Old Dominion University

NIH Research Projects · FY 2026 · 2026-06

Project Summary: The development of new, concise, and modular approaches toward physiologically important natural products that are synthetically flexible and amenable to late-stage modification is important for accessing new lead compounds and novel structural motifs for investigation as therapeutics to treat human disease. Provided that from 1981-2020 roughly 65% of all FDA approved drugs were influenced by natural products research, the unique molecular scaffolds found in nature are excellent starting points for the discovery of new lead therapeutics. While there are many factors to consider when developing a lead compound, structural complexity has become an increasingly important factor for clinical success in recent years. This work will advance new operationally-simple catalytic protocols employing bench-stable cobalt(III) complexes to build synthetically useful motifs through carbon-carbon bond formation reactions including carbonyl additions and allylboration reactions that are high utility for the synthesis of biologically relevant natural products, agrochemicals, and pharmaceuticals. The methods advanced in this work will contribute fundamental knowledge on the reactivity of first-row transition metal catalysts to help facilitate their use in new, cost-effective, and safer chemical processes. The utility and enabling power of the protocols developed with cobalt(III)-based catalysts in this work will be demonstrated through applications in the total synthesis biologically relevant triterpenoid natural products. This will lead to the first total synthesis of the sodwanones A-C and sodwanone H, which exhibits 2 nM nanomolar activity against A549 lung cancer cell lines and has the potential to serve as a lead compound for further biochemical investigations. The newly prepared compounds have a high potential to serve as a new class of antibiotics and sporicidal agents against C. difficile providing a new treatment for this common hospital acquired infection, thereby having a direct impact in solving an unmet challenge in healthcare. The pursuit of these aims will provide and expand research and training opportunities for 8 undergraduate students and one graduate student at Old Dominion University. Student trainees will develop important skillsets through cross-training in synthetic method development, total synthesis, organometallic chemistry, computational chemistry, medicinal chemistry, and biochemistry.

NIH Research Projects · FY 2026 · 2026-06

Pulsed electric fields (PEFs) are widely used in medical applications ranging from neuromodulation and neuromuscular stimulation to tissue and cancer ablation. The primary effect of PEF treatments is charging of the cell plasma membrane for cell activation. However, excessive charging disrupts the membrane through a process known as electroporation or electropermeabilization. Electroporation can be caused by routine electrostimulation; it is a significant adverse effect of defibrillation and a pathogenic factor in electrical trauma. Conversely, controlled electropermeabilization enables gene electrotransfer, pulsed field ablation for atrial fibrillation, cancer ablation, and electrochemotherapy. PEF treatments must be tailored to each application, grounded in fundamental knowledge of how the cell membrane responds to electrical stress. Understanding the kinetics of membrane charging and relaxation, lesion formation and repair mechanisms, their dependence on PEF parameters and cell physiology will create a framework for fine-tuning PEF effects – either for efficient stimulation without damage, or for effective ablation with minimal neuromuscular activation. Our team has long been involved in studying electropore properties and impact on cell function, excitability, and survival. We recently advanced the field by pioneering dynamic imaging of single electropores in live cells and by developing pulsed laser strobe microscopy to capture membrane charging and relaxation kinetics on the nanosecond timescale. This research proposal will explore membrane response to PEF at the single-pore level and with nanosecond resolution, to systematically characterize the primary mechanisms of complex electropermeabilization phenomena. We will design and validate novel mechanisms-based PEF protocols for medical applications. Aim 1: Quantify the formation of membrane lesions (diffuse permeabilization, transient and persistent focal pores) with respect to PEF duration (nano- to milliseconds) and the electric field strength (0.1-10 kV/cm). Compare effects of single PEFs and PEF trains at 10–90% duty cycles. Correlate lesion types with membrane charging and relaxation kinetics in cells of different shape and excitability. Characterize membrane charging by MHz compression of nsPEF bursts and bipolar cancellation mechanisms. Aim 2: Characterize the lifecycle, permeability, and current-voltage function of single electropores and their dependence on cell physiology, environment, and PEF protocols. Investigate electropore association with lipid rafts, voltage-gated channels, and structural proteins. Analyze whether membrane proteins form electropores and/or contribute to electropore structure and longevity. Test the electrodeformation mechanism of electropermeabilization. Aim 3. Examine the protective role of the adaptive electropore conductance and analyze the underlying mechanisms. Design novel nsPEF protocols to control electropermeabilization. Quantify cellular protection conferred by the activation of voltage-gated ion channels, lanthanide ions, and poloxamers. Primary membrane effects of PEF determine diverse downstream physiological effects and cell survival. A detailed understanding of these early events is key to developing effective treatments and minimizing adverse effects.

NIH Research Projects · FY 2026 · 2026-06

PROJECT SUMMARY Wound healing complications following injury to the oral tissues from surgery or trauma can lead to significant pain and need for revision surgeries. Cleft palate occurs in 1:1000 live births all undergo palate surgery; how- ever, 60% develop complications including oronasal fistula (ONF) formation which often recur. ONF also occur after traumatic oral injury or dental surgery representing a critical unmet need in clinical care. The physical con- sequences of ONF formation lead to pain, poor feeding and poor speech. Currently, there are no regenerative options to mitigate the complications of oral injury or surgery leading to significant pain and cost due to need for multiple revision ONF surgeries. Our group has developed a novel murine phenocopy of ONF, and identi- fied that FTY720, an active biolipid, targets the sphingosine pathway to preferentially attract proregenerative immune cells to improve ONF healing and reduce pain. Our long-term goal is to optimize FTY720 delivery as an immunoregenerative therapy to improve oral wound healing and reduce pain. The overall objective in this application is to determine the mechanism of FTY720-guided ONF healing and optimize FTY720 delivery. The central hypothesis is that FTY720 reduces the inflammatory neutrophils, monocytes and macrophages during oral cavity wound healing through genetic, epigenetic, and sphingolipid pathways and that the chemical struc- ture of FTY720 can be optimized to attract pro-regenerative immune cells leading to improved wound healing and reduced pain. The rationale for the proposed research is that a comprehensive and mechanistic under- standing of FTY720’s immunoregenerative function during ONF healing will provide mechanistic insights into oral wound healing and allow the optimization of FTY720 as the first oral regenerative therapy. Guided by strong preliminary data from the parent grant, including a paper demonstrating the immune cell changes during FTY720-guided ONF healing, the hypothesis will be tested by pursuing two specific aims: 1) Investigate FTY720-based therapeutic mechanisms during ONF healing via integrated spatiotemporal metabolic and tran- scriptomic profiling; 2) Optimize FTY720’s delivery using novel formulations to enhance its immunomodulatory and analgesic properties in vitro and in vivo. In Aim 1 we will define the genetic, epigenetic and sphingolipid signaling mechanisms through which FTY720 is altering the immune system and regenerating the oral tissues. In Aim 2, we will engineer the delivery of FTY720 to optimize bioavailability and therapeutic efficacy to improve ONF healing and reduce pain. The proposed research is innovative by mechanistically characterizing FTY720’s ability to heal ONF as a model for all oral wound healing and optimization of FTY720 towards the first immunoregenerative therapy for oral wound healing. The proposed research is significant because FTY720, an FDA-approved drug approved to treat multiple sclerosis, could be fast tracked to clinical trials to allow ONF healing, improve pain and reduce opioid dependence.

NIH Research Projects · FY 2026 · 2026-04

SUMMARY. Rickettsiae are obligate intracellular bacteria transmitted by arthropods. Many rickettsiae are pathogenic to humans, causing infections that range from severe, as is the case of Rocky Mountain spotted fever (R. rickettsii), Mediterranean spotted fever (R. conorii), or epidemic typhus (R. prowazekii), to mild, such as those caused by R. parkeri or R. africae. In contrast, others have not been associated with disease (e.g., R. montanensis). What underlies these dramatic differences in pathogenicity among rickettsiae is still not known. Growing evidence provided by us and others has led to the emerging hypothesis that macrophage permissiveness to rickettsial infection acts as a key virulence factor and a strategy employed by pathogenic Rickettsia to escape host immune defenses, and promote organ colonization. However, fundamental gaps in knowledge remain regarding the molecular details governing Rickettsia-macrophage interactions and how induced alterations contribute to immune evasion. Rickettsiae are strictly dependent on host nutrients/metabolites to survive and proliferate. This massive burden must require tight control of host metabolic/proteostatic systems. Our data points to mitochondria as a central hub used by Rickettsia to sustain a viable niche. We hypothesize that pathogenic SFG Rickettsia target macrophage mitochondrial function, acting both as clients – through the generation of metabolites, ATP, and transporters actively required by Rickettsia – as well as regulators, by redirecting host metabolism-dependent signaling to sustain a viable niche and fine-tune immune defenses to evade innate immunity. We will address our hypothesis with 2 Aims: 1. To determine how pathogenic SFG Rickettsia remodel mitochondrial dynamics in macrophages; and 2. To define regulation of rickettsiae-induced interferon responses at the mitochondria. In Aim 1, we will determine how highly pathogenic (R. conorii) and mildly pathogenic (R. parkeri) differentially modulate mitochondrial dynamics through the evaluation of the different components of the fusion/fission machinery. In Aim 2, we propose to determine the mechanism(s) by which highly pathogenic (R. conorii) and mildly pathogenic (R. parkeri) differentially regulate interferon responses at the mitochondria and modulate innate immune signaling. Innovation: (1) Mitochondrial fusion is a still unclear and uncommon response to infection. We propose that pathogenic SFG Rickettsia target mitochondria by stimulating fusion by an as-of-yet unknown mechanism, and this will reveal a new “Achilles heel” in infection. (2) We propose that pathogenic SFG Rickettsia evade immune surveillance through differential regulation of RLR/MAVS signaling, a key antiviral defense.

NIH Research Projects · FY 2025 · 2025-09

PROJECT ABSTRACT Conventional systemic drug delivery methods suffer from non-specific distribution, which can lead to reduced efficacy, severe side effects, and the development of drug resistance. Drug-induced liver injury is the main cause of acute liver failure, while drug-induced renal injury is the cause of around 20% of acute renal failure. Antimicrobial resistance contributed to 4.95 million global deaths in 2019, while around 90% of metastatic cancer patients experience resistance to chemotherapy. Localized delivery approaches have been implemented to resolve these challenges. However, limited drug penetration, specifically for the treatment of biofilm infections, results in poor outcomes. In the United States, more than 5 million patients suffer from superficial infections, with an annual financial burden of ~$11-28 billion. Microneedle arrays (MNAs) can resolve the penetration issue in localized drug delivery by bypassing the barriers against drug exposure to the cells and microbes. However, “prefabricated” MNAs have limited translational capability due to poor tissue penetration, challenges associated with their adaption to irregularly shaped large wounds, and limited compatibility with controlled spatiotemporal distribution of different drugs. Furthermore, the limited number of needles in MNAs is insufficient to effectively disrupt the biofilm and expose the microorganisms. We have recently developed a novel drug delivery strategy based on intralesional printing of drug-eluting biomaterials depots to resolve these challenges of prefabricated MNAs for drug delivery applications, and here we propose its evaluation in the treatment of wound infections. Our hypothesis is that intralesional patterning of drug-eluting depots has a strong chance of treating various defects including infected wounds through disruption of the physical barriers such as biofilms and delivery of drugs with controlled spatiotemporal distribution. The ultimate goal of this proposal is to develop an intralesional drug delivery strategy that enables maximum drug exposure to the target cells or microorganisms and enable spatiotemporal control over drug distribution, with effectiveness in treatment of infected wounds. Our objective is to in situ fabricate defect-specific and biodegradable gelatin-based drug depots carrying antimicrobial and pro- regenerative drugs directly into the defect and compare their effectiveness with topical delivery for the treatment of infected wounds. We will test our hypothesis in vitro, ex vivo, and in vivo, and achieve our objective through the following specific aims: (1) characterization of the in situ formed microneedle-like drug depots and drug release kinetics in vitro; (2) development and evaluating the effectiveness of sustained drug delivery from in situ fabricated drug depots in the treatment of biofilms in an ex vivo porcine wound biofilm model; and (3) assessing the effects of in situ fabricated drug depots on the healing of biofilm-challenged diabetic wounds in vivo. Completion of this work will demonstrate the feasibility of the technology and enable us to evaluate its efficacy in patient-derived microorganisms and pre-clinical large animal models of infected wounds in an R01 application.

NIH Research Projects · FY 2025 · 2025-09

Summary: The U.S. Department of Homeland Security (DHS) has identified over 200 chemicals of concern (CoCs) that threaten public health, with approximately 25% posing respiratory risks. Notably, nitrogen mustard (NM) and ammonia (NH3) are significant contributors to lung injury. Currently, there are no effective medical countermeasures to mitigate NM- or NH3-induced lung damage, therefore, the search for an effective therapeutic or prophylactic treatment persists. The endocannabinoids and their lipid-related mediators contribute to the modulation of oxidative stress and lipid peroxidation and 2- Arachidonoylglycerol (2-AG), is the most abundant endocannabinoid. 2-AG is produced ‘on demand’ and rapidly degraded, and monoacylglycerol lipase (MAGL or MgII) is the main enzyme responsible for 2-AG catabolism. The hydrolysis of 2-AG by MAGL terminates the activation of CB receptors, releasing free AA that serves as a precursor for the synthesis of pro-inflammatory eicosanoids. Enhancing the 2-AG signaling displays an anti-inflammatory response to proinflammatory and mechanical insults. Our preliminary findings indicate that acute exposure to NM or NH3 results in a significant increase in MAGL in mouse lung tissue. The NM-induced increase in MAGL was significantly higher in lung immune cells compared to alveolar epithelial and endothelial cells. A similar increase in MAGL was observed in human alveolar macrophages (HAMs) in response to NM exposure. Furthermore, our pilot study showed that the administration of JZL184, a potent and highly selective inhibitor of MAGL, resulted in a significant reduction in NM- and NH3-induced acute lung injury and immune cell infiltration in mice. Given these promising indications, this project aims to further investigate how augmenting the 2-AG signaling by inactivation of MAGL could confer protection against NM- or NH3-induced lung injury. Aim 1: To determine the extent to which MAGL inactivation/inhibition changes NM- or NH3-induced lung injury, inflammatory response, and toxicity. Aim 2: To determine the cellular and molecular responses of MAGL inactivation on NM or NH3-induced lung injury and inflammatory response. Aim 3: To delineate the mechanism by which MAGL inhibition/inactivation exerts the protective effects in NM or NH3-induced acute lung injury and inflammatory response.

NIH Research Projects · FY 2025 · 2025-08

Project Summary/Abstract Chronic ankle instability (CAI) is a common disorder that can lead to reduced physical function, joint health, and physical activity. Similar to healthy aging populations, balance and ankle joint health of individuals with CAI decline from young adulthood to middle adulthood. Age-related changes in balance, walking ability, reaction time, and strength can both contribute to and result from reduced physical activity. Impairments in function and declining physical activity are known to increase the likelihood of developing comorbidities and elevate risk of all-cause mortality in older adults. Older adults with CAI have reduced balance performance compared to healthy individuals of the same age, indicating that declines in the CAI population are more pronounced. With respect to ankle joint health, talar cartilage thickness is reduced in approximately 80% of individuals who have CAI for at least 10 years. However, whether CAI-related declines from young through older adulthood are steeper than healthy aging adults is unknown. No studies have examined the effects of CAI on physical activity or ankle joint health in populations aged beyond early adulthood through middle and older ages. Also, no CAI research has tracked physical activity, ankle joint health, or physical function longitudinally to examine declines over time. The purpose of this proposed study is to determine the extent to which CAI and aging independently and interactively affect joint health, physical activity, balance, gait, reaction time, muscular strength, and perceived function. To accomplish this objective, we will use a longitudinal study design in which 180 participants will be equally divided across two independent variables: age (20-39, 40-59, and 60-79 years) and injury history (CAI & control [CON]). The CON group will be matched to the CAI group based on sex and age. After enrollment, participants will undergo three separate laboratory testing sessions: Time Point 1 (baseline) Time Point 2 (4 weeks after Time Point 1), and Time Point 3 (12 months after Time Point 1). In each laboratory session, participants will undergo tests of ankle joint health, balance, gait, reaction time, strength, and perceived function. Each laboratory testing session will be followed by 7 days of physical activity tracking using a wearable monitor. To analyze the effects of CAI and aging cross-sectionally and longitudinally, we will employ a combination of Generalized Linear Model (GLM) and Analysis of Variance (ANOVA) models that as our primary statistical analyses. Each model will control for potentially relevant covariates such as injury and disease comorbidities. When appropriate, Tukey post hoc analyses will be performed, and effect sizes will be calculated. We expect that CAI and higher age will lead to worse performance cross-sectionally and greater declines longitudinally on measures of joint health, physical activity, balance, gait, reaction time, strength, and perceived function versus those without CAI and/or of lower age.

NIH Research Projects · FY 2026 · 2025-08

Abstract Maternal RNA clearance is an essential process that occurs in the absence of transcription in all sexually reproducing species examined. Stored maternal transcripts sustain early embryonic development prior to activation of the embryonic genome. Perturbations of maternal mRNA degradation can either halt or irreparably alter the maternal-to-zygotic transition, leading to early embryonic demise. Oocytes are unique cells in which translation and RNA clearance are coupled, in the absence of new transcription. Maternal mRNA is eliminated via translationally coupled mRNA degradation, a process that includes recruitment of mRNA to the ribosome, shortening of the poly(A) tail, decapping, and degradation by both 5′ and 3′ exonucleases. Mechanisms that regulate translationally coupled mRNA degradation are poorly understood. Recent studies have established RNA modifications as regulators of maternal mRNA clearance. Our previous work established that inosine RNA modifications within the coding region of mRNA can impact maternal mRNA stability through a translation mechanism. Here, I describe two research directions I will pursue in the next 5 years that address fundamental questions about the role of RNA modifications in maternal mRNA clearance. The first direction will investigate the relationship between inosine mRNA modifications in translation. The second direction will focus on identifying the relationship between inosine RNA modifications and other RNA modifications, and how this combined “RNA modification code” can regulate RNA stability and translation during maternal mRNA clearance. We will use innovative molecular analyses, novel RNA-sequencing approaches, proteomics, in vivo imaging, and combined single molecular/single oocyte assays to test how inosine impacts translation, alters the occurrence of amino acid substitutions, and how this facilitates maternal mRNA clearance. In addition, I will catalog the “RNA modification code” on whole, individual transcripts in oocytes to determine the relationship between multiple RNA modifications and maternal mRNA clearance. While these studies will significantly advance the understanding of RNA processing in oocytes, the discovered principles will also apply to other cells. In fact, inosine RNA modifications were first discovered in oocytes (Xenopus laevis), and this novel discovery led to a change in the entire way we view RNA processing mechanisms. The utility of the oocytes as a model will enable the impact of RNA modifications on the fate of the RNA to be amplified, and will advance our appreciation of the biological roles RNA modifications have within cells, organs, and organisms. These studies will provide a powerful framework of understanding that will build the research focus of the Brachova lab for the future, eventually leading to an understanding of the relationship between RNA modifications and embryo genome activation.

NIH Research Projects · FY 2025 · 2025-08

Summary: This proposal builds upon our recent completion of an NIH grant titled “Development of an Integrated 3D Human Osteo-Mucosal Model,” for which our team stands as the sole entity to have successfully developed this model. During that project, we engineered an osteo-mucosal construct, overcoming various challenges, particularly in establishing robust adhesion between the soft and hard tissue sections. Now, with this proposal, we aim to bridge the gap between our previous grant and future directions. More specifically, given that the osteo-mucosal construct is inherently critical-sized, the challenge of vascularization hinders the advancement of this research for pre-clinical examinations. It is evident that non-vascularized constructs are prone to failure when implanted into critical-sized defects. Therefore, this proposal is dedicated to addressing this challenge by focusing on the pre-vascularization of the previously developed osteo-mucosal construct and evaluating its effectiveness in vivo. To achieve this goal, we will implement key angiogenesis strategies that synergistically activate pre- vascularization. More specifically, we will harness the synergistic effects of intracellular and extracellular mechanisms to further promote pre-vascularization. Specifically, we will utilize FG-4592 (Roxadustat), a hypoxia- inducible factor prolyl hydroxylase inhibitor (HIF-PHI), known for its intracellular mechanism. FG-4592 stabilizes HIF-1α by inhibiting HIF-prolyl hydroxylases, thereby preventing its protease degradation within the cells. This process facilitates the expression of relevant genes and promotes angiogenesis. Notably, FG-4592 is currently undergoing global Phase III clinical trials for the treatment of anemia. In addition to FG-4592, our model incorporates an aptamer targeting an extracellular mechanism. This aptamer binds effectively to both VEGF receptors 1 and 2 (VEGFR-1 and -2), with a notably stronger affinity for the latter, thereby facilitating the promotion of angiogenesis. The osteo-mucosal construct will be created using 3D-printed scaffolds loaded with these pro-angiogenic factors and seeded with HUVECs and supporting cells. The research involves comprehensive in vitro testing to promote blood vessel formation, followed by in vivo studies using a rat alveolar bone model with overlaying gingiva to evaluate the construct's integration with host tissue, and formation of well-defined and interconnected vascular networks. The success of this approach will be measured by the density and functionality of the blood vessels with capillary density of 50-100 vessels/mm² in bone tissue and 30-60 vessels/mm² in mucosal tissue. The osteo-mucosal complex presents one of the most challenging multi-tissue defects. It represents a key example of critical-sized defects, and its successful regeneration can establish a robust approach for treating other critical-sized defects involving both hard and soft tissues. Furthermore, a laboratory-grown osteo-mucosal construct derived from a patient's own cells proves immensely beneficial for oral surgeries. Moreover, it offers an alternative to animal models for studying biomaterial-oral tissue interactions and for screening oral diseases.

NIH Research Projects · FY 2026 · 2025-07

Abstract In the era of combination antiretroviral therapy, people living with HIV (PLWH) have a similar life-expectancy to the HIV-negative population. However, the life-quality of PLWHIV is still deeply compromised due to the prevalence of neurological symptoms (NeuroHIV). Recently, a novel subtype of Mg, called lipid-droplet- accumulating Mg (LDAM), has been identified in both mouse and human aging brains as well as in Alzheimer’s disease. LDAM have dysregulated lipid metabolism and are characterized by increased reactive oxygen species (ROS) production, low levels of inflammation, and defective phagocytosis. Previous investigations have shown that HIV infection and abused drugs could affect brain lipid metabolism which contributes to NeuroAIDS. However, whether and how HIV/HIV proteins and abused drugs affect microglial lipid metabolism and whether and how LDAM are involved in NeuroHIV in the context of HIV and abused drugs have never been explored. We initiated an investigation and obtained these preliminary data : (1) HIV-TAT and cocaine individually increases lipid droplets (LDs) formation in vitro and in vivo; (2) HIV-TAT and cocaine individually upregulates the activity of sterol regulatory element binding proteins (SREBPs) - mediated pathway in time- and dose- dependent manners in vitro; (3) SREBP2 inhibitor 5-O-methylembelin blocks HIV-TAT-mediated LDs formation and Mg activation; (4) The co-exposure of cocaine and HIV-TAT exerts combined effects on Mg lipid metabolism in vitro and in vivo; (5) HIV-inducible(i) TAT mice both show increased LDAM in the brains compared to age-matched counterparts; (6) SIV (+) macaque brains showed increased LDAM compared to their age-matched uninfected counterparts. Based on these preliminary data and the critical roles of LDAM in multiple neurodegenerative diseases, we hypothesize that dysregulated lipid metabolism serves as the master mechanism responsible for Mg activation in the context of HIV/HIV proteins and cocaine, and that targeting on LDAM could mitigate NeuroHIV in PLWH who use cocaine. We will test this hypothesis in the following two specific aims (SA) through complimentary in vitro and in vivo approaches: SA1: Determine the role of dysregulated lipid metabolism in Mg activation and neuronal injuries in the context of HIV-TAT/HIV and cocaine in vitro. SA2: Determine the potential therapeutic effects of inhibiting lipid metabolism on NeuroHIV symptoms. We will correlate lipid dysregulation and Mg activation with neurological behaviors to validate the therapeutic effects of targeting lipid metabolism on NeuroHIV in PLWH using cocaine.

NIH Research Projects · FY 2026 · 2025-04

ABSTRACT Gastrointestinal (GI) toxicity remains a significant contributor to mortality following exposure to ionizing radiation (IR), leading to GI Acute Radiation Syndrome (GI-ARS). Current lack of effective therapy for GI-ARS underscores the critical need for medical countermeasures (MCM). This study aims to investigate the mechanisms regulating the renin-angiotensin system (RAS) and reactive oxygen species (ROS) signalling through the AT1 receptor to mitigate GI-ARS. The research introduces a novel, selective and potent Tempol-ARB conjugate, YK-4-250, targeted to the AT1 receptor. Preliminary data show that oral administration of 20 mg/kg YK-4-250 at 24, 48 and 72 hours after exposure to 14.3 Gy partial body irradiation (PBI) significantly mitigated GI-ARS in mice, leading to GI-recovery by day 7 and an 88% overall 30-day survival. The hypothesis postulates that IR activates RAS, elevating Ang II levels, causing oxidative damage and triggering pro-inflammatory cytokine release, ultimately contributing to acute GI-ARS. YK-4-250 is proposed as an effective radiation mitigator, with specific aims including assessing improved safety vehicle formulations, measuring basic in vivo safety, defining pharmacokinetics, determining pharmacodynamics, and evaluating survival efficacy in GI-ARS and pulmonary fibrosis models. Our final aim explores the concept that AT1R knockout mimics YK-4-250 in mitigating GI injury and prolonging post-radiation survival. The study seeks to perform formulation development for YK-4-250 for use in vivo studies, identify molecular mechanisms enhancing cytoprotective function, and investigate its utility in mitigating IR-induced lung fibrosis. Successful completion of these studies is anticipated to advance YK-4-250's development as a effective radiation mitigator for GI-ARS, potentially expanding its application as a broader medical countermeasure.

NIH Research Projects · FY 2024 · 2024-09

Abstract Microglia (Mg), the brain residential macrophage, play critical roles in maintaining adult brain homeostasis. Mg can function as multi-players such as housekeepers, guards, and warriors under both physiological and pathological conditions. Accumulating evidence reveal that Mg also play vital roles in brain aging. Reversing or eliminating senescent Mg and replenishing with new-born Mg have been suggested as treatment for aging and neurodegenerative disease. Recently, Mg depletion and repopulation (MgDR) has been tested as a potential therapeutic approach for acute brain injury and Alzheimer’s diseases. Mechanically, the repopulated Mg show homeostatic phenotype with restoring BDNF signaling or increasing the activity of IL6 pathway to improve brain functions. It has been well-accepted that sustained and lower levels of Mg activation promote Mg senescence and contribute to HAND pathogenesis. Several anti-neuroinflammatory drugs have been proposed as alternative approach for HAND therapy. However, whether MgDR could be a novel therapeutic approach to mitigate neurological deficiency in chronic HIV (+) individuals have never been explored. We initiated pilot studies and obtained these preliminary data: (1) Both HIV transgenic (Tg) rats and HIV-inducible (i) mice show increased LDAM in the brains compared to age-matched counterparts; (2) HIV-iTAT mice show dysregulated lipid profile in the brain hippocampus; (3) MgDR by PLX3397 restored locomotion coordination ability in HIV-iTAT male mice. Based on these observations, we hypothesize that LDAM contribute to the pathogenesis of HAND and neuropsychiatric symptoms and MgDR can exert therapeutic effects on brain dysfunctions in the context of chronic HIV infection. The hypothesis will be tested in the following two specific aims (SA): SA1: Investigate the effects of MgDR on HAND and neuropsychiatric symptoms in HIV-Tg26 mice. SA2: Explore the detailed mechanisms underlying the effects of MgDR on brain pathology in vivo. This proposal will explore the therapeutic effects of MgDR on brain pathology in the context of chronic HIV infection. If succeed, we could open a new research direction to identify effective therapy to improve the life-quality of chronic HIV (+) individuals.

NIH Research Projects · FY 2024 · 2024-09

Summary Atherosclerosis is the major etiological process responsible for 25% of global deaths. Numerous reports indicate both the adaptive and innate immune systems are involved in atherogenesis. In addition to well- established risk factors such as age, hypertension, high circulating levels of low-density lipoprotein cholesterol, and type 2 diabetes, the quality and quantity of sleep are now recognized as important factors for atherogenesis. Sleep is vital for life. In the USA, ~70 million people report insufficient sleep and/or fragmented sleep due to work responsibilities, sleep apnea, caregiving, and lifestyle choices. Changes in sleep are a part of the normal aging process, leading to increased sleep fragmentation (SF), nighttime awakenings, and a greater tendency for daytime sleep. Dysregulation of normal sleep negatively affects homeostatic functions and is associated with an increased risk of chronic diseases, including atherosclerosis. Long-term SF may lead to endothelial dysfunction, oxidative stress, and altered vessel wall structure. SF has a negative impact on atherogenesis through the regulation of myelopoiesis, highlighting the complex relationship between sleep, the vascular system, and the immune system. One of the remaining outstanding questions in the field is the extent to which SF affects the vulnerability of plaques. Another important question that remains to be investigated is whether the restoration of sleep quality might reduce atherosclerosis development and improve the phenotype of the plaques. Our results indicate that SF supports neutrophil functions such as ROS and NET production. We detect increased circulating levels of LPS that might serve as a neutrophil activator during SF. Competitive homing experiments clearly demonstrated that SF directs neutrophil recruitment into the aorta. Importantly, neutrophil depletion improves plaque phenotype of SF mice. One of the strongest phenotypes observed in the circulation and the gut was a significant degree of oxidative stress, cell death, and neutrophil activation. Gut inflammation was also supported by alterations in the intestinal immune composition. In this application, we propose the hypothesis that disturbed sleep negatively influences gut-associated inflammation, triggering LPS-induced oxidative stress, and activating neutrophils, which, in turn, plays a key role in the formation of accelerated vulnerable plaques. Here, we propose to investigate the role of NADPH-dependent oxidative stress in vulnerable atherosclerotic plaque formation in response to SF in HFD-fed via neutrophil or intestinal epithelial cell-specific NADPH-deficient Apoe-/- mice (Aim 1). In Aim 2, we will test to what extent restoring sleep quality would improve the phenotype of atherosclerotic plaques and reduce atherogenesis. The findings from this proposal could lead to the development of new treatments aimed at preventing gut-associated and neutrophil- induced oxidative stress and suppressing accelerated atherogenesis induced by SF.

NIH Research Projects · FY 2025 · 2024-09

Summary In the past decade, we have witnessed revolutionary progress in the attainable resolution of macromolecular assemblies via cryo-electron microscopy (cryo-EM) and in the development of deep learning algorithms (such as AlphaFold) that reliably predict atomic structures that can be fitted to cryo-EM maps. Whereas single- particle cryo-EM today is capable of directly solving the atomic structures of biomolecular assemblies in isolation, cryo-electron tomography (cryo-ET) is widely used in unstained frozen-hydrated samples to capture the 3D organization of supramolecular complexes in their native (organelle, cell, or tissue) environments. The increasing availability of high-quality maps and corresponding atomic models enables the validation of computational strategies, yielding rigorous and reproducible modeling technologies for the future. In this proposal, we have identified research areas for the next five years and beyond, leveraging our computational modeling experience (historically rooted in pre-revolution multi-scale approaches) to offer the biggest value to today’s post-revolution EM community. Our vision is to combine the converging advancements in cryo-EM, cryo-ET, structure prediction, and rigorous validation of modeling methods into a comprehensive research strategy. We will quantitatively measure the fitness of an atomic model in local density regions and characterize the fitness of maps with reliable reference structures. This will lead to new breakthroughs in the flexible fitting and refinement of AlphaFold2 models as well as secondary structure prediction for medium- resolution maps, which have been our key research areas in recent years. Medium- to low-resolution maps are still widely used in EM and can be of significant biological importance. This is particularly true in the case of cryo-ET maps, which are harder to read than single-particle cryo-EM maps because they often exhibit considerable noise, anisotropic resolution, and anisotropic density variations due to the low dose requirements and the missing wedge in the Fourier space. As automated segmentation algorithms in cryo-ET continue to improve, validation of these approaches has become more incumbent. Having a known ground truth on which to base predictions is crucial to reliably testing predicted structures and modeling approaches. We propose a software tool for the realistic simulation of “phantom” cryo-ET maps. We describe current and future applications in the validation of cytoskeletal filament tracing methods. The collaborative efforts supported by this grant will include the refinement of cytoskeletal actin filaments, molecular motors, bacterial chemoreceptor arrays, and hair cell stereocilia. The algorithmic and methodological developments will be distributed freely through the established Internet-based mechanisms used by the Situs and Sculptor packages and as plugins for the popular UCSF Chimera graphics program.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY / ABSTRACT Despite significant progress made in preventing perinatal mother-to-child-transmission of HIV, 2022 saw about 130,000 new HIV infections in children globally, a vulnerable group that also accounts for 13% of AIDS-related deaths. Postnatal infection through breastfeeding remains a significant contributing factor for mother-to-child transmission of HIV, and often derives from untreated/undiagnosed mothers living with HIV or poor adherence to antiretrovirals (ARVs). Currently available prophylaxis options for newborns with the potential for perinatal exposure to HIV is ARV therapy. However, ARV formulations (oral and injectable) and regimens meant for pediatric use are limited, difficult to adhere to, and potentially accompanied by multiple undesirable side effects. Broadly neutralizing antibodies (bnAbs), currently under investigation in clinical trials, represent an innovative and safer way to deliver perinatal HIV prophylaxis by directly engaging and reinforcing host immunity. Despite the promise, these highly potent Abs have demonstrated in adults and children, in their current form, they often require multiple injections and cold chain storage and transportation, which is burdensome to health care systems and a considerable expense in low-resource settings. A user-friendly transdermal microneedle (MN) patch containing thermally stabilized bnAbs would circumvent these problems through the safe and easy delivery of these potent Abs for protection against HIV-1, even outside of a clinic setting. The MN patch design proposed here is aimed to accomplish co-delivery of a cocktail of long-acting bnAbs offering long-lasting protection without the burden of frequent and adverse-effect laden pediatric ARV formulations or potentially painful and cumbersome antibody infusions for newborns. In this proposal, we will test this innovative delivery platform for the co-delivery of three different bnAbs currently in advanced clinical development (VRC07-523LS, PDGM1400LS and PGT-121LS). Rigorous design and fabrication of MN patches with in vitro and ex vivo characterization of formulated bnAbs will precede their in vivo preclinical assessment of safety in young rabbits, pharmacokinetics (PK) in rats, and subsequent testing in neonate Rhesus macaques to characterize the PK profile and efficacy against oral challenges with SHIVSF162P3. Together, these findings will inform the characteristics and viability of an innovative bnAb delivery platform that could significantly curtail the persistent health crisis of pediatric HIV infections.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY/ABSTRACT Benign prostatic pathological changes (collectively known as Benign Prostatic Hyperplasia/BPH), characterized by lower urinary tract symptoms (LUTS), is a prevalent condition that significantly impacts the quality of life in aging males. Current medical treatments for LUTS/BPH are largely ineffective, necessitating a deeper understanding of the underlying pathogenesis to develop novel therapeutic strategies. Our prior investigation identified that lipid accumulates in the prostate in BPH as well as macrophages enter the prostate lumen and become lipid-laden foam cells. However, we have very limited information on the etiology and consequences of these processes. Therefore, through the utilization of clinical specimens, a well-established mouse model of prostatic disease, and the creation and testing of novel mouse models, we will investigate the intricate interplay between prostatic inflammation, lipid accumulation and urinary dysfunction. Accordingly, aim 1 will elucidate the role of CXCL17 as an epithelia-derived cytokine in remodeling the prostatic immune environment in mice as well as will identify candidate cytokines that drive the same process in humans via a comprehensive transcriptomic and proteomic approach. Aim 2 will generate a mouse model with inducible upregulation in lipid synthesis pathways in the prostate epithelium to assess pathological and functional consequences. Aim 3 will decipher the impact of foam cell-derived factors via transurethral instillation. Our research team consists of experts in urology, immunology, and proteomics research, ensuring a comprehensive and multi-faceted approach to address the research questions. The anticipated outcomes of this research project include a better understanding of the pathogenesis of LUTS, the identification of novel biomarkers for disease diagnosis and monitoring, and the development of innovative therapeutic strategies targeting foam cell formation and lipid accumulation. In conclusion, this study holds significant promise for advancing our understanding of benign prostate disease and ultimately improving clinical outcomes for patients suffering from lower urinary tract symptoms. By elucidating the role of lipid accumulation and foam cells, we aim to pave the way for the development of effective interventions to alleviate the burden of LUTS in the aging male population.

NIH Research Projects · FY 2025 · 2024-08

Project Summary Social isolation (SI) and loneliness are associated with adverse health outcomes and are serious public health risks throughout life. In early life, SI has well known deleterious effects on socioemotional and cognitive development, and on the developing brain. SI and loneliness also can increase drug and alcohol usage in adolescent humans and post-weaning SI increases cocaine and methamphetamine self-administration in rats. The mechanism by which SI impacts cognition and addictive behavior are mostly unknown, but they may involve disturbed sleep and neuroinflammation. Disturbed sleep has been suggested to be important in the adverse effects of SI and loneliness, and sleep disturbances and the risk for SI and loneliness are common in adolescence. SI and loneliness can increase sleep disturbances and disturbed sleep negatively impacts cognitive performance. Disturbed sleep also heightens risks for substance use disorders (SUDs). SI can alter microglia activity, which determines neuroinflammation levels. However, there have been few studies of how early SI, and degrees of isolation, impact cognition and addictive behavior. There also is minimal information as to the role disturbed sleep and neuroinflammation play in SI-induced changes in cognition and addictive behavior in adolescence and potential persistence into adulthood. We will employ a paradigm comparing rats single-housed in standard transparent cages (SI) and rats single-housed with opaque barriers between cages to prevent visual contact (ESI, enhanced SI) to pair housed (PH) rats. In adult rats, chronic ESI, a putative model of loneliness compared to SI alone reduced sleep, reduced EEG delta, produced different levels of neuroinflammation, and altered astrocyte morphology. There also were significant sex differences in sleep and behavior. We will use this model in our planning project to develop methods to assess the effects of SI and ESI during development on neurocognitive function and addictive behavior in rats. We have assembled a multidisciplinary team with expertise in translational SUD research/addiction, neurocognitive testing, neural signal processing, sleep neurobiology, neurohistology and circadian rhythms to determine how SI and ESI impact drug seeking and consummatory behavior, cognition and neurocircuit activity and the role that sleep may play in altered cognition and addictive behavior. We will develop methods and paradigms to assess, in adolescent and adult male and female rats, the effects of early life SI and ESI compared to PH on cognitive performance and drug self-administration. We will also develop methods to record neural activity during cognitive testing and to assess sleep and neuroimmune function as mediators of SI- and ESI-induced effects. Our goal is to identify, and develop methods to pursue, promising lines of research that will increase understanding of the effects of early life SI and loneliness on subsequent cognition and addictive behavior.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Cystic Fibrosis (CF) has been a pediatric disease, but with improved treatment and care it has become a chronic disease extending the life expectancy of patients. The characteristic chronic pro-inflammatory process in CF accelerates cellular senescence and further complicates this disease. Therefore, novel therapeutic strategies are needed to combat cellular senescence in the CF lung. Cellular senescence is a key process underlying aging, which leads to an accumulation of epigenetic noise. Such noise distorts the epigenetic landscape and dysregulates gene expression, resulting in declined tissue function and age-related diseases. We and others have established that epigenetics are involved in the pathogenesis of age-related diseases. We have shown that anti-aging methods improve mucociliary clearance in the CF bronchial epithelium. Cellular senescence is regulated via gene transcription through the interplay between promoters, enhancers, and cis-acting regulatory elements bound by transcription factors. Bromodomain- containing protein 4 (Brd4) is an epigenetic reader protein. It binds to acetylated histone and recruits transcriptional factors for actively transcribed genes. Transcription factor AP-1 was recently reported to mark the senescence enhancer landscape and drive the transcriptional program in senescent cells. Brd4 is critical for many senescence-associated secretory phenotype related inflammatory gene expression profiles. However, the role of Brd4 and its interactions with AP-1 to control the senescent related gene expression in the CF bronchial epithelium has not been explored. Our preliminary data showed that blocking Brd4 down-regulated multiple senescent genes, and reduced the recruitment of AP-1 to those genes. We hypothesize that blocking Brd4 in the CF bronchial epithelium will epigenetically reduce cellular senescence in the CF lung ultimately preserving lung function. To test our hypothesis, we will first determine if Brd4 regulates the chromatin accessibility and is required for senescent-related gene expression in the CF bronchial epithelium; we will also explore the underlying mechanisms and the interactions of Brd4 and AP-1 in regulating senescent related gene expression; lastly, we will determine the efficacy of Brd4 inhibition in CFTR responsive and unresponsive CF rat models. Results from this study will offer new insights of epigenetic regulation in the CF bronchial epithelium and provide support for targeting senescent airway cells in CF lung disease as a novel therapeutic strategy.

NIH Research Projects · FY 2025 · 2024-02

PROJECT SUMMARY Idiopathic pulmonary fibrosis (IPF) is a fatal lung disorder of unknown etiology. IPF is characterized by altered epigenetic state. Epigenetic alterations are potentially reversible, thus are attractive therapeutic targets. Chromatin structural remodeling through histone post- translational-modifications control transcriptional responses. Acetylated histone marks as well as some transcriptional factors are recognized by bromodomains (readers). Bromodomain- containing protein (Brd) 4 is a member of the bromodomain and extraterminal (BET) family, which binds to cell type-specific enhancers and promoters. Brd4 has been reported to be essential for enhancer-mediated pro-fibrotic genes expression in many organ fibrosis. However mechanisms of how Brd4 regulates genome-wide pro-fibrotic responses and its interaction with other his acetyltransferase, such as p300, is not clear. Fibrotic responses involve many cellular processes, including epigenetic alterations. Our preliminary data show that by blocking Brd4, multiple profibrotic genes can be downregulated; inhibition of Brd4 can disrupt the association of p300 and H3K27ac with profibrotic genes promoter region. We hypothesis that Brd4 affects chromatin accessibility, mediates the up-regulation of profibrotic genes through interaction with p300 by acetylating active enhancer mark H3K27ac during lung injury and repair process. Our aims are: 1. Determine the effects of Brd4 inhibition on profibrotic responses in lung fibroblasts; 2. Determine if Brd4 through p300 mediates histone acetylation to regulate profibrotic genes expression, 3. Determine the in vivo targeting of Brd4 inhibition in pre-clinical models of lung fibrosis. Results from this research will make a significant impact on our understanding of the role of Brd4 in epigenetic regulation in the pathobiology of IPF.

NIH Research Projects · FY 2023 · 2023-09

Project Summary: Osteo-odonto-keratoprosthesis (OOKP) or Tooth-in-Eye surgery is an exceptional procedure that can save severely injured corneas. The OOKP procedure is effective and capable of treating corneas after multiple corneal transplantation failures, or when the cornea is severely damaged by Stevens–Johnson syndrome, pemphigoid, chemical burns, trachoma and multiple corneal graft failure. The success rate in OOKP is high, however, it is an aggressive procedure, time consuming and involves partial sacrifice of the patient's oral cavity. This proposal aims to simplify this surgery by making a novel OOKP synthetic prosthesis. At the first stage of the OOKP surgery, a patient's tooth and surrounding tissue are harvested to make an autograft prosthesis called an osteo-odonto-lamina into which an optical cylinder is placed. This construct is then implanted into a submuscular pouch for 2–4 months to be vascularized. At the second stage of the surgery, the vascularized osteo-odonto-lamina is inserted into the eye to act as a frame for holding a PMMA optical cylinder. In this proposal, we build a special keratoprosthesis for the OOKP surgery as a replacement of the osteo-odonto- lamina so the surgeons do not need to harvest teeth and surrounding jawbone of the patients. For this purpose, we will follow the general design we have proposed in our recently issued patent. Although the design and idea of the keratoprosthesis have already been proposed in our patent, it has not been produced, and the details of the construct are yet to be configured in this proposal. More specifically, we are going to figure out the material, density, porosity, and pore-size of the different parts of the prosthesis in a way to mimic the details of the natural osteo-odonto-lamina structure, and particularly mimic the features of it that are accountable for the success of OOKP surgery. We will use a titanium alloy and 3D-printing technique to fabricate this keratoprosthesis. By considering our criteria for mimicking such construct, we will make 18 samples. They will go through a full physical, mechanical, and in vitro characterizations to find the most optimized synthetic OOKP keratoprosthesis. In sum, OOKP surgery is a multi-disciplinary, labor-intensive, and invasive procedure requiring both dental and ophthalmology expertise. Through the use of our new prothesis, the OOKP procedure can eliminate one very aggressive stage of the surgery that harms patients and can reduce the overall length of the treatment by a few months. It will considerably enhance the treatment of patients with severely injured corneas.

NIH Research Projects · FY 2024 · 2023-09

Project Summary Youth trajectories of tobacco use are of critical importance in the future of public health. Most tobacco use begins in adolescence and adolescents are particularly vulnerable to long-term tobacco use. While cigarette smoking significantly declined among adolescents over the past decade, overall tobacco/nicotine use actually increased by some measures, primarily due to increased rates of use of electronic nicotine delivery systems (ENDS). These changes in population levels of adolescent tobacco use are of critical importance in predicting tobacco control challenges and opportunities in the next decade. As the scientific debate unfolds regarding ENDS health effects, adolescents nationwide are learning about tobacco products and attempting to rectify conflicting information to create and refine their own beliefs regarding these products. At the same time, youth are increasingly living their lives filtered through social media account participation. Marketers are well aware of this fact and act to influence youth to purchase their products. Tobacco marketers are certainly no exception. Social media marketing is particularly important to examine as it is a prevalent source of adolescent exposure that is amenable to policy change. Social media marketing differs from other forms of marketing in that it exploits participatory facilitation, algorithmic coordination, context- specific promotions, real-world enmeshment, and seamless integration with purchasing opportunities. Although some research has examined this issue, there are important methodological concerns that may lead to flawed or incomplete understanding of the relationships involved and hinder the development of policies or other approaches to reduce the influence of social media marketing on youth ENDS use. We will address limitations in prior research by using both longitudinal data-adaptive, semiparametric estimators and a causal inference approach. We will focus on two risk perceptions: harmfulness and addictiveness. The degree to which risk beliefs act as mediators of the effects of social media marketing on use is unclear. Effective ENDS control strategies for adolescents require a more comprehensive understanding of how attitudes change over time and the potential impact of social media marketing on these changes The Population Assessment of Tobacco and Health (PATH) dataset provides a unique opportunity to examine youth beliefs about tobacco products, marketing exposure, and patterns of tobacco use among adolescents in the United States longitudinally. The current study will examine underutilized, recent Waves to examine the impact of online ENDS marketing on use with longitudinal data-adaptive, semiparametric estimators and to examine risk perceptions as potential mediators explaining the effects of adolescent marketing exposure on ENDS use with a causal inference approach.

NIH Research Projects · FY 2023 · 2023-09

ABSTRACT Infertility is a major reproductive health issue that affects ~12% of reproductively aged women in the United States. Approximately 1-3% of infertile or subfertile women have oocytes that arrest in meiosis or shortly after fertilization due to genetic variants. Unfortunately, there are no therapies for women experiencing infertility due to oocyte arrest. Strategies to restore oocyte maturation in women with oocyte arrest are of dire need in order to give these women fertility options. An emerging class of therapies, called mRNA therapeutics, utilize in vitro synthesized mRNA as a treatment for diseases and for vaccines such as the SARS-CoV-2 mRNA vaccines, but the safety and efficacy has not been explored in infertility. Microinjection of RNA into oocytes is an established tool that has enabled discovery of critical aspects of oocyte biology, but it could also be used as a therapeutic, particularly in women with oocyte arrest. Two recent studies successfully generated blastocysts in oocytes from women with genetic variants causing oocyte arrest, following the injection of in vitro synthesized wild-type RNA during assisted reproductive procedures. RNA therapies represent a novel treatment strategy for women experiencing oocyte arrest, however, rigorous testing is needed before they become an assisted reproductive technology. Considering the unique RNA processing and transcriptional quiescence of fully grown oocytes it is critical to understand how oocytes process exogenous RNA therapeutic molecules. Furthermore, synthetic therapeutic mRNA contain RNA modifications that promote RNA stability, translation, and reduce immune stimulation. Recently, our work and others have implicated RNA modifications as playing an important regulatory role in RNA stability and translation in oocytes. However, the impact of multiple RNA modifications on RNA stability, translation, and oocyte maturation has not been examined. Our goal here is to test how RNA modifications impact the function of mRNA therapeutics designed to rescue oocyte maturation defects. To understand how RNA therapeutics are processed by the oocyte and how they impact oocyte maturation and fertility, we will use a genetic knockout mouse model of the Protein Associated with Topoisomerase II Homolog 2 (Patl2 gene), which results in oocyte maturation arrest. Mice lacking PATL2 protein phenocopy women with genetic defects in Patl2, and have oocytes that fail to mature, so we predict that microinjection of an RNA therapeutic for Patl2 will restore oocyte maturation, fertilization, and birth. We will determine the effects of RNA modifications on stability and translation of therapeutic Patl2 RNA. Our studies have the potential to reveal novel aspects of RNA modifications in oocyte RNA processing and translation, as well as establish groundwork for future studies testing the safety and efficacy of RNA therapeutics to treat female infertility due to oocyte arrest.

NIH Research Projects · FY 2024 · 2023-07

Abstract Despite extensive studies, environmental cues and signaling circuits regulating onset of autoimmune diseases are not completely understood. Here we report that a receptor of the TGF-β cytokine family, Bone Morphogenetic Protein Receptor 1α (BMPR1α, Alk-3) has important immunoregulatory functions. BMPR1α is upregulated by activated effector and Foxp3+ regulatory CD4+ T cells (TR cells) and modulates functions of both of these cell types. BMPR1α regulates inflammation by inhibiting generation of Th17 cells and sustaining TR cells. Abrogation of BMPR1α signaling in TR cells resulted in a gradual loss of Foxp3 expression and upregulation of transcription factors and cytokines specific for Th effector lineage including Rorgt, Batf, Hif1a, IL-17 and IFN-g. This data suggests that BMPR1α controls phenotypic stability of TR cells and regulates Th17/TR balance, critical for maintenance of peripheral tolerance and protection from autoimmune diseases. Cells which downregulate Foxp3 convert into effector Th cells (exTR cells) which produce proinflammatory cytokines and contribute to disease pathology in a number of autoimmune diseases. A recent RNA-seq transcriptome analyses of BMPR1α-deficient TR cells in situ showed upregulation of a number of epigenetic modifiers and transcription factors specific for effector Th cells including Med14, Supt16H, Setbp1, Tbx21 (Tbet) and Maf. These new data demonstrate that BMPR1α-deficient TR cells in unmanipulated mice are predisposed to follow epigenetic and transcription programme to dedifferentiate to exTR cells when subject to antigenic stimulation and inflammation. To gain mechanistic insight on the immunoregulatory role of BMPR1α we will combine ATAC-seq and RNA- seq analyses of epigenetic profile and transcriptomes to identify molecules involved in TR/exTR transition. We will test if TR/exTR transition can be regulated by pharmacological modulation of BMPR1α dependent signaling pathways. We have found that at the molecular level BMPR1a deficiency in TR cells led to upregulation of Kdm6b (Jmjd3) demethylase, an antagonist of polycomb repressive complex 2 (PRC2). We will examine if phenotype of BMPR1a deficient TR cells could be sustained by Kdm6b inhibitor. We will also generate mouse models to test additional compounds for their ability to impact BMPR1a signaling and modulate TR cell stability. In summary, proposed research will contribute to our understanding of immunoregulation and has the potential to outline new therapeutic strategies for inflammatory and autoimmune diseases.

NIH Research Projects · FY 2025 · 2023-06

Electrostimulation (ES) is a versatile and efficient tool for interrogating, altering, and manipulating neural activities in health and disease. Deep brain ES delivered with implanted electrodes requires an elaborate neurosurgery and carries risks of tissue damage, bleeding, stroke, infection, and inflammation. This limits the use of deep brain ES for disease diagnostics and conditions that may not justify the risks. Non-invasive targeted deep brain ES has long been a major quest, with countless potential applications. The challenge is avoiding stimulation near surface electrodes, where the electric field is the strongest, while stimulating at a depth by a (much) weaker electric field. One way to stimulate at a distance is by temporal interference (TI) of two high-frequency sine waves delivered with a small frequency shift. The interference of two such waves creates an amplitude-modulated stimulus at the target. Assumed demodulation of this signal by neurons leads to their excitation at the modulation frequency. Here, we introduce an entirely different concept of the temporal interference, based on (a) complete cancellation of identical frequency carrier signals at the target, and (b) on the introduction of transient distortions in one or both these signals. The distortions, such as a brief frequency or phase shift, will be concealed by the strong periodic signal near the stimulating electrodes and will not lead to excitation at the surface. However, these distortions will add up at the remote target location. They will stand out from the “silent” background and will readily lead to excitation despite the attenuation of the electric field with distance. We will perform mechanistic studies which support this next generation TI (NG-TI) stimulation paradigm. We will continue with the design and experimental evaluation of different NG-TI protocols in vitro, in comparison with the “standard” TI. We will systematically analyze the impact of TI stimulation parameters, to achieve targeted tuning and modulation of individual neurons and neuronal circuitry. We hypothesize that NG-TI can be improved for more focal stimulation, with much better penetration. It will have lower electric charge stimulation threshold and enable better steerability than the standard TI. The most efficient NG-TI protocols will further be validated by in vivo animal experiments. We will qualitatively compare targeting, possible off-site effects, current consumption, and steerability of NG-TI and the standard TI. We will also define the feasibility and model the electric field parameters for NG-TI stimulation at distances useful for medical applications. The effects will be linked to dielectric and physiological properties of neurons and neural tissue, to build predictive models for non-invasive deep brain stimulation in large animal and human trials. This project will lay the ground to translate the NG-TI technology for disease diagnosis and treatment.

NIH Research Projects · FY 2024 · 2023-05

PROJECT SUMMARY This exploratory proposal will be the first to characterize glycosylated RNAs in the context of human prostate cancer (PCa). PCa is the most prevalent form of non-skin cancer in males and second leading cause of cancer-related deaths among men. 20% of men diagnosed with PCa will progress to fast-growing, advanced disease. There are no curative treatments for PCa that has spread to distant sites and the 5-year survival rate for metastatic disease is only 30%. As high-grade PCa often leads to poor prognoses, it is imperative to better understand these pathologies and improve patient stratification methods as well as therapeutic treatment options to increase patient survival. Noncoding RNAs are widely misexpressed in PCa patients and act as key tumor suppressor genes, pro-oncogenic factors in the prostate. Noncoding RNAs harbor a large range of post- transcriptional modifications (m6A, inosine, pseudouridine, 2'-O-Me, A-to-I editing, 3’ uridine tailing) that are important for RNA maturation, folding, expression, and nuclear transport. Dysregulation of these post- transcriptional modifications are associated with tumor growth, invasion, angiogenesis, immune response and disease recurrence. Noncoding RNAs were serendipitously discovered to carry glycosylation modifications when cultured human and mouse cells were metabolically labeled using bioorthogonal chemistry methods normally employed for glycosylated protein and lipid enrichment. Glycosylation is an intricate process that typically involves the covalent attachment of carbohydrates onto proteins and lipids as the biomolecules move through the secretory pathway. Aberrant protein/lipid glycosylation contributes to tumor growth, metastasis, and immunosurveillance evasion and is being utilized as cancer biomarkers. We hypothesize that RNAs require glycosylation for cell signaling to maintain prostate homeostasis and cancer prevention and therefore the glycosylated state of RNA will correlate with prostate tumorgenicity. In support of this rationale, we confirmed using click chemistry and northern blotting that glycoRNAs exist and with differing abundance in human prostate cells. Specific Aims: This proposal will use two independent methods, metabolic labeling with azide click chemistry and lectin-based purification, to classify glycoRNA distribution from a panel of human prostate cell lines differing in their metastatic potential and hormone sensitivity. Aim 1 will unbiasedly determine if small versus large noncoding RNAs are preferentially glycosylated in the prostate, if these glycoRNAs share features for N- or O-linked sugars, and identify these species using RNAseq. In Aim 2, fractionation methods and chemical inhibitor studies will determine the PCa-associated glycoRNA subcellular localization, possible exosome enrichment and characterize these carbohydrate moieties via mass spectrometry. In Aim 3, the Ptenpc-/- Smad4 pc-/- double knockout mouse model will be employed to characterize glycoRNAs from living animals throughout a prostate cancer progression time course. This work will lead to novel insights into how RNA modifications impact PCa progression and identify first-in-class clinical tools to improve patient outcome.