants, detected in food and drinking sources, and can bioaccumulate in humans. Epidemiological data indicate that PFAS are associated with a variety of negative health effects including chronic kidney disease (CKD) and hypertension. Moreover, the deleterious effects of PFAS may be exacerbated in individuals already at risk for CKD and hypertension. Yet, the direct effects of PFAS on kidney function and disease remain poorly understood, especially in susceptible populations. The goals of this proposal are to investigate the direct effects of PFAS on renal hemodynamics and mechanisms of renal injury in rat strains (Sprague-Dawley (SD) and Dahl Salt-Sensitive (SS)) with large differences in susceptibility of developing CKD and hypertension. Our overarching hypothesis is that PFAS cause proximal tubule injury, renal vasoconstriction, hypertension, and ultimately ischemia-induced acute kidney injury (AKI), which is a major cause of CKD. As a corollary to our central hypothesis, we propose that the deleterious effects of PFAS will be exacerbated in Dahl SS rats, which are susceptible to developing CKD and hypertension. Aim 1 will test the hypothesis that PFAS cause renal vasoconstriction and AKI over time. The direct effects of PFAS on arterial blood pressure, renal blood flow, and glomerular filtration rate will be assessed in conscious, chronically instrumented rats. Serum and urine concentrations of PFAS will be measured by mass spectrometry to assess renal handling of PFAS and body burden levels. In addition to renal pathology, the off-target pathological effects of PFAS will be assessed in liver, heart, and spleen tissue. Aim 2 will test the hypothesis that pathogenic signaling and renal pathology will evolve over time during the transition from initial proximal tubule injury to the subsequent development of AKI. We will use an unbiased mass spectrometry/proteomics approach to identify PFAS-induced pathogenic signaling pathways associated with the initiation of proximal tubule injury, independent of changes in renal hemodynamics, and the subsequent development of ischemia-induced AKI. Renal hemodynamics, proximal tubule injury, renal pathology, and proteomics-based signaling pathways will be assessed at the same time points to assess relationships between pathophysiology, pathology, and molecular mechanisms. Preliminary data show that our experimental model is well-suited to address these questions and that the experiments are feasible. Our approach involves an investigative team with expertise in renal hemodynamics (Dr.'s Polichnowski and Griffin), AKI (Dr.'s Polichnowski and O'Connor), PFAS and proteomics (Ms. Grindstaff and Dr. Lau), and renal pathology (Dr. Youngberg), which will provide novel insights regarding the pathophysiologic and molecular mechanisms mediating PFAS-induced kidney disease and bioaccumulation.

NIH Research Projects · FY 2025 · 2025-09

Project Summary/Abstract Ethanol consumption has been on the rise throughout the COVID-19 pandemic and one of the major consequences has been a surge in alcohol-associated liver disease (ALD), especially severe alcoholic hepatitis (sAH). sAH is a life-threatening condition with 30-day patient mortality greater than 30%, limited treatment options, and often requires a liver transplant. There is an urgent need to identify novel mechanisms of ALD progression that are potential targets for treatment. The ethanol metabolite acetaldehyde (AcH) is known to form adducts on lysine residues within proteins, which affect protein function. Many proteins are known to bind to AcH, but no one has characterized the overall composition or downstream effects of these protein modifications on the pathogenesis of ALD. Albumin is one of the major proteins known to bind to AcH, and these AcH adducts mostly occur on the many exposed lysine residues throughout the albumin molecule. The fact that a single albumin can bind many AcH molecules combined with albumin being the most abundant protein in the liver and circulation means that albumin may act as a “sponge” for excess AcH. Therefore, any decrease in albumin levels may lead to increased modification of other proteins, having deleterious effects on ethanol-induced organ injury. My preliminary data shows that plasma albumin levels are decreased in heavy drinkers compared to control subjects in the absence of liver dysfunction. In albumin-deficient mice, ethanol feeding leads to decreased lymphocyte accumulation in the liver, but the mechanism is unknown. This project is designed to test the overarching hypothesis that albumin alters the distribution of AcH-protein adducts after ethanol consumption, modulating lymphocyte function in ALD. In Aim 1 (K99 phase) I will receive training from my mentor, Dr. Bin Gao, to determine how AcH and albumin regulate lymphocyte function. In Aim 2 (K99 phase), I will receive training in proteomic methods and analysis from my committee member Dr. Fritz to characterize the AcH-protein adductome in immune cells and albumin-deficient mice. In Aim 3 (R00 phase), I will use the training from the K99 phase to analyze public proteomic datasets and identify specific AcH-protein adducts that are present in patients with ALD. I will utilize cellular models to determine how these modifications impact immune cell and hepatocyte functions in the context of ALD. This project will provide a framework for how AcH-protein adducts modulate ALD using albumin as a model protein. The training provided by this grant will provide the PI with a strong foundation to achieve his long-term goal of identifying systemic mediators of ethanol-induced liver injury to develop therapeutics that protect against alcohol-induced injury in multiple ways. The NIAAA will provide an ideal environment for cross-disciplinary training and the necessary resources to transition to independence.

NIH Research Projects · FY 2025 · 2025-09

The ongoing opioid crisis, exacerbated by synthetic opioids like fentanyl, has been further complicated by rising co-use of stimulants such as methamphetamine. Despite the increasing prevalence of polysubstance use, current research largely focuses on single-drug models, limiting understanding and treatment options for polysubstance use disorders. Methamphetamine and fentanyl both induce neuroinflammation, but the combined effects of these co-used drugs on neuroinflammation and related behaviors remain unexplored. This project aims to address this gap by investigating the functional significance and mechanism of polysubstance-induced neuroinflammation utilizing a novel rodent model of polysubstance dependence and withdrawal using co- administered fentanyl and methamphetamine, with particular focus on the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) and the neuropeptide hypocretin/orexin (HCRT). HCRT influences a wide array of physiological functions, including those relevant to drug dependence and neuroinflammation, although the function of HCRT in drug-induced neuroinflammation is not yet well-understood. The overarching hypothesis is that during polysubstance withdrawal, HCRT functions as a pro-inflammatory agent in withdrawal-sensitive brain regions of the extended amygdala and the dorsal hypothalamus, contributing to neuroinflammation (including increases in TNFα) associated with polysubstance dependence. Aim 1 will determine TNFα’s role in motivated polysubstance use and stress-induced reinstatement of drug-seeking using neuropharmacological techniques with a small-molecule TNFα-inhibitor. Aim 2 will define the role of HCRT signaling on polysubstance-induced neuroinflammation by measuring cytokine response to HCRT manipulations in polysubstance dependent rats. Findings from this research could significantly advance our understanding of the neuroimmune mechanisms driving polysubstance dependence and lead to the development of more effective treatments for addiction. Students will play an integral role in conducting hands-on, rigorous research as aligned with current NIH priorities and under the direct supervision of the PI and a team of collaborators.

NSF Awards · FY 2025 · 2025-09

This award supports experimental research focused on high-precision measurements of neutron properties. These measurements will advance our understanding of the weak nuclear force, which is one of the four fundamental forces of nature. When not bound into an atomic nucleus, the neutron will soon decay into a proton, an electron, and an anti-neutrino. The rate at which the neutron decays, along with the direction of the emitted electrons, offers critical insights into the formation of elements in the early universe, the processes driving fusion in stars, and the interactions among fundamental particles. In addition, searches for the neutron’s permanent electric dipole moment, a tiny separation of internal charges within a neutral particle, may shed light on why the universe is dominated by matter rather than antimatter. The PI and a team of undergraduate researchers will contribute to three major experiments at the Los Alamos Ultracold Neutron Source that explore these questions. Their efforts will focus on improving the sensitivity of electron and neutron detection systems used across all three experiments. This research will also provide students with hands-on training in both hardware and software, preparing them for future roles in the national STEM workforce. The primary goal of this work is to deepen our understanding of the Standard Model of particle physics and to search for phenomena that lie beyond it. Three ongoing experiments at the Los Alamos Ultracold Neutron Source aim to enhance the sensitivity of key measurements related to the neutron lifetime, beta asymmetry, and electric dipole moment. Both UCNA+ and UCNtau+ build upon the success of their predecessor experiments, targeting improvements in the elements that previously limited precision. UCNA+ will focus on measuring the beta-asymmetry parameter in neutron beta decay with a target sensitivity better than 0.2%. Achieving this level of precision requires minimizing the trigger threshold of the electron detectors; to that end, the PI will develop custom FPGA firmware and amplifier hardware. Preliminary studies suggest that lowering the trigger threshold will reduce the uncertainty due to electron backscattering by a factor of two. In parallel, a new room-temperature search for the neutron EDM is under development, with a goal sensitivity below 10⁻²⁷ e·cm. The PI and their research team will contribute by designing and implementing a robust data acquisition and mirroring system to support collaboration-wide access and redundancy. They will also continue developing particle transport simulations to evaluate systematic effects and contribute to data analysis across all three experiments. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2026 · 2025-08

PROJECT SUMMARY/ABSTRACT Stimulant and opioid dependence are major public health problems and the number of people illicitly using drugs of abuse in combination is rising. In fact, opioid and opioid co-involved overdose related fatalities regrettably reached a devastating new high of more than 107,000 deaths in 2021. Co-use of stimulant and opioids, or polysubstance use, is increasingly reported in clinical survey data, highlighting the need to study the pharmacodynamics of polysubstance use. Uncovering the mechanisms and neurocircuitry underlying polysubstance use is essential to understanding and treating co-existing stimulant and opioid use disorders. Yet, current research primarily targets stimulant or opioid dependence in isolation, thereby limiting discovery of interventions for polysubstance use disorders. The hypocretin/orexin neuropeptide system has been widely implicated in mechanisms of drug addiction, across all classes of drugs and alcohol. Given the universal effect of hypocretin-receptor blockade on mitigating highly motivated drug-taking in animal models, the hypocretin system is a logical target for treatment of polysubstance use disorders in humans. Nonetheless, the role of hypocretin signaling in combined methamphetamine and fentanyl use remains to be examined. The overarching hypothesis of this proposal is that additive opioids, such as fentanyl, enhance methamphetamine self- administration, and that hypocretin signaling within stress-sensitive brain regions of the extended amygdala and the lateral habenula is a key modulator of the enhanced motivation associated with polysubstance use and dependence. Thus, this proposal aims to elucidate the pharmacodynamic nature of polysubstance use across the development and trajectory of dependence utilizing a rat model of concomitant methamphetamine and fentanyl intravenous self-administration (Aim 1) and to systematically examine the hypocretin-related neurobiological mechanisms and neurocircuitry underlying polysubstance dependence using neuropharmacological and chemogenetic DREADDs techniques (Aims 2 & 3). The data obtained from these studies will broaden our understanding of the underlying mechanisms of drug dependence and identify novel therapeutic interventions for treatment of polysubstance use disorders.

NIH Research Projects · FY 2025 · 2025-08

Project Summary/Abstract This proposal aims to characterize the potential beneficial effect of focal chemogenetic stimulation of sympathetic preganglionic neurons in the recovery of sympathetic regulation of blood pressure after left surgical hemisection in the rat. Spinal cord injury (SCI) can cause serious cardiovascular dysfunction. One of the most common dysfunctions is orthostatic hypotension, which is the inability to achieve an upright posture without fainting. Sympathetic regulation of blood pressure is driven by preganglionic neurons located in the lateral horn of the thoracic and upper lumbar spinal cord. Orthostatic hypotension after SCI is caused by a loss of supraspinal regulation of spinal sympathetic preganglionic neurons. Considering that the most significant rate of improvement in motor function occurs within the first six months after the SCI, after which progress plateaus, early and effective physical therapy is imperative. As such, orthostatic intolerance is a significant barrier for those with SCI to participate fully in rehabilitation, thereby impeding recovery. We have previously mapped the spinal pathways in the rat responsible for baroreflex-mediated activation of sympathetic preganglionic neurons to regulate blood pressure. We observed that over eight weeks, rats recover some degree of baroreflex function after severe SCI as long as some baroreflex-related spinal pathway is spared. This recovery is likely due to an increased efficacy of the remaining spinal pathways after the injury. Considering that approaches for the repair and/or regeneration after SCI have been elusive, this discovery provides an opportunity to determine the mechanistic action in the neuroplasticity after SCI for improving sympathetic regulation of blood pressure. Advances in rehabilitation, particularly spinal cord stimulation, have improved sympathetic regulation of blood pressure. While the mechanism of action is not fully known, animal models suggest that plasticity is due to engaging previously dormant neural tissue. However, it is unknown if it activates sympathetic preganglionic neurons directly or indirectly via stimulation of spinal networks that synapse on sympathetic preganglionic neurons. This is important because a better understanding of the neural mechanisms will provide better- targeted therapeutics. We propose using a novel targeted chemogenetic approach in transgenic rats to exclusively stimulate only sympathetic postganglionic neurons independent of the surrounding spinal network. In doing so, we will determine if chronic focal stimulation of sympathetic preganglionic neurons after SCI improves the recovery of sympathetic baroreflex function. We will also determine if this stimulation improves the reorganization of spinal networks involved in the sympathetic regulation of blood pressure. Thus, the goal of this small research project is to provide proof of concept for a novel approach to stimulate sympathetic neurons after SCI. This project will provide a data-derived path for more extensive studies to better understand the regulation of sympathetic activity and neuroplasticity, improve blood pressure regulation, and ultimately improve the ability to participate in rehabilitation after SCI.

NIH Research Projects · FY 2025 · 2025-05

Project Summary/Abstract We are applying for funds to purchase the Vevo F2 imaging system from VisualSonics (Fujifilm) to be used by the investigators at Quillen College of Medicine (QCOM), College of Public Health and College of Arts and Sciences at East Tennessee State University (ETSU). This imaging system will replace our current ultrasound system, the Vevo 1100 from VisualSonics (Fujifilm), which is nearly ten years old and is no longer supported by the manufacturer. The failure of the current system will leave our institution without an ultrasound imaging system. We currently have many cardiovascular and cancer researchers who use the Vevo 1100 and have published images acquired on this machine for nearly a decade. The Vevo F2 imaging system boasts new and updated features compared to the Vevo 1100, which will make the system useful for our eight major and four minor users across multiple disciplines of heart, vasculature, and oncology research. Features specific to the Vevo F2 that will be particularly useful to our investigators include HD (high definition) image processing to generate clearer images, advanced software that includes artificial intelligence algorithms for image analysis, 4D imaging to enable dynamic changes over time, and cardiac strain analysis. The Vevo F2 imaging system will primarily be used by NIH-funded investigators, but it will also provide ultrasound imaging access to junior faculty who are currently seeking NIH funding. The Vevo F2 imaging system will enhance our imaging quality and speed to make us more competitive in our research efforts. The Vevo F2 imaging system will be housed in a central location to ensure access to all users. An abundance of institutional support will ensure the successful use of this imaging system at ETSU. Provisions have been made with the Dean of QCOM, the Associate Dean of Research and Graduate Education (QCOM), and the Department of Biomedical Sciences (QCOM) to fund two computers to be dedicated to the Vevo F2 imaging system, a supplemental Vevo analysis software package, a stipend for our dedicated trainer, salaries of the Principal Investigators and Manager, an anesthesia system to accompany the Vevo F2, and four years of a service contract with the VisualSonics, Fujifilm company after the included one-year service contract expires. The Vice Provost for Research and Chief Research Officer (ETSU) has endorsed the acquisition of the Vevo F2 imaging system. His office will provide necessary administrative and logistical support for the set-up and maintenance of the new system. An advisory committee has been established to oversee training, calendar appointments, animal care, and overall usage of the instrument. Our users would like to stress that no other options for ultrasound imaging capabilities are available to us at ETSU if the retired Vevo 1100 imaging system goes down. Thus, the Vevo F2 imaging system would satisfy a significant research need at our institution.

NIH Research Projects · FY 2026 · 2025-01

Project Summary (30 lines): Aging is a complex, multifactorial process wherein transcriptional reprogramming is accompanied by perturbations in the epigenome. We have performed experiments to determine the histone expression profile in the aging RPE and discovered an age-dependent loss of histone expression that was specific to the RPE. It is not known whether loss of histone expression during RPE aging alters gene expression, induces cell death, or leads to the progression of age-related macular degeneration (AMD). AMD is a blinding disease that is hallmarked by the death of retinal pigment epithelium (RPE), choroidal neovascularization, and loss of the overlying photoreceptors in the central retina. Several recent scientific advances have revealed an array of molecular mechanisms that result in RPE atrophy including disinhibition of the alternate complement pathway, inflammasome activation, and the expression of pro-apoptotic signaling components Tight histone which serve expression to histone be regulatory regulation of expression is essential to maintain chromatin structure and appropriate gene expression profiles are critical to cell viability and function. Moreover, post-translational modifications (PTM) of histones as critical mechanisms for epigenetic regulation of gene expression. Decreased histone gene and altered histone PTMs have been identified as a molecular hallmark of aging. The Objective is define signatures of histone expression in the aging RPE and determine the cellular effects of loss of expression in RPE homeostasis and AMD progression. The Hypothesis is that histone depletion may a key factor contributing to RPE aging and senescence and that therapeutic targeting of key histone mechanisms ma serve as a valuable approach to reverse aging and senescence in the RPE. Using RNA-seq, ChIP-seq, high-throughput screening of histone PTMs, Western blotting, and immunofluorescence SA1 homeostasis blotting, RPE histone whether and further in and evaluates histone expression profiles and marks to identify critical regulatory factors o histone in the aging mouse RPE. Using RNA-seq, high-throughput screening of histone PTMs, Western and immunofluorescence, and immunohistochemistry, SA2 i nterrogates in vitro models of human aging, aged normal human eyes and eyes with advanced dry AMD for loss of histone expression, modifications, and molecular mechanisms of histone related cell death. Further work will demonstrate restoration of specific histone isoforms or histone regulatory factors will reverse aging, senescence restore RPE cell viability in mouse and cell culture models. These data will be critical to developing scientific insight into the critical role of tightly regulated histone gene expression in the aging RPE and AMD progression while offering a novel therapeutic approach to maintaining RPE health, genomic fidelity, cellular integrity. f

NIH Research Projects · FY 2025 · 2024-06

Abstract For many years it was thought that “immunologic memory” cannot be induced within the innate immune system. However, recent research challenges this long held dogma. There is now compelling evidence that the innate immune system can be “trained” to respond more rapidly and effectively to pathogens. These findings challenge the existing paradigm that innate immunity cannot develop “memory” or be “trained” to respond more effectively to infection and indicate that “trained immunity” can be harnessed to increase resistance to infection. Trained immunity elicits broad resistance to infection that persists for weeks to months and is not specific with respect to the causative agent. The development of immune training drugs would make it possible to harness the potential of trained immunity for the treatment of disease. β-Glucan, a fungal cell wall constituent, confers resistance to infection with Gram-negative and Gram-positive bacteria as well as viral and fungal pathogens. Numerous studies have shown that β-glucans will induce innate immune training. In fact, β-glucans are now recognized as the “gold standard” for induction of the immune trained phenotype. However, β-glucan is a natural product that is isolated from fungal sources. Large scale isolation of natural product β-glucans is plagued by QA/QC problems and reproducibility issues. This is due in large part to the fact that all natural product β-glucans are a distribution of glucan polymers with varying polymer lengths and, in some cases, differences in branching frequency, length of side chain branches and solution conformation. What was needed were methods for the complete chemical synthesis of (1→3,1→6)-β-glucans with specific structural characteristics including polymer size, side chain branching frequency, side chain branch length, etc. We have successfully developed convergent synthetic approaches for the de novo synthesis of (1→3,1→6)-β-glucans with specific structural characteristics. The goals of this research are to: i) evaluate the ability of synthetic glucan glycomimetics to induce the immune trained phenotype and ii) to compare and contrast the bioactivity of different glucan glycomimetic formulations to natural product glucans. We hypothesize that synthetic glucan glycomimetics will induce the trained immune phenotype similar to natural product glucans. To critically evaluate this hypothesis we propose the following specific aims. Aim 1. Assess the ability of glucan glycomimetics to induce the immune trained phenotype. Aim 2. Examine the immune training ability of glucan glycomimetics conjugated to silica nanoparticles. A successful conclusion to this research will result in the identification of synthetic (1→3,1→6)-β-glucan glycomimetics that will induce the immune trained phenotype in human immunocytes. This will lead to the development of glycomimetic drug candidates that can prophylactically and/or therapeutically induce immune training and alter the course of disease. In addition, a successful conclusion to this research will result in new and novel data on the structure activity relationships of glucans in trained immunity.

NIH Research Projects · FY 2025 · 2024-05

Project Summary New targets to combat Pseudomonas aeruginosa is paramount given the difficulty of treating Pseudomonas infections and increasing drug resistance of this pathogen. A major goal has been to target the type III secretion system that directly delivers toxins into host cells. However, this has yet to result in a clinical therapeutic. We recently discovered an unannotated regulator, PA1627, that controls expression of the type III secretion system. In this proposal, we will determine the regulation of PA1627 and determine how PA1627 controls the type III secretion system. Understanding this new level of control of the type III secretion can lead to new possibilities for combatting P. aeruginosa.

NIH Research Projects · FY 2026 · 2024-04

SUMMARY - Engineered exosomes carrying synthetic gRNA/Cas9 targeting HBV-infected cells Despite universal vaccinations against hepatitis B virus (HBV), chronic HBV infection remains a public health threat in the United States and worldwide. The current antiviral treatments, using various nucleos(t)ide analogs (NAs), can block the HBV life cycle but cannot eliminate integrated HBV DNA and have little effect on HBV covalently closed circular DNA (cccDNA), which sustains viral replication. Thus, novel curative strategies are urgently needed to eliminate HBV cccDNA from infected hepatocytes. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9)-mediated gene-editing is an appealing approach to tackle this problem. However, the major hurdle in the application of this technology is how to deliver gene-editing drugs to target cells and elicit specific antiviral activities without causing off-target effects. Notably, the current CRISPR/Cas9 delivery technologies often require viral vectors, which pose safety concerns for therapeutic application in humans. Synthetic guide RNA (gRNA)/Cas9 ribonucleoproteins (RNPs) represent a novel non-viral formula with excellent features, including rapid DNA cleavage activity, low off-target effects, no risk of insertional mutagenesis, easy production, and readiness for clinical use. We have designed and tested a series of gRNA/Cas9 gene-editing RNPs targeting HBV and selected the most specific and potent gRNA/Cas9 RNP candidates to abolish HBV replication in HBV cellular models. However, the existing viral and non-viral delivery systems for gRNA/Cas9 RNPs in vivo use face several challenges, such as non-specific (off-site) delivery, limited drug-loading capacity, low biocompatibility, poor stability, cytotoxicity, and potential of immunogenicity. These challenges are major bottlenecks, limiting the use of synthetic gRNA/Cas9 RNPs for in vivo applications. To address these limitations, we developed a novel exosome- based delivery platform engineered to deliver the HBV gene-editing RNPs specifically to human hepatocytes. These engineered exosomes are designed in such a way that they carry an HBV pre-S1-derived peptide (binding to HBV receptor) on the surface of exosomes so that they can more specifically deliver and intracellularly release our synthetic gRNA/Cas9 RNPs to HBV target cells. In this proposal, we aim to evaluate the capability of these exosome- based HBV gene-editing gRNA/Cas9 RNPs (herein called Exo-HBV-Eliminator) in targeting HBV-infected hepatocytes and investigate their antiviral efficacy and potential cytotoxicity using both cellular and animal models. We hypothesize that our Exo-HBV-Eliminator will specifically target HBV DNA and thus efficiently abolish HBV replication and elicit minimal cytotoxic effects both in vitro and in vivo. We propose two specific aims to test our hypothesis. Aim 1 will determine the biophysical and biological properties of our Exo-HBV-Eliminator in vitro. Aim 2 will evaluate the antiviral and off-target effects of our Exo-HBV-Eliminator in HBV-infected, liver-humanized mice in vivo. The objective of this study is to develop and test a novel gene therapy (engineered exosomes carrying synthetic gRNA/Cas9 RNPs) targeting HBV cccDNA in HBV-infected hepatocytes that are incurable by the current antiviral regimens, to lay the foundation for achieving our long-term goal of curing chronic HBV infection.

NIH Research Projects · FY 2026 · 2024-04

Abstract: Sepsis is a life-threatening condition caused by an uncontrolled host response to infection and is a leading cause of death in intensive care units. Liver dysfunction contributes significantly to the morbidity and mortality associated with sepsis. Liver sinusoidal endothelial cells (LSECs) play a critical role in hepatic immune and metabolic functions, but their specific role in bacterial infection and sepsis remains poorly understood. Our study focused on HSPA12B, a member of the HSP70 family, shows distinct expression in liver sinusoidal endothelial cells (LSECs), being prominent in periportal LSECs and reduced in midzonal LSECs. Interestingly, mice lacking endothelial cell HSPA12B (eHSPA12B-/-) exhibit disrupted hepatic Kupffer zonation and impaired hepatic gluconeogenesis even under normal conditions. Furthermore, we induced sepsis in these mice and observed severe LSEC capillarization, along with increased bacterial load and lactate accumulation compared to WT septic mice. These findings highlight the importance of eHSPA12B in maintaining LSEC phenotype and as well as hepatic immune and metabolic function during sepsis. Further investigations revealed that eHSPA12B is necessary for GATA4 nuclear translocation in LSECs. Endothelial cell-specific GATA4 deficiency resulted in an abnormal LSEC phenotype, impaired Kupffer cell zonation, and dysfunction of hepatic gluconeogenesis. These data suggest a cooperative role of eGATA4 and HSPA12B in maintaining the specialized phenotype and function of LSECs. Patients with NASH/cirrhosis are more susceptible to bacterial infections and sepsis. Our study found decreased HSPA12B expression and impaired GATA4 transcriptional activity in LSECs of NASH/cirrhosis patients, suggesting their contribution to NASH/cirrhosis associated hepatic immune and metabolic dysfunction and increased susceptibility to bacterial infections. Based on our novel findings, we hypothesize that eHSPA12B serves as a novel co-transcriptional factor of GATA4 to maintain the unique phenotype and function of LSECs, and that functional LSECs play a crucial role in maintaining Kupffer cell zonation, enhancing bacterial clearance, and regulating hepatic gluconeogenesis during bacterial infections and sepsis. To critically evaluate this hypothesis, we propose the following aims. Aim 1. Investigate how HSPA12B and GATA4 cooperatively regulate LSEC phenotype and function during sepsis. Aim 2. Define the mechanisms by which LSECs regulate hepatic Kupffer cell zonation and bacterial clearance during sepsis. Aim3. Investigate the mechanisms by which LSECs regulate hepatic gluconeogenesis. Successful completion of these studies will provide novel insights into the novel role of LSEC in regulating hepatic immune and metabolic function. These findings could be an important foundation for the development of innovative therapeutic approaches to enhance hepatic hepatic immune and metabolic function, leading to improved outcomes in patients with bacterial infections and sepsis.

NIH Research Projects · FY 2025 · 2024-01

SUMMARY – Engineering exosomes for new gRNA/Cas therapeutics to eliminate HBV infection Chronic hepatitis B virus (HBV) infection is a common public health problem in the United States and worldwide. The current antiviral treatments, using various nucleos(t)ide analogs (NAs), can block the HBV life cycle but cannot eliminate integrated HBV DNA and have little effect on HBV covalently closed circular DNA (cccDNA), which sustains viral replication. Thus, novel curative strategies are urgently needed to eliminate HBV cccDNA from reservoir cells. CRISPR/Cas-mediated gene-editing is an appealing approach to tackle this problem. However, major hurdles in the application of this technology lie in selecting the most potent guide RNAs (gRNAs) to form a specific therapeutic regimen and delivering CRISPR/Cas therapeutics to the target cell, to elicit on-target gene-editing without causing off-target effects. Compared to Cas9 which often requires 2 or 3 gRNAs to avoid viral escape/resistance, Cas12 is a programmable and more potent DNA endonuclease and only requires a single gRNA for targeted gene-editing. Unlike Cas9 and Cas12 which primarily edit DNA, Cas13 edits RNA and can be used together with Cas9 and Cas12, for both DNA and RNA targeting. Additionally, the current CRISPR/Cas expression and delivery systems often require viral vectors, which pose safety concerns for therapeutic applications in humans. Synthetic ribonucleoproteins (RNPs) are a novel non-viral formula with excellent features, including rapid DNA cleavage activity, low off-target effects, low risk of insertional mutagenesis, easy production, and readiness for clinical use. We have designed and tested a series of gRNA/Cas9 gene-editing drugs targeting HBV cccDNA and selected the most specific and potent gRNA/Cas9 candidates to abolish HBV replication in HBV cellular models. We have also developed a novel exosome-based delivery platform engineered to specifically deliver these HBV gene-editing drugs to human hepatocytes. These engineered exosomes are designed to carry an HBsAg pre-S1-derived peptide (binds to HBV receptor) on the surface of exosomes so that they can specifically deliver our gRNA/Cas therapeutics to HBV reservoir cells. In this proposal, we will compare the capacity and specificity of gRNA/Cas12 and gRNA/Cas13 (with gRNA/Cas9 as a positive control) to eliminate HBV infection in HBV cellular models to select the most potent gRNA/Cas regimen in vitro (R21 phase). Then, we will evaluate the capability of our engineered exosomes to deliver our HBV gene-editing gRNA/Cas therapeutic (herein called Exo-HBV-Eliminator) for eliminating HBV infection using an HBV-infected, liver-humanized animal model (R33 phase). We hypothesize that our Exo-HBV-gRNA/Cas therapeutics will specifically and efficiently eliminate HBV infection and elicit minimal cytotoxic effects both in vitro and in vivo. We propose two specific aims to test our hypothesis. Aim 1 will select the most specific and potent gRNA/Cas9/12/13 candidates and test a combination regimen in vitro. Aim 2 will evaluate the antiviral efficacy and off-target effects of the Exo-HBV-Eliminator in HBV-infected, liver-humanized mice in vivo. The objective of this study is to develop a novel gene therapy capable of targeting HBV cccDNA, which is incurable by the current NA treatment, and thus this research will lay the foundation for achieving our long-term goal of curing chronic HBV infection.

NIH Research Projects · FY 2026 · 2023-12

Sepsis represents a life-threatening disorder caused by a dysregulated host response. Sepsis survivors frequently have long-term immune dysfunction that contributes to high mortality from opportunistic infections. Clinical data shows that lactate levels strongly and positively correlate with severity, morbidity and mortality in sepsis5-8. It is unclear whether lactate contributes to sepsis impaired immune response and susceptibility to subsequent infection. To address this important question, we performed preliminary studies and observed that enhanced lactate levels markedly increased mortality. We also observed that lactate suppresses macrophage phagocytic function during sepsis. Our findings suggest that lactate exerts a previously unknown biological function contributing to the mortality of sepsis and re-infection of sepsis survivors. Cellular senescence is a fundamental mechanism of age-related organ dysfunction. Our RNA-seq and other preliminary data shows that lactate markedly induces macrophage senescence during sepsis. our preliminary data also shows that splenic macrophage senescence is significantly greater in macrophage specific YAP/TAZ deficient (mYAP/TAZ-/-) sham and septic mice than in WT controls. Our finding suggests that YAP is required for the protection against cellular senescence during sepsis. Interestingly, we found that lactate induces lactylation of YAP. We reported that lactate induces HMGB1 lactylation in macrophages25, suggesting that lactate could induce lactylation of non-histone proteins. Indeed, we made a novel observation in our preliminary studies that lactate induces Keap1 lactylation and increases Nrf2 activation. Keap1 is an important suppressor for activation of Nrf2 to upregulate the expression of genes related to suppressive Immune responses. Our findings indicate that lactate alters macrophage immune response during sepsis that may be mediated by promoting macrophage senescence and inducing lactylation of important transcription factors and co-effectors. Based on our novel findings, we hypothesize that lactate is a novel endogenous mediator which contributes to impaired macrophage immune function during sepsis via lactylation of transcription factors and effectors and promotion of cellular senescence. To critically evaluate this hypothesis, we propose three specific aims. Specific aim 1. Investigate the role of lactylation of YAP/TAZ in macrophage immune dysfunction during sepsis. Specific aim 2. Define the role of Keap1 lactylation and Nrf2 activation in macrophage immune dysfunction during sepsis. Specific aim 3. Investigate whether lactate promoted senescence of macrophages will contribute to impaired macrophage immune function during sepsis. Successful completion of the proposed studies will result in a wealth of new and novel data showing that lactate plays an important role in the regulation of immune responses during sepsis.

NIH Research Projects · FY 2024 · 2023-09

Candida auris is a fungal pathogen that has been identified as an emerging infectious disease and a public health threat. C. auris is notable for its resistance to antifungal therapy, its transmissibility, its high mortality rate as well as its ability to colonize patients, healthcare personnel and healthcare environments. C. auris strains are typically resistant to fluconazole and approximately half of C. auris strains are resistant to two or more anti-fungal drugs. C. auris causes nosocomial outbreaks of invasive candidiasis with mortality rates of ~60%. There are five distinct clades of C. auris, all of which appear to have evolved since 1996. In addition to its drug resistance, C. auris can colonize skin, spread from person-to-person and survive in healthcare environments for long periods of time. These features are unique to C. auris. C. auris is resistant to many standard decontamination reagents and protocols, which has resulted in the rapid spread of this pathogen throughout the world. This makes it a particularly dangerous emerging fungal pathogen. Thus, it is not surprising that C. auris is the first fungal pathogen that has been identified as a public health threat. What is needed is a C. auris specific vaccine that can prevent and/or ameliorate the morbidity, mortality and dissemination of this emerging fungal pathogen. The research outlined in this proposal directly addresses this pressing need. We have discovered that the mannan which coats the outermost surface of C. auris clinical strains is both structurally and biologically unique, i.e. it contains two unique acid labile Mα1-PO4 side chains that are not found in other fungal mannans or other fungal pathogens. Thus, C. auris mannan distinguishes it from virtually all other fungal pathogens. This suggests that C. auris mannan represents a target of opportunity for the development of a C. auris vaccine. We hypothesize that the dual Mα1-PO4 mannan structure can be used to develop a C. auris vaccine that will be effective against multiple C. auris strains. The goals of this R21 proposal are to: i) evaluate the efficacy of the vaccine in a murine model of C. auris infection and ii) evaluate the anti-fungal immune response to the vaccine. To address these objectives we propose two specific aims. Aim 1. Evaluate the efficacy of a C. auris mannan based vaccine formulation against systemic C. auris infection. Aim 2. Assess innate and adaptive immune responses elicited by the C. auris mannan vaccine.

NIH Research Projects · FY 2024 · 2023-09

Abstract Age is a major risk factor for cardiovascular associated diseases which are leading causes of death globally. Accordingly, 80% of all cardiovascular deaths occurred in patients aged 65 and over. Endothelial cell senescence, characterized by cell cycle arrest and a senescence-associated secretory phenotype (SASP), is a major contributor to age related cardiovascular dysfunction. However, the mechanism by which endothelial cells are subjected to senescence is still unclear. We have recently made novel findings that endothelial cell HSPA12B deficient (eHSPA12B-/-) mice exhibit cardiac endothelial cell senescence accompanied by severe cardiac hypertrophy, fibrosis, and persistent inflammation when compared with age- and gender-matched WT controls. Our findings suggest that senescent endothelial cells play an important role in the regulation of cardiac hypertrophy and fibroblast activation and that endothelial cell HSPA12B limits senescence of endothelial cells. Therefore, understanding the mechanisms by which HSPA12B regulates endothelial cell senescence would be important for seeking an approach to prevent or reverse senescent endothelial cells, thus reducing age-related cardiovascular disease. To elucidate how HSPA12B limits senescence of endothelial cells, we examined the effect of HSPA12B on ATF6 (Activating transcription factor 6) transcriptional activity and its target gene MANF (Mesencephalic Astrocyte Derived Neurotrophic Factor) expression. ATF6 is an important transcriptional factor that regulates the expression of genes involved in proper protein folding and cellular senescence. MANF is an evolutionarily conserved protective modulator for the maintenance of tissue immune and metabolic homeostasis. Interestingly, we observed that HSPA12B is critical for sustaining ATF6 transcriptional activity and MANF expression. The data suggest that ATF6 and MANF may be involved in the HSPA12B mediated protective effect on endothelial cell senescence. To investigate how endothelial cell senescence contributes to cardiac hypertrophy, we examined cardiac mitochondrial glucose oxidation (MGO) which is one of the major contributors to myocardial energy production. Compromised mitochondrial glucose oxidation leads to the development of cardiac hypertrophy and eventually heart failure. We observed that eHSPA12B-/- resulted in decreased Acetyl-CoA and increased lactate accumulation. The data indicate that eHSPA12B-/- impairs cardiac mitochondrial glucose oxidation. Our observation also suggests that senescent endothelial cells induce cardiac hypertrophy via impairment of cardiac myocyte MGO (mitochondrial glucose oxidation). To address how endothelial cell senescence impairs cardiac MGO, we induced endothelial cell senescence by ETO (etoposide), collected the medium as the senescent conditioned medium (SCM), and treated adult cardiac myocytes with the SCM. We observed that SCM promotes metabolic reprogramming from glucose oxidation to glycolysis in adult cardiac myocytes by increasing PDK4 (Pyruvate Dehydrogenase Kinase 4) and decreasing TFAM (Mitochondrial transcription factor A). PDK4 is a key enzyme in the regulation of glucose oxidation by inhibiting the conversion of pyruvate into Acetyl-CoA via promoting PDH phosphorylation and inactivation. TFAM is a core mitochondrial transcription factor for mitochondrial biogenesis by regulating mtDNA replication and transcription. This application is to decipher the role of HSPA12B in age-related endothelial cell senescence and how endothelial cell senescence results in cardiac hypertrophy and dysfunction. Based on our novel findings, we hypothesize that: i) HSPA12B limits endothelial cell senescence is mediated by activation of ATF6/MANF signaling and that; ii) endothelial cell senescence contributes to cardiac hypertrophy and dysfunction via impaired mitochondrial glucose oxidation in cardiomyocytes. To critically evaluate this hypothesis, we propose the following specific aims. Aim 1. Define the mechanisms by which HSPA12B regulates ATF6 transcriptional activity and limits endothelial cell senescence. Aim 2. Investigate the role of endothelial cell senescence in age-related cardiac hypertrophy and its underlying mechanisms. Successful completion of the proposed studies will result in a wealth of novel data showing the novel role of HSPA12B mediated ATF6 in the regulation of endothelial cell senescence. The senescent endothelial cells contribute to age-related cardiac metabolic disorders and hypertrophy. These new findings will be the basis for the development of innovative therapies for age-related cardiomyopathy.

NIH Research Projects · FY 2024 · 2023-08

Project Summary Invasive fungal infections are a serious public health threat and are associated with high mortality rates, demonstrating that current antifungal therapies are inadequate. While innate immunity is known to be critical for host defense against fungi, these complex host-pathogen interactions remain poorly elucidated. Recently, a novel behavior of neutrophils, a key innate immune cell for antifungal defense, has been characterized, that of neutrophil swarming. Swarming is thought to play a role in the containment of pathogenic microbes, but its role in antifungal defense is poorly characterized, representing a significant gap in knowledge in neutrophil function. The hypothesis driving this research application is that the early events in swarming are critical determinants of if the pathogen will be successfully contained and that characterization of these pathways will highlight novel therapeutic options for optimizing neutrophil function during infection and inflammation. Unfortunately, detailed study of these host-pathogen swarming interactions has been hindered by the shortcomings of current experimental assays. To address this, we have developed and optimized a novel microscale device to allow us to characterize human neutrophil swarming to live fungal pathogens. This microspotting assay allows us to pattern live microorganisms in large arrays, with direct access both visually and to supernatants for molecular analysis. The objective of this application is therefore to leverage novel microscale tools to allow rigorous investigation of the dynamic interactions between host immunity and live fungi during swarming to expand our understanding of swarming biology. We will do this by via the pursuit of two specific aims (1) Identify the molecular mechanisms by which neutrophils decide to initiate swarming behavior and (2) Elucidate the molecular pathways that enhance swarming mediated fungal killing. In the short term, this research is expected to generate critical knowledge on the role of neutrophil swarming in antifungal defense. Leveraging these tools and the knowledge they generate, the long term goal is the development of improved and novel therapeutic options for patients with invasive fungal infections. The work will also provide a strong foundation which the candidate can use to achieve his immediate career goals of attaining an independent, tenure-track faculty position and to progress towards his long term career goals of a tenured research faculty position with a unique academic research program studying innate host- pathogen interactions during fungal infection. In order to attain these career goals, the candidate will also assemble an effective mentoring and consulting team to promote the successful completion of research and the continuing improvement of grantsmanship and lab management skills. These career goals and the proposed research are therefore fulfil NIAID’s mission to pursue and identify novel therapeutic strategies to combat infection and to support the transition of junior scientists into independent faculty positions via the K22.

NIH Research Projects · FY 2025 · 2023-07

The primary goal of the present proposal is to explore the role of pathway-specific RE neurons in the synchronized activity of the prefrontal-hippocampal circuit (mPFC-HC) and memory-related rhythms in wake (episodic-like sequence memory) and in sleep states (REM/NREM). To accomplish this, Aim 1 focused on determining if and how excitation of RE neurons could drive memory-related mPFC-HC coherent states. The K99 phase experiments tested a range of relevant frequencies in RE pathway-specific neurons to identify stimulation capable of driving mPFC-HC coherent modes in freely behaving rats. It was found that activating RE neurons elicited a beta-driven coherence between the prefrontal cortex and hippocampus, similar to that of memory-driven brain states, and this effect was independent of frequency or optogenetic stimulation type (pulse or sinewave) delivered in RE. As I transition to my independent career (R00), Aim 2 will explore the role of pathway-specific RE neurons in driving theta- and delta-related sleep oscillations (REM/NREM) using a closed-loop setup. During the K99 training phase, I learned to run a sophisticated rodent sequence memory task capable of testing multiple RE-dependent memory dimensions and developed a cutting-edge closed-loop optogenetic control system capable of detecting neurophysiological modes (NREM/REM) between the hippocampus and medial prefrontal cortex in real time to trigger RE optogenetic stimulations at relevant behavioral states (wake/sleep). My scientific training in anatomy, optogenetics, multisite electrophysiological recordings, closed-loop approaches, and my theoretical background on RE-related mnemonic processes and arousal provide me with the foundation and technical skills necessary to pursue the goals of the R00 phase of this award. The training received during the K99 phase has prepared me to enter the independent stage of my career with the theoretical, technical, methodological, networking, and laboratory management skills necessary to answer scientific questions at the circuit and network level, establish my own laboratory, and independently pursue future scientific directions. Overall, this research will shed new light on the mechanisms of mPFC-HC circuit interactions that support memory and are modulated by the midline thalamus, specifically RE. This fundamental understanding will advance knowledge of RE function in relation to pathophysiological issues such as memory and sleep symptoms observed in many psychiatric and neurological disorders.

NIH Research Projects · FY 2026 · 2022-07

Project Summary Olfactory sensory neurons (OSNs) in the olfactory epithelium (OE) are continuously replaced from basal stem cells and grow their axons to the olfactory bulb to maintain the sense of smell. Failure to reconstitute the OE after injury, infection or aging, causes olfactory dysfunction which is a safety and a quality of life issue. No treatments are available. Defining the signals that regulate olfactory neuroplasticity would reveal new therapeutic targets to improve olfactory deficits. Ciliary neurotrophic factor (CNTF) is highly expressed in horizontal basal cells (HBCs) and olfactory ensheathing cells (OECs), while the CNTFRα receptor is expressed in the neighboring neuronal progenitor globose basal cells (GBCs). We found that CNTF is suppressed by focal adhesion kinase (FAK) and that intranasal application of an FAK inhibitor promotes OE neurogenesis via CNTF. Importantly, FAK inhibitor further enhances CNTF expression caused by OE injury with methimazole. We will use genetic, pharmacological, and behavioral approaches in male and female mice to test the hypothesis that FAK inhibition promotes olfactory neuroplasticity following injury by increasing CNTF expression. Aim 1 will define the FAK-CNTF-CNTFRα pathway underlying GBC proliferation by first determining whether FAK inhibition induces CNTF in HBCs, OECs or both. We will also determine whether CNTF is released to activate CNTFRα signaling in stimulating GBC proliferation, and whether FAK inhibition acts through this intercellular mechanism. To increase the relevance of our findings, Aim 2 will determine whether FAK inhibition can increase olfactory neurogenesis via CNTF following acute OE injury. Thus, we will use acute injury with methimazole and determine whether injury increases CNTF in HBCs and/or OECs which leads to increased GBC proliferation and neurogenesis. The effect of FAK inhibitor treatment following methimazole may be within HBCs and/or OECs, something we will test. To prepare for additional studies, we will define an optimal dose of FAK inhibitor and then test whether CNTF mediates the effect of FAK inhibitor to promote OE neurogenesis after acute injury. Chronic olfactory inflammation inhibits HBC proliferation and increases FAK signaling in HBCs, suggesting that CNTF might be suppressed. To broaden the relevance to more types of olfactory injuries, Aim 3 will use a refined chronic olfactory inflammation mouse model to determine whether FAK inhibition increases CNTF and promotes GBC proliferation and olfactory neurogenesis. Aim 4 will determine the ability of FAK inhibition to promote OSN axonal growth and olfactory function recovery following acute and chronic types of OE injury, using genetic axon tracing methods combined with behavioral tests. This proposal will define the role of FAK and CNTF and validate the therapeutic potential of FAK inhibitors to improve olfactory function after injury. FAK inhibitors are well-tolerated in cancer clinical trials and intranasal administration avoids systemic side effects.

NIH Research Projects · FY 2024 · 2022-05

PROJECT SUMMARY The contribution of the autonomic nervous system to the development of inflammation is an important new subject that has massive potential for clinical applications. It has been demonstrated that α7 nicotinic acetylcholine receptor (α7nAChR) is a critical component of anti-inflammatory cholinergic pathway that protects an organism during infection, arthritis, diabetes and atherosclerosis. So far, the protective role of α7nAChR was established only on the prevention of expression of pro-inflammatory cytokines, such as TNFα and IL-6. However, the mechanism of α7nAChR contribution to inflammatory response seems to be more comprehensive. One of the most critical steps of the inflammatory response is macrophage migration to the site of inflammation. The goal of this project is to determine the mechanism of macrophage migration regulated by the autonomic nervous system. We hypothesize that α7nAChR activation affects macrophage migration during inflammation by changing the expression of cell adhesive receptors, that modifies the overall α7nAChR- mediated anti-inflammatory response. Guided by strong preliminary data, this hypothesis will be tested by pursuing three Specific Aims: 1. Determine the contribution of α7nAChR to macrophage accumulation within the site of inflammation utilizing in vivo inflammatory models. 2. Evaluate the effect of α7nAChR on macrophage adhesion and migration using in vitro assays. 3. Define the critical molecules that regulate α7nAChR-mediated leukocyte migration. Under the first aim, the effect of α7nAChR-deficientcy on macrophage accumulation during endotoxemia and atherosclerosis will be evaluated. Under the second aim, the contribution of α7nAChR to macrophage motility will be assessed using in vitro adhesion and migration assays. Under the third aim, α7nAChR-dependent expression of adhesion and chemokine receptors on macrophages will be evaluated and analyzed. The significance of our study resides in providing a new insight into the function of neuro-immune synapse during inflammation. The direct contribution of α7nAChR cholinergic receptor to monocyte/macrophage migration proposes a new mechanism for the regulation of inflammatory response though the vagus nerve. This proposal is innovative because most studies of cholinergic anti- inflammatory mechanisms have focused on the ability of nicotinic agonists to suppress the synthesis and release of inflammatory cytokines from activated macrophages. We propose the role of neuro-immune synapse in macrophage migration. The results of our studies will provide completely new information regarding the role of α7nAChR in inflammation. These findings will be of significant therapeutic interest for targeting α7nAChR to regulate macrophage migration and to control inflammation in several disorders. The development of anti- inflammatory treatments is an objective of our further investigations.

NIH Research Projects · FY 2026 · 2018-08

1 Project Summary 2 3 Vagal nerve stimulation (VNS) therapy is approved for three central nervous system disorders. Despite promising 4 preclinical evidence of success amelioration of heart failure, several large patient trials have failed to meet 5 designated endpoints. Remarkably many recommended clinical protocols for VNS therapy appear to violate 6 strong preclinical evidence as well as approved therapies that rely on vagal afferent activation of central nervous 7 mechanisms. Low intensities of clinically tolerated VNS activate myelinated vagal afferents and these activate 8 neurons within the nucleus of the solitary tract (NTS). Preclinical data on vagal NTS neurons outline multiple 9 mechanisms controlling successful vagal transmission but indicate that current clinical VNS protocols likely 10 engage substantial frequency dependent transmission depression within NTS. Our global hypothesis is that VNS 11 drives central nervous system (CNS) pathways that contribute to beneficial therapeutic outcomes. Our goals are 12 to identify the mechanistic basis for processes contributing to optimal VNS therapy. Activation of the CNS by 13 VNS requires successful activation of action potentials in NTS neurons. We found that midcollicular knife cuts 14 eliminate pathways above the medulla and profoundly reduced VNS activation of NTS neurons. Our work will 15 examine the contribution of the paraventricular nucleus of the hypothalamus (PVN). Our experimental design 16 assays cellular mechanisms of NTS activation in brain slices as well as single NTS neurons in intact animals. 17 Hypotheses related to PVN will center on potential mechanisms of NTS modulation including roles for the 18 neuropeptides, vasopressin and oxytocin. New optogenetic experiments will incorporate transfections of PVN 19 neurons that are activated by VNS to expression ChR2 or halorhodopsin to control these neurons via light. Our 20 Aims focus on weak-intensity, vagal afferent activation of the CNS. Our approach relies on our direct assays of 21 NTS neuron activation – both in the intact brain as well as brain slice, intracellular recordings of NTS in vitro. We 22 propose Specific Aims supported by Preliminary Results that will identify the mechanisms for amplification and 23 spread of VNS excitation. Aim 1 focuses on potential mechanisms within NTS with emphasis on vagal timing 24 and synaptic depression while Aim 2 assesses mechanisms related to PVN modulation of NTS neurons with a 25 particular focus on oxytocin and vasopressin. Aim 3 assesses vagal afferent transmission in heart failure animals. 26 Aim 4 evaluates the therapeutic efficacy of VNS to relieve heart failure in a rat model. This proposal embraces 27 an assessment of whether optimizing activation of NTS neurons will enhance VNS efficacy in a heart failure 28 model. This work addresses a fundamental knowledge gap of neuromodulation therapy. Greater knowledge of 29 visceral afferent processing should drive new stimulation approaches in all clinical applications. We aim to 30 provide a knowledge foundation necessary for deliberate, evidence-based criteria for VNS therapy to improve 31 clinical outcomes.