Wake Forest University Health Sciences

NIH Research Projects · FY 2024 · 2023-09

Project Summary/Abstract Rural Americans experiencing an ST-Elevation Myocardial Infarction (STEMI) are eight times less likely to receive timely definitive treatment than their urban counterparts. This disparity exists even after percutaneous coronary intervention (PCI) times are adjusted for distance to the hospital and exposes rural patients to excess morbidity and mortality. A major obstacle to timely rural STEMI care is a lack of tools available to assist paramedics in providing a consistent evidence-based approach to prehospital STEMI. Our proposal will translate evidenced-based rural Emergency Medical Services (EMS) STEMI best practices into a multifaceted, digital, clinical decision support tool to address this obstacle. This study builds on our team’s foundational mixed methods research that identified (i) previously poorly quantified complexities of rural EMS STEMI care, (ii) barriers to timely care, and (iii) opportunities for improvement. The Rural STEMI Application will be developed, implemented, and refined using an open-source, cross-platform mobile application developed internally by our team. Initially, we will develop and test the usability of the Rural STEMI App in a rural North Carolina County EMS agency to improve prehospital providers’ ability to reduce first medical contact (FMC) to reperfusion (PCI or thrombolytic) times. This will be the first smart device application to provide real-time, evidence-based, guideline-driven, patient-specific treatment assistance for EMS patients with STEMI. We anticipate that the final App will incorporate specific real-time data, including EMS arrival on scene, ECG time, map integrations of nearby emergency departments and catheterization labs, and catheterization lab availability. This novel digital tool will assist the EMS team by providing a scene time countdown, hospital activation metric countdowns, and EMS-specific route navigation assistance to further decrease FMC to reperfusion time. Thus, our application will incorporate critical parameters needed to predict FMC to reperfusion time and identify patients that are better treated with initial thrombolytic administration instead of PCI. Through phased implementation of the Rural STEMI App in seven additional rural EMS agencies, we will evaluate its feasibility and preliminary effectiveness to reduce FMC to reperfusion time. This application will also address the need for improved STEMI encounter communication by providing an automatically generated STEMI feedback report to all key stakeholders. Finally, this proposed study will be the first to apply a mixed methods approach to characterize implementation facilitators and barriers among rural EMS agencies in the care they provide to STEMI patients. By engaging field providers in semi-structured interviews, the study will emphasize quality improvement efforts, EMS administration support, and interdisciplinary collaboration in the care of EMS patients with STEMI. This proposal directly addresses a critical gap regarding how to improve rural prehospital FMC to reperfusion times, which in turn will reduce disparities in morbidity and mortality. We will subsequently test the Rural STEMI App in a large multisystem hybrid effectiveness-implementation trial.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY The number and variety of Highly Toxic Chemicals (HTCs) that pose a health risk to the civilian population is extensive. The Department of Homeland Security has identified close to 200 HTCs as credible public health and safety threats. HTCs comprise diverse chemical classes and toxicity mechanisms including acids, alkylating agents, vesicating agents, metabolic poisons, cellular respiration inhibitors, and many with understudied toxicity and mechanisms. However only a small subset of known HTCs have been well-characterized, and there remains an urgent unmet need to improve our understanding of the physiological mechanisms involved in the initiation and downstream events of injury following exposure understudied HTCs. Relevant to this proposal, we have developed micro-physiological 3D human airway Organ Tissue Equivalent (OTE) platform for modeling pulmonary toxicity resulting from exposure to chlorine gas and for identification of novel mechanisms of injury and for testing of potential medical countermeasures (MCMs). Our HTC exposure system allows safe delivery of a broad range of gas, vapor or nebulized liquid HTCs to lung OTEs with high precision and accuracy. We have established assays rapidly determining dose/toxicity relationships, physiologically relevant chemical, biological and functional evaluation of mechanisms of toxicity and transcriptomic analysis for the discovery of novel toxicity pathways and MCM targets. Our overall hypothesis is that our established airway OTE - HTC delivery system and transcriptomic bioinformatic capabilities can be applied to different classes of HTCs to characterize mechanisms of toxicity and define potential molecular targets for MCM intervention. If successful, this proposal promises to improve our understanding of the initiation and downstream events of injury on acute exposure of a broad range of understudied HTCs. Rapidly defining dose/toxicity relationships and mechanisms of action of understudied HTCs will have a major impact on understanding potential risks for mass HTC exposure events. Finally, the potential to identify common molecular pathways of injury in response to a range of HTC types could have a significant impact in identifying and deploying effective medical countermeasures with broad application across unidentified or understudied HTCs. Future work will accelerate MCM discovery, repurposing and development with broader applicability across the pulmonary threat spectrum.

NIH Research Projects · FY 2025 · 2023-08

Multimorbidity, the coexistence of two or more chronic and often aging-related diseases, affects the majority of older adults. Chronic inflammation has been implicated in multimorbidity and individual aging-related diseases including cardiovascular disease, diabetes, cancer, Alzheimer’s disease, and osteoporosis. Anti-inflammatory therapy targeting IL1β reduces risk for cardiovascular disease, but clinical benefits of modulating inflammation remain controversial. Animal studies suggest that the infiltration of immune cells including T helper cells into the arterial wall, adipose, and other tissues may be the central mechanism underlying chronic inflammation and subsequent diseases. Most human studies, however, rely on inflammatory biomarkers such as IL6 and CRP, which lack mechanistic specificity. The paucity of human studies directly characterizing the immune cells’ role in the inflammatory process is a major gap retarding the translation of animal-based mechanistic work. We propose that T helper cells are an important contributor to both chronic inflammation and subsequent diseases because of their regulating immune response of both adaptive and innate immune cells including activation of monocytes/macrophages. This view is supported by our preliminary data from the Multi-Ethnic Study of Atherosclerosis (MESA) which shows the associations of pro-inflammatory signatures of T helper cells with multimorbidity, chronic inflammation, and inflammatory response of monocytes. While these microarray data support the role of T helper cells in chronic inflammation and multimorbidity, large longitudinal studies are needed to establish whether cellular features precede disease development. The proposed study will leverage the unique resource of isolated human T helper cells in MESA (N=1,900). We hypothesize that cellular features that coordinate the inflammatory response in peripheral T helper cells contribute to subsequent chronic inflammation, inflammatory changes in peripheral monocytes, and multimorbidity. To test this hypothesis, we will examine the following specific aims: 1) to examine whether omics profiles of T helper cells predict 11-year changes in multimorbidity in 1,900 MESA participants, 2) to evaluate whether omics profiles of T helper cells predict 6-year changes in omics profiles of monocytes and circulating pro-inflammatory biomarkers in 1,900 individuals, and 3) To test effects of pharmacological modulation of T helper cells from MESA participants on inflammatory responses. The proposed study will for the first time investigate the longitudinal relationship between omics profiles of T helper cells and multimorbidity in humans. The results from the proposed study will improve our understanding of inflammatory processes in humans, particularly their cellular features, and accelerate the development of more precisely targeted anti-inflammatory interventions for preventing multimorbidity.

NIH Research Projects · FY 2024 · 2023-08

Project Summary The quantity of opioid prescriptions dispensed in the United States (US) is so high that every other person could receive one opioid prescription. Many of these opioids go unused and are kept in homes rather than disposed after ceasing use. The primary source of prescription opioids for nonmedical use is relatives or friends; which suggests that diversion of excess and retained prescription opioids contribute significantly to nonmedical use and associated consequences. Without effective strategies to facilitate their secure storage and disposal, prescription opioids will remain in communities, increasing the likelihood for nonmedical use- related morbidity and mortality. Our long-term goal is to prevent nonmedical prescription opioid use by decreasing accessibility of unused medications in the home. The overall objective of this R03 application is to assess (1) the extent to which disposal boxes have been implemented in pharmacies across the US, (2) accessibility of disposal boxes to diverse community members, and (3) determinants of implementation. To achieve this objective, we propose two specific aims: (1) generate prevalence estimates of disposal box implementation in US pharmacies and examine place-based health disparities in implementation, and (2) examine determinants of pharmacy-based disposal box implementation using the Consolidated Framework for Implementation Research (CFIR). For Aim 1, we will sample 1,000 of the nation's approximately 60,000 retail pharmacies, geocode locations, and link with disposal box presence and neighborhood characteristics. We will conduct logistic regression with clustering to examine inequities in neighborhood characteristics that are associated with disposal box implementation (primary outcome). For Aim 2, we will conduct semi-structured interviews with a sample of 60 pharmacies who have and have not implemented medicine disposal boxes and will link geocoded neighborhood characteristics with qualitative data. The proposed research study is innovative, in our opinion, in that it applies an approach that has been piloted by the PI in a single state to study pharmacy-based disposal boxes by (1) developing nationwide prevalence estimates of disposal boxes across the US, (2) examining place-based health disparities associated with implementation, (3) assessing determinants of implementation using a theoretical framework, and (4) uniquely linking interview themes and neighborhood characteristics. The proposed research is significant in that findings will advance the understanding of inequities in and determinants of disposal box implementation at pharmacies and pave the way for comparative efficacy studies. The proposed study is relevant to NIDA's priorities to implement and evaluate theoretically-based, prevention interventions that can be implemented in healthcare settings to elicit population-level impacts to address the ongoing opioid crisis.

NIH Research Projects · FY 2025 · 2023-07

PROJECT ABSTRACT: Treatment of genetic disorders by in utero transplantation (IUTx) has safely been performed for decades in humans. The first IUTx cure, in the US, used hematopoietic stem cells (HSC) and corrected a child with X-SCID. Since this groundbreaking moment >25 yrs. ago, >50 patients have now been treated with this procedure for 14 different genetic disorders. However, for reasons that are still not well understood, full therapeutic success has only been achieved in X-SCID patients. Thus, a better understanding of the mechanisms by which HSC engraftment is hindered after IUTx is required, so that strategies can be developed to achieve therapeutic levels of HSC engraftment in other genetic disorders, such as hemoglobinopathies, that could benefit from IUTx. We and others have identified several characteristics of the developing fetus that may negatively impact its ability to serve as an amenable HSC recipient. Among these factors are competition from highly proliferative host HSC, more significant fetal immune barriers than initially known, and the degree of maturity and receptivity of nascent BM niches required for engraftment of donor (adult) HSC. Here, using fetal sheep as a model, we propose to: (Aim1) define the nature of, and overcome, the barriers to engraftment by using non-genotoxic conditioning to dissect the role that niche availability, host HSC competition, and fetal immunity play in the engraftment of adult donor HSC following IUTx, and (Aim 2) determine the impact of the phenotype and functionality of the donor HSC on the levels of engraftment following IUTx. We hope that, upon completion of these first 2 Aims, we will not only have identified the mechanisms involved in resistance to HSC engraftment, but we will also have achieved a minimum target of 20-25% HSC engraftment, which would allow IUTX to become a viable therapeutic approach for hemoglobinopathies. Among these, sickle cell disease (SCD) is the most common inherited blood disorder in the US, and one of the diseases that could benefit from IUTx, since even though the fetus is protected from sickling by the presence of fetal hemoglobin (Hb), clinical manifestations of SCD start during early infancy, placing the child at risk of complications such as stroke, splenic crisis, pain episodes, life-threatening infections, and episodes of acute chest syndrome, which can cause permanent lung damage. Of direct relevance to SCD, sheep exhibit the same developmental pattern of fetal to adult Hb switching as humans. Recently, using CRISPR/ Cas editing and subsequent somatic cell nuclear transfer, we produced SCD sheep with a disease phenotype mirroring that of human patients, displaying sickled cells in blood smears, positive Hb solubility test, and HbS detected by Hb electrophoresis. In Aim 3, we propose to validate the sheep SCD model by monitoring the animals over time, determining the stressors that induce sickle cell crises, and defining acute and chronic disease complications. In addition, we will test the therapeutic efficacy of IUTx for treating/curing SCD. Upon completion, we hope these studies will contribute to the development of novel strategies to achieve curative levels of HSC engraftment after IUTX and will validate a highly clinically relevant model for the SCD community in general.

NIH Research Projects · FY 2025 · 2023-07

This proposal seeks funding for an NRSA Institutional Predoctoral Training Grant from the Systems and Integrative Biology Program of the National Institute for General Medical Sciences. This grant will support predoctoral training in Redox Biology and Medicine (RBM) leading to a Ph.D. degree at Wake Forest University. The RBM Training Program is a multi-disciplinary, multi-departmental program that has as its goal the training of researchers working at the interface of biomedical sciences, biology, chemistry and physics to communicate and work across disciplines to delve into the specialized chemistry of oxidation and reduction reactions underlying the pathophysiology of many diseases. The 34 members of the RBM training faculty are highly collaborative, and include faculty from Biochemistry, Cancer Biology, Biomedical Engineering, Internal Medicine, Microbiology and Immunology, Chemistry, Biology, and Physics. Seven physician or veterinarian scientists (with MD, MD/PhD or DVM/PhD degrees) are also among the training faculty, encouraging translational science. Participating faculty represent a balance of career stages, including Assistant (10), Associate (11) and full Professors (13). Major scientific themes of the RBM Training Program include the overlapping areas of (i) oxidative modifications regulating metabolism and signal transduction pathways, (ii) redox regulation of nucleic acid metabolism and cell cycle, (iii) reactive nitrogen species biochemistry and biophysics relating to human health, and (iv) redox-linked mechanisms of disease initiation, progression and treatment. The RBM training program has a flexible curriculum built around a common core of required courses (Fundamentals of Redox Biology and Medicine, MCB 715; Omics Data: Generation, Analysis & Exploration, CABI 740; and at least one technology-specific minicourse or workshop), electives, as well as the discipline-specific courses to meet the requirements of the Ph.D. programs of the trainees. Students in the program are trained in such areas as chemical biology, omics (proteomics, lipidomics, metabolomics), biomedical engineering, enzymology, animal models of disease, mitochondrial biology and computational biology / bioinformatics as fits best to the research area of the individual and their Individual Development Plan. The training program takes advantage of the activities driven by the Center for Redox Biology and Medicine in coordination with other basic research and clinical centers at the institution, including a well-developed system of interdisciplinary seminars and retreats to promote interaction among members of the program. Students participate in trainee-focused Chalk Talks which foster development in the cross-discipline communication skills so critical to the multi-disciplinary research in this field. We also offer specific training in skills for a wide variety of potential careers for our Ph.D. graduates, including careers in academia, biotechnology and pharmaceutical industries, intellectual property, and science communication and policy.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Our team has demonstrated availability of evidence-based supportive care services in community oncology to support informal (unpaid/ family) caregivers in caring for themselves and their care recipient undergoing cancer treatment (e.g., psychosocial support, self-care/ wellness classes, medical task training), but connection to these services is abysmal. A scalable and systematic process is critically needed to identify caregiver needs and connect caregivers with services. This may be especially valuable for lung cancer caregivers who report exceptional supportive care needs and high levels of caregiver burden. We previously demonstrated strong feasibility and acceptability of Caregiver Oncology Needs Evaluation Tool (CONNECT) in a pilot randomized clinical trial with 40 lung cancer caregivers at one academic medical center. We also observed significant improvements in caregiver burden and a clinically significant decrease in depression in the intervention group relative to control group. We propose in this next step of multi-site intervention planning, to also include navigation to address barriers that may hamper caregiver supportive care use. The overall goal of this program of research is to develop innovative and scalable programs to improve supportive care delivery for cancer caregivers in the community. In this application, we propose to gather essential data for a future multi-site efficacy trial of CONNECT in collaboration with the Wake Forest NCI Community Oncology Research Program Research Base (WF NCORP RB) and key stakeholders. For Aim 1, we will survey NCORP sites to determine outpatient oncology practice (N=60) interest and capacity to test the CONNECT Intervention across the broad NCORP network. For Aim 2 and Aim 3, we will conduct a multi-site randomized pilot trial. For Aim 2, we will assess the multi-site feasibility (accrual, participation, and retention) of the CONNECT Intervention enrolling 120 lung cancer caregiver-patient dyads (CONNECT, n=40; Standard Care, n=40; Generic Resource List, n=40) with the goal of informing methods for the future trial. For Aim 3, we will obtain preliminary data on site training needs, time needed for site participation, and communication processes between participating sites and study personnel to optimize multi-site intervention delivery in the future fully powered trial. Pending demonstration of feasibility, we will conduct a future fully powered efficacy trial using the NCORP network.

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY/ABSTRACT The prevailing dogma in the alcohol field is that the rewarding properties of ethanol (EtOH) result from enhancement of ventral tegmental area (VTA) dopamine (DA) neural activity and accompanying DA release in the mesolimbic reward system. In preliminary studies, we will demonstrate that some of EtOH’s effects on midbrain neurons and NAc DA release are mediated by peripheral substrates including DA D2-subtype 2 receptor (D2R) expressing monocyte-derived macrophages (MDMs). These findings suggest a neuroimmune interaction for acute EtOH use and challenge the dogma that EtOH has exclusively central effects on DA neuronal activity, release, and reward. Our proposed studies constitute a focused investigation into the role of neuroimmune interactions in EtOH effects on VTA neurons, DA transmission, and EtOH reward and consumption. The core thesis is that acute EtOH enhancement of mesolimbic DA transmission and EtOH reward is mediated by EtOH enhancement of blood DA, subsequent activation of D2R-expressing MDMs, and subsequent cytokine modulation of VTA neurons, that are responsible for chronic adaptations in VTA GABA neurons and DA release. Prior and preliminary evidence supporting our hypothesis include: 1) Peripheral DA increases the activity of DA neurons and NAc DA release, reduces locomotor activity, and promotes reward via peripheral D2Rs; 2) EtOH enhances blood DA, inhibits VTA GABA neurons, enhances brain DA, and reduces intoxication via peripheral D2Rs; 3) EtOH induces microglia activation and enhances D2 receptor expression on monocytes, neurons, and microglia; 4) Depletion of MDMs reduces EtOH effects on VTA GABA neurons and DA release; 5) Select cytokines enhance VTA neuron excitability and DA release; 6) Last, we show preliminary evidence of DA and ATP co-release from DA terminals, and motility effects of EtOH on microglia, which indicate further study for potential NAc EtOH immune interactions in vivo. These data will provide new, fundamental knowledge on the neurobiology of EtOH reward and dependence and the role of peripheral substrates that may help improve drug development efforts. To test the hypotheses, we propose two Specific Aims: 1) Define the role of peripheral neuroimmune interactions in EtOH effects on VTA GABA neurons and NAc DA release, and related behaviors; 2) Describe effects of EtOH on NAc DA terminals and microglia, co-release of DA and ATP. We will use wild-type and transgenic mouse models (GAD67-GFP knock-in; VGAT-Chr2, VGAT-Cre/GAD67-GFP; and MaFIA mice) and MDM depletion to study neurochemical and electrochemical recordings of DA release. Cytometry techniques will be used to determine cytokine factors involved in mesolimbic alterations. Multiphoton microscopy approaches to study microglia chemotaxis in the context of DA and ATP as measured by fast scan cyclic voltammetry. Multiphoton microscopy will be used in vivo through endoscopic relay gradient index lenses to study GFP labeled satellite microglia surveillance while measured dopamine release using a red shifted optical sensor for detecting DA release, which will be performed on mice undergoing chronic intermittent EtOH (CIE) induction, thus describing neuroimmune activity from first exposure to EtOH, through to dependence. Scientific rigor is high considering the use of conventional behavioral, pharmacological, electrochemical, microscopy and molecular tools.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Currently 14.5 million people suffer from alcohol use disorder (AUD) in the United States. Identifying early risk factors that increase the likelihood of developing AUD could help elucidate potential models for preventative care and mitigate the impact AUD has on society. One highly studied risk factor associated with developing AUD is early life stress (ELS). The mechanisms that facilitate ELS's impact on the development of AUD is poorly understood. One consistent finding in ELS literature shows a role of microglia in the development of a host of neurological disorders including AUD. Over the past several years, new evidence has been uncovered identifying a role of microglia in the development and maintenance of AUD. To expand this field of work and discover novel targets for treatment, my project seeks to implement a model of early life stress: periadolescent social stress (PSS) and a model to promote voluntary binge-like consumption of ethanol: drinking in the dark. Using these models, I will identify how microglia respond to these factors in isolation and when combined. Over the past two years, I have run experiments to optimize the PSS paradigm and drinking in the dark protocol. I have performed immunohistochemical staining and run qPCR to determine how microglia respond to these stimuli. I have found that combined PSS and binge ethanol consumption decrease microglia number, increase microglial reactivity, and alter microglia morphology in the ventral hippocampus. In addition, markers related to homeostasis are reduced following PSS and binge ethanol consumption. While these results are interesting, evidence suggest that microglia are present heterogeneously throughout the brain and serve a variety of functions. To better characterize the effects of early life stress and ethanol consumption on microglia function, my proposal aims to identify key regions that demonstrate alterations in microglia count, reactivity, and phenotype; and identify how these changes relate to changes in microglial RNA. To determine these key regions, I will use light sheet microscopy. This approach will provide us an opportunity to create an unbiased 3D reconstruction of the whole brain with single microglia resolution. I will use this technique to identify how microglia are distributed throughout the brain and phenotypically altered due to these environmental exposures. Focusing on the regions identified with light sheet microscopy, I will perform targeted purification of polysomal mRNA sequencing (TRAP-seq). Using a CX3CR1CreER transgenic mouse line that primarily labels for microglia in GFP, I will sort microglia from other cell types and identify changes in microglial RNA expression due to PSS and ethanol exposure. Following each of these main experiments, I will determine whether microglia inhibition during PSS will prevent changes in binge-like ethanol consumption and microglial adaptations. This work will mark a significant advancement in the field and will call for the consideration of microglia function as a target for treatment of ELS and prevention of AUD.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Metastatic colorectal cancer (mCRC) remains highly lethal despite decades of optimizing 5-fluorouracil (5-FU)- based combination chemotherapy regimens that are central to treatment. The causes of treatment failure with 5-FU-based regimens include decreased metabolism to FdUMP, the primary active metabolite, and overexpression of thymidylate synthase (TS), the molecular target of FdUMP. To improve outcomes and overcome resistance we have developed a nanoscale FP polymer, CF10, that is comprised of FdUMP monomers linked in a single-stranded DNA backbone that releases FdUMP in a single step. CF10 displays markedly improved anti-tumor activity with low systemic toxicity relative to 5-FU and is a candidate for clinical translation. We hypothesize that nanoformulation of CF10 to increase plasma retention and actively target malignant tissue will improve anti-tumor activity and specificity. Lipid nanoparticle (LNP) formulation has proven to be a robust delivery strategy for multiple nucleic acid drugs, and in collaboration with NanoVation Therapeutics (NTx) in Aim 1 we will test two CF10:LNP formulations designed to differentially target CF10 to liver and more broadly to target metastatic tissue while protecting CF10 from degradation in plasma. In Aim 2, we will investigate PEGylation of CF10 together with active targeting to tumor tissue through conjugation with a cyclic RGD peptide that targets integrin V3 expressed specifically in malignant tissue. We will test our novel nanomaterials for improved anti- metastatic activity using: (i) a novel genetically engineered mouse model, iKAP, that forms tumors specifically in the colon with metastatic progression to the liver and lung; and (ii) a rat model of established colorectal liver metastases (CRLMs). Pharmacokinetic profiling (PK) will be evaluated using LC/MS/MS. In Aim 3, we will develop a 3rd generation FP polymer that includes 5-ethynyl-2’-deoxyuridine (EdU), a thymidine analog that is cytotoxic through complementary mechanisms to FdU. We will use the optimal LNP formulation and PEGylation strategies determined for CF10 in Aims 1 and 2 to develop a highly novel nanomaterial with exceptional potential for improved treatment of mCRC.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY Many congenital and acquired conditions resulting in the abnormality of the vagina often require reconstructive surgery to achieve anatomical and physiological function. Unfortunately, reparative procedures are challenged by the availability of vaginal tissues. Unfortunately, the use of non-native vaginal tissues and biomaterial substitutes has contributed to various complications, including mechanical, structural, functional, or biocompatibility problems. It is evident that native vaginal tissue, with its inherent functional properties, is most suitable for surgical reconstruction. Recent advances in tissue engineering and regenerative medicine have provided a solution to create autologous tissues for various clinical applications, including skin, bone, cartilage, urethra, and bladder. Applying the principles of tissue engineering, we have previously applied bioengineered autologous neovaginal tissues to create a functionally normal vaginal vault in pediatric patients born with vaginal aplasia. Due to the varying levels of pathologic conditions in each patient, manufacturing the target bioengineered vaginal tissue construct for reconstruction is challenging, requiring a better solution to meet the clinical needs. 3D bioprinting technology has emerged as a solution to develop patient-specific personalized tissue constructs with precision and reproducibility, addressing the current translational limitation of biomanufacturing for wide reconstructive applications. To address this unmet clinical need, our central hypothesis is that developing a bioprinting workflow will permit the fabrication of personalized autologous vaginal tissue constructs for clinical use. Thus, the objective of this study is to establish a clinically applicable 3D bioprinting workflow to manufacture personalized autologous vaginal tissue constructs that consist of patient-derived vaginal epithelial cells (EPCs) and smooth muscle cells (SMCs) for vaginal tissue reconstruction. The central hypothesis will be tested by pursuing three Specific Aims: 1) Develop and optimize a bioprinting workflow to bioengineer a readily implantable vaginal tissue construct; 2) Validate the individualized bioprinted vaginal tissue constructs using a preclinical animal model; 3) Establish a process development and batch record for regulatory approval. Successful completion of the proposed work will provide a standardized 3D bioprinting workflow that generates personalized vaginal tissue constructs with the required precision for vaginal tissue reconstruction. Using personalized autologous bioengineered tissue constructs will significantly impact patient care and change how we approach vaginal reconstruction in growing children and adults.

NIH Research Projects · FY 2026 · 2023-05

ABSTRACT – OVERALL The proposed program project, A Multidisciplinary Assessment of Risks of Vaping in Early Life (Project MARVEL), takes an integrated approach to assessing vaping behavior and the emergence of dependence; elucidating the impact of vaping on adolescent health; and generating empirically-tested messages to reduce adolescent vaping. In 2020, 19.6% of high school students, representing over 3 million adolescents, reported past month vaping. Moreover, the proportion of high school students who report frequent vaping has increased substantially. Significant gaps exist in understanding current patterns of youth vaping, the relationship between vaping and dependence, the impact of vaping on health, and how best to stem the surge in adolescent vaping. The overarching aims of this program project are to: (1) Assess the use of nicotine-containing vaping products and the relationship between vaping and the emergence of symptoms of dependence, including individual, interpersonal, and environmental factors that may moderate the impact of vaping; (2) Determine how vaping disrupts health and well-being through its impact on key physiological and psychological systems that are still developing during adolescence; and (3) Evaluate the potential impact of vaping prevention messages, developed based on emerging evidence of health harms from Project MARVEL and focused on the ways in which vaping affects adolescents’ bodies and lives. Four projects are proposed, supported by four cores. Project 1 will assess the relationship between vaping and the emergence of dependence, describe how vaping impacts the development of adolescent well-being, and determine how adolescents perceive the impact of vaping on their daily lives. Project 2 will examine the association between vaping, neurodevelopment, autonomic regulation, and cognitive/psychological skills. Project 3 will determine how vaping impacts respiratory health by examining symptoms, function, and early molecular alterations in airways. Project 4 will develop, optimize, and evaluate messages about novel health harms of vaping, identified in Projects 1-3 and the extant literature, that resonate with high school students. The Administrative Core will manage operations for Project MARVEL, facilitate interactions between projects, and coordinate study participation. The Biostatistics Core will ensure our designs and analyses maximize validity, generalizability, and efficiency. The Adolescent Research Support Core will address the unique challenges of conducting research with a large number of adolescents from multiple schools, facilitate recruitment and retention, ensure protection of human subjects, and maintain and leverage community partnerships. Finally, the Biomarker and Product Evaluation Core will characterize the most commonly used products and provide objective markers of exposure and biological effect. Project MARVEL has the potential to greatly improve our understanding of how vaping impacts adolescent health and how best to intervene to reduce vaping during this critical developmental period.

NIH Research Projects · FY 2024 · 2023-05

PROJECT SUMMARY/ABSTRACT Given persistent racialized and socioeconomic inequities in neighborhood tobacco retailer availability (TRA), decreasing TRA is an important intervention for decreasing inequities in tobacco use and tobacco-related disease. To address inequities in TRA, several cities have implemented licensing-based policies such as limiting the number of retailers that may possess a license to sell tobacco in a neighborhood. However, local licensing-based policies may be difficult to implement in states with preemption, which prohibits local governments from enacting tobacco control policies such as retailer licensing. To date, 14 states preempt local governments from regulating tobacco retail licensing. Oklahoma (OK) is one of the most comprehensively preempted states in the nation, and the state has exclusive authority over tobacco retailer licensing. However, an examination of OK’s statutes reveals that the state does not preempt localities from regulating zoning or land use. Use-based zoning may be able to reduce TRA by regulating the land (i.e., parcels) where retailers are permitted or prohibited from selling tobacco products (e.g., residential, industrial). This mixed-methods study (based in Oklahoma City and Tulsa) will be the first to explicitly evaluate the impacts of use-based zoning on eliminating racialized and socioeconomic inequities in TRA in a comprehensively preempted state. The two Specific Aims include: 1) Evaluate the equity impacts of policies that cap the number of tobacco retailers in certain zones and 2) Engage with local key stakeholders to identify facilitators and barriers to the dissemination of Aim 1 findings and implementation of zoning-based tobacco control policies. We will use geographic information systems and spatial epidemiology methods to identify the relationships between specific zones, TRA, and neighborhood sociodemographic characteristics. We will then simulate policies that limit the number of tobacco retailers within certain zones and compare changes in observed neighborhood inequities in TRA (e.g., weakening of inequities; percent and absolute decreases in TRA) pre- vs. post-policy. We will then use a semi-structured interview guide to explore key stakeholder recommendations for improving the dissemination of study results and to better understand perspectives on facilitators and barriers to implementing zoning- based tobacco control policies in OK, focusing on key implementation outcomes. This innovative and equity- focused study fits within NCI’s mission to “support cancer research across the nation to advance scientific knowledge and help all people live longer, healthier lives” with the long-term objective to reduce inequities in tobacco-related cancers. This study is grounded in dissemination and implementation science best practices and may produce evidence of reductions in TRA and pro-equity effects, which may open the door for a new suite of policy tools that can promote tobacco control and health equity, especially in preempted states.

NIH Research Projects · FY 2026 · 2023-04

Development of novel therapeutics targeting cellular and molecular mechanisms underlying cancer health disparities is among the top priorities in the oncology field. Non-small cell lung cancer (NSCLC) patients treated with immune checkpoint blockade (ICB) at the Atrium Health Wake Forest Baptist Comprehensive Cancer Center (AHWFBCCC) have shown up to 30% objective response rates (ORR) with Black NSCLC patients displaying better ORR than White patients. Building on this observation, we posit that increasing ICB efficacy through rational drug combinations could result in increased clinical benefit and a further reduction in overall cancer health disparities. Our preliminary single-cell RNA sequencing (scRNA-seq) studies using a race-balanced cohort of White and Black NSCLC patients show greater numbers of exhausted CD8+ T cells expressing high levels CTLA-4, LAG-3, and 4-1BB checkpoints (ICB targets), fewer myeloid and natural killer cells, and enriched redox and fatty acid metabolic processes in Black NSCLC patients. We and others have also shown that MCL-1 and TP53, both critical regulators of fatty acid metabolism, display higher rates of amplification and mutations, respectively, in Black NSCLC patients. These findings together with publications showing redox and lipid metabolite-based regulation of immune cell reprogramming and tumor immune cell balancing support the following two central hypotheses: (a) In Black NSCLC patients, unique redox and fatty acid metabolic features give rise to dysfunctional immune cell microenvironment and (b) these features can be exploited by innovative redox and lipid metabolism-targeting therapeutics to further improve response to ICB. Lung cancer is the most frequent cancer diagnosis at AHWFBCCC. Among our patients, 14% are Black, and epidemiologic data show that Black Americans in our region have lung cancer incidence and mortality rates 15.1% and 15.5% higher, respectively, than rates among Black Americans in the United States. We are in a unique position to investigate the molecular events that support high ICB response rates in Black NSCLC patients and to develop effective personalized therapeutic strategies that increase ICB efficacy. We will achieve these goals through three Specific Aims: 1) to generate high-resolution spatial single-cell expression profiles of the tumor microenvironment (TME) in Black and White NSCLC patients, 2) to generate supporting data linking specific genomic events in Black NSCLC patients to reprogrammed redox and fatty acid metabolism; 3) to generate pre-clinical data demonstrating that targeting mitochondrial redox and fatty acid metabolism can reshape the NSCLC TME and improve response to ICB. We will use NSCLC patient-derived ex vivo models and MCL1-overexpressing and p53-mutated NSCLC cell lines in humanized mouse models to determine whether MCL1 inhibitors (fatty acid metabolism modulators) and Devimistat (redox modulator) can reprogram the TME and increase response to ICB. Results from the proposed research will inform future preclinical studies and guide potential clinical trials aimed at enhancing treatment response for NSCLC patients.

NIH Research Projects · FY 2025 · 2023-04

PROJECT SUMMARY Current care patterns for the 7 million patients visiting Emergency Departments (EDs) in the United States with chest pain are heterogenous and not sustainable from a healthcare quality or economic perspective. Chest pain is the second most common cause of ED visits and most common reason for short-stay hospitalizations. During these hospitalizations patients undergo in-depth evaluations (stress testing, computed tomography coronary angiography, or invasive angiography), but ultimately <10% are diagnosed with acute coronary syndrome (ACS). These evaluations cost $3 billion annually and strain health system resources without clear evidence of improved health outcomes or patient experience. Our preliminary data suggest that moderate risk patients (35% of patients with chest pain) can be safely managed as outpatients using a cardiovascular ambulatory rapid evaluation (CARE) strategy as an alternative to hospitalization. In the CARE strategy, patients are discharged from the ED and receive outpatient clinic follow-up within 72 hours focused on medical management for cardiovascular risk factors (e.g., hypertension, diabetes) and determining whether further cardiac testing is needed. This strategy aims to enhance patient-centered outcomes while safely and equitably decreasing hospital resource utilization. However, equipoise exists between outpatient and hospitalization strategies for moderate risk patients. There is a paucity of prospective data evaluating the efficacy and patient-centeredness of outpatient chest pain evaluation strategies in moderate risk patients. Thus, it is unclear whether use of an outpatient chest pain management strategy (CARE) will safely reduce healthcare utilization and be preferred by patients compared to a traditional hospitalization strategy. To address this key evidence gap, we propose the first prospective multisite randomized trial testing outpatient vs hospitalization strategies in moderate risk patients. Our experienced research team will randomize 502 patients 1:1 to the CARE or hospitalization management arms at three ED sites with a history of high recruitment rates and productive collaborations in cardiovascular clinical trials. The primary outcome will be hospital-free days (HFD) over a 30-day period. Patient-centered outcomes, such as patient satisfaction and experience and out-of-pocket cost will be assessed at 30-days. Additional endpoints include HFD over 1-year, 30-day and 1-year cardiovascular HFD, rates of noninvasive and invasive cardiac testing, cardiovascular rehospitalizations, and cardiovascular repeat ED visits. Patients will be monitored for safety: the composite of cardiovascular death, myocardial infarction, and unplanned coronary revascularization at 30-days. This novel trial addresses a key evidence gap by providing guidance on how best to evaluate moderate risk ED patients with acute chest pain. Without this guidance care patterns are likely to remain heterogeneous, inefficient, non- patient-centered, and unguided by the highest level of evidence. We hypothesize that data from this trial will support widespread implementation of a CARE strategy, which could improve the quality and value of care for millions of patients in the U.S. and beyond.

NIH Research Projects · FY 2026 · 2023-04

Project Summary/Abstract Alzheimer’s disease (AD) is an aging-related multifactorial neurodegenerative disorder characterized by cognitive impairment and synaptic failure. Elucidation of the molecular signaling pathways that go awry in AD could provide insights into etiology and potential therapeutic strategies for this devastating neurodegenerative disease. Mounting evidence has linked abnormal over-activity of glycogen synthase kinase 3 (GSK3) to multiple aspects of AD pathophysiology. Meanwhile, the development of therapies based on GSK3 inhibition has been hindered by the lack of understanding of isoform-specific neuronal effects, particularly for GSK3α, which has been largely overlooked compared to GSK3β. Challenges in developing isoform-selective inhibitors for GSK3 present another layer of difficulties since inhibition of both α and β isoforms could result in significant toxicity. This project is aimed to investigate the isoform-specific roles of GSK3 in AD pathogenesis. We have selectively suppressed GSK3α or β isoform in the brain of AD model mice using genetic approaches. Moreover, we will test the first-in-class isoform-selective antagonists of GSK3 in AD models. Based on previous work and preliminary studies, the central hypothesis to be tested is that disruption of GSK3 isoforms, particularly GSK3α, represents a key molecular signaling mechanism underpinning AD-associated cognitive impairment. There are three specific aims. Aim 1 seeks to elucidate roles of GSK3α isoform in AD-associated synaptic plasticity impairments. Aim 2 is to determine the effects of GSK3 isoform-specific inhibition on cognitive impairments in AD model mice. Aim 3 is to identify molecular mechanisms associated with GSK3 isoform-specific roles in AD. The project includes in-depth analyses using multiple experimental approaches, including synaptic electrophysiology, pharmacology, imaging, mouse genetics, proteomics/phosphoproteomics, and behavioral tests. Data derived from the proposed studies could uncover previously unrecognized isoform-specific roles of GSK3 signaling dysregulation in AD etiology. The multidisciplinary approach shall enable us to identify detailed mechanisms associated with aberrant GSK3 isoform signaling in AD pathogenesis, thus providing insights into effective therapeutic targets and diagnostic biomarkers for AD and other aging-related cognitive syndromes.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY Major depressive disorder (MDD) and alcohol use disorder (AUD) are comorbid. Acute alcohol decreases both anxiety- and depressive-like behaviors. Chronic alcohol exposure increases both of these negative emotion- like behaviors. In fact, decades of epidemiological data suggest that individuals use alcohol to alleviate a negative affective state. Whether the negative emotional state is due to MDD or repeated alcohol exposure (AUD), researchers are now beginning to examine how mechanisms that are initially positive and rewarding shift toward negative affect/anhedonia during withdrawal. For example, much is known about the neural circuitry and neurotransmitter/peptide systems underlying acute and chronic effects of alcohol on anxiety-like behaviors. Surprisingly, much less attention has been directed at the negative affective (anhedonia) component. Our preclinical work suggests that acute ethanol treatment produces an antidepressant-like effect and hijacks the same biochemical pathway as NMDAR antagonists. Pivotal to ethanol and NMDAR antagonists’ antidepressant efficacy is the dynamic expression of the RNA-binding protein Fragile X Mental Retardation Protein (FMRP). Reduction in FMRP expression allows for the upregulation of transsynaptic proteins that promote new synapse formation and antidepressant-like effects. Consistent with a shift from positive to negative emotion-like behaviors, FMRP is upregulated during withdrawal in ethanol-dependent animals. Using molecular, biochemical, and electrophysiological techniques, combined with a novel synapse detection assay that we developed in our laboratory (DetectSyn), and the expertise of our collaborators in the Weiner laboratory in preclinical models of alcohol dependence, we will determine in Specific Aim 1 if acute ethanol regulates the binding of FMRP to transsynaptic mRNAs, regulates the synaptic protein synthesis of these target mRNAs, and promotes new synapse formation. Moreover, in Specific Aim 2 we will determine if the FMRP-regulated antidepressant signaling pathway is muted or suppressed during the WD/negative affect state, and if acute ethanol treatment can reverse the biochemical and behavioral depressive phenotypes. Furthermore, we will isolate and sequence FMRP target mRNAs that are repressed during WD but released due to treatment with acute ethanol during WD. Using a computational/genomic approach such as the Library of Integrated Network-Based Cellular Signatures – a method that has shown promise in AUD research – these data will facilitate future studies using these targets to predict effective, repurposed FDA-approved drugs to treat AUD and MDD. These studies will lay the foundation for future pharmacological intervention to treat negative affect during WD in alcohol-dependent individuals with comorbid MDD.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY / ABSTRACT The number of cancer patients and cancer survivors continues to increase while the prevalence of obesity also continues to increase in the US. Obesity is associated with a greater risk for developing 40% of cancers and two of the four most prevalent cancers (i.e. breast and colon), are tightly linked with obesity. 5 fluorouracil (5FU) remains the first line of treatment for colon cancer despite 5FU’s well established toxicities - cytopenia, mucositis, anorexia, weakness, and fatigue. These toxicities contribute to reduction in relative dose intensity, increase patient susceptibility to infection, and lead to debilitating functional impairments that not only burden the patient, but also the patient’s support system. Given the increased prevalence of obesity in the US, it is increasingly more likely that those needing to undergo anti-cancer treatment will be obese. While it is common practice to apply a dosing cap, the current recommendations for the treatment of obese cancer patients are to give full body surface area dosing regimens, despite some evidence suggesting obese patients have exacerbated drug toxicities and reduced survival. This evidence is not ubiquitous as certain investigations have highlighted better prognosis and survival with increasing BMIs. I have discovered that obese mice are unable to sustain 2-3 cycles of 5FU. This I have attributed to a reduction in dihydropyrimidine dehydrogenase (DPD), the enzyme responsible for catabolizing 5FU in the liver. This has highlighted the need for mechanistic inquiry into the impact of obesity on 5FU’s toxicities; my K99/R00 proposal addresses this unmet need and will serve as a platform to launch my independent career in this domain. The overall goal of my proposed K99/R00 is to: 1) to understand the impact of obesity on 5FU’s anti-cancer efficacy and 5FU’s off-target effects and 2) provide critical training to facilitate my transition to independence. My central hypothesis is that obesity induced non- alcoholic fatty liver disease (NAFLD) contributes to disrupted 5FU catabolism and increased toxicity through reduced DPD resulting in reduced functional quality of life and survival. I am proposing a mechanistic aim (1), an exploratory aim (2), and a treatment/intervention aim (3) to test this hypothesis: in aim 1, I will investigate the role of DPD in the metabolism and toxicity of 5FU with obesity (K99); in aim 2, I will explore the impact of 5FU on skeletal muscle and immune cell -omics with obesity (R00); and in aim 3, I will examine the utility of manipulating dietary macronutrients on 5FU’s efficacy and off-target toxicities with obesity (R00). My research aims are complemented by my four training aims: 1) Obesity Phenotyping and Specialized Diet Formation, 2) Plasmid Preparation, CRISPR/Cas 9 Utilization, and –omics, 3) Drug Metabolism (5FU metabolite analysis), and 4) Professional Development and Lab Management. I expect that my findings will provide paradigm shifting evidence for how obese patients should be dosed and monitored to limit chemotherapy’s off-target effects. Additionally, the results from these studies will serve as the foundation for a pathway to independence to continue examining the contributing factors underlying cancer patient life quality and survival.

NIH Research Projects · FY 2026 · 2023-03

My objective for the K25 award is to establish myself as an independent neuroimaging statistician, with expertise in whole-brain network analyses and an integral member of multidisciplinary research teams devoted to addressing diseases of the brain. Attaining these goals will require didactic training and research guidance. Research We will develop new methodology to improve whole-brain dynamic connectivity analyses of normal and abnormal brain function, which is vital for understanding various brain disorders, such as Alzheimer’s Disease, and may help identify biomarkers and inform early prevention and treatment. Previous studies are largely based on one average network constructed using data from an entire brain scan (i.e., static connectivity), but emerging evidence suggests network topology exhibits meaningful variations on the second to minute scale, creating a gap in understanding unless these variations are quantified. While several methods have been proposed to address this new direction in the field, there does not yet exist a unifying framework that accurately estimates whole-brain networks, as well as the dynamics of network change across a functional magnetic resonance imaging (fMRI) experiment, while a) accounting for variables of interest and motion- induced artifacts and b) allowing for individual estimates of dynamics. The novel methods proposed here will address these needs and provide a set of tools for future dynamic brain network analysis research. This research, along with my proposed training plan, will facilitate my progression toward becoming an independent neuroimaging statistician with expertise in brain network analysis. Training The proposed training program involves four components: 1) career guidance and neuroscience and network analysis training from a mentoring committee; 2) an educational component to establish fundamental knowledge in computational neuroscience and image analysis; 3) performing innovative research using the skills gained from the proposed training plan and; 4) participating in the exchange of knowledge and ideas with other statisticians and neuroscientists through workshops, conferences, seminar series, and journal clubs. The training will enable me to shift from an early career statistician to an established, independent, neuroimaging statistician with expertise in whole-brain network analyses. The training in computational neuroscience and image analysis will allow me to become a multidisciplinary research team scientist dedicated to studying the human brain. The growth gained through this 5-year period will lead to a skill set, and a confidence, that allows me to be more well-versed in the neuroscience and biology behind the data I am analyzing. This will ultimately lead to more effective communication with neuroscientists and clinicians, improved study design, more informed statistical analyses, and a more comprehensive interpretation of the results in my future work.

NIH Research Projects · FY 2026 · 2023-02

(<30 lines) Youth-onset type 2 diabetes (YO-T2D) is increasingly prevalent in parallel with the obesity epidemic, yet effective treatment and prevention strategies are limited. The physiologic increase in insulin resistance occurring during puberty, in combination with obesity-related insulin resistance, enhances the risk of T2D. Yet, it remains unclear why some youth progress through puberty with intact β-cell function, while others do not, despite similar phenotypic and metabolic characteristics. More information is needed regarding the unique events during puberty to better understand 1) the basic pathophysiology of glucose control, insulin sensitivity, β-cell function, and T2D risk in youth, 2) differences among girls and boys, populations at highest risk, and urban and rural geographies, and 3) the potential contribution of other risk factors including psychological, behavioral, and social and external contexts. Importantly, this research needs to address the timeline of pathophysiology and progression from normoglycemia or prediabetes to YO-T2D. The DISCOVERY of Risk Factors for Type 2 Diabetes in Youth (DISCOVERY) study provides a unique opportunity to characterize the risk progression profile and mechanisms underlying the development of YO-T2D, and evaluate the effects of modifiable and non-modifiable risk factors. Ultimately, the results of this study will establish a basic pathophysiology to inform future studies aimed at achieving target glycemia, improving insulin sensitivity, preserving β-cell function, and/or preventing YO-T2D. To address this goal, DISCOVERY will recruit, enroll, and follow a nationally representative cohort of 3,600 at-risk obese youth in early puberty; extensively phenotype them as they transition through puberty; and characterize the course of decline and dysfunction in pathophysiological indicators that lead to YO-T2D. The expected duration of the DISCOVERY is 5 years, including planning, recruitment, follow-up, analysis, and reporting. In addition, DISCOVERY will store longitudinal biospecimens and genetic material with the intention of acquiring additional ancillary funding to pursue analysis of emerging indicators. Wake Forest University School of Medicine/Health Sciences has experience in multicenter and diabetes-related investigations and will contribute to DISCOVERY through the recruitment of approximately 240 at-risk youth, implementation of the IRB-approved consensus protocol, participation on DISCOVERY committees, and collaboration on the analyses and dissemination of the findings from DISCOVERY.

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY Gene therapy (GT) clinical trials using AAV vectors are poised to fulfill the promise of a safe, affordable, lifelong correction of bleeding disorders following a single treatment. Still, clinical trials using AAV vectors to treat hemophilia A (HA) in adults have underscored the hurdles, such as the presence of pre-existing AAV antibodies, and unexpected risk of hepatoxicity in these patients. Importantly, this toxicity was not seen in preclinical animal studies, highlighting the dangers of extrapolating data from animal models to humans. Since the next step for GT to treat severe HA will be implementation of this approach in children, it is crucial to predict, as accurately as possible, unforeseen risks in this population. Currently, is unknown whether the unexpected immune/ inflammatory responses seen are due to the use of AAV as a delivery vehicle, or they are caused by the forced expression of FVIII within hepatocytes, which are not the native site of FVIII production. However, since similar toxicity has not been seen in AAV clinical trials for hemophilia B (hepatocytes are the natural site of FIX production), it is rational to posit that ectopic FVIII expression likely plays a role. In addition, preclinical data have also shown that, at the high doses used, AAV, long assumed to be largely episomal, may exhibit significant levels of host genome integration that could potentially drive clonal expansion and hepatocellular carcinoma (HCC), the risk of which increases as a result of hepatocyte proliferation. These are critical questions to safely extend the use of these potentially curative treatments to the pediatric population, in whom the higher proliferation and more primitive state of the liver may increase these risks. The overall goal of the present proposal is to utilize a human liver tissue equivalent (hLTE) platform to answer these questions and to determine the impact recipient age has on these variables. We will use hLTE to test the overall hypothesis that FVIII expression can be improved, the pre-existing immunity to AAV overcome, and the toxicity seen in clinical trials avoided, by optimizing the codon usage and/or sequence of the fVIII transgene to minimize the unfolded protein response and ER stress and/or by targeting transduction to hepatic endothelium, the native site of FVIII synthesis. Specifically, we will use a physiologically relevant hLTE platform to: 1) define age-dependent impact of AAV transduction vs. hepatocyte-targeted FVIII expression on human liver biology and function, the potential to trigger innate immunity, and whether optimizing the codon usage and sequence content of the fVIII transgene can prevent this undesired immune/inflammatory response; 2) test whether targeting AAV transduction to hepatic endothelium will improve FVIII expression, prevent hepatic inflammation/immunity, preserve liver function, and protect AAV from existing anti-capsid immunity; and 3) investigate if genomic integration frequency will be higher at younger ages, due to increased cell cycling, and whether targeting hepatic endothelial cells will decrease the potential for genotoxicity. It is hoped that these studies will identify the means to maximize the efficacy and safety of human liver-targeted AAV GT for HA and thereby pave the way for its use in pediatric patients.

NIH Research Projects · FY 2026 · 2023-02

Project Summary Infective endocarditis (IE) has high morbidity and mortality. Upwards of 30% of cases of IE are caused by oral bacterial species that enter the bloodstream and colonize heart valves. Frequent episodes of bacteremia from dental biofilm (plaque) are likely to be significant risk factors for development of IE. Our prior work demonstrates that: 1) there are only informal guidelines for prevention in 90% of people at risk for IE; 2) tooth brushing results in a high incidence of bacteremia of IE-causing species; 3) the risk of such bacteremia increases with the level of dental plaque and calculus present; and 4) patients with IE have a higher burden of dental plaque and calculus than matched non-IE controls. Although millions of people in the U.S. are at risk for IE because of pre-existing cardiac conditions, there are no prospective studies or solid evidence to show that improving oral hygiene reduces bacteremia from routine daily activities (e.g., toothbrushing). Evidence does show, however, that bacteremia is a strong surrogate marker for risk of IE. The lack of supporting data means that longstanding speculation about the importance of oral hygiene and gingival inflammation as risk factors for IE have had too little impact on practice guidelines on prevention, clinical practice, or funding for preventive care. The next step is to determine if professional scaling and oral hygiene instruction significantly reduce the incidence and duration of IE-causing bacteremia from toothbrushing. We plan to enroll 320 people at risk for IE into a clinical trial. Enrollees will be randomized to professional scaling and oral hygiene instruction versus routine oral care. We will test the steps in the hypothesized causal pathway from improved oral hygiene to decreased bacteremia from IE-causing species by: 1) determining the impact of professional scaling and oral hygiene instruction on the incidence and duration of bacteremia with IE-causing species during and following toothbrushing; 2) comparing oral hygiene and gingival health measures between randomized treatment groups and testing whether improvement in these measures is associated with reduced incidence and duration of bacteremia from toothbrushing; and 3) determining the degree to which reduction in bacteremia incidence and duration, and improvement in oral hygiene and gingival inflammation scores, are maintained following the intervention. This study will provide novel, important data to inform the healthcare community, guideline committees, and health funding agencies of the importance of improving oral hygiene and reducing gingival inflammation as primary preventive measures for all people at risk of IE.

NIH Research Projects · FY 2026 · 2023-02

Project Summary The opioid epidemic currently affecting the United States has entered a new wave of mortality, with combined use of opioid and psychomotor simulants as a major contributing factor to the number of overdose deaths. Combined use of fentanyl and methamphetamine may be due to a number of factors, including greater rewarding effects, decreased negative side effects, and/or feelings that combined use is somehow ‘safer’ than use of fentanyl alone. For those reasons, there is a clear need to examine the differences in the behavioral and neurobiological alterations that occur following chronic use of fentanyl, methamphetamine, and these two substances in combination. My preliminary findings suggest that there is enhanced hypodopaminergia following combined use of fentanyl and methamphetamine (COMBO) when compared to fentanyl use alone. I have determined that 1) in both male and female rats, elicited DA release was decreased in COMBO animals compared to fentanyl alone animals and; (2) male COMBO animals had greater response rates and responded more on a progressive ratio schedule of reinforcement. Therefore, this study aims to investigate the scarcely studied combination of fentanyl and methamphetamine via a three-pronged approach examining behavioral and neurobiological alterations to the DA system through self-administration, microdialysis and fast scan cyclic voltammetry, and the use of the novel photosensor dLight during behavioral responding. Collectively, the proposed aims will provide insight into the neurobiological and behavioral differences elicited by combined use of fentanyl and methamphetamine, an under-studied topic that is highly relevant for the current state of the opioid epidemic in the United States.

NIH Research Projects · FY 2026 · 2023-01

SUMMARY Food is perceived and enjoyed through the multisensory quality of “flavor”, which involves the senses of taste and smell, but is not easily dissociated into its components. Flavor perception has a major influence on food choice and is thus directly linked to health. However, the underlying neural mechanisms remain mysterious. Previous work in humans has suggested that the brain actively combines taste and smell inputs to create our sense of flavor, and that this process depends heavily on eating experience. However, human imaging techniques lack spatial and temporal resolution to provide a mechanistic understanding of how neural circuits produce multisensory flavor representations. Moreover, people’s highly subjective eating history precludes a systematic understanding of how experience drives the development of flavor processing. To overcome these issues, the present proposal takes a unique approach to the study of flavor. Using the awake, tasting rat as an animal model allows us to directly access to the neural computations underlying flavor processing at the cellular level, and complete control over the individual’s flavor experience. The proposed hypotheses build directly on our own recent findings on cross-modal flavor processing in rat olfactory cortex, as well as decades of work on the development of multisensory computations in cortical and subcortical regions of other multisensory systems. The project comprises a coherent series of experiments that systematically seeks to provide mechanistic understanding of the neural circuits that integrate flavor-related sensory information. Specifically, we will answer the following questions: 1) What are the guiding principles by which olfactory cortical circuits integrate taste and smell inputs? To address this, we will use electrophysiological techniques to record responses from olfactory cortical neurons to taste-smell mixtures as well as their unisensory components in awake adult rats; 2) How are the multisensory operations performed by olfactory cortical circuits shaped by experience with specific flavors? To address this, we will experimentally manipulate rats’ experience with specific taste-smell mixtures, and record responses from ensembles of olfactory cortical neurons to congruent, incongruent and unexposed taste-smell mixtures as well as their unisensory components; and 3) How does the ability to integrate cross-modal inputs develop across the lifespan? To address this, we will track uni- and cross-modal responsiveness of olfactory cortex in awake rats across different stages of early postnatal development; To successfully achieve these objectives, our unique team of investigators brings together expertise in flavor processing and awake rodent olfactory cortex physiology (PI Maier), the computational basis of multisensory interactions (Co-I Rowland), and the development of multisensory systems (Co-I Stein).

NIH Research Projects · FY 2026 · 2022-12

SUMMARY Treatment of glioblastoma (GBM) represents an unmet need in medicine. We have been pursuing a therapeutic approach of delivering potent targeted and specific cytotoxins using continuously evolving convection-enhanced delivery. Patients with GBM over-express interleukin 13 receptor alpha 2 (IL-13RA2), EphA2, EphA3 and EphB2 receptors that are present in various pathophysiological compartments of GBM and all four are expressed in tumor cells of the core of tumor, and in locally-infiltrating tumor cells, while EphA2 is also found in tumor neovasculature. Further, IL-13RA2, EphA2, and EphA3 are associated with, and play crucial roles in, the pathobiology of glioma stem-like cells. Finally, the EphA3 receptor are found in M2 GBM- associated macrophages. Thus, collectively, IL-13RA2, EphA2, EphA3 and EphB2 are over-expressed in principal GBM compartments shown to be involved in tumor progression and/or resistance to therapies. In a first-of-kind approach, we performed Phase I clinical trial in dogs with spontaneous gliomas, which represents a faithful model of human disease, using a cocktail of cytotoxins targeting IL-13RA2 and EphA2 receptor. We observed exceptional anti-tumor responses, including several near complete regressions, prolongation of survival and excellent quality of life in this dose-finding trial, at no toxicity. In addition, we found evidence for immune system activation during the therapy. Encouraged by these results, we pursued the novel idea of targeting all four receptors instead of two with one pharmaceutical compound. One of the Eph receptor ligands, ephrinA5 (eA5), binds EphA2, EphA3 and EphB2 receptors. We have thus generated an agent based on eA5 and IL-13 mutants targeting all four receptors using an IgG1 scaffold (QUAD). In our initial experiments, the QUAD was conjugated to derivatives of Doxorubicin (Dox) or a derivative of Pseudomonas exotoxin A, PE38QQR, to generate single pharmaceutical agents and these drug conjugates retained their binding affinities towards the targeted receptors while demonstrating prominent killing activity on GBM cells. QUAD- Dox and QUAD-PE38QQR conjugates have already shown prominent, long-lasting anti-tumor effects in dogs with spontaneous glioma at no toxicity: 60, 88, and 91% of tumor volume regression in the treated dogs, respectively. Recently, we have conjugated QUAD to DM1, a microtubule-disrupting agent. The QUAD-DM1 is extremely potent on GBM cells with IC50s in low femtomolar range, ~50x better than the Dox/PE conjugates. Therefore, we will continue this exciting line of research through Specific Aims as follows. In Specific Aim 1, we will treat dogs with spontaneous newly diagnosed and recurrent high-grade gliomas with QUAD-DM1. In Specific Aim 2, we will examine immune responses and the phenotype and genotype of recurring tumors in the course of QUAD-DM1 therapy. Our approach addresses crucial issues of inter- and intra-tumoral heterogeneity and evokes an in situ vaccination or so called “tumor inflaming” effect. We envision that this all-out assault, termed by us “molecular resection”, will result in a more effective management of GBM.