Virginia Commonwealth University

NIH Research Projects · FY 2026 · 2024-05

Opioid use disorder (OUD) is a leading cause of death in the United States, with overdose-related mortality rates remaining high nationally. Medications for OUD are life-saving, and increasing their utilization and reten-tion in treatments for OUD is the most effective pathway forward to combat this public health crisis. Comorbid conditions common among people with OUD, such as insomnia, hinder OUD treatment retention and benefit, rendering patients at persistent overdose risk. Insomnia is prevalent in over half of patients receiving medica-tion treatment for OUD, representing an ideal target for adjunctive treatments to medications for OUD aimed to improve health and recovery outcomes. Cognitive Behavioral Treatment for Insomnia (CBT-I) is considered the gold-standard treatment for insomnia, yet its evidence is limited in OUD populations. Historically, behavioral therapies integrated into OUD treatment have demonstrated improved effectiveness when they are adapted to patients’ specific needs. The objectives of this R34 application are to develop, refine, and preliminarily test the effects of a novel, integrated approach to treating insomnia in adults with comorbid opioid use disorder and in-somnia. We will develop, pilot test, and refine a Cognitive Behavioral Therapy for Insomnia and Opioid use dis-order, called CBT-IO. The proposed study will tailor CBT-I to OUD patient-reported needs and preferences gained from preliminary qualitative data. We will adapt the CBT-I content (OUD specific information, such as anchoring sleep schedules to administration of OUD medication) and its delivery (utilizing telehealth vs. in per-son options for sessions based on factors that impact treatment engagement). We will take a multi-disciplinary approach to the intervention’s development and testing, incorporating an expert panel review, as well as pa-tient pre-testing using both qualitative and quantitative methods to evaluate feasibility, acceptability, and en-gagement outcomes. We will then examine the preliminary effects of the newly developed and refined interven-tion in a sample (N=56) of adults with comorbid insomnia and OUD using a clinical trial design. Outcomes will include both sleep and substance use outcomes. Specific Aims include: AIM 1: Develop and refine a CBT-I intervention (CBT-IO) for individuals with OUD, which includes adapted content and a decision aid to guide in-dividualizing treatment delivery route (telehealth vs. in person) based on patient-reported factors that impact treatment engagement; AIM 2: Evaluate preliminary efficacy of CBT-IO to improve insomnia and OUD out-comes for individuals receiving medication treatment for their OUD. Results will inform large scale studies to determine best methods for treating insomnia and OUD in treatment seeking adults along with supporting NIDA’s goal of advancing personalized medicine in addictions.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY Periodontitis is one of the most common human health conditions and is a chronic inflammatory disease affecting the tooth-supporting structures resulting from the actions of polymicrobial communities of organisms that induce destructive immune responses. Progression of periodontitis is associated with a microbial population shift and synergistic interactions between pathobionts such as Porphyromonas gingivalis and Treponema denticola. T. denticola emerges as one of the most abundant bacteria in diseased periodontal sites and is predictive of the severity and progression of periodontitis. The mechanisms that allow T. denticola to thrive in the diseased periodontium remain poorly defined. Two-component signal (TCS) transduction systems are ubiquitous sensory transduction systems in bacteria that often sense environmental stimuli to mediate cellular responses via genetic regulation. Our scientific premise is that TCSs allow T. denticola to sense and respond to changing periodontal environments, promoting persistence, interactions with other oral bacteria, and pathogenicity. We have initiated the characterization of the AtcS/AtcR and Hpk2/Rrp2 TCS of T. denticola, which represents half of the TCS encoded in the genome. We have characterized the AtcR binding motif and identified genes that contain the AtcR binding motif within their promoter. Interestingly, the promoter regulating the expression of the Hpk2/Rrp2 TCS is bound by AtcR. Yet, AtcR-mediated regulation of gene expression and the impact on T. denticola cellular processes are untested. We have observed that Hpk2 kinase activity is regulated by oxygen. However, the role of Rrp2 as a transcription factor and its regulon remains unstudied. Our ongoing studies indicate that both AtcR and Rrp2 may co-regulate the expression of genes with the alternative sigma factor, s54. However, no study has ever explored the role of s54 in T. denticola. The published literature supports our preliminary data suggesting that AtcR likely contributes to community interactions with P. gingivalis, and both AtcR and Rrp2 likely impact the fitness and virulence of T. denticola. Here, we propose three complementary Specific Aims that will fill these gaps in knowledge. Aim 1 will determine how phosphorylation of AtcR impacts DNA binding kinetic, affinity, and stoichiometry, while knockout of atcR in T. denticola will characterize the role of AtcR in gene regulation and physiology. Aim 2 will define the Rrp2 and s54 binding sites, demonstrate Rrp2 interacts with s54 to regulate gene expression and determine if Rrp2-mediated regulation responds to changes in environmental oxygen. Aim 3 will determine if AtcR and Rrp2 contribute to T. denticola colonization of the gingiva, induction of inflammatory markers, and alveolar bone loss in murine models of periodontitis. We will then determine if AtcR contributes to synergistic growth and pathogenicity with P. gingivalis. Completing this study will define half of the sensory transduction systems of T. denticola and elucidate the complex genetic regulatory mechanisms that allow for T. denticola to be a successful periodontal pathogen.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY In this revised R01, we propose to use novel synthetic transcription factors (TFs) delivered to the mouse nucleus accumbens (NAc), a key brain reward region, to uncover and manipulate the cell-type-specific brain molecular processes that distinguish stimulant versus opioid addiction. This will enable us to generate knowledge critical for the design of the next generation of drug-specific, anti-addiction medications. An existing body of literature suggests that, in response to use of addictive drugs, the function of key TFs within the cells that comprise brain reward regions drive transcriptional adaptations and lasting changes in drug use behaviors. Preliminary and published data point to NAc ZFP189, a member of the KRAB-domain containing zinc-finger (KZFP) TF family, as highly sensitive to cocaine use, and causal in worsening stimulant- but not opioid-related behaviors. This suggests that the NAc ZFP189 TF is uniquely sensitive to, and contributes to, the brain adaptations that worsen stimulant addiction. Here, we have reprogrammed ZFP189 to create synthetic TFs, each possessing functional moieties that exert distinct forms of transcriptional control at in vivo target genes. This enables us to uncover how the stimulant versus opioid use experience differentially primes the epigenetic status of NAc cells to facilitate the function of drug-specific TFs. In Aim 1 we will virally deliver synthetic ZFP189 TFs to the NAc of mice, dose with repeated stimulant or opioid treatments, and perform multiome single nuclei ATAC and RNA sequencing. This will reveal the biological mechanisms through which stimulant versus opioid exposure enables drug-specific TF function, the transcriptional adaptations that facilitate specific drug addictions, and the NAc cell-types in which this occurs. In Aim 2, we will virally deliver synthetic ZFP189 TFs to mouse NAc and investigate their contribution to stimulant versus opioid conditioned and drug self- administration reward-related behaviors. This will reveal how drug-specific transcriptional neuroadaptations may differentially drive a worsening of stimulant- versus opioid-related behaviors. Lastly, in Aim 3, we will introduce novel synthetic TF co-factors, capable of re-programming the in vivo gene-regulatory function of the entire KZFP TF family, to interrogate how distinct KZFP members are recruited to worsen stimulant- versus opioid addiction. To accomplish this, we will virally deliver variants of the KZFP co-factor, TRIM28, to simultaneously dysregulate members of the KZFP TF family within the NAc. We will perform self-administration for stimulants and opioids and subject tissues to single nuclei RNA sequencing. This will uncover the collective contribution of NAc KZFPs to the worsening of stimulant and opioid drug behaviors and reveal the KZFPs, and their regulated genes, that most drive these damaging effects. Together, this research will provide new approaches to identify the TF functions at the core of specific drug addictions and yield refined gene candidates as targets for future drug-specific medications.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Animals can modulate learning in response to differing internal and external environments. Layering context onto learned information can enhance the diversity and specificity of memories, and can allow efficient access to situation-specific behaviors. In state-dependent learning (SDL), information learned by an animal while it is in a particular internal “state” is most effectively recalled when the animal is tested in the same internal state. Drug intoxication-induced SDL can be observed across diverse animal species, including humans. We will employ a model of ethanol intoxication-induced SDL that we have developed in the nematode Caenorhabditis elegans to define the molecular mechanisms that are required for SDL to occur. The simple and highly conserved nervous system of C. elegans provides an excellent model in which to study the molecular events that underlie SDL. When worms experience an attractive olfactory cue in the absence of food, they can learn to associate the odorant with starvation and attenuate their response to it in a process called olfactory learning (OL). We have shown that OL can become state-dependent; when animals undergo OL while they are intoxicated, they only express the learned behavior when they are tested in the same intoxicated state. We have shown that the internal intoxication state is encoded by a secreted peptide signal and its receptor during OL, but, surprisingly, this signal does not encode intoxication state during recall of OL. Here, we will identify the mechanisms by which state is encoded during recall of state-dependent OL. We will define the neural circuit in which SDL occurs and the inputs into it that signal state during both learning and recall. We will distinguish between two explanations for the lack of expression of state-dependent OL when the animals are tested in a non-intoxicated state: forgetting (loss of the learned information) or failing to recall (the learned information remains, but the memory is only accessible when the animals are intoxicated). We will determine if other drugs besides ethanol can confer state-dependency on OL. Finally, we will probe the generalizability of SDL beyond OL by asking if learning paradigms that use other neural circuits and signaling systems can become state dependent. Successful completion of these aims will provide novel molecular insight into how context cues are added to learned information.

NIH Research Projects · FY 2026 · 2024-04

HIV associated neurocognitive disorder (HAND) affects ~50% of people living with HIV (PWH), and concurrent opioid use disorder (OUD) can enhance symptoms. Since CNS pathology in HIV associated neurocognitive disorder (HAND) bears similarities to Alzheimer’s disease (AD) and other ‘tauopathies’ in which accumulation of abnormally phosphorylated Tau protein (pTau) is considered a critical factor in the development/progression of cognitive symptoms, it is reasonable to think that pTau may have a role in driving cognitive dysfunction in HIV/HAND. Compared to AD, cognitive changes in HAND are measured earlier and only ≤5% result in dementia. While post-mortem studies have documented pTau species within HAND+ and OUD brain tissue, and we see pTau deposition in animal models of HAND/OUD that exhibit cognitive decline, the question of whether pTau triggers deficits in neuron function that underlie HAND/OUD is not clear. The studies proposed here will clarify whether pTau has a causal role. Our preliminary and published work shows that multiple pathologic forms of pTau are present in specific brain regions of a murine model of HAND, and that pTau levels are increased by 2- 8 wks of concurrent morphine exposure. These findings coincide with cognitive issues and motor dysfunction, as well as in situ physiological changes in neurons in specific brain regions, including hippocampus. We propose functional studies to connect these interrelated findings, testing the hypothesis that tau/pTau is a critical driver of cognitive dysfunction in HAND and the exacerbation by opioids. Studies evaluate (i) CA1 pyramidal cell (PC) output; (ii) a CA1 microcircuit of interneurons that are vulnerable to Tat and express µ-opioid receptor. Functional assessments use (i) patch-clamp physiology, and (ii) miniscopes to follow activity of individual neurons for 12 weeks in awake, behaving mice (inscopix.com). Physiological (patch clamp), histological and biochemical damage are evaluated on tissue from individual behaving mice so that all outcomes can be directly correlated. While pTau is elevated in several brain regions, we focus on hippocampus, an area critical to normal cognitive processes which is clearly involved in a multiple conditions of cognitive decline. Two transgenic models of HAND in virally suppressed PWH that exhibit cognitive dysfunction (inducible CNS expression of HIV Tat; Tg26) are used to suggest relative contributions of Tat vs other HIV proteins in HAND. Longitudinal, 12 week studies evaluate gain- (Cre/AAV) and loss-of-pTau function on cognitive behavior and neuron dysfunction/damage in both sexes. The loss-of-pTau studies employ 2 models. The primary model is treatment with TRx023, an anti- pTau drug with significant effects in a Phase III clinical trial (TauRx.com). Because it is given during the disease process it is clinically relevant. The second model, judiciously undertaken based on TRx0237 results, is to create a new line of Tat+ or Tg26 mice crossed with a Tau-deficient transgenic (Tau-/-) that will eliminate Tau/pTau completely and constituitively. Overall, the studies represent a comprehensive approach that tests the role of pTau in HAND/OUD using a design for direct correlations between tissue level damage and cognitive decline.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Estrogen receptor (ER) signaling is the main driver of tumorigenesis in ER+ breast cancers by inducing proliferation and survival through genomic and non-genomic means. Therefore, inhibition of ER signaling has been a mainstay of treatment for decades. While primary ER+ breast cancer has a relatively good initial prognosis, ~30% of patients will develop treatment-refractory recurrence or metastasis within their lifetime, with bone metastasis being twice as likely for patients with hormone receptor positive disease. Despite the frequency of diagnosis, breast-to-bone metastasis of ER+ breast cancer remains an incurable condition. Treatment includes administration of ER antagonists like Fulvestrant or aromatase inhibitors, often in combination with a CDK4/6 inhibitor, which negatively regulates the cell cycle downstream of ER activity. These treatments are designed to slow progression of disease but are incapable of eliminating metastatic cells. A new endocrine- targeted therapy, Elacestrant, has been approved by the FDA for the treatment of ER+, ESR1-mutant advanced or metastatic breast cancer. Interestingly, this compound shows selective agonist activity in the bone microenvironment, and antagonist activity in cancer cells. Although Elacestrant has been approved by the FDA, the compound is understudied and is only approved for a fraction of patients. This proposal will utilize in vitro, in vivo, and ex vivo models of breast-to-bone metastasis of ER+ breast cancer to evaluate the efficacy of Elacestrant versus the standard of care, as well as dissect the transcriptional landscape which is promoted by its ER modulation in the treatment setting. Intra-Iliac artery injection of PDX cells in pre-clinical rodent models will be used to model metastasis and evaluate progression free survival following treatment with Elacestrant as a mono-agent, in combination with CDK4/6 inhibitors, as well as in novel therapeutic combinations. Because Elacestrant-induced ER-controlled transcriptional regulation is context dependent, NanoString GeoMx spatial transcriptomic sequencing, ER ChIP-seq, and rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) will be used to begin to determine how one drug can act as an antagonist and agonist in different contexts. The overarching hypothesis of this proposal is that Elacestrant will extend progression free survival in pre-clinical models of bone metastasis because it displays context dependent agonist and antagonist activity. This study will not only lead to a more complete understanding of ER biology in the bone metastatic microenvironment but will also provide insights which could lead to improved patient outcomes. This project will also afford me the outstanding training experience to work with multidisciplinary leaders across multiple fields of biology while I work to complete my PhD training at Virginia Commonwealth University.

NIH Research Projects · FY 2025 · 2024-04

Stigma, distress with pubertal changes, and societal expectations of beauty, are factors that can affect gender minority (GM) youths’ relationships with their bodies, eating, and exercise, and lead to disparities in eating disorders and other mental health conditions. GM youth are at higher risk of disordered eating behaviors (DEB) and experience unique cognitions that contribute to DEB; however, they have historically been excluded from DEB research. Additionally, extant DEB research with GM youth is limited due to a lack of representation of subpopulations of GM youth participants. These gaps in research limit the field’s ability to provide specific clinical care to a group with unique health disparities associated with DEB. To advance this care, we urgently need to extend DEB research to include GM youth. A novel approach to address these knowledge gaps is to implement community-engaged research methods to develop and evaluate a tailored measure of DEB in GM youth. The proposed health disparities research has three aims: 1. Obtain qualitative data from a sample of GM youth on facilitators of and barriers to research participation to improve recruitment protocols for the remaining aims, 2. Use focus groups and cognitive interviews to develop and refine a measure of DEB in GM youth, 3. Evaluate the psychometric properties of the DEB measure and assess correlations between DEB and GM-specific risk and protective factors. A GM youth advisory board and community consultant with expertise in DEB among GM youth will actively participate in research protocols throughout the 4-year grant proposal. Research activities include designing recruitment matrices, conducting qualitative interviews and analyses, and disseminating results. The data from this innovative study will inform a longitudinal study on changes in DEB throughout: 1) eating disorder treatment in GM youth with DEB and 2) pubertal development in GM youth. Dr. An Pham is an Adolescent Medicine physician with clinical expertise in eating disorders and working with youth with histories of health disparities like GM youth. This K23 Career Development Award will fill gaps in her training and provide her with the necessary mentorship and skills to become an independent investigator on youth-engaged DEB research. Specifically, Dr. Pham will work closely with her mentorship team and community partners to advance her knowledge and skills in qualitative and mixed methods community-engaged research, recruitment and retention of youth populations, and advanced statistical approaches to measure development and evaluation.

NIH Research Projects · FY 2026 · 2024-03

Emerging adulthood (18-25 years of age) represents a critical developmental window for lifestyle intervention given high rates of obesity, proinflammatory behaviors (e.g., dysregulated sleep, intake of highly processed foods, declines in physical activity), life stressors, and psychological symptoms—all of which serve to perpetuate chronic low-level inflammation, thereby increasing risk for cardiometabolic disease and many cancers. Despite this, few behavioral obesity management interventions have developed specifically for emerging adults (EA). Those that do exist have produced modest treatment effects, with EA women experiencing considerably less benefit than EA men. EA women are also at disproportionate risk for psychological dysregulation in the form of depressive symptoms and psychological stress. Further, our preliminary data suggest that exposure to life events, insufficient sleep, perceived stress, and depressive symptoms interfere with weight loss treatment response in this population. Importantly, this does not appear to be driven by differences in self-regulation behaviors; rather, we posit that the cognitive processing required for effective self-regulation is disrupted by inflammatory cytokine activity. Extant behavioral weight loss programs are insufficient to address this underlying behavioral/psychological dysregulation, and do not promote behavior change through the lens of inflammation. Thus, a new intervention model is needed to optimize clinical impact in this high-risk population. Our overarching hypothesis is that explicitly targeting psychological functioning and proinflammatory behaviors could not only enhance weight loss outcomes, but also confer greater improvements in inflammation and cardiometabolic health among EA women than those achieved by weight loss alone. We developed and tested an integrated lifestyle intervention (ILI) in a single arm proof-of-concept trial and observed clinically and statistically significant improvements in depression, anxiety, perceived stress, as well as weight. ILI consisted of BWL content adapted to meet the needs of EAs with an increased focus on changing discrete behaviors linked to inflammation in the absence of calorie goals, all intertwined with training in empirically-supported strategies to improve psychological function (e.g., cognitive restructuring, distress tolerance). Building upon these promising results, we propose a pilot randomized clinical trial of ILI compared to our developmentally adapted behavioral weight loss (BWL). We will recruit 32 EA women age 18-25 with BMI 25-50kg/m2 and randomize them to ILI or BWL. All participants will receive digital tools to facilitate self-monitoring and contact schedule will be identical between arms—the initial 4-month program will consist of group sessions via Zoom (8 weekly, 4 bi-weekly) with weekly tailored e-coaching, followed by monthly boosters through 12 months. Assessments will occur at 0, 4, 8 and 12 months to assess the preliminary efficacy of ILI to improve adiposity (Aim 1) and biomarkers of inflammation and cardiometabolic risk (Aim 2), and to explore putative psychological mechanisms of action (Aim 3).

NIH Research Projects · FY 2025 · 2024-03

Project Summary/Abstract Cells can perform their functions by spatiotemporally enriching biomacromolecues to form membrane-less condensates through liquid-liquid phase separation (LLPS). The formation of phase-separated condensates allows the cell to concentrate regulatory proteins with interaction partners to accelerate biological processes not only for cellular homeostasis but also for disease progression. Targeting condensates that promote dysregulated cellular processes has been suggested as a novel approach to developing new drugs. Studies have linked LLPS to super-enhancers (SEs), a transcriptional mechanism driven by an extremely high density of master regulators and Mediator complex that maintains the high expression of cell-type specific genes to determine cell identity, disease states, and most notably, oncogenesis. It has been implicated that the formation of phase-separated condensates of master regulators with Mediators is critical for activating the transcription of cell identity genes driven by SEs. However, how the master regulators undergo LLPS at SEs to maintain key oncogene expression is still largely under investigation, especially in HNSCC. In addition, no therapeutics are available for targeting SEs in cancer treatment, indicating the need for alternative strategies to be developed. We recently discovered that FOSL1 serves as a master regulator to promote the malignant progression of HNSCC through establishing SEs with Mediator complex (MED1) at key oncogenes. However, how FOSL1 drives key oncogene expression without a transcriptional activation domain is still not fully understood. Furthermore, the crystal structure of FOSL1 still remains unsolved and the structure-based drug design for FOSL1 inhibitors has been severely stifled. Currently, there are no FOSL1 inhibitors for cancer treatment. Using the AlphaFold program, we identified two intrinsically disordered regions (IDRs) in FOSL1. The presence of IDRs in a protein is frequently diagnostic of its ability to phase separate. We found that purified FOSL1 recombinant proteins can phase separate at in vitro conditions. Moreover, phase-separated condensates containing FOSL1 can be visualized in live HNSCC cells. Based on these results, we hypothesize that FOSL1 associates with MED1 to undergo phase separation to establish super-enhancers at key oncogenes and maintain their high expression and consequent malignant phenotype of HNSCC. We proposed the following two specific aims to validate our hypothesis. In Aim 1, we will determine whether FOSL1 cooperates with MED1 to assemble phase-separated condensates at in vitro conditions and also in live HNSCC cells. Furthermore, we will also examine these condensate formations at key oncogenes driven by SEs in HNSCC cells and patient-derived xenografts. In Aim 2, we will explore whether the deletion of IDRs in FOSL1 will abolish condensate formation to disrupt SEs to suppress the expression of key oncogenes and the consequent malignant phenotype of HNSCC. The successful accomplishment of this study will provide solid evidence to link LLPS with SEs and key oncogene expression in HNSCC, support a notion that targeting FOSL1 phase separation suppresses HNSCC tumorigenesis, and lay the foundation for future studies.

NIH Research Projects · FY 2026 · 2024-02

New cancer screening technologies with great promise for reducing cancer death are emerging. These technologies require large-scale controlled clinical trials to evaluate their effectiveness and assess the feasibility of adopting and implementing them into routine practice if effective. Furthermore, the National Academy of Medicine and US Preventive Services Task Force have identified dozens of evidence gaps in current cancer screening approaches that will need similar large scale randomized controlled trials and longitudinal studies.1-3 To address this, the National Cancer Institute (NCI) is creating a Cancer Screening Research Network (CSRN) with a network of Accrual, Enrollment, and Screening Site (ACCESS) Hubs capable of conducting clinical trials and longitudinal studies, a central Statistics and Data Management Center, and a central Coordinating and Communication Center. We propose to create the Virginia CSRN ACCESS Hub from long-standing, inter-institutional partnerships to assist in this important national effort. These partnerships include 1) two cancer centers – Massey Comprehensive Cancer Center at Virginia Commonwealth University (VCU), an NCI-designated cancer center, and Inova’s Schar Cancer Institute which includes a novel Saville Cancer Screening and Prevention Center; 2) two medical schools – VCU and the Eastern Virginia Medical School; 3) the NIH-funded Wright Regional Center for Clinical and Translational Science; 4) three health systems – VCU, Inova, and Sentara Healthcare; and 5) a state-wide primary care practice-based research network with over 500 member practices (Virginia Ambulatory Care Outcomes Research Network, ACORN). These entities have an established track record of working together for several decades; collectively care for a sizable proportion of state residents including the largest safety net health system providing both urban and rural services to a majority of Virginia’s communities; demonstrated expertise in recruiting and retaining a broad range of patients and communities in trials; and national expertise in cancer prevention, informatics, recruitment across communities, and dissemination and implementation science. The Virginia CSRN ACCESS Hub will initially recruit and retain at least 2,000 participants in the NCI-coordinated, three-arm randomized controlled Vanguard study to test two novel multi-cancer detection (MCD) tests compared to usual cancer screening care. We will measure our hub’s success by generalizable recruitment, timeliness and adherence to the diagnostic workup and treatment protocols, and participation in all national CSRN efforts. Additionally, we will conduct a novel mixed methods analysis of our work using a consolidated framework for implementation research (CFIR) constructures to demonstrate our hub’s feasibility and sustainability for future CSRN trials and to assess factors necessary for widespread adoption of MCD tests, if proven effective. Our hub will meaningfully contribute to the Vanguard study and will bring unique partnerships, a generalizability lens, and dissemination and implementation expertise to the broader CSRN effort.

NIH Research Projects · FY 2025 · 2024-02

ABSTRACT Human papillomaviruses (HPV) are causative agents in ano-genital and head and neck cancers and are responsible for around 5% of cancers worldwide. In order to identify and develop novel approaches for targeting these viral diseases, we must enhance our understanding of the viral life cycle. HPV16 is the most common type that causes cancer (high-risk, HR-HPV). During its life cycle, the virus exists as an 8kbp DNA plasmid. A viral protein that mediates viral replication is E2, a DNA binding protein that forms homodimers that bind to 12bp palindromic DNA target sequences. E2 has three functions during the viral life cycle: first, it binds to target DNA sites around the viral origin of replication and recruits the viral helicase E1 to the origin. E1 then forms a di-hexameric complex that initiates viral replication in association with host cellular factors. Second, E2 mediates segregation of the episomal viral genome into daughter nuclei during cell division by simultaneously interacting with viral and host chromatin acting as a bridge to locate the viral genomes to daughter nuclei. Third, E2 can regulate viral and host transcription. The focus of this proposal is on how E2 regulates transcription from the host genome to facilitate the viral life cycle. Using RNA-seq data from isogenic N/Tert-1-Vec (Vector control), N/Tert-1+E2 (expressing the E2 protein only) and N/Tert-1+HPV16 (containing the episomal viral genome, this model demonstrates several aspects of the HPV16 life cycle in organotypic raft cultures) we demonstrated that E2 regulates host transcription that is important during the viral life cycle. This proposal will focus on E2 repression of innate immune gene transcription (IIG) and suppression of epithelial to mesenchymal transition (EMT) via repression of TWIST1. The repression of IIGs is via DNA methylation and here we demonstrate that E2 is in a cellular complex with DNMT1, providing a potential mechanism (E2 recruitment of DNMT1 to IIG promoters) for E2 repression. E2 represses TWIST1 via a direct interaction with the TWIST1 promoter; DNA methylation is not involved. The TWIST1 promoter is activated by STAT3 and here we demonstrate that E2 interacts with STAT3, providing a potential mechanism of how E2 is repressing TWIST1. In this proposal, we will investigate the repression of IIG IFIT1. The IFIT1 protein can bind to and disrupt HPV18 E1-E2 replication, and we have demonstrated that it binds to HPV16 E1, and preliminary studies demonstrate it can attenuate E1-E2 replication. Using novel CRISPR technology we have increased the levels of IFIT1 in a host of HPV16 and E2 positive cells. We will investigate whether elevated TWIST1 interferes with the HPV16 life cycle. We will carry out similar experiments with TWIST1. Recently we demonstrated that E2 sensitizes cells to cisplatin, an important process that could contribute to the better outcomes for E2 expressing HPV+ cancer patients. We will investigate the roles of IIGs and TWIST1 in this process. Overall, our results will advance our understanding of how E2 regulates host transcription, and provide potential novel therapeutic approaches for combatting HPV16 infections and cancers.

NIH Research Projects · FY 2025 · 2024-01

Type IV pili (T4P) are extraordinarily widespread surface appendages made by many species of extant archaea and bacteria as well as by the last universal common ancestor. T4Ps are assembled by a complex, multi-component machine that consumes energy from ATP hydrolysis. Many human pathogens require T4P for virulence. Volunteer studies have proven that T4P of enteropathogenic Escherichia coli (EPEC) and Vibrio cholerae, which make type IVb pili (T4bP) that diverged from other T4P early in evolutionary history, are required for illness in people. Despite remarkable progress, the molecular details of T4P assembly remain obscure. The overall structure of the T4P machinery is remarkably conserved, but closer investigation reveals fundamental differences among systems. Which findings from convenient model systems can be extrapolated to pathogens remains unclear. For example, we recently solved the structure of the BfpD extension ATPase from EPEC to 3.0 Å resolution using cryo-EM. In contrast to the two-fold symmetry and complex enzyme kinetics of the distantly-related extension ATPases from thermophiles, the BfpD ATPase has six-fold symmetry and simple kinetics, consistent with a concerted mechanism of catalysis rather than the symmetric rotary mechanism proposed for its counterparts. In the current brief, circumscribed multi-investigator proposal, we aim to determine to what extent the structure and mechanism of catalysis exhibited by BfpD is representative of other potentially lethal human pathogens. To do so, we have purified the extension ATPases from four other pathogens. Since initial (A0) submission of this revised (A1) proposal, we collected cryo-EM images of the PilB extension ATPase from Pseudomonas aeruginosa. The 2D classes from these images demonstrate unambiguous two-fold symmetry like the structures obtained from thermophiles with which it is closely related. Furthermore, the structure of one such enzyme, which was derived from X-ray crystallography, fits well into our 3D PilB model, currently at 3.4 Å resolution. In the first aim of the current proposal, we will use cryo-EM to determine the structure of the TcpT ATPase from Vibrio cholerae, which is more closely related to EPEC BfpD than to those from thermophiles or P. aeruginosa. This structure will reveal whether it has two-fold, six-fold, or perhaps even three-fold symmetry. In Aim 2 we will perform careful enzyme kinetic studies to determine whether each of the four additional extension ATPases that we purified exhibits classic Michaelis-Menten monophasic kinetics like BfpD or complex multiphasic kinetics like the extension ATPase described for a thermophilic bacterium. Differences among diverse T4P systems indicate that efforts to develop new approaches to prevent and treat lethal bacterial pathogens will benefit from a better understanding of which essential features of T4P are specific to a subset of the organisms and which are universal. As the extension ATPase is a potential target for anti-virulence therapeutics, the efforts described herein will provide important information regarding its mechanism of action in pathogens.

NIH Research Projects · FY 2026 · 2024-01

Project Summary: High-risk neuroblastoma (NB) is responsible for ~15% of pediatric cancer-related deaths, with nearly half of these characterized by MYCN amplification. The addition of anti-GD2 antibodies and retinoids to maintenance therapy has improved the outcomes of these patients, but still more than 50% perish. Omics studies in NB tumors has revealed that most NBs are driven by an adrenergic (ADRN) core regulatory complex (CRC) of transcription factors that can be driven by MYCN and includes GATA-3, TBX2, HAND2, PHOX2B, ISL1 and ASCL1. Genomically targeting the CRCs has been demonstrated to be highly effective, however a pharmaceutical way to target the CRCs and in particular one that would be tolerated by the patient has not been developed. Here we demonstrate a remarkable therapeutic vulnerability of MYCN-amplified NB to SUMOylation inhibition. We provide evidence that toxicity involves disruption of the ADRN CRC which requires the presence of MYCN. This pathway has become actionable, with the advent of the SUMOylation inhibitor, TAK-981 (Subasumstat) now in multiple clinical trials. Thus, we propose we have uncovered an ADRN CRC inhibition strategy that is effective in MYCN-amplified NB, suggesting a real therapeutic window. Specific Aims Specific Aim 1: Test a diverse set of well-annotated high-risk patient-derived xenograft (PDX) models for efficacy and safety of SUMOylation inhibition in neuroblastoma alone and combined with maintenance therapy Specific Aim 2: Investigate the mechanism of adrenergic (ADRN) core regulatory complex (CRC) disruption following SUMOylation targeting Study Design: We will further characterize the high sensitivity of TAK-981 in high-risk MYCN-amplified NB patient-derived xenograft (PDX) models either alone or in combination with the anti-GD2 therapy, dinutuximab. Through a series of genomic and proteomic studies, we will further categorize the mechanism of sensitivity to SUMOylation inhibition. As TAK-981 induces NK cell activation, it is a rational inducing partner with the anti- GD2 immunotherapy, dinutuximab. We will also investigate this combination in NB mouse models. In all, we will attempt to gather the preclinical evidence that TAK-981 should be clinically evaluated in MYCN-amplified NB patients.

NIH Research Projects · FY 2025 · 2024-01

Summary Latently infected brain myeloid cells including microglia (MG) and perivascular macrophages can serve as HIV reservoirs, contributing to NeuroHIV persistence, chronic neuroinflammation and HIV-associated neurocognitive disorders (HAND). Strategies aimed at eliminating HIV reservoirs are highly promising to cure HIV, even in the presence of effective anti-retroviral therapy. Extensive studies including FDA-approved phase I clinical trial have demonstrated the therapeutic potential of CRISPR/Cas genome editing to cure HIV. However, a major barrier to the clinical application is the lack of effective and specific delivery to the targeted disease-relevant tissues and/or cells in vivo, particularly in NeuroHIV. The overall objective of this proposal is to develop AAV-mediated stealth cargo delivery of miniature Cas12f genome editor to the HIV cellular reservoir in the brain. We will utilize novel PEG10-mediated endogenous retrovirus-like particle (ERVLP) technology that relies on endogenous PEG10 and syncytin-A for Cargo(Cas12f) mRNA transfer into MG. This approach will harness the benefits of the most promising AAV gene therapy. Several AAV serotypes such as AAV1, 2, 5, 6 can transduce MG (AAV-M) with >80% efficiency in vitro and in vivo, but cannot cross the blood-brain barrier (BBB). In contrast, the currently available BBB-penetrating AAV serotypes (AAV-B) such as AAV9, PhP.B, PhP.eB, F, B10 or B22 have low efficiency in transducing MG both in vitro and in vivo. Therefore, novel AAV serotypes that effectively cross the BBB and transduce MG (AAV-BM) are urgently needed. We hypothesize that AAV-B can offer a one-time injectable systemic delivery of stealth cargo (cDNA) into astrocytes and/or neurons thatin turn serve as relay stationsfor sustained mRNA/sgRNA transfer to MG. This stealth AAV cargo will also include a designer exosome transfer into cells (EXOtic) device via CD63 linked with MG-specific peptide (CD63M). We expect that PEG10-mediated ERVLP and CD63M-mediated EXOtic will synergistically boost the endogenous spreading of HIV eradicator to MG. To accomplish this, we will first use the Cre-LoxP system for proof of concept that MG-targeted exosome-enveloped ERVLP system (Exo-ERVLP) via AAV-B can efficiently deliver Cargo(Cre)-mRNA in vivo from transduced astrocytes/neurons to non-transduced MG in LoxP-STOP- LoxP (LSL)-tdTomato reporter mice (Aim 1). Then, we will assess MG-targeting and genome editing efficiency of multiplexed Cas12f mRNA/sgRNA sustained delivery in LSL-tdTomato mice and HIV Tg26 transgenic mice (Aim 2). Finally, we will explore the therapeutic potential of Exo-ERVLP AAV-B-Cas12f systemic injection in HIV Tg26 transgenic mice (Aim 3). This high-risk high-reward proposal brings together several advancing technologies and established teams with complementary expertise. The all-in-one multiplexed Cas12f/sgRNA transgene is delivered via AAV-B, PEG10 cargo and CD63M EXOtic for sustained targeting and HIV eradication. The expected positive outcomes will offer a novel tool to systemically deliver CRISPR/Cas editor to MG, and provide new avenues for therapeutics development for multiple MG-related diseases.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY AND RELEVANCE The sphingolipid metabolites, sphingosine-1-phosphate (S1P) and its precursors sphingosine and ceramides, have emerged as pleiotropic signaling molecules that regulate diverse cellular responses. Previously, we have shown that in response to numerous stimuli, S1P produced intracellularly by two sphingosine kinases, SPHK1 and SPHK2, is exported out of cells via a specific transporter Spinster homolog 2 (SPNS2) to activate its receptors S1PRs. While this process that we coined “inside-out signaling by S1P” is responsible for many of its known functions, it has long been suspected that SPHK and the sphingolipids it regulates, S1P, sphingosine, and ceramide, also have intracellular functions. This is an important gap that our research and the MIRA proposal are aimed at filling as lower organisms express the evolutionary conserved sphingolipid metabolic enzymes and although sphingolipid metabolites regulate important biological functions in these organisms, they do not have S1PRs. Moreover, these enzymes are present in distinct subcellular compartments, suggesting that the location(s) where sphingolipid metabolites are produced dictates their functions. The MIRA proposal is focused on the roles of these sphingolipid metabolites in governing membrane contact sites (MCS) between the ER network and late endocytic organelles versus the plasma membrane to control the movement of cholesterol and sphingolipids between distinct cell membranes and their metabolism. Our research will also address the involvement of these sphingolipid metabolites in establishment of lysosome-mitochondria MCS and consequence to mitochondrial function and bioenergetics and define how they regulate localization of GRAMD1s family, evolutionarily conserved ER-anchored cholesterol transfer proteins. Another goal of the proposed research is to understand the functional transport mechanism of SPNS2, which remains controversial, and could have important ramifications as SPNS2 has been linked to metabolism, cancer metastasis, and auditory, inflammatory, and immune disorders. The new conceptual groundwork in this proposal will alter our understanding of sphingolipid metabolites signaling and will reveal their ancient but understudied intracellular roles in regulation of membrane dynamics and contact sites as sensors that integrate and coordinate sphingolipid and cholesterol metabolism, and potentially glucose homeostasis. Although this area of research is just in its infancy, we believe that our proposed studies will shed new light on enigmatic functions of bioactive sphingolipids appropriate to their name associated with the riddle of the Sphinx. It should also provide deeper understanding of how perturbations of these fundamental biological processes contribute to human diseases.

NIH Research Projects · FY 2026 · 2024-01

Obligate intracellular bacteria in the order Rickettsiales cause emerging and potentially fatal infections. Orientia tsutsugamushi, an etiologic agent of scrub typhus, is one of the deadliest rickettsial pathogens to humans. Yet, it is severely understudied due, in part, to a lack of genetic manipulation tools. We have made important progress on this front by developing protocols for Tn5 transposon mutagenesis and allelic exchange in O. tsutsugamushi. We employed both approaches to insert a cassette that expresses mCherry and chloramphenicol acetyl transferase into the O. tsutsugamushi chromosome. Using chloramphenicol selection and enrichment via fluorescence activated cell sorting, we recovered a Tn5 mutant pool and an allelic exchange mutant in which we knocked out a virulence factor. The Tn5 mutant pool and knockout mutant can be maintained under antibiotic selection. In the R61 phase, we will further develop these genetic tools. In Aim 1, we will transform O. tsutsugamushi with Tn5 transposomes to generate an insertional mutant library that we will catalog. In Aim 2, we will complement this random mutagenesis approach by using allelic exchange to delete selected single-copy O. tsutsugamushi genes. For this purpose, we prioritized a set of virulence factors whose functions we previously defined to provide us with phenotypes to characterize in downstream R33 functional studies. While transposon and allelic exchange mutagenesis are exciting “firsts” for this organism, neither can be used to target essential genes in any other obligate intracellular bacterium. Therefore, in Aim 3 we will develop a riboswitch system that allows us to control protein expression in transgenic O. tsutsugamushi using a diffusible ligand. This approach can be used to block expression of any O. tsutsugamushi protein at any point during infection. Even if the protein is essential, the mutant can be maintained in the presence of inducer to ensure viability. To establish proof-of- principle for the riboswitch, we will target a gene that we have determined to be non-essential for viability. In the R33 phase, we will use the newly developed tools to study O. tsutsugamushi pathogenesis. Up to four allelic exchange mutants and the riboswitch mutant will be assayed alongside their wild-type counterparts for the ability to modulate host cell functions. The knockout mutants will also be assessed for pathogenesis in the mouse model. Up to two Tn5 mutants, prioritized based on the predicted functions of the genes that are disrupted, will be included in the in vitro and in vivo studies. Overall, we will establish the genetic basis of virulence in O. tsutsugamushi for the first time and provide valuable tools for the field. We envision that the proposed work will bolster the field by encouraging an influx of researchers to study scrub typhus.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY Few therapies have yet to achieve durable remission of advanced stage triple negative breast cancer (TNBC). One promising approach to improve clinical outcomes is to combine multiple chemotherapies to stimulate an antitumor response. An underlying premise for this approach is that select chemotherapies enhance TNBC immunogenicity, thereby stimulating the anti-tumor immune response improving tumor growth control. With this strategy in mind, we have discovered that inhibiting the epigenetic regulator BPTF, of the nucleosome remodeler NURF, either genetically or pharmacologically, is a novel approach for improving TNBC immunogenicity. In this proposal we now show using two different TNBC tumor models that when BPTF depletion is combined with the DNA methyl transferase inhibitor (DNMTi) Guadecitabine (Guad) we further stimulate tumor cell immunogenicity and dramatically improve tumor growth control in immune competent, but not immune compromised mice. Genome wide analysis shows that the combination therapy enhances immune stimulatory pathways, with an enrichment for immune stimulatory cytokines. Thus, using a novel combination approach, we hypothesize that BPTF depletion/inhibition will synergize with immune stimulating therapies targeting epigenetic factors (Guadecitabine) for TNBC treatment improving therapeutic outcomes. In Aim1, will identify the immune reactive effector cells required for the antitumor effects of Guad + BPTF depletion using the mouse E0771 TNBC tumor model. Additional flow cytometry and ex vivo immune cell assays will characterize and further define the immune cells required for these antitumor activities. The BPTF inhibitor BZ1 + Guad will be used in parallel tumor studies using the E0771 model to determine if it has in vivo activity, and in molecular studies (RNA-Seq and ATAC-Seq) from FACS sorted E0771 tumor cells to determine if it has similar effects to that of BPTF KO. Humanized mouse models and TNBC PDX samples will validate the translatability of these findings. In Aim2, we will optimize the drug-like properties of our second generation lead BPTF inhibitor. Validating the therapeutic potential for BPTF inhibition has been significantly limited due to a lack of potent inhibitors for BPTF. Our first generation BPTF bromodomain inhibitor, AU1, while effective for initial validation of BPTF inhibition in cells, and in vivo, its poor physiochemical properties and modest activity in vivo have limited further validation of BPTF as a drug target. Using structure-based design we have now developed a second and highly potent inhibitor series. Here, we focus on optimizing its drug-like properties, using both established biophysical and cell-based assays to translate our findings to our in vivo model system in Aim 1. The completion of these Aims will deepen our understanding of cancer cell epigenetics, and cancer cell immunogenicity by characterizing the effects of inhibiting the chromatin factor NURF in combination with DNMTi. Our approach is further designed to enhance antitumor immunity to TNBC by improving therapy induced tumor immunogenicity through a combination epigenetic therapy.

NIH Research Projects · FY 2026 · 2023-12

SUMMARY Orientia tsutsugamushi is a genetically intractable obligate intracellular bacterium that causes scrub typhus, a globally emerging infection with a high fatality rate. Disease progression depends on bacterial-driven modulation of host antimicrobial responses that affords O. tsutsugamushi the ability to survive in leukocytes and endothelial cells. The bacterial mechanisms responsible are largely unknown, highlighting a gap in our knowledge of host- pathogen interactions that influence scrub typhus outcome. A family of eukaryotic-like effectors called Anks are key O. tsutsugamushi virulence factors. Most consist of an N-terminal ankyrin repeat (AR) domain that mediates protein-protein interactions with host targets and a C-terminal F-box that recruits the host SCF E3 ubiquitin ligase complex to ubiquitinate the AR-bound proteins. The interacting partners and cellular processes that the Anks modulate are mostly unknown. We discovered that O. tsutsugamushi Ank13 is a nucleomodulin. Gene expression profiles in cells ectopically expressing Ank13 recapitulate many of those observed for O. tsutsugamushi infected host cells, indicating that Ank13 contributes to the pathogen’s ability to modulate cellular processes at the transcriptional level. Both infected and Ank13-expressing cells exhibit down-regulation of genes involved in immune responses and other processes regulated by the Notch signaling pathway. A yeast two- hybrid screen coupled with co-immunoprecipitation identified host MIB1 as an Ank13 binding partner. MIB1 is a positive regulator of canonical Notch signaling. MIB1 levels are reduced in O. tsutsugamushi infected cells, and this is phenocopied in cells ectopically expressing Ank13 or an Ank13 mutant with a functionally inactivated F- box. These data suggest that Ank13 sequestration of MIB1 promotes its auto-ubiquitination and proteasomal degradation during infection. Notch ligand surface presentation on infected cells is altered and Notch-related gene expression is quiescent in these cells, indicating that O. tsutsugamushi impairs Notch signaling. Notably, these same genes are significantly downregulated in cells ectopically expressing Ank13. A preliminary yeast toxicity suppressor screen implicated yeast proteins that have human homologs involved in host transcription regulatory pathways, including ribosome and cell cycle modulation, and non-canonical Notch signaling, as being modulated by Ank13. Thus, Ank13 alters Notch-dependent and -independent transcription to manipulate multiple eukaryotic processes. Aim 1 will interrogate the hypothesis that O. tsutsugamushi Ank13 promotes MIB1 auto- ubiquitination/degradation to impede Notch-stimulated processes. As a complementary approach, Aim 2 will comprehensively define the cohort of host targets and cellular processes that Ank13 modulates during infection. Specifically, we will couple unbiased yeast suppressor screening and affinity proteomics assays to identify host proteins/pathways targeted by Ank13 and will investigate their relevance to O. tsutsugamushi pathogenesis. Overall, this proposal will advance our fundamental understanding of nucleomodulin biology and define novel pathways targeted by O. tsutsugamushi, together providing a powerful impact to the bacterial pathogenesis field.

NIH Research Projects · FY 2026 · 2023-12

The Translational Biomedical Sciences (TBS) CTSA Pre-doctoral T32 Program at Virginia Commonwealth University (VCU) will provide trainees with in-depth topic expertise as well as crucial, non-discipline specific knowledge, skills, and abilities to advance clinical and translational science research. TBS trainees will engage in research experiences that provide a strong foundation in the mechanistic paradigms of disease pathology as well as practice more contemporary skills such as team science, community engagement, and research dissemination and implementation. Specifically, the TBS Program will 1) implement a novel interdisciplinary mentoring approach with mentors who bring varied perspectives to the trainees’ research and career development; 2) develop trainees knowledgeable about translational science who are proficient in study design and conduct, data analysis, and publishing in accordance with best regulatory and ethical practices; 3) cultivate trainees’ career development skills to prepare them to be productive biomedical researchers; and 4) connect trainees with collaborators from across VCU as well as from other universities and with community stakeholders to encourage interdisciplinary, scientifically rigorous, and clinically-relevant research. The TBS Program will be co-directed by three, highly-regarded translational researchers with leadership roles in clinical and translational research education at VCU and housed within the Wright Regional Center for Clinical and Translational Science to provide trainees with a breadth of training and career development opportunities as well as exposure to a transdisciplinary, collaborative, and translational research environment. A cadre of mentors comprised of select faculty from 15 different departments at VCU whose research addresses all stages of the translational spectrum as well as Translational/Community advisers will create a fertile training environment that encourages the development of researchers who are confident working with a variety of collaborators. During the funding period, two highly-qualified trainees will be appointed to the TBS Program annually (total of 10 trainees over the 5-year funding period). In summary, the TBS Program will provide a comprehensive educational experience that prepares a broad group of trainees for successful, independent research careers in the broad area of translational science as well as equip them with the skills to work across disciplines and with stakeholders to design, disseminate, and implement their research.

NIH Research Projects · FY 2025 · 2023-09

Abstract Firearm-related violence is a major public health problem in the United States. Between 2019 and 2020, firearm- related homicide increased by nearly 35%, and 2021 saw the highest gun-related homicide rates since 1993. The effects of the pandemic have impacted the rates of violence, and we can expect to see these lasting effects for many years. Nationally, young adults (18-35 years old) are the most at risk to die from firearm-related violence. This is a risk that is not evenly shared among subpopulations of adults, with African American men being 20 times more likely to die from firearm-related homicide than Caucasian men. Further, once discharged from a hospital, violently injured adults are at an increased risk of violent re-injury and mortality, as well as 88 times more likely to engage in retaliatory violence. The hospital has become a critical location for violence prevention. Unfortunately, patients who are most at risk of retaliatory violence are often difficult to engage in intervention, which is why new and innovative strategies are needed to meet patients where their interests and needs are. The goal of the present proposal is to develop and evaluate a virtual reality (VR) violence intervention, Brief Violence Intervention VR (BVI-VR), to prevent incidents of firearm-related violence perpetration, re-injury, and mortality. BVI-VR provides patients with culturally relevant immersive stories and gameplay while providing them with psychoeducation and intervention to increase resiliency to retaliatory firearm-related violence across five steps: (1) emotion regulation and managing trauma, (2) conflict resolution and non-violent alternatives, (3) gun safety, (4) future goals and aspirations, and (5) community resource connection. This will be the first VR intervention targeting adult firearm-related violence, injury, and mortality. BVI-VR provides the connection between an immersive individual-level intervention and exposure/connection to community resources tailored to the patient’s local community. The present study addresses Funding Option B of this funding announcement, which supports research projects that collect new data and/or implement prevention activities. This study will conduct a randomized control trial including 220 violently injured adults to assess the effectiveness of BVI-VR for preventing firearm-related violence, injury, and mortality. The study also aims to identify risk and protective factors that are most malleable (mediators) to change in response to BVI-VR. Lastly, we will assess the economic efficiency of BVI-VR as a brief hospital-based gun violence prevention strategy. The results from this study may support the use of BVI-VR as a cost-effective strategy for preventing firearm-related violence, injury, and mortality among victims of violence.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY / ABSTRACT The overarching goal of this 5-year K23 proposal is to support Taylor Crouch, PhD, in her development as an independent investigator in the overlapping areas of chronic pain and addiction, with a focus on enhancing uptake of evidence-based nonpharmacological chronic pain interventions and reducing reliance on long term opioid therapy (LTOT). Evidence-based behavioral interventions for pain are among the most effective and safe treatments, but utilization remains low, especially among patients on LTOT. The risks of LTOT, including opioid use disorder and overdoses, are well-established, but complex dependence can limit patients’ readiness to try something different. Two key barriers to uptake of nonpharmacological interventions have been identified as low motivation and logistical barriers to care. Two existing evidence-based interventions to enhance motivation and support behavior change, Motivational Interviewing (MI) and Contingency Management (CM), have promise for enhancing readiness to change among patients with chronic pain displaying risks on LTOT, and eHealth interventions can improve access and reach of interventions. Therefore, the objective of this proposal is to develop a MI/CM eHealth intervention to increase readiness to utilize nonpharmacological treatments and reduce reliance on opioids among individuals displaying risk on LTOT. Specific Aims are: Aim 1: To inform intervention approach, conduct exploratory testing with providers and patients to clarify needs/barriers, consolidate existing theory/evidence using the Medical Research Council framework, and complete initial testing of intervention ingredients. Aim 2: Develop an alpha prototype MI/CM eHealth intervention aimed at enhancing motivation to utilize nonpharmacological treatments and reduce opioid reliance, using the Computerized Intervention Authoring System, and assess usability. Aim 3: Develop beta and conduct a pilot randomized clinical trial to assess feasibility and acceptability. At the completion of the pilot trial in Aim 3, the intervention will be ready to be tested in an R01 to evaluate its efficacy in enhancing not only motivation, but action toward reduced opioid use and utilization of evidence- based non-opioid CP interventions. This proposal represents a 5-year comprehensive mentoring, training, and research plan to transition the candidate, Dr. Crouch, to a career as a successful independent investigator. Dr. Crouch has background training and expertise in behavior change, motivation, addictive behaviors, and behavioral chronic pain interventions, and through her comprehensive training plan she will be positioned to become a leader in these areas and further develop her expertise in implementation science and clinical trials.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY In this F31 proposal resubmission, I use novel synthetic transcription factors (TFs) delivered to the mouse medial prefrontal cortex (PFC) to uncover and manipulate the brain molecular processes that govern complex social behaviors. Social behaviors are central to the health of society and the individual, and are disrupted in a number of psychiatric illnesses. However, the neurobiological origins of social behaviors are incompletely understood. Here, I present synthetic biology approaches to re-program the function of ZFP189, a Kruppel-associated box (KRAB) zinc finger TF whose expression and function in the mouse PFC was previously determined to be protective against stress-induced social deficits. To interrogate gene targets and nuanced behaviors controlled by ZFP189, my laboratory and I developed novel synthetic ZFP189 TFs, each capable of exerting distinct forms of transcriptional control at in vivo ZFP189 target genes. We replaced the endogenous repressive KRAB moiety of ZFP189WT with a synthetic transcriptional activator in ZFP189VPR. Viral delivery of these synthetic TFs allows me to knowingly augment ZFP189-mediated transcription in the PFC of awake and behaving mice. In data presented in this application, I observe that virally delivering ZFP189VPR to the PFC of mice ablates social behaviors, without other observable behavioral deficits. By performing RNA sequencing (RNAseq) on manipulated tissues, I identify that the ZFP189 TFs of opposing function exert divergent impact on genes relating to adaptive immune response. These preliminary findings are in agreement with earlier published work proposing a co-evolutionary link between social behavior and the molecular cascades associated with anti-pathogen response in PFC pyramidal neurons. Other published evidence suggests that KZFPs bind repetitive DNA elements, including transposable elements (TEs), with are self-replicating sequences of DNA. My data shows that some of the transcripts most activated by ZFP189VPR are TEs. Based on my data and previous literature, I believe ZFP189 represents an evolutionally ancient link between regulation of brain TEs and the immune cascades that drive pro-social behaviors. Completion of this project will resolve the impact of synthetic ZFP189 TFs in governing social behaviors and social cognition necessary to function in a social group (Aim 1). Additionally, this project will illuminate the brain transcriptional mechanisms regulated by ZFP189 by performing RNAseq of virally manipulated PFC tissues (Aim 2). Together, this work will provide new approaches to identify the TF functions and illuminate the molecular origins of complex social behaviors. The skills I develop through this project and fellowship will position me for a competitive and successful career as a scientist.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Down Syndrome Regression Disorder (DSRD) is a serious neuropsychiatric syndrome that is of growing interest among the Down syndrome (DS) community and the INCLUDE Down Syndrome Research Plan. Adolescent and adult DSRD is associated with a subacute decline in cognitive and adaptive functioning, mood, and catatonia, with the highest risk period between 10 and 30 years old. Symptoms cause severe disability in the form of loss of ability to participate in activities of daily living and cause a great burden on parents and caregivers. Without prompt treatment, DSRD may persist for years. Recommended medical evaluation is costly and specialists are few and far between, making appropriate treatment difficult for some and inaccessible for others. There are also significant gaps in research on the prevalence and causes of DSRD, due in part to the lack of scalable assessments in large DS cohorts. Validated low-burden assessments are needed to identify patients, monitor treatment response, and improve research. This study aims to assemble a cohort of adolescents and young adults with DS and develop the DS- Regression Rating Scale (DSRRS), a parent-report form, which will aid in assessment and advance systematic research on the etiology and treatment of DSRD. To accomplish this, we will use a stakeholder- informed approach to assemble a cohort of 600 adolescents and young adults with DS, with and without DSRD. We will develop the DSRRS through an iterative process of focus groups, pre-testing, and item reduction. We will validate the newly created scale against well-validated assessments of symptoms associated with DSRD (e.g., stress, adaptive functioning, catatonia), examine the test-retest reliability, and examine sensitivity and specificity to optimize recommended cutoff scores. This project is in line with several priorities identified in the INCLUDE Down Syndrome Research Plan (pp. 13 – 14). These tools will lay the groundwork for vital research for years to come aimed at identifying the prevalence and etiologies, the gene-brain-behavior connections underlying DSRD, and most importantly, the identification of prevention and intervention strategies to improve the lives of people with DS at risk for DSRD.

NIH Research Projects · FY 2025 · 2023-09

Even though it is well established that oral health is integral to overall health, economic productivity, and well-being, it remains the most common unmet health need in the US. Low-income individuals carry a significantly high burden of dental disease, highlighting a lack of access to oral health care. A large proportion of these groups rely on Medicaid, but unlike children, federal law does not mandate dental coverage for adults, and coverage varies by state, leaving many uninsured or underinsured. Starting July 2021, Virginia expanded dental coverage for all Medicaid-enrolled adults (up to 138% of FPL), providing access to comprehensive dental care to thousands of adults for the first time. This dental policy change provides a unique opportunity, a 'natural experiment,' to gain a deeper and clearer understanding of the effect of implementing a comprehensive dental benefit on the oral health of Medicaid enrollees and fill a critical gap in oral health services research. The purpose of the proposed K08 research project is to evaluate the role of Virginia's comprehensive adult Medicaid dental policy in improving and advancing dental care utilization and access among the Medicaid population. The project will: 1) examine the demand for dental services among Medicaid-enrolled adults following the implementation of the dental benefit, 2) evaluate the impact of dental benefit on the use of the emergency department (ED) for dental visits by comparing adults enrolled in Medicaid with those having private insurance, and 3) assess the effect of the Medicaid adult dental benefit on children's dental care use. Data sources will include multi-year claims data from the Virginia Medicaid program and Virginia All Payers Claims Data that will be linked with the county-level workforce and community data on social factors from multiple sources. Causal inference methods and advanced econometric and data science approaches, such as difference-in-difference and interrupted time series, will be used to isolate the policy's effect on measured oral health outcomes. The primary goal of this K08 application is to advance the candidate's (Dr. Naavaal) research career by protecting her time to acquire training in health policy, econometrics, and data science and gain research experience in applying these methods to conduct rigorous oral health services and health policy research. This goal will be accomplished through executing proposed research supported by carefully planned training objectives and intensive career development activities. The proposed research outcomes will generate the evidence needed to inform strategies and multi-level approaches needed to advance oral health for all and maximize oral health gains. The expertise and guidance of the multi-disciplinary mentoring and advisory teams, in addition to the wealth of resources and commitment to training provided through the institutional environment, will ensure the success of the candidate's research project and enable her to establish a robust research program as an independent research investigator.

NIH Research Projects · FY 2024 · 2023-09

Project Summary/Abstract One way in which chronic alcohol exposure produces neuronal adaptations is by changing gene expression. These changes can influence alcohol-drinking behavior and may lead to the development of alcohol use disorder (AUD). Additionally, during alcohol withdrawal, gene expression changes can contribute to the development of negative affective states, such as anxiety and depression, which makes it challenging for individuals to stop consuming alcohol. Mounting evidence from many species indicates that chronic a lcohol exposure also leads to alternatively spliced transcripts in different brain regions. Yet the molecular mechanisms by which alcohol alters RNA splicing remains unknown. This K99/R00 award includes a comprehensive career development and research plan based on Dr. Luana Carvalho’s preliminary data showing that withdrawal from chronic alcohol exposure in male rats increases the expression of genes encoding components of the RNA splicing machinery and leads to changes in RNA splicing. My preliminary data shows increased expression of the splicing factor Poly r(C) binding protein (PCBP1) in the hippocampus (HPC) of ethanol withdrawn rats that present with anxiety and depression-like behavior, as well as in the postmortem HPC of subjects diagnosed with AUD. I also found that PCBP1 is implicated in the mis-splicing of the Hapln2 gene, an important regulator of neuronal conductivity in which loss of function could negatively impact neurotransmission in the context of alcohol use. The scientific goal of this K99/R00 is to investigate RNA splicing, with a focus on PCBP1, as a mechanism by which chronic alcohol exposure and withdrawal leads to molecular alterations and contributes to the emergence of negative affective states. I will manipulate PCBP1 expression using viral-mediated gene delivery to test its behavioral relevance during alcohol withdrawal. I will also perform RNA immunoprecipitation with a PCBP1 antibody, followed by next generation sequencing to identify PCBP1-targets. Finally, I will perform full-length transcriptome sequencing to identify the portfolio of alternatively spliced transcripts in the HPC during chronic alcohol exposure and withdrawal. During my K99 phase, I will gain additional technical training in cutting-edge molecular, bioinformatic and statistical approaches. I will also enhance my leadership skills and receive considerable training in grant writing, oral presentations, and ethics that will be crucial to my success as an independent researcher. During the R00 phase, I will apply all training received to continue this project and further explorer PCBP1 targets in the HPC of humans diagnosed with AUD. Collectively, this work will provide insights into the molecular mechanisms underlying alcohol withdrawal-induced changes on RNA splicing and negative affective states, reveal novel targets and testable hypothesis for future functional studies, and facilitate translational research for finding new targets for AUD treatment.