Virginia Commonwealth University

NIH Research Projects · FY 2026 · 2023-09

Cystolic DNA (cyDNA), which is acquired in somatic cells, is emerging as an instigator/integrator of cellular functions associated with aging, yet the causes/consequences of cyDNA are poorly understood. Do individuals have a genetic predisposition to develop cyDNA or is its frequency most heavily influenced by environmental factors? Is cyDNA an early trigger for the acquisition of other age-related biomarker hallmarks, or does it arise in response to perturbations involving a subset of these hallmarks? To answer these primary questions, we will complete a longitudinal study (10 to 15 years timeframe) of 100 twin pairs [70 identical (MZ) and 30 fraternal (DZ) twin pairs; 200 individuals] who are discordant (35 MZ; 15 DZ) or concordant (35 MZ; 15 DZ) for cyDNA frequencies. The twin pairs will vary in age (currently 22 to at least 80 y.o) to allow us to chronicle associations between aging hallmarks and the acquisition of cyDNA. For each time point we will determine: (a) cyDNA levels, (b) chromosome specific-telomere/subtelomere lengths, (c) senescence markers, and (d) DNA methylation patterns in cells from two different tissues (blood and buccal mucosa cells [to assess potential soma-related differences]). Two measures of cyDNA will be quantified: (1) micronuclei (MN) frequency; and (2) extrachromosomal circular DNA (eccDNA) frequency. The MN frequencies will be identified for each of the 24 human chromosomes using a novel assay we developed that combines spectral karyotyping and fluorescence in situ hybridization technologies. The genetic contents of the eccDNA will be determined using our rolling circle amplification and sequencing protocol. Chromosome-specific telomere and subtelomere lengths will be determined using our Q-FISH method and our newly developed nanomapping method that exploits atomic force microscopy, CRISPR-Cas9, and our novel genome sequence algorithm to provide unprecedented resolution of telomere/subtelomere measures. We will also use “state of the art” tools we developed/optimized, to quantify telomere dysfunction; senescence (SADS, classical, and transcriptome studies), and genome-wide DNA methylation patterns. Using a method of robust variance component estimation (implemented in the FISHER quantitative genetics package), this study will provide the first measure of the extent to which individual differences in cyDNA and subtelomere lengths (which are associated with TERRA) are determined by additive genetic, common environmental, and specific environmental effects. We will also use “state of the art” statistical modeling and bioinformatic tools that we developed/optimized to analyze biomarker patterns within individuals, between co-twins, and among twin pairs to determine the stability of patterns with aging, and to identify temporal, as well as driver/mediator, relationships among cyDNA and other aging hallmarks (telomeres/subtelomeres, DNA methylation, senescence). The information gained from this study could also lead to the development of a health screening test(s) and/or identify new therapeutic targets that could transform our approach for developing treatments to alleviate symptoms of age-related health conditions.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Alcohol use disorder (AUD) and pain are major public health problems affecting millions of people in the United States. More than 14 million Americans met the diagnostic criteria for AUD in 2019, and 50 million people in the United States were estimated to have a chronic pain condition. These public health crises are related, as people experiencing pain often report using alcohol to manage pain symptoms, and people with chronic pain are at an elevated risk for developing AUD. Drinking alcohol to alleviate pain is a maladaptive strategy and may lead to withdrawal-induced hyperalgesia, peripheral neuropathy, and AUD. While alcohol confers analgesic effects in a dose-dependent manner, genetic differences mean that not every person experiences this analgesia to the same degree. Previous research has revealed moderate-to-strong heritability (h2=0.42) for ethanol-induced analgesia in C57BL/6J and DBA/2J mice, but the genes underlying this relationship are still poorly characterized. Studies in rodent models have revealed evidence that opioid receptor systems, GIRK2 potassium channels, and GABAergic signaling pathways all have roles in ethanol analgesia, but no whole- genome interrogations have been conducted. This project aims to identify candidate genes modulating alcohol- induced analgesia using complementary genetic, behavioral and transcriptomic experiments in genetically informative BXD mouse populations. By mapping quantitative trait loci (QTL) for both basal and post-ethanol hot plate latency with BXD strains, we will identify genomic loci linked with variation in ethanol-induced analgesia. As an additional genome-wide approach, prefrontal cortex and periaqueductal gray samples from high- and low-ethanol analgesia BXD strains will be studied by RNA-seq. We will perform weighted gene co- expression network analysis (WGCNA) to identify differentially expressed genes in these two analgesia groups. By combining evidence from behavioral, statistical, and molecular methodologies, we will select the strongest ethanol analgesia candidate gene for experimental verification using either overexpression or knockout mouse models. This proposal comprises an investigative progression from identifying candidate genes by QTL mapping, to elucidating gene expression patterns in critical brain regions, to validating ethanol analgesia candidate genes. It represents an outstanding training opportunity for the candidate to design and perform a suite of systems genetics and animal behavioral experiments and contribute to the scientific understanding of substance-induced analgesia.

NIH Research Projects · FY 2024 · 2023-09

Project summary/ Abstract The overarching goal of this K99/R00 proposal is to explore aggregate genetic liability and neurocognitive mechanisms in the co-occurrence of alcohol use/problems and suicidal thoughts and behaviors (STB). Excessive alcohol use is prevalent worldwide and represents a significant burden to human health; it is associated with medical and psychopathological problems such as STB. STB are a critical public health concern, with a continuing increase in suicide attempts and deaths every year in the US. The prevalence of STB is particularly high among individuals with alcohol problems and studies have tackled this question from different perspectives. Research indicates that alcohol problems could causally impact risk of STB; there is also empirical support for a shared genetic liability between alcohol use/problems and STB. This shared genetic liability underscores the existence of possible common mechanisms that would be involved in both alcohol use/problems and STB. Decision-making (DM) has been observed in relation with adolescent drinking behavior and alcohol use disorder, while both have been associated with STB. In STB research, DM difficulties have been described and may be characteristic of impulsive suicide attempts, but current findings largely rely on self-reports and lack objective evaluations. Additional knowledge could be gained by relying on a theoretical conceptualization of DM, a systematic evaluation of its underlying neurocognitive mechanisms, and a consideration of the role of genetic factors. Capitalizing on genetics and neuropsychology, we will explore which DM mechanisms play a role in alcohol use/problems and STB co-occurrence, how genetic liability is involved in this association, and whether environmental factors may influence the development of DM and its relation with alcohol use/problems and STB. An improved understanding of these processes will contribute to prevention and intervention efforts by advancing our ability to target potentially modifiable mechanisms according to the influence of genes and environment. This proposal delineates a series of training aims to advance our understanding of the co-occurrence between alcohol use/problems and STB: 1) the candidate will establish expertise in the assessment of aggregate genetic risks and other genetic models, and in advanced statistical methods that will lay the foundation of her independent career; 2) different suicide phenotypes will be used and distinguished according to their association with alcohol use, neurocognitive, and genetic characteristics; 3) the K99/R00 proposal gathers experts in the disciplines of genetics and neuropsychology that will support the development of the scientific project and the pathway to independence. The environment at the Virginia Institute for Psychiatric and Behavioral Genetics is ideal for the candidate’s goal of developing a comprehensive program in genetics, alcohol, and STB research, and the proposed project represents an important contribution toward advancing the understanding of alcohol use/problems and STB through a combination of genetics and neuropsychological methods, consistent with NIAAA’s missions.

NIH Research Projects · FY 2024 · 2023-09

Project Summary This new R61/R33 application responds to NOT-NS-22-095: “Development and Validation of Pain-Related Models and Endpoints to Facilitate Non-Addictive Analgesic Discovery.” In 2017, the National Institutes of Health launched the HEAL initiative, and one major goal of this initiative has been to develop new, non-addicting analgesics. Analgesic drug discovery depends on preclinical models of pain and analgesia for early-stage evaluation of candidate therapeutics, but conventional models that rely on reflex-withdrawal responses elicited by thermal or mechanical stimuli have significant deficits in face and predictive validity. To address these deficits, we have spent more than a decade developing and refining novel behavioral endpoints that focus on pain-related depression of normal behavior. Assays of pain-depresse behavior improve face validity because behavioral depression and functional impairment are cardinal signs of pain diagnosis, and restoration of normal function and behavior is often a major goal of pain treatment. These assays also significantly improve predictive validity by eliminating false-positive effects with drugs that produce motor impairment. This application seeks funding for further validation of a new procedure for use in mice that measures unconditioned locomotor activity in a complex environment as a new type of assay for pain-depressed behavior. Preliminary data suggest that the procedure elicits high and replicable baseline behavior, reliable depression by an acute visceral pain stimulus, and reversal of pain effects by clinically effective analgesics as positive controls but not by two prominent classes of non-analgesics as negative controls. The procedure also has attributes to promote replicability of results, efficiency of experimental design, assessment of sex as a biological variable, and quantitative objectivity of data collection. Proposed studies for further validation would proceed in two phases. In the first R61 phase (Year 1), we propose further studies to meet three milestones. (1) Further validate our procedure with additional positive and negative controls against our visceral acute pain model. (2) Evaluate selectivity of drug effects to restore behavioral depression by acute pain in comparison to behavioral depression by a non-pain treatment (lithium chloride). (3) Evaluate sensitivity of behavioral endpoints to models of more sustained cutaneous and visceral inflammatory pain (intraplantar complete Freund’s adjuvant, Ipl CFA; post-surgical abdominal-incision, AI). In the second R33 phase (Years 2-3), we propose to collaborate with colleagues at VCU and Wake Forest University to scale up and extend the model in three ways. (1) Extend from ICR outbred mice to an inbred strain commonly used to generate gene-altered mice (C57BL/6J). (2) Extend to three additional disease models of chronic pain (mononeuropathy pain using spared nerve injury, SNI; irritable bowel disease, IBD; sickle-cell anemia, SCA). (3) Evaluate independent replication of key results at a partner academic institution (Wake Forest University). Successful completion of the project would provide a comprehensive empirical foundation for broad use of this procedure in efforts to discover non-opioid non-addicting analgesic drugs.

NIH Research Projects · FY 2025 · 2023-09

A key problem in substance use disorders (SUD) is their etiological and functional heterogeneity, which is not well captured by the current psychiatric nosology. An influential neuroscience-based heuristic framework, Addictions Neuroclinical Assessment (ANA), proposes that to address this heterogeneity, the assessment of addictions should be multi-dimensional and focus on three key domains: executive function (EF), incentive salience (IS), and negative emotionality (NE), assessed with comprehensive batteries of self-report and neurobehavioral tasks. While computational tools have increased the knowledge extracted from these tasks, there are surprisingly few high-quality assays for monitoring and characterizing these domains. The burden of administration of current assessment batteries may take up to 10 hours and most assessment instruments lack precision in identifying underlying etiological mechanisms. Critically, most neurobehavioral and neuroimaging tasks have low test-retest reliability, which limits their utility for biomarker discovery. To address these limitations, we propose to apply Bayesian adaptive design optimization (ADO; Myung & Pitt, 2009) to established tasks that index the three ANA domains, with the goal of developing rapid, robust, and reliable neurobehavioral probes of these domains. ADO is a general-purpose computational machine-learning algorithm that optimizes data collection and extracts the maximal information from participant responses in the fewest possible trials. Our preliminary data show that ADO led to 0.95 or higher test-retest reliability of the delay discounting rate in under 1-2 minutes of testing, captured approximately 10% more variance in test-retest reliability, and was 3-5 times more precise and 3-8 times more efficient than conventional assessment methods (Ahn et al., 2020). The current study proposes to develop and evaluate a battery of ADO-based tasks, software, and mobile apps using state-of-the-science computational approaches that will significantly reduce the time for neurocognitive task administration, while increasing task reliability, precision, and efficiency. To capture the heterogeneity of addiction, this battery will be tested with neurotypical individuals and several populations with different types of SUD (opioid, stimulant, alcohol, and tobacco) in three countries (USA, South Korea, Bulgaria) where we have developed infrastructure for this type of research. This value-added perspective would be useful for out-of-sample validation of our models and allow us to address not only the generalizability of the ANA domains to different types of SUD, but also the cross-cultural generalizability of the domains, which has not been examined. The specific aims of the study are to: (1) Develop a battery of reliable and efficient ADO-based neurobehavioral tasks of the ANA domains and assess its test-retest reliability in neurotypical individuals; (2) Assess the predictive utility of the newly developed ADO tasks for SUD outcomes by testing patients with different types of SUD; and (3) Design web-based platforms and mobile apps for measuring cognition with the newly developed ADO tasks, and open-source software platforms with the ADO and other computational methods we develop.

NIH Research Projects · FY 2025 · 2023-09

Project Summary: Apoptosis is a type of programmed cell death and has for a long time been appreciated to be a hallmark of cancer cells. In recent years, drugs targeting the apoptotic pathway, such as the FDA- approved BCL-2 inhibitor, venetoclax, have revolutionized therapy in cancers which have a particular vulnerability to targeting this pathway. A different programmed cell death pathway, ferroptosis, has recently been discovered. Understanding which cancers may be vulnerable to the induction of ferroptosis and which targetable molecules are involved could lead to a new wave of successful cancer therapy. MYCN-amplified neuroblastoma (NB) is one of the deadliest subtypes of pediatric cancer. Here in, we demonstrate that amplified MYCN drives an aberrant iron capture program in NB and increases intracellular cysteine biosynthesis and selenocysteine dependence through multiple mechanisms to detoxify reactive oxygen species (ROS) accumulation as a result of high cellular iron. The consequence of these MYCN-directed changes is a synthetic lethality to genetic or pharmaceutical targeting of the glutathione/glutathione peroxidase 4 (GPX4) pathway resulting in ferroptotic cell death. This grant aims to expand our understanding of how MYCN alters cysteine and selenocysteine production and ferroptotic inducing pathways to sustain an antioxidant defense and how these pathways may be exploited pharmaceutically to improve therapeutic responses in this recalcitrant tumor type. Specific Aims: Aim 1: Characterize the ability of MYCN to suppress ferroptosis in neuroblastoma Aim 2: Identification of synthetic lethal ferroptosis resistance mechanisms in MYCN-amplified neuroblastoma Aim 3: In MYCN-amplified neuroblastoma mouse models, evaluate novel ferroptotic combination therapies Study Design: Using well characterized isogenic cell lines and patient-derived xenograft cell cultures, we will mobilize expertise in selenocysteine biosynthesis (Copeland), pantothenate kinase inhibitors (Rock), and genomic screening of ferroptotic pathway modifiers (Olzmann) to better define the ferroptotic vulnerability in MYCN-amplified NB and to uncover novel sensitizers to ferroptotic inducers in MYCN-amplified NB. The goal of these experiments is to not only better understand how the MYCN oncogene hijacks cysteine for selenocysteine production to mount a defense against an oxidized phenotype, but to create new therapeutics to create better anti-ferroptotic approaches in MYCN-amplified NB. To this end, we will work with our preclinical mouse model expert (Koblinski) and a NB clinical investigator (Glod) to build the preclinical evidence of synthetic lethal new therapies into the clinic for refractory NB patients.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT Organophosphate (OP) compounds include pesticides and chemical warfare nerve agents. They are highly toxic and can produce a cholinergic crisis that rapidly progresses into status epilepticus (SE) and even death without emergency care. The standard-of-care (SOC) treatment with atropine, pralidoxime, and midazolam has dramatically improved survival after OP intoxication. Yet, many survivors of OP-SE exhibit brain injury, cognitive impairments, and spontaneous recurrent seizures (SRS). In addition, both acute and protracted neuro-inflammation and increased expression of pro-inflammatory cytokines have been reported following OP SE. These persistent neuroinflammatory changes are thought to underlie neurodegeneration, network hyper- excitability, and maladaptive plasticity, leading to cognitive dysfunction and SRS. Thus, mitigating neuro- inflammation is a primary target in alleviating neuronal injury and behavioral morbidities following OP SE. Verapamil (VPM) is a water-soluble calcium-channel blocker for high blood pressure and angina treatment. Recent findings have also demonstrated the potent neuroprotective and anti-inflammatory action of VPM in various CNS injuries. Our preliminary results showed that intramuscular VPM (10 mg/kg, i.m.) was safe and produced significant neuroprotection and decreased neuroinflammation in multiple brain regions when administered after the termination of DFP SE. It was also associated with decreased pro-inflammatory and upregulation of anti-inflammatory cytokines. Finally, this effect had a functional outcome since VPM improved anxiety and cognitive impairment at eight weeks post-DFP SE. Thus, this UG3-UH3 will investigate and optimize a VPM therapy as a potential countermeasure for treating OP SE that could be rapidly administered in the field. Studies will employ DFP-SE rat model to conduct the Specific Aims: In Aim 1, the safety of repeated i.m. injections will be assessed along with an assessment of pharmacokinetic parameters and the stability of VPM formulation. In Aim 2, the effects of VPM treatment on reducing neuronal death and neuroinflammation after DFP SE will be evaluated utilizing Fluoro-Jade C along with Glial Fibrillary Acidic Protein and Ionized calcium-binding adaptor molecule-1 immunohistochemical staining, respectively. We will also assess the effect of VPM therapy on pro- and anti-inflammatory cytokine expression. In Aim 3, VPM therapy will be optimized by studying the effects of various VPM doses and treatment durations on neurodegradation and neuroinflammation. In Aim 4, the functional outcomes of optimized VPM therapy on long-term anxiety, cognitive impairment, and SRS will be tested using a battery of rodent behavioral assays and EEG techniques. We will also draft a preliminary target product profile (TPP) by the end of our studies. These studies will provide further insight into the role of neuroinflammatory mechanisms in mediating OP morbidities and optimizing a VPM-based anti-inflammatory therapy as a novel countermeasure drug to alleviate OP toxicities.

NIH Research Projects · FY 2025 · 2023-09

/// PROJECT SUMMARY \\\ Alcohol use disorder (AUD) is a medical condition characterized by an impaired ability to stop or control alcohol use despite adverse social, occupational, or health consequences. AUD is common with a 12-month and lifetime prevalence in the US of 13.9% and 29.1%, respectively. Gene expression studies provide promising opportunities to better understand AUD etiology. As cells differ in their function, gene expression will typically also differ across cell-types. When studying bulk tissue, failure to account for cell diversity has a detrimental impact on the ability to detect disease associations. For example, case-control differences will be “diluted” if they affect only one cell-type, may cancel out if the differences are of opposite signs across cell-types, and may be undetectable if the differences involve low abundance cells. Furthermore, identifying the specific cell- types from which the association signals originate is key to formulating refined hypotheses of AUD etiology, designing proper follow-up experiments and, eventually, developing novel clinical interventions. Our overall goal is to perform a rigorous study in post-mortem brain samples to detect differentially expressed genes in AUD at a fine-grained cellular level, and identify the genetic elements that regulate these differences. To achieve this goal, we propose a strategy that has become available with the recent evolution of single nucleus RNA sequencing (snRNA-seq) technology that can characterize the expression levels of thousands of individual nuclei with a single reaction. Our pilot data shows that our lab is able to produce snRNA-seq data of the highest quality and illustrates the promise of snRNA-seq to detect biologically relevant findings. Using careful selection procedures, we obtained a unique collection of relatively severe cases that were diagnostically homogenous while having minimal comorbidities, and that were matched to the controls on key variables. We will focus on three brain regions that are heavily implicated in AUD and capture different aspects of the disease. Study design features were guided by a series of power analyses that are grounded in empirical observations from our pilot study. To ensure robust results, findings will be replicated using snRNA- seq data from independent individuals and validated with a different technology. Finally, we propose a series of follow-up analyses aimed at identifying potential regulators of replicating findings, (functionally) characterizing existing robust AUD GWAS associations, and detecting blood biomarkers of AUD disease processes in brain. Successful completion of this project will yield (i) unprecedented insights into AUD disease mechanisms, (ii) possible blood biomarkers of genes differentially expressed in brain, and (iii) lay the foundation for functional follow-up studies and, eventually, novel highly specific clinical interventions for improving AUD treatment.

NIH Research Projects · FY 2024 · 2023-09

Project Summary According to the American Cancer Society, lung cancer is the second most commonly diagnosed cancer in men and women. Outside of the sex-specific cancers, lung cancer and melanoma have the greatest sex disparity, though the exact mechanisms behind these differences are not well understood. In the United States, men have a higher lifetime risk of developing lung cancer and are more likely to develop severe disease than women. Additionally, there are sex disparities in responses to treatment, such as men having better responses to immunotherapies over women. There are multiple factors contributing to this sex disparity including lifestyle choices, sex hormones, and differences in immune response. Our preliminary data shows that two mouse tumors models of lung cancer, CMT-167 and Lewis Lung Carcinoma (LLC), two models of non-small cell lung cancer (NSCLC), grow slower in female than in male mice. This sex difference is dependent on the ovary as tumors in ovariectomized female mice grow equivalent to those in male mice. Innate immune cells (macrophages and NK cells) and more specifically NKG2D receptor activity are required for reduced tumor growth in females. Multi- parameter flow cytometry analysis shows significant sex differences in NSCLC tumor resident innate immune cells. We show that these sex-disparities extend to several chemotherapy and anti-PDL1 immunotherapy treatments. In preliminary in vivo data we show that the sex-disparity in NSCLC tumor growth and the response to chemotherapy requires NK cells. Ex vivo NK cell killing assays show that preincubation of NSCLC cells with female serum, but not male serum or serum from ovariectomized females, stimulates enhanced NK cell activity utilizing the secreted pro-apoptotic factor TRAIL. This ex vivo NK cell assay provides a means to identify bioactive molecules. Based upon this preliminary data, we propose two Aims for our future studies. Aim 1 we will fractionate serum from female mice to identify a biological molecule from female serum required for LLC and CMT-167 sensitization to NK cell killing via TRAIL. The sensitization of LLC and CMT-167 tumor cells in female mice to the effects of TRAIL are proposed to contribute to the observed sex–differences in tumor growth and sensitivity to therapies (both chemotherapy and immunotherapy). For Aim 2, we will determine if the response to standard of care chemotherapies or anti-PD1 immunotherapy using orthotopic CMT-167 and LLC tumor models is dependent on sex. If a sex difference in response is observed, we will characterize the immune responses of male and female mice to lung tumors in the context of these drugs. In summary, this project aims to gain an understanding of the biological mechanisms behind the sex disparities in NSCLC progression and response to treatment, with the hopes of gaining knowledge to inform clinic decisions and treatment of the human disease.

NIH Research Projects · FY 2024 · 2023-09

ABSTRACT Organophosphate (OP) compounds include pesticides and chemical warfare nerve agents. They are highly toxic and can produce a cholinergic crisis that rapidly progresses into status epilepticus (SE) and even death without emergency care. The standard-of-care (SOC) treatment with atropine, pralidoxime, and midazolam has dramatically improved survival after OP intoxication. Yet, many survivors of OP-SE exhibit brain injury, cognitive impairments, and spontaneous recurrent seizures (SRS). In addition, both acute and protracted neuro-inflammation and increased expression of pro-inflammatory cytokines have been reported following OP SE. These persistent neuroinflammatory changes are thought to underlie neurodegeneration, network hyper- excitability, and maladaptive plasticity, leading to cognitive dysfunction and SRS. Thus, mitigating neuro- inflammation is a primary target in alleviating neuronal injury and behavioral morbidities following OP SE. Verapamil (VPM) is a water-soluble calcium-channel blocker for high blood pressure and angina treatment. Recent findings have also demonstrated the potent neuroprotective and anti-inflammatory action of VPM in various CNS injuries. Our preliminary results showed that intramuscular VPM (10 mg/kg, i.m.) was safe and produced significant neuroprotection and decreased neuroinflammation in multiple brain regions when administered after the termination of DFP SE. It was also associated with decreased pro-inflammatory and upregulation of anti-inflammatory cytokines. Finally, this effect had a functional outcome since VPM improved anxiety and cognitive impairment at eight weeks post-DFP SE. Thus, this UG3-UH3 will investigate and optimize a VPM therapy as a potential countermeasure for treating OP SE that could be rapidly administered in the field. Studies will employ DFP-SE rat model to conduct the Specific Aims: In Aim 1, the safety of repeated i.m. injections will be assessed along with an assessment of pharmacokinetic parameters and the stability of VPM formulation. In Aim 2, the effects of VPM treatment on reducing neuronal death and neuroinflammation after DFP SE will be evaluated utilizing Fluoro-Jade C along with Glial Fibrillary Acidic Protein and Ionized calcium-binding adaptor molecule-1 immunohistochemical staining, respectively. We will also assess the effect of VPM therapy on pro- and anti-inflammatory cytokine expression. In Aim 3, VPM therapy will be optimized by studying the effects of various VPM doses and treatment durations on neurodegradation and neuroinflammation. In Aim 4, the functional outcomes of optimized VPM therapy on long-term anxiety, cognitive impairment, and SRS will be tested using a battery of rodent behavioral assays and EEG techniques. We will also draft a preliminary target product profile (TPP) by the end of our studies. These studies will provide further insight into the role of neuroinflammatory mechanisms in mediating OP morbidities and optimizing a VPM-based anti-inflammatory therapy as a novel countermeasure drug to alleviate OP toxicities.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Alcohol use disorder (AUD) and posttraumatic stress disorder (PTSD) commonly co-occur, and this comorbidity is associated with higher consumption, treatment dropout, and risk for relapse. Urban populations of low socioeconomic status are particularly at risk for AUD and PTSD. Research on the etiology of co-occurring AUD and PTSD is needed in these understudied and low resourced populations to help address a disparity in the knowledge base. Directional models of comorbidity exist, self-medication and susceptibility, although there are major gaps (e.g., few studies testing direction of causation and or bidirectionality effects, lack of specificity of assessment, lack of test of sex effects). Additionally, comorbidity could be influenced by genetic risk as both AUD and PTSD are moderately heritable, overlap in latent genetic risk, and are genetically correlated in large genome wide association studies (GWAS; rG=0.35). Black persons are underrepresented in genetics research, and thus, genetically informed studies in this population are critically needed for equity in knowledge gained in this area. The current multi-method study will fill these gaps by conducting a genetically informative ecological momentary assessment (EMA) study using a longitudinal measurement burst design. Participants recruited through the Grady Trauma Project (GTP), which consists of high-risk inner-city residents. We will enroll a sample of 400 individuals and they will be asked to provide: clinical interview diagnostic data on PTSD, AUD, and comorbidities, detailed self-report measures including trauma history, social determinants of health, other risk and protective factors, and a saliva sample for GWAS. The EMA protocol will capture the temporal relations between PTSD and alcohol use phenotypes (e.g., consumption, binge, AUD symptoms, craving) and clarify not only who is at risk, but when the risk behaviors occur. Analyses will simultaneously test all three models of comorbidity (i.e., self-medication, susceptibility, shared risk) and will test for sex specific pathways. Following this initial period of EMA, a measurement burst design consisting of three EMA bursts, each spaced two months apart, will occur to examine the impact of time varying social determinants of health (e.g., new trauma, financial stress, racial discrimination) on the functional relationships found in the first aim. Lastly, the exploratory aim will conduct genome wide analyses with a focus on a novel multivariate genetic method, genomic Structural Equation Modeling (gSEM), which will be used to produce polygenetic risk scores (PRS) that index genetic risk for comorbidity of PTSD and AUD, and unique risk for each condition. PRS indexing shared risk between AUD- PTSD, unique to AUD, and unique to PTSD, will be incorporated into the best fitting models from the EMA analyses to determine if the phenotypic relations found are influenced by genetic risk. This study will advance our understanding of risk underlying co-occurring AUD and PTSD, which is imperative to the development of effective prevention and treatment programs, particularly among racially minoritized inner-city residents who are at increased risk for trauma exposure and subsequent AUD and PTSD.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY/ABSTRACT Decreased muscle and bone mass and strength resulting from musculoskeletal unloading (disuse osteosarcopenia) has long been associated with increased fracture risk, impaired bone healing and worse patient outcomes. Current disease modifying drugs are centered primarily on bone targeted therapies (anti- resorptives and PTH), and remain ineffective at targeting muscle loss that appears crucial for healthy bone repair and reducing fall risk. Although early reambulation and physical rehabilitation following bone injury is known to be beneficial for fracture healing and muscle recovery, there remains a gap in our knowledge of the appropriate mechanical loading regimens following osteosarcopenic fracture due to limited knowledge of how disuse affects fracture healing mechanobiology. In preliminary work, we have a developed a murine model of fracture healing during disuse by hindlimb unloading, with and without remobilization. This model recapitulates many clinical features of bone repair during disuse (decreased skeletal muscle mass, decreased radiographical callus formation) with new findings such as altered callus vascularity and osteoclastogenesis that are attenuated with reambulation. The aims outlined in this proposal seek to greatly expand upon our preliminary studies by using non-invasive loading modalities targeting muscle and or bone directly to determine the critical cellular and molecular mediators of callus mechanobiology during disuse. In the mentored K99 portion of this grant, we will utilize non-invasive optogenetics and direct tibial loading to determine optimal mechanical inputs to increase callus healing, biomechanical integrity, and muscle mass during disuse (Aim 1). Next using high-throughput techniques (RNAseq and flow cytometry), we will investigate the potential underlying mechanisms by which non-invasive loading affects callus mechanobiology during disuse (Aim 2). During the R00 phase, Dr. Buettmann will leverage recent mechanistic findings to determine the conditional role of mechanosensitive molecules in coordinating load-induced alterations in fracture healing during disuse (Aim 3). These insights will help bridge a significant gap in our understanding of how disuse alters callus mechanobiology and how mechanically-regulated molecules can be leveraged to improve fracture healing and rehabilitation in osteosarcopenic “high risk” patients. These findings, owing to the preclinical model’s translatability, could also have far-reaching implications for other pathologies associated with impaired fracture healing and altered mechanosensation such as aging, obesity/diabetes, and hormonal deprivation. Dr. Buettmann has assembled a mentoring team and collaborators with expertise in bone regeneration/osteoimmunology (Drs. Olivares-Navarrete), optogenetics and muscle-bone mechanoregulation (Dr. Megan Killian), musculoskeletal bioinformatics (Dr. Charles Farber), biomechanics (Dr. Hannah Dailey) and mechanobiology (Dr. Henry Donahue). This project will prepare Dr. Buettmann for an independent research career in musculoskeletal research by acquiring the necessary training and research data for an R01 equivalent award.

NIH Research Projects · FY 2025 · 2023-08

1 PROJECT SUMMARY 2 A primary goal of this K01 proposal is to equip the candidate, Dr. Alexander Lucas, with the expertise to become 3 an independent investigator who is able to utilize state-of-the-art imaging techniques to evaluate subclinical 4 outcomes that predict future cardiovascular (CV) events in cancer patients, and that respond to behavioral 5 adaptations (i.e., exercise). Prostate cancer (PC) patients undergoing androgen deprivation therapy (ADT) 6 experience deleterious changes in CV risk factors (body composition, exercise intolerance, metabolic 7 dysfunction). Exercise training interventions are an effective non-pharmacological approach for managing these 8 negative effects of treatment. Furthermore, the use of non-invasive methods to determine CV and 9 musculoskeletal contributors to exercise capacity can inform the design of more precise exercise prescriptions 10 to address specific functional deficits in men initiating ADT. However, while supervised exercise training is 11 effective, many men lack access to such programs and establishing whether home-based behavioral exercise 12 programs can effectively improve exercise capacity (peak VO2) is important. Dr. Lucas’s preliminary data from 13 PC patients and other populations of cancer survivors support the feasibility and potential of exercise training to 14 limit treatment side effects. The proposed research project will provide critical data important for determining 15 whether exercise training leads to improvements in aerobic fitness (peak VO2), and whether central and/or 16 peripheral factors and components of body composition are key determinants of peak VO2 among men 17 undergoing treatment with ADT. Our primary hypothesis is that a combined aerobic and resistance training 18 intervention will improve peak VO2 by maintaining cardiovascular function, enhancing oxygen extraction at the 19 muscle, maintaining lean body mass, and reducing accumulation of fat mass. We propose a 2-arm, randomized 20 controlled trial to test a 12-week home-based and remotely delivered exercise intervention among 21 60 men undergoing ADT for intermediate to high-risk PC (Aim 1). We will use highly sensitive, non- 22 invasive cardiac magnetic resonance (CMR) imaging techniques with cardiopulmonary exercise testing 23 (CPET) to test effects of the intervention. We will also determine the relative contribution of central and peripheral 24 components of peak VO2 and their association with body composition (Aim 2). Surveys and qualitative semi- 25 structured interviews will be conducted with men who complete or dropout of the intervention to assess 26 intervention acceptability and to refine both content and delivery of a future intervention to be tested in a larger 27 RCT. This work focuses on optimizing cardiovascular health in men diagnosed with prostate cancer – a growing 28 population. The proposed K01 will provide essential training for the candidate, who will work closely with an 29 expert mentoring team with regard to study design and execution and the Career Development Plan. A specific 30 goal of training is to also gain advanced skills in design and conduct of clinical trials thus supporting the 31 implementation of future programs aiming to improve health outcomes in diverse communities.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Racial discrimination has been identified as the primary social stressor for Black youth1 where 90% of Black youth (age 8-16) reportedly experienced at least one racial discriminatory encounter.2 As a result of these encounters, Black adolescents experience racial stress, or race-related transactions between individuals and their environment that are perceived as taxing or threaten well-being (e.g., racial microaggressions, threats of harm or injury, and witnessing harm to Black individuals).3,6 Racial stress has been associated with an increase in depression, anxiety, and psychological distress.4 The prevalence of racial stress among Black adolescents is alarming as it increases the likelihood of youth engaging in risky sexual behavior and substance abuse.16To address the pervasive and ubiquitous public health concern of the presence of racial stress in the lives of Black adolescents and their subsequent adjustment, the overarching goal of this NRSA proposal is to utilize a multi-method approach to (1) understand the nature of racial coping among Black adolescents, (2) identify cultural parenting processes that predicts racial coping among Black adolescents, and (3) examine the attenuating effects of racial coping on the association between racial discrimination and mental health (i.e., symptoms of depression, anxiety, and stress) among Black adolescents. Present understanding of racial coping among Black adolescents is limited due to (1) the lack of inclusion of physiological processes in racial coping measurement, (2) the use of variable-centered approaches in investigations of racial coping, and (3) the lack of inclusion of emotion socialization in parents’ racial coping socialization. Anticipated findings can ultimately inform the enhancement and tailoring of current and future racial coping and mental health interventions for Black adolescents. The current project leverages data from an existing longitudinal study on Black adolescents’ emotion regulation processes during middle school (grades 6-8) in Richmond, Virginia: Emotion Regulatory Flexibility among African American Adolescents Study (ERFAA; PI: Lozada, NSF CAREER Award: 2046607). The ERFAA study collects relevant data to test the specific aims of this proposal using the assessment of physiological and self-reported arousal and regulation while watching a video vignette of racial discrimination and self-reported measures of racial discrimination experiences depression, anxiety, and stress symptoms. Utilizing a person-centered approach will advance the literature by enhancing understanding of: (1) individual differences in racial coping and (2) how those individual differences contribute to the ways racial discrimination is associated with Black adolescents’ mental health. Additionally, I will examine a novel observational measure of parents’ socialization to examine how Black parents' messages about behavioral and emotional coping with racism predict Black adolescents’ racial coping profiles. Results from the proposed research can ultimately inform the enhancement and tailoring of current and future racial coping and mental health interventions for Black adolescents. Anticipated findings have implications for mental and physical health development research among Black adolescent populations and align with the key goals of the NIMH, and the NIMHD. Specifically this proposal includes the integrative investigation of biological, behavioral, and experiential factors during an important developmental period that can inform an understanding of the developmental pathways of health and psychopathology.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Atrial fibrillation is a highly prevalent condition resulting in disordered contraction of the atria (top chambers of the heart). It affects one in four individuals during their lifetime. It is known that the arrhythmia has a bidirectional relationship with dysfunctional atrial contraction, a condition which itself is becoming understood as a means by which individuals may develop heart failure. However, the majority of published literature to date involves examining atrial function while lying supine at rest, thus incompletely characterizing the varying states of exertion that one experiences during activities of daily living. Exercise-based cardiovascular magnetic resonance imaging is a powerful non-invasive diagnostic modality that can measure heart function during exercise and has the potential to uncover key mechanisms by which patients with atrial fibrillation may progress to overt heart failure – and whether current treatment techniques (namely, catheter ablation) may alter that trajectory. The broad goal of this application is to facilitate training of the principal investigator, Dr. Cory Trankle, MD, for a career as a patient-centered independent researcher who uses exercise-based diagnostic modalities (including magnetic resonance imaging), with a specific interest in characterizing mechanisms of exercise limitations such as left atrial dysfunction and identifying them as potential biomarkers and targets for therapeutic interventions. In addition to didactic coursework and subspecialty seminars, Dr. Trankle will lead a multidisciplinary team conducting a prospective clinical study investigating the effects that catheter ablation for atrial fibrillation has on atrial function during exercise. He will address three specific aims in patients with atrial fibrillation undergoing catheter ablation: (1) determine the effects of ablation on the ability of the left atrium to augment its function during exercise, (2) determine the impacts ablation on left ventricular performance during exercise, and (3) determine whether those changes experienced in left ventricular performance are indeed associated with changes in left atrial function. A better understanding of the impact of catheter ablation, a common treatment for atrial fibrillation, has on these cardiac structures during exercise has the potential to identify those at highest risk for heart failure incidence and lead to more targeted treatment in this highly prevalent disease.

NIH Research Projects · FY 2025 · 2023-08

Project Summary: Synovial Sarcoma (SS) is driven by the SS18-SSX oncofusion, and SS18-SSX is the only reoccurring mutation in SS. SS normally metastasizes, resulting in a 15-year overall survival rate of less than 50%. This presents a particular problem as 1/3 of the patients that are diagnosed are under the age of 30. Polychemotherapy has a modest and variable effect on patients, immunotherapy activity is unremarkable, and there are currently no targeted therapy options to combat SS. SS18-SSX remains undruggable despite clearly being the driving event in these cancers. Thus, SS requires entirely new therapeutic approaches. In light of this, we tried to identify potential novel therapies by assessment of full genome short-interfering (si)-RNA screen data deposited into the DepMap database and subsequent cell culture experiments with SS cell lines and patient-derived xenograft cell cultures. Through these efforts, we have identified a clinically actionable synthetic lethality with SSX-SS18 in SS, namely disruption of the post-translational modification, SUMOylation. We find SS18-SSX activates the SUMOylation, and disruption of this pathway with the in-clinic SUMOylation inhibitor, TAK-981, disrupts SS18- SSX function, induces DNA damage and shrinks SS tumors in mice. Specific Aims Specific Aim 1: Test a diverse set of synovial sarcoma mouse models for efficacy and safety of SUMOylation inhibition Specific Aim 2: Investigate the relationship between SS18-SSX and the SUMOylated proteome in synovial sarcoma Study Design: We will further characterize the sensitivity of TAK-981 (alone and with chemotherapy and BRD9 degrader) in SS cell culture models including patient-derived organoids, and in vivo, in orthotopic patient-derived xenograft (PDX) models and genetic mouse models of SS. Through a series of proteomic and biochemical experiments, we will further categorize the mechanism of SS18-SSX-dependent toxicity stemming from disruption of the SUMOylation pathway. We will further examine the role of SS18-SSX in activating the SUMOylation pathway and the mechanism of action of TAK-981 in SS, which involves disrupting the SS18-SSX- ncBAF transcriptome. In all, we will attempt to gather the preclinical evidence supporting the translation of SUMOylation inhibitors to treat SS.

NIH Research Projects · FY 2025 · 2023-08

Project Abstract Traumatic brain injury (TBI) produces significant pathology, including post-injury inflammation (particularly within the thalamus), that can lead to long-term morbidities. Microglia, the innate immune cells of the brain, are critical mediators of neuroinflammation that can have either neurotoxic or neurotrophic effects. Progress has been made investigating TBI-induced neuroimmune responses in rodents and therapies showing great promise have moved to clinical trials but failed to translate into beneficial interventions for humans suffering TBI. However, therapies targeting processes that occur in higher order mammals, with immune responses, cytoarchitecture, and metabolic rates similar to humans, such as pigs, would be more likely to translate to the clinic successfully. The neuroinflammatory progression following brain injury in pigs, however, is not well- understood. Our preliminary data demonstrated that microglial processes converge onto injured axonal swellings (microglial process convergence; MPC) in the thalamus of micro pigs, that is not recapitulated in rats, following diffuse TBI but does appear to occur in humans. Studies in mice indicate that this microglial process convergence (MPC) requires functional P2Y12R, however, the mechanisms behind MPC in higher order mammals is currently unknown. Both our preliminary data and previous studies indicate that MPC may be an ameliorative process, promoting axonal outgrowth acutely post-injury. Therefore, the goal of this study is to assess the roles of microglial changes on pathological progression in a pig model of diffuse TBI. Studies indicate that males have greater pro-inflammatory responses and less axonal outgrowth compared to females. However, there are no known studies evaluating sex as a biological variable in MPC. Accordingly, the current study will address the following specific aims 1) to evaluate the effects of inflammatory and P2Y12R modulation on MPC, neuronal survival and axonal outgrowth/retraction following injury and 2) to assess the prevalence of MPC in human brain tissue following diffuse TBI and other CNS injuries/diseases. To address these aims we will complete quantitative 3D assessments of multiplexed immunohistological samples for microglial-axonal interactions in vitro and in pigs to determine the degree of MPC in relation to axonal outgrowth/retraction changes, P2Y12R expression, and sex-related variability. As the prevalence of MPC following brain injury in the human population is currently unknown, we will also probe for the degree of MPC onto injured axonal swellings, neuronal survival, axonal outgrowth/retraction, and spatially resolved RNA profiles in a unique cohort of postmortem brain tissue. This study is significant because understanding microglial-neuronal interactions and repair mechanisms in higher order species and humans of both sexes will translate into therapeutics strategies for the treatment of TBI.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT – OVERALL The United for Health Equity-Living PDX Program (U4HELPP) seeks to develop >500 new patient-derived xenograft (PDX) models of cancers originated from the breast, pancreas, colon, liver, lung, and other advanced peritoneal cancers. These PDXs will be made from >60% of patients from underrepresented populations which our safety-net clinical system serves. To develop such a large number and diverse set of PDXs, we have enlisted the support of a set of surgeons that specialize in each cancer type. Once extracted, the tumors are immediately provided to the U4HELPP PDX Core which functions to propagate, distribute, and cryopreserve them. Successfully generated PDXs will be subjected to genomic characterization and quantitative ancestral profiling by the U4HELPP Bioinformatics Core so that we can better understand how ancestry contributes to genetics and responses to targeted therapeutics. The PDXs will be provided to two Research Projects, that will focus on either pancreatic cancer or breast cancer. The Pancreatic Cancer project will generate large-scale proteomics insights from over 200 PDXs to refine subtypes of the disease and identify more effective therapeutics from Black and White patients. The Breast Cancer project will utilize 20 PDXs that are Basal-like breast cancers, characterize their metastatic profiles, define proteogenomic pathways that mediate metastatic growth, and target metastases with synergistic investigational new drug combinations. The information from these PDXs and Projects will be shared with the U4HELPP Pilot Projects and Trans-Network Activities Core which will award Pilot Project grants to utilize the PDXs generated by us and our partnering PDXNet institutions. All of the PDXs and insights gained will be shared with the NCI with the goal of reducing cancer disparities. Through this team effort we are confident that we will be able to develop these unique resources and generate significant preliminary data that will support investigator initiated clinical trials at the Massey Cancer Center.

NIH Research Projects · FY 2025 · 2023-07

Colorectal, lung, and female breast cancers are major public health burdens and there are clear geographic and racial/ethnic differences in their incidence. Each of these cancers has been linked to neighborhood factors including socioeconomic deprivation, the built environment, and environmental pollutants in attempts to explain existing disparities in risk. While associations exist, most previous studies considered neighborhood exposures at only one time (e.g., time of diagnosis) and used only single measures of neighborhood exposures (e.g., neighborhood deprivation or singular environmental pollutants), which is a simplification of the multifactorial nature of cancer. As a result, the relative importance of exposure domains is unknown, effects may be underestimated, and a cumulative assessment of risk factors is lacking. Increasing interest in the exposome calls for a more thorough assessment of neighborhood exposures over time that could better explain the factors leading to differences in cancer risk. Therefore, we propose to study comprehensive neighborhood disadvantage (ND), a combination of socioeconomic deprivation, historical housing patterns, environmental pollutant, and built environment domains, to provide stronger evidence of neighborhood associations and identify risk factors that could be modified to eliminate geographic and individual-level variations. The important limitations of existing work that this proposal overcomes are 1) the temporality and extent of exposures (i.e., earlier life exposure, later life exposure, or cumulative lifetime exposure), 2) the identification of key neighborhood exposure variables for cancer incidence, and 3) consideration of different domains of the neighborhood exposome over time both independently and in combination to more comprehensively consider relationships with cancer. Our specific aims are to estimate exposure effects for ND domains over time and study the trajectories of these domain exposures and effects by cancer site and individual characteristics. We will bring together data from the Virginia and Pennsylvania state cancer registries, population based controls, residential histories, and exposure data documenting historical disadvantage indicators. Highly innovative aspects of our approach include: 1) examination of historical levels of exposure to several important neighborhood disadvantage domains and 2) the application of novel Bayesian statistical methods that our team has been refining for estimating neighborhood disadvantage and its effects. We hypothesize that our novel approaches to estimating neighborhood disadvantage will better explain variation in cancer incidence than existing methods and will identify the most influential exposure variables over time for each cancer. This study is highly significant as it is the first study to estimate neighborhood disadvantage effects for multiple cancers that considers cumulative risk from several historic exposure domains using residential histories. The expected outcomes of this research will be the identification of historic neighborhood disadvantage exposures associated with significant cancer risk to target for policy development and interventions and to help reduce variations in cancer risk.

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY In this new R01 application that is directly responsive to RFA-DA-23-015, our research team proposes to study the cell type specific molecular mechanisms regulated by fentanyl and methamphetamine polysubstance use at both the initiation and withdrawal stages of the substance use trajectory. We propose to overcome current limitations in polysubstance research by utilizing preclinical assays of drug-vs.-food choice procedures in male and female rats, which more fully capture the severity of polysubstance use seen in humans by modeling the behavioral misallocation and decision making between concurrently available addictive drugs and alternative non-drug reinforcers. We will combine these enhanced behavioral models with single nuclei RNA sequencing (snRNAseq) of the medial prefrontal cortex (PFC) and nucleus accumbens (NAc), key brain regions implicated in drug reinforcement and drug-taking, to capture and characterize the exact drug-induced molecular adaptations that occur in specific cell types, including non-neuronal cells. We will directly test the hypothesis that the synergistic action of combined fentanyl and methamphetamine use produces enhanced drug use behaviors and brain molecular adaptations that are distinct from what is achieved by either fentanyl or methamphetamine use alone; that this polysubstance synergy involves unique transcriptional adaptations by brain region, accumulates as a function of drug experience, and contributes to the behavioral misallocation towards drug use over more beneficial rewarding activities that is the hallmark of drug addiction. We will test this overarching hypothesis in two Aims. In Aim 1, we will uncover the impact of fentanyl/methamphetamine polysubstance use during the withdrawal phase of the substance use trajectory. We will use drug-vs.-food self- administration choice procedures for saline, fentanyl alone, methamphetamine alone, and fentanyl/methamphetamine combinations to uncover how an extended history of polysubstance use synergizes to increase somatic withdrawal effects and drug taking behavior while experiencing withdrawal. We will then perform snRNAseq in the PFC and NAc to interrogate the brain cell type specific transcriptional adaptations in these rats. In Aim 2, we will similarly perform drug-vs.-food choice procedures and snRNAseq of these brain regions to explore the emergence of behavioral and transcriptional adaptations at the of initiation of drug use experience. We will go on to compare our snRNAseq data from Aims 1 and 2 to understand how the fentanyl/methamphetamine polysubstance cell type transcriptional profile changes over the substance use trajectory. This project will reveal how fentanyl and methamphetamine synergize to produce maladaptive drug choice behaviors and brain cell type specific transcriptional responses at distinct stages of the substance use trajectory that are common barriers to recovery. Results gained from this project will inform the discovery of novel and more efficacious pharmacological agents to treat the core decision making process that is uniquely disrupted by polysubstance use.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract This project is focused on reactivation of p53 mutants by peptidase D (PEPD). p53 tumor suppressor is the most frequently mutated protein in cancer. Most p53 mutations are missense mutations, causing a single amino acid change in each mutant, and are clustered within its DNA binding domain. p53 mutations nullify its tumor suppressor functions and/or endow oncogenic functions. PEPD, also known as prolidase, is a dipeptidase important for collagen metabolism. However, we recently found that PEPD binds to both wild- type p53 (p53WT) and various mutants via their proline-rich domain (PRD) and that disrupting the binding by PEPD knockdown (KD) not only activates p53WT but also reactivates its mutants. This is a novel function of PEPD, which does not require its enzymatic activity. Our long-term goal is to advance the understanding of regulation and function of p53WT and its mutants. The objective of the present proposal is to delineate the reactivation of oncogenic hotspot p53 mutants by PEPD KD and to assess the tumor-suppressing activities of the reactivated p53 mutants. The central hypothesis of the proposal is that PEPD binds to nearly half of each p53 mutant in cells and that, while PEPD is not required for their oncogenic activities, disrupting PEPD binding to p53 mutants induces post-translational modifications (PTMs) of the mutants that cause their refolding and reactivation. We will test the hypothesis in three specific aims: 1) to determine binding of p53 mutants to PEPD and their reactivation by PEPD KD; 2) to determine the molecular mechanism by which PEPD KD reactivates p53 mutants, focusing on the roles of PTMs, K373 acetylation in particular, in driving refolding and reactivation of the mutants; 3) to determine the tumor-inhibitory activities of the reactivated p53 mutants. We will pursue these aims by focusing on some of the most common oncogenic p53 mutants in cancer, including conformation mutants (R175H, G245C, and R249S) and contact mutants (R248Q, R273H, and R280K). Cell lines and mouse tumor models, including patient-derived xenografts, will be used. PEPD KD will be achieved using siRNA and a doxycycline-regulated system. The proposed research is significant, because it may bring about a paradigm shift in understanding of the biology and regulation of p53 mutants, which in turn may offer innovative cancer treatment strategies. The expected outcome of this project includes: 1) showing that PEPD binds to nearly half of each p53 mutant in the nucleus and cytosol; 2) showing that disrupting the PEPD-p53 mutant complex by PEPD KD frees the mutant for PTMs which drive refolding and reactivation of the mutant, whether it is a conformation mutant or a contact mutant, and K373 acetylation by p300/CBP is key to this process; and 3) showing that the tumor-suppressive activities of the reactivated p53 mutants are similar to that of activated p53WT. As such, our research will bring to light a critical intrinsic reactivation mechanism of p53 mutants, which may have far-reaching implications in p53 research and may break new ground for developing novel cancer therapeutic strategies.

NIH Research Projects · FY 2025 · 2023-07

Hypoxemia and acute respiratory distress syndrome (ARDS) arising from direct lung injury are associated with a dysfunctional lung surfactant system; however, large clinical trials of surfactant replacement therapy have been unsuccessful in this population. The method employed for surfactant delivery in these unsuccessful trials was liquid bolus instillation, which often requires intubation, use of large liquid volumes (~500 ml) and subsequent mechanical ventilation often late in the progression of ARDS. A successful dry powder aerosol synthetic lung surfactant product would provide the advantages of early surfactant administration, potentially before the need for invasive mechanical ventilation (IMV), rapid and high dose delivery to the alveolar region, and improved efficacy compared with instillation based on preliminary animal model findings. The goal of this study is the preclinical development of a synthetic lung surfactant dry powder aerosol product (including delivery strategies, formulations and devices) for administration to adults experiencing hypoxemia or ARDS from direct lung injury in a rapid, efficient and safe manner while receiving different modes of ventilation support. Aerosol delivery strategies and devices will be developed and optimized for high efficiency aerosol administration during high flow nasal cannula (HFNC) therapy, noninvasive positive pressure ventilation (NPPV) and IMV. High efficiency aerosol administration will be enabled by a combination of a highly dispersible spray- dried powder formulation, a new positive-pressure dry powder inhaler (DPI), and an excipient enhanced growth (EEG) aerosol delivery strategy. Aerosolization performance and lung delivery efficiency will be established and optimized using a concurrent approach of realistic in vitro experiments and computational fluid dynamics (CFD) modeling. Animal experiments (in rats) will be implemented to determine appropriate levels of the surfactant protein analog and assess in vivo efficacy of the lead synthetic surfactant dry powder formulation in different models of direct lung injury. Specific aims of the project are as follows: Specific Aim 1. Develop a synthetic surfactant dry powder aerosol formulation that can be easily dispersed into a small particle aerosol with low air volume, exhibit hygroscopic growth, and enable stable product storage. Specific Aim 2. Develop and optimize surfactant delivery strategies and devices that enable safe, efficient and rapid aerosol administration to adults receiving HFNC, NPPV or IMV. Specific Aim 3. Test the efficacy of the lead synthetic surfactant formulations administered with an animal- version of the air-jet DPI using in vivo rat models of acute lung injury (ALI) mimicking bacterial infection, viral infection, and ventilator-induced lung injury. Outcomes and Impact. The proposed advances directly address multiple previous failure mechanisms related to instilled and aerosolized (liquid and powder) surfactants and are necessary to make aerosolized surfactant therapy for hypoxemia or ARDS with the option of early treatment a viable option in adults.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Low-value care, or the provision of care without net benefit, is associated with patient harm and billions of dollars in unnecessary medical costs.18,19 One example is the prescription of antibiotics for viral upper respiratory infections (URIs). It is estimated that over 50% of antibiotic prescriptions for respiratory infections are unnecessary and cost over $700 million per year.20 Antibiotics can increase antimicrobial resistance, and cause adverse events in 10% of patients.21 The Choosing Wisely initiative has been joined by over 80 specialty societies, including the American Academy of Pediatrics, to develop recommendations against low-value care. Despite these recommendations, low-value care continues to affect one in ten children every year.22 To plan for future de-implementation research involving claims-based audit and feedback, we selected three types of pediatric low-value care based on high prevalence22 and the ability to distinguish low-value care using claims data: 1) antibiotics for viral URIs, 2) vitamin D screening in healthy children and 3) antacid medications for infant reflux. It is not well understood to what extent providers are familiar with these recommendations against low- value care. Even if a provider is aware of recommendations against low-value care, there can be other barriers to de-implementation such cognitive biases that favor historical practices, discomfort with diagnostic uncertainty or unease in discussing low-value care with families.23 In Aim 1, we will first evaluate the patient (age, race, sex, insurance type), provider (medical specialty) and system-level factors (clinical setting, healthcare system, rurality) associated with the three different types of low-value care. In Aim 2, we will query providers about 1) knowledge of, 2) agreement with, and 3) actions regarding the three Choosing Wisely recommendations. We will use the Consolidated Framework for Implementation Research (CFIR) framework24-26 to interview a subset of providers about barriers to de-implementation. We will use this information to design a targeted and effective de-implementation strategy that can be used across the Commonwealth of Virginia. This proposal builds on Dr. Wolf's KL2 study related to the delivery of evidence-based, high-value care and her clinical training in general pediatrics. The Center for Clinical and Translational Research (CTSA) will support use of the All-Payers Claims Database (APCD) and mentorship by Drs. Krist and Sabo who have expertise in patient- oriented health services research and implementation science. During the study period, Dr. Wolf will address critical gaps of knowledge about what drives the delivery of low-value care in children and what prevents providers from de-implementing low-value practices. By engaging providers in her research, she will design an intervention that is consistent with their values, preferences, and goals. Ultimately, this proposal will allow Dr. Wolf to build a larger research program to investigate system-oriented, patient-centered solutions to reduce low- value care for children.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract New antibiotic targets are needed to combat antibiotic-resistant bacteria. A new potential drug target is a phage-related ribosomal protease (Prp), which is used by a variety of Firmicutes pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, and Clostridioides difficile. These pathogens use Prp for ribosomal maturation. Prp is a cysteine protease that cleaves an N-terminal extension of the ribosomal protein L27, and in the case of S. aureus, Prp is essential for survival. Since most bacteria do not contain this N- terminal extension on L27 and do not encode Prp, targeting it should lead to less killing of commensal bacteria that are important for human health. Different pathogens that use Prps have distinctive L27 cleavable sequences. In this proposal, we will study the cross-selectivity and kinetics of L27 sequence cleavage by different Prps. We will identify the minimal L27 N-terminal sequence required for recognition and processing by Prp, and the L27 N-terminal sequence length required for cross-species Prp selectivity. These lessons will be applied to the synthesis of Prp activated prodrugs of ciprofloxacin and eperezolid. These prodrugs will be tested for activity and selectivity against the Prp-containing pathogens S. aureus, S. pneumoniae, and C. difficile, as well as the Prp-containing commensal bacteria Lactobacillus rhamnosus. These studies will greatly increase our knowledge of the selectivity and reactivity of Prps, and allow for the targeted killing of bacteria that use them.

NIH Research Projects · FY 2026 · 2023-07

Alcoholic liver disease (ALD) remains the most common chronic liver disease worldwide. Despite extensive studies, no FDA-approved therapy is available for any stage of ALD due to the limited understanding of disease pathogenesis. Therefore, the unmet need to identify novel targets for developing effective therapeutics against ALD is urgent. Alcohol not only alters hepatic lipid and bile acid metabolism but also disrupts the gut microbiome and intestinal barrier function, which results in a leaky gut and bacterial translocation as well as activation of systemic and hepatic inflammation. We have previously reported that conjugated bile acids activate sphingosine- 1 phosphate receptor 2 (S1PR2), which further activates ERK1/2 and AKT. S1P is one of the most studied sphingolipids and is synthesized from sphingosine by either sphingosine kinase 1 (SphK1) or SphK2. S1P can regulate various fundamental cellular responses either as an intracellular signaling molecule or a ligand for five GPCRs, S1PR1-5. SphK2-generated nuclear S1P is a potent natural inhibitor of histone deacetylases (HDAC1/2). Activation of the stimulator of interferon genes (STING) also has been identified as critical signaling in ALD. A recent study reported that SphK2-mediated production of nuclear S1P in CD11b+ macrophages is a strong inhibitor of STING and suppresses the inflammatory response in alveolar macrophages. We have reported that SphK2-/- mice developed more severe fatty liver and hepatic injury in the NIAAA ALD mouse model. Hepatic SphK2 expression levels were markedly reduced in both ALD cirrhotic patients and ALD mouse models. Alcohol-feeding significantly increased intestinal permeability and bacterial translocation in SphK2-/- mice. Our preliminary data further showed that 1) alcohol-feeding induced more severe liver injury in the global SphK2-/- mice than hepatocyte-specific SphK2 knockout (SphK2Hep-/-); 2) intestinal epithelial cell-specific SphK2 knockout (SphK2IEC-/-) mice were more prone to alcohol-induced liver injury compared to SphK2fl/fl mice; 3) RNAseq analysis showed that chronic alcohol feeding significantly disrupted hepatic sphingolipid, fatty acid, and bile acid metabolism and activated inflammatory and fibrotic responses; 4) deletion of SphK2 inhibited the growth of intestinal organoids. Based on these key findings, we HYPOTHESIZE that disruption of SphK2/S1P-mediated signaling pathways in the gut-liver axis plays a critical role in alcohol-induced liver injury. Two specific aims are proposed to test the hypothesis. 1) To investigate the role and mechanisms of SphK2 in modulating hepatic lipid metabolism and inflammatory response under the conditions of acute and chronic alcohol-induced stress. 2): To define the role of SphK2 in modulating intestinal barrier function and to identify the cellular mechanisms by which SphK2 modulates the gut liver axis in response to alcohol-induced stress. Accomplishing these specific aims will significantly advance our current understanding of the tissue and cell-type-specific roles of SphK2/S1P-mediated signaling pathways in ALD, which holds great promise for developing novel therapeutic interventions not only for ALD but also for other related metabolic diseases.