Univ Of North Carolina Chapel Hill

NIH Research Projects · FY 2025 · 2024-07

The University of North Carolina (UNC) Molecular and Cellular Biophysics Program (MCBP) is a successful interdisciplinary graduate training program with a mission to train graduate students to conduct biomedical research, probing the molecular and cellular processes of life and disease using biophysical approaches. The MCBP has three core objectives: 1. Attract a cohort of talented graduate students to apply the methods and concepts of the quantitative and mathematical sciences to problems in biology; 2. Provide training in fundamental biophysical principles and techniques important for developing molecular-level descriptions of complex biological systems and processes; 3. Provide professional training to enable and potentiate career trajectories in the biological sciences. This proposal requests 10 training slots to support 5 students in their second year and 5 students in their third year of graduate school. The MCBP fulfills a specific need at UNC, uniting graduate students and faculty across the campus under a comprehensive molecular and cellular biophysics training program. The program selects students from a variety of UNC graduate school-admitting portals including biological sciences, chemistry, and physics, that align with the academic interests of our 36 faculty, who are drawn from 9 departments from across the School of Medicine, the School of Pharmacy, and the College of Arts and Sciences. Our students and faculty share an interest in characterizing and modeling the biophysical behavior of macromolecules and cellular systems. By promoting interactions across biology, physics, and chemistry, MCBP students gain proficiency in cross-disciplinary science and communication. Students complete rigorous coursework in small classes rooted in the theory, and application of biophysics. Students become trained in the responsible conduct of research and rigor and reproducibility. Specific training includes core classes in the principles of the molecular and thermodynamic behavior of macromolecules, elective courses in methods to investigate macromolecular structure and function and behavior, both in vitro and in cells, and a seminar class in biophysics where students are trained in scientific presentations, communication and critique, attend seminars, and meet with visiting speakers. The MCBP further promotes student development through the Biophysics Colloquium, by hosting a biennial North Carolina Biophysics Symposium, a class in written communication, career panels with program graduates, and via student advising. Over its 28-year history, MCBP graduates have used the skills they developed in the program to become leaders in research and education across academics and industry.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT People with serious mental illness (SMI) are substantially overrepresented in the US criminal justice system. In an effort to reduce recidivism among this vulnerable population, average effects of administering single interventions to all have been studied in recent years. These interventions include ones that target SMI symptoms (such as mandated psychiatric services) and others that target criminogenic risk factors (such as cognitive behavioral therapy). However, justice-involved people with SMI are diverse in their characteristics, needs, and risk factors; there is no “one-size-fits-all” solution to prevent recidivism. Decreasing adverse criminal outcomes for this population requires recognizing its heterogeneity. Therefore, the broad objective of this proposal is to understand which interventions work best for which justice-involved people with SMI using state-of-the-art precision medicine methods drawn from causal inference, statistical theory, and machine learning. This is a step toward the long-term goal of identifying optimal ways to reduce criminal behavior in this population by matching interventions to those who need and benefit from them. Supported by a dedicated and talented mentorship team, the training portion of this grant will fill gaps key to achieving this goal: 1) subject-matter training and field experience on SMI in the criminal justice system, 2) development of novel technical methods in precision medicine, 3) skills in translating technical findings to high-impact recommendations for practice and policy, and 4) professional skills development. This rich training will provide a foundation for accomplishing the following research aims to reach the proposal's broad objective: 1) understand the differential pathways that situate this population at risk for re-offending, 2) identify tailored rules for administering interventions to those who benefit from them based on individual characteristics, and 3) assess whether and how the identified rules for matching interventions generalize across the US. This research will leverage randomized controlled trial data, federal data from the Administrative Office of the US Courts, and state data from the North Carolina Department of Public Safety to achieve these aims. Importantly, research from this grant will result in novel statistical precision medicine methods for transporting rules to settings different than those on which they are learned. The aims of this grant are well-aligned with NIMH's strategic goal of striving for prevention and treatments among people with SMI, including, but not limited to, the objectives of: a) advancing methods to match interventions to individuals and populations (especially marginalized and underserved communities) and b) strategies for scaling up interventions for the greatest public health impact. By learning who benefits from which interventions using the latest advances in precision medicine methods, this work has the potential to help stop the so-called “revolving prison door” for justice-involved people with SMI. Further, this research will advance the toolkit for data-supported precision medicine strategies that most efficiently and effectively increase public health and safety.

NIH Research Projects · FY 2025 · 2024-07

PROJECT ABSTRACT Focal epilepsy is the most common form of refractory epilepsy. Focal seizures are often associated with malformations of cortical development (MCD) but can also occur in the absence of a radiographically-detected lesion. Patients will often require surgical resection of the epileptogenic lesion to control their seizures, as many patients develop a drug resistance to the numerous antiepileptic drugs available. The availability of resected tissue has allowed for the study of the role of somatic variation in focal epilepsy. Somatic variation arising during embryonic brain development is increasingly recognized as a major contributor to genetic risk of focal epilepsy. Somatic variants localized to the brain have been readily identified in hemimegalencephaly and focal cortical dysplasia (FCD) type II; furthermore, evidence suggests that somatic variants also contribute to FCD type I and radiographically nonlesional focal epilepsy. While there has been substantial progress in our understanding of the genetic architecture underlying focal epilepsy, more work is needed to characterize the remaining cases lacking a genetic cause. Continued work to identify novel genes and the types of variants involved in focal epilepsy is essential to further advance understanding of the underlying mechanisms of disease and fuel the development of novel therapeutic approaches. The overall goal of my dissertation and postdoctoral research is to advance novel gene discovery in focal epilepsy and identify pathways that cause focal seizures to better inform drug discovery strategies and widen the scope of treatment. I have demonstrated my ability to identify putative pathogenic somatic variation affecting known epilepsy genes, as shown in my preliminary data in Aim 1. I have also identified a disease-causing variant in genes not yet associated with focal epilepsy. In Aim 1, I propose using deep exome and targeted sequencing approaches to identify novel genes and variants contributing to intractable focal epilepsy. Additionally, I propose using duplex sequencing to detect low abundance somatic variants that may have been missed by exome and panel sequencing. In the K00 Phase, I plan to expand these studies to in vivo models to probe the functional consequence of pathogenic somatic variation in the zebrafish model system. The proposed research provides opportunities for developing technical expertise in gene discovery, functional characterization of disease-causing somatic variation and drug discovery. I will rely heavily on the support of my sponsors to contribute to the development of my skillset in experimental design, scientific communication, and grantsmanship; as well as advancing my search for a postdoctoral training environment that is aligned with my research interests and career goals. The training plan outlined in this proposal integrates scientific and professional development that will put me on a trajectory to emerge as a well-rounded and independent investigator.

NIH Research Projects · FY 2025 · 2024-07

Project Summary Abstract Alteration of essential metabolic pathways is a major mechanism by which oncogenic KRAS promotes tumor development and growth in pancreatic ductal adenocarcinoma (PDAC). KRAS-driven PDAC is dependent on macropinocytosis (MP) and autophagy to fuel the high metabolic demand of rapid proliferation. Thus, these metabolic processes are attractive targets for the development of treatments for PDAC. Genetic depletion of KRAS results in downregulation of MP in PDAC. Additionally, our lab demonstrated that KRAS depletion or inhibition of ERK-MAPK signaling decreased glucose uptake and glycolysis but increased autophagy, thereby enhancing dependency on autophagy for essential nutrient supply. Accordingly, dual RAS-pathway and autophagy inhibition via chloroquine (CQ) synergistically enhanced efficacy in PDAC. Clinical data demonstrated that resistance to this treatment arises over time through unknown mechanisms. Preliminary data indicates that following RAS or ERK inhibition, both autophagy induction and MP downregulation are transient—with autophagic and MP activity returning to/surpassing basal levels after prolonged treatment. Recent work has suggested that these nutrient scavenging processes are able to compensate for loss of each other. We demonstrate an inverse temporal relationship between autophagy and MP following depletion or inhibition of the KRAS-ERK MAPK pathway. Furthermore, we show that CQ, commonly thought to inhibit lysosomal acidification, does not prevent degradation of MP-derived proteins. We hypothesize that there is compensatory regulation between autophagy and MP in the context of RAS-pathway inhibition. Thus, during prolonged RAS-pathway and autophagy inhibitor treatment, PDAC cells upregulate MP, consequently abrogating dependency on autophagy and reducing sensitivity to autophagy inhibition. Aim 1 will test the hypothesis that upregulation of MP confers resistance to dual autophagy and RAS-pathway inhibition in vitro and in vivo. Additionally, we present the unexpected observation that prolonged inhibition of the ERK-MAPK pathway results in the upregulation of MP. Previous PDAC studies have employed genetic depletion of KRAS to demonstrate the role of oncogenic KRAS in driving MP with no evaluation of inhibitors of KRAS or the ERK-MAPK pathway. Given the dependence of PDAC on macropinocytic uptake for tumorigenic growth, it is imperative to understand how these inhibitors regulate MP. Initial Aim 2 studies will be dedicated to comprehensive characterization of MP regulation in the context of genetic and pharmacological inhibition of KRAS and ERK over time. Subsequent work in Aim 2 will determine whether the changes in MP via RAS-pathway inhibition corresponds with changes in uptake and efficacy of nab-paclitaxel. Further understanding of RAS-driven regulation of these essential metabolic pathways in PDAC will inform future development of RAS-pathway inhibitor combinations. These studies will require my application of a diverse spectrum of experimental approaches, advance my understanding of key steps in anti- cancer therapeutic development, and foster my career development as an independent cancer researcher.

NIH Research Projects · FY 2025 · 2024-07

SUMMARY ABSTRACT Clostridioides difficile is among the most common causes of nosocomial infections with disease ranging from antibiotic-associated diarrhea to pseudomembranous colitis. Secreted toxins are largely responsible for disease development, yet many aspects of C. difficile physiology and virulence remain poorly understood. Recent work has revealed that C. difficile produces two colony morphotypes—a rough colony variant and a smooth colony variant—and can reversibly switch them. This phenomenon is conserved among diverse C. difficile strains. The two morphological variants differ in several ways. Bacteria from rough colonies are longer and often found in chains, exhibit greater surface motility and diminished swimming motility, produce less biofilm biomass, and show greater pathogenicity in animal models compared to the smooth colony counterpart. Our prior work linked colony morphology and the correlated phenotypes to the expression of genes encoding a signal transduction system, CmrRST, but the molecular mechanisms by which C. difficile develops rough and smooth colony morphologies have yet to be defined. The objective of this study is to identify genes required for formation of each colony morphotype and to determine the roles of these genes in cell morphology, motility, biofilm formation, and virulence. In Aim 1, we propose three complementary yet independent mutagenesis and screening strategies to identify genes required for rough and smooth colony development. This work takes advantage of mutants designed to yield only one colony morphotype. The in vitro phenotypes of the mutants obtained through the genetic screens will be evaluated to determine the broader impact of the identified genes on C. difficile physiology and virulence traits. In Aim 2, we will examine how one identified mutation results in strictly rough colonies and assess its ability to colonize and cause disease in a mouse model of C. difficile infection. Similar strategies will be used to characterize additional mutants obtained in Aim 1. Pilot studies with the proposed approaches have identified multiple candidate genes predicted to affect cell division. As such, these gene products may serve as new targets for therapeutic development, yet almost none of these genes have been previously studied. The proposed research will identify factors contributing to disease relevant phenotypes including cell division proteins, facilitating efforts to combat C. difficile infection.

NIH Research Projects · FY 2025 · 2024-07

ABSTRACT This K08 application is to support the research and career development of Dr. Adam Lietzan, a periodontist and research assistant professor at the UNC Adams School of Dentistry. This award will provide Dr. Lietzan an intensive mentored experience in order for him to complete the transition to an independent clinician-scientist with a research program focused on understanding the dynamic immune responses to microbial dysbiosis during periodontitis. As such, a training plan including didactic, hands-on, and career development experiences has been put forth to enable this transition. During this award, Dr. Lietzan will gain skills/knowledge in: 1) host- pathogen interactions, 2) cutting-edge techniques to evaluate immunological responses, 3) proposal development, scientific writing, and oral presentations, and 4) leadership. This will allow Dr. Lietzan to develop collaborative opportunities with scientists and clinicians across multiple disciplines and a high-quality independent research program. Dr. Lietzan has established a multi-disciplinary team to provide expertise in research training and mentorship in career development. Together this team will provide foundational training in immunology, cell-based techniques, and animal models as well as help advise with critical aspects of the career development. This will be coupled with a strong research plan. The onset of inflammation is facilitated by a cascade of signaling molecules released by both canonical (e.g. macrophages) and noncanonical immune cells (e.g. epithelial cells). The highly potent interleukin (IL)-1 family of signaling molecules primarily induces inflammatory damage and is heavily implicated in periodontitis development and severity. Interleukin-37, an IL- 1 family member, conversely suppresses inflammation and has been shown to be dysregulated in severe forms of periodontitis. The oral epithelial cell (OEC), which acts as a physical, chemical, and immunological shield against infection, highly expresses IL-37 when exposed to microbial stimuli. Despite this knowledge, the impact of IL-37 on OECs and the molecular interactions that underlie its anti-inflammatory bioactivity in periodontitis remains largely uncharacterized. Here Dr. Lietzan seeks to fill this knowledge gap by dissecting the cellular pathways that IL-37 modulates during microbial challenge of OECs using in vitro and in vivo model systems. The molecular interactions between IL-37 and its receptors, which are highly expressed on OEC, will also be delineated. Lastly, the relevance of glycosaminoglycans (GAGs) within the periodontal extracellular matrix on IL- 37 bioactivity will be investigated. Successful completion of this proposal will inform the development of new and/or improve existing IL-37-centric interventions aimed at modulating chronic inflammation, such as that associated with periodontitis. Together these training and research activities will be a significant step towards Dr. Lietzan achieving his long-term goal of becoming an independent clinician-scientist who can bridge the gap between basic science discovery and clinical care advances through a multi-disciplinary approach.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY We propose MomGenes Fight PPD: Expanding to Increase Participation from Diverse Ancestries to systematically investigate the genomic risk factors for postpartum depression (PPD) and their interplay with major environmental contributors. PPD affects ~500,000 women annually in the US, with an increased prevalence among black, indigenous, people of color (BIPOC) mothers. The effects of PPD are not only present in the mothers, but also can be seen in offspring at a critical developmental period. Despite the public health issues PPD poses, it is under-studied and its precise etiology is unknown. There is pressing need to develop methods for early detection of PPD for all women to mitigate severe outcomes and identify features of the disorder which can lead to personalized therapies. The need for diversity is evident across biomedical research and healthcare. Within genomics, increasing representation of BIPOC individuals results in improved locus discovery, fine-mapping, and genetic score accuracy. However, as we diversify research participants, we must also capture environmental contributors to PPD that disproportionately affect BIPOC women: adverse life events (ALE), which increase risk for PPD, and discrimination. Combined with our proposed increase in phenotyping and genetic analyses, we can have a more complete understanding of risk factors contributing to PPD. In 2016, we began the MomGenes Fight PPD study (formerly called PPD ACT) to power genome-wide association studies (GWAS) for PPD. Using our existing ascertainment platform, MomGenes will: Aim 1) expand the study to recruit 8,000 new samples (4,000 PPD cases and 4,000 ancestry-matched controls) from across the United States, focusing on BIPOC women and recontact ~12k existing MomGenes participants to collect deeper phenotypes; Aim 2) conduct trans-ancestry GWAS meta-analyses for PPD and a pre-planned set of secondary analyses using newly ascertained samples and existing PPD samples (~51,000 cases, ~141,000 controls from first GWAS and eight new cohorts); Aim 3) examine the impact of ALE (natural disaster, sexual abuse, physical abuse) and discrimination (racial, gender, religion, sexual orientation, weight) on PPD risk. This proposal expands on our team’s success in PPD genomics research. Samples collected in our initial MomGenes study contributed to the first PPD GWAS meta-analyses. With the proposed work, we will continue to build on this success, identifying genetic and environmental factors that increase risk for PPD. Ultimately, this can lead to actionable findings that aid all women suffering from PPD.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY In chronic infection and cancer, CD8 T cells differentiate into an epigenetically distinct, dysfunctional lineage referred to as exhausted T cells. Through use of in vivo genetic screens and genetic mouse models, we have generated preliminary evidence that epigenetic regulators of the bromodomain and extraterminal domain (BET) family orchestrate CD8 T cell differentiation and exhaustion during infection. The BET family is composed of highly druggable chromatin readers BRD2, BRD3, BRD4, and germ cell-restricted BRDT. First-generation pan- BET inhibitors (BETi) have been evaluated clinically for cancers and inflammatory diseases, and have garnered considerable interest for the treatment of infections. However, BETi clinical trials have, so far, yielded mixed results. A critical roadblock to improving BETi therapy is the lack of a detailed molecular understanding of the function of each individual BET protein in the immune system. This proposal seeks to define how BET proteins intrinsically regulate T cell exhaustion, which is crucial for advancing our fundamental understanding of epigenetic regulation of T cell differentiation and for optimizing BETi strategies to reprogram exhaustion. Accordingly, our preliminary data indicate that BET proteins have discrete, non-redundant roles in regulating CD8 T cell differentiation and stemness in vivo. We have also found that a tailored, finite BETi treatment strategy may allow for efficient exhaustion reprogramming and optimal T cell function. Here, we will use inducible genetic depletion strategies to pinpoint the intrinsic role of each BET protein in controlling T cell fate during chronic infection, and we will investigate whether precisely timed treatment with second-generation BETi targeting individual BET proteins effectively modulates exhaustion. Last, despite widespread therapeutic interest in epigenetically reprogramming exhausted cells, epigenetic mechanisms governing T cell exhaustion remain to be fully elucidated. Based on preliminary findings, we will investigate whether BRD4 promotes exhaustion through remobilizing to exhaustion-specifying enhancer regions. We hypothesize that BRD2, BRD3, and BRD4 coordinate CD8 T cell differentiation into an exhausted state, and BETi treatment can be optimized to restore function to exhausted cells during chronic infection. We propose to: Aim 1, determine the role of BRD4 in controlling the differentiation and stability of exhausted CD8 T cells during chronic infection. Aim 2, define the role of BRD4 in regulating the function of exhaustion-specific enhancer elements in CD8 T cells. Aim 3, assess BRD2 and BRD3 as novel regulators of T cell exhaustion and investigate the impact of BET inhibition on CD8 T cell differentiation during chronic infection. Resolving the roles of BET proteins in exhaustion has broad implications for understanding fundamental epigenetic control of T cell differentiation, the immunological consequences of BETi, and may reveal strategies for genetic or pharmacological reprogramming of exhaustion.

NIH Research Projects · FY 2026 · 2024-06

ABSTRACT Cannabis is the third most commonly used substance in the United States (US), with almost half of all US adults reporting lifetime use. Cannabis use is associated with physical harms (e.g., respiratory diseases), psychological harms (e.g., cognitive deficits), and other types of harms (e.g., car crashes). Despite these harms, many adults underestimate or do not know about the risks of cannabis use. Warning labels on cannabis packages are a population-level intervention that can increase risk perceptions, knowledge, and recall of cannabis harms, however, there is a lack of research on how cannabis warnings can be improved. Currently, most US states with legalized cannabis require text-only cannabis warnings, but they are often placed on the back of packages, are small (e.g., 6-point font), are wordy (e.g., more than 100 words), and read like a legal disclaimer. As a result, current cannabis warnings are unnoticed, hard to read, and confusing. Accordingly, the long-term goal of this research is to develop cannabis warnings that inform people about cannabis harms and that states can implement into their cannabis warning regulations. The overall objective of this research is to rigorously examine the current landscape of cannabis warning regulations and experimentally determine which warning characteristics most effectively communicate the harms of cannabis use. The central hypothesis is that large cannabis warnings with characteristics found to be promising (e.g., have icons, include colors) will increase risk perceptions of cannabis harms. To accomplish our long-term goal and objective, the proposed study will include three specific aims: Aim 1: Examine the legal and regulatory landscape of US cannabis warnings; Aim 2: Develop a set of evidence-based cannabis warnings and identify which warning characteristics increase perceived warning effectiveness; and Aim 3: Experimentally determine if large cannabis warnings with characteristics found to be promising in Aim 2 increase risk perceptions, knowledge, and recall of cannabis harms. Included within these aims are rigorous and innovative methods: a comprehensive legal analysis, key informant interviews with cannabis regulators, an expert panel review, online experiments, and a discrete choice experiment. Throughout the project, we will disseminate findings from our study to regulators so that they can implement evidence-based warnings and impact change in the cannabis regulatory landscape. This project is significant because each state that legalizes cannabis is tasked with developing regulations on warning labels, but there is a lack of rigorous published research, especially from the US, on the effectiveness of different cannabis warning themes (e.g., content), characteristics (e.g., text length, color) and format (e.g., size). Therefore, our project can help meet a critical need for a comprehensive and rigorous evaluation of cannabis warnings. The proposed study directly addresses NIDA’s priorities by assessing the impact of state-level policies on cannabis use as well as a Notice of Special Interest on public policy effects on cannabis-related outcomes.

NIH Research Projects · FY 2025 · 2024-06

Cardiac fibroblasts (CFs) are the major cardiac cell type responsible for producing extracellular matrix (ECM) proteins, forming a structural scaffold crucial for supporting cardiac tissue during development and homeostasis. CFs are also known for their high plasticity, enabling them to swiftly respond to injuries and pathological conditions. Under such circumstances, CFs are rapidly activated and become transdifferentiated into myofibroblasts that produce and secrete an excessive amount of ECM components, ultimately leading to fibrotic scarring that disrupts tissue compliance and accelerates the progression toward heart failure. The transformation of CFs into myofibroblasts requires wholesale programming of the CF transcriptome. Yet, in addition to transcriptional regulation, post-transcriptional regulation by RNA-binding proteins (RBPs) has emerged as a critical regulatory layer for controlling gene expression. RBPs actively regulate every step of mRNA life cycle, including splicing, stability, and translation. In our preliminary studies, we found that one of RBPs, Ybx1, was significantly upregulated during CF to myofibroblast conversion and further discovered a potentially important role of this RBP in myofibroblast formation. Specifically, we found that Tcf21:MerCreMer mediated ablation of Ybx1 inhibits the transformation of CFs into myofibroblasts and reduces cardiac fibrosis. While the conversion CFs to myofibroblasts is inherently pathological, the extensive pool and plasticity of resident CFs has been recently harnessed for cardiac regeneration whereby CFs are reprogrammed into induced cardiomyocytes (iCMs) by local delivery of three cardiac transcription factors (TFs) - Gata4, Mef2c, Tbx5 (abbreviated as GMT). Building upon our intriguing finding that depletion of Ybx1 attenuates CF to myofibroblast conversion and reduces cardiac fibrosis, we posed the question whether Ybx1 ablation enhances GMT-mediated iCM reprogramming. Indeed, our preliminary study indicates that, compared to MGT-mediated iCM reprogramming, delivery of GMT with Ybx1 depletion enhances iCM induction, further attenuating cardiac fibrosis and improves heart function following MI. Combining these two lines of investigation and the corresponding preliminary data, in this proposal we will leverage our series of unique tools, reagents, and animal models to address our hypothesis that Ybx1 activity exerts a significant influence on the fate switch involving CFs, specifically the transformation of CFs into myofibroblasts and the reprogramming of CFs into iCMs, which can be leveraged for reducing fibrotic scarring and regenerating lost myocardium after MI.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY/ABSTRACT This NCI K08 Mentored Career Development Award will prepare Dr. Daniel Richardson to become an independent R01-level investigator in patient-centered cancer care delivery for patients with acute myeloid leukemia (AML). AML treatment paradigms are changing for older patients. After almost 40 years without any new treatments, 10 new therapies have been approved since 2017. These approvals have introduced clinical equipoise into treatment decision-making for many older patients. Choosing the “best” treatment option often depends on what each patient values most: optimizing quality of life or prolonging survival. Clinicians and patients can engage in shared decision-making to clarify patient values and help patients arrive at informed preferences about therapy. Multiple stakeholders have called for the development of new tools to support patients and clinicians in this complicated process. Dr. Richardson plans to work under the guidance of his mentors to evaluate a novel values elicitation tool called “PRIME” (Preference Reporting to Improve Management and Experience). PRIME generates a personalized values report in real-time to inform shared decision-making based on best-worst scaling (BWS), a simple yet robust values clarification method. This proposal builds on Dr. Richardson’s previous research with his mentors to develop values elicitation surveys for AML patients. The objective of this proposal is to evaluate the feasibility of using PRIME to improve treatment decisions and optimize its implementation for a RCT to evaluate efficacy. The aims of this study are (1) to determine the feasibility of using PRIME to improve treatment decision-making and (2) to identify barriers and facilitators of implementing PRIME into clinical workflows to inform development of appropriate implementation strategies. Success in this project will advance innovative, rigorous methods to capture patient values that may have broad applicability across oncology. This project will also promote Dr. Richardson’s long- term research goal of improving the integration of patient values into treatment decision-making. Dr. Richardson is supported by a mentorship team with expertise in values elicitation (John Bridges, PhD), implementation science (Stephanie Wheeler, PhD), and cancer care delivery trials (Ethan Basch, MD, Antonia Bennett, PhD). As part of this K08 award, he will develop expertise in cancer care delivery trial design and execution, values elicitation methodology, and implementation science.

NIH Research Projects · FY 2026 · 2024-06

The latent infection of human immunodeficiency type 1 virus (HIV) is the major hurdle to the eradication of HIV. A better understanding of the molecular basis of HIV latency is essential for the development of proper strategies to attack such stable HIV reservoirs. HIV latency is largely controlled by epigenetic regulations surrounding the chromatin proximal to the HIV promoter, i.e. HIV long terminal repeats (LTR). However, our understanding of epigenetic regulation of HIV transcription remains incomplete. This is evidenced by the fact that an effective reduction of HIV reservoirs has not been achieved in people with HIV (PWH) by the inhibition of histone deacetylase alone or in combination with other latency reversal reagents. We recently found that HIV transcription was activated from latency when crotonylation is induced. This was associated with enhanced histone crotonylation and acetylation but reduced histone methylation at the HIV LTR. Crotonylation induction also enhanced latency reversal elicited by the activation of NF-κB signaling pathway. Importantly, while crotonylation is controlled by the same enzymes stimulating acetylation to activate gene transcription (e.g. p300), crotonylation and its downstream signaling are regulated by distinct mechanisms, which are independent of histone acetylation. Furthermore, unlike the inhibitory role of acetylation reader BRD4 in HIV transcription, the crotonylation reader ENL is essential for HIV latency reversal elicited by the induction of histone crotonylation. Of interest, the opposite may also hold. For example, although crotonylation is reversed by the same enzymes regulating deacetylation to induce latency (e.g. HDACs), decrotonylation-efficient but deacetylation-deficient HDAC3 directly suppresses the Tat transactivation of HIV. Lastly, we discovered several selective HDCR inhibitors (HDCRi) that prefer to induce histone crotonylation over acetylation in both T cells and brain myeloid cells. These selective HDCR inhibitors did not dock into the deacetylation enzymatic pocket at the HDAC3 crystal structure predicted by Google AlphaFold. Instead, they docked just outside the enzymatic pocket, distinct from the nonspecific HDACi SAHA. Of note, one such HDCRi prevented HIV entry to latency. The overall objective of this application is to determine the molecular mechanism of histone decrotonylation underlying HIV latency. We hypothesize that distinct from histone deacetylation, decrotonylation plays an important role in the establishment of HIV latency, and this can be applied to our current efforts to control HIV latency by preventing HIV entry into latency. Our goals will be achieved through 3 specific aims, directed at the following premises: Aim 1: Crotonylation is distinct from acetylation to regulate HIV transcription. Aim 2: Decrotonylation uniquely regulates HIV latency. Aim 3: Pharmacologic modulating decrotonylation prevents the establishment of HIV latency.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY Rates of potentially life-threatening IgE mediated food allergy continue to rise, and therapeutic options are limited. Understanding how to alter immune tolerance in patients with food allergy is paramount to the development of new therapies. Two types of food allergen immunotherapy, oral immunotherapy (OIT) and sublingual immunotherapy (SLIT), rely on the administration of gradually increasing amounts of allergen to induce tolerance. For some children, these therapies can induce a state described as remission, where the protection against allergic reactions lasts weeks to months after stopping therapy. While current research has focused on the adaptive immune responses to immunotherapy, the immunologic milieu necessary for these responses is not known. Using metabolomic profiling, the large-scale quantification of metabolites by mass- spectrometry, we identified 3 major pathways (bile acids, arachidonic acids, and histidine metabolites) with known roles in modulating T cell biology that differentiate children who develop remission versus those who do not. The overall goal of this proposal is to determine the role that immunomodulatory metabolites play in the remission of food allergies induced by immunotherapy. In Aim 1, we will validate the presence of protective metabolomic signatures of bile acids, arachidonic acids, and histidine pathways in remission using samples from an interventional trial of OIT in young children. We will then assess how these metabolites change with immune biomarkers of remission (serology, basophil reactivity, and T cell cytokines). While there are many similarities in the mechanisms of OIT and SLIT, there is growing evidence for differences in the pathophysiology of remission induced by these two types of immunotherapy. Based on this, we will study the immunometabolism of remission in SLIT in Aim 2. Using samples from an interventional trial of SLIT, we will characterize the presence of these three immunomodulatory metabolite pathways in remission and assess how these pathways relate to immune biomarkers of remission. Finally, we will compare the metabolomic profiles of SLIT and OIT to identify shared and disparate pathways between the two types of therapy. We anticipate that this work will identify mechanisms of allergic tolerance in immunotherapy and inform the development of new therapies.

NIH Research Projects · FY 2025 · 2024-06

PROJECT ABSTRACT Family psychoeducation for adults living with psychotic disorders in Tanzania (KUPAA Trial) Psychotic disorders such as schizophrenia contribute to the global burden of disease in low- and middle- income countries (LMICs) yet mental health services have been severely neglected. Family Psychoeducation is an evidence-based practice from high-income countries that could have a significant impact in settings such as Africa and other regions worldwide where resources are limited and family involvement in treatment is fundamental. In response to PAR-21-130, this R01 will evaluate the impact of a culturally tailored version of family psychoeducation called KUPAA (meaning “to soar” in Swahili) that is group-based and family-involved in a low-income country context. The KUPAA intervention was recently pilot tested with adults living schizophrenia-spectrum disorders and their families in Tanzania. The proposed hybrid effectiveness- implementation clinical trial would individually randomize patient-caregiver pairs to either the group-based intervention or the standard of care across 14 health facilities in four regions of Tanzania. The overall objective is to improve recovery outcomes for individuals with schizophrenia-spectrum disorders. Aim #1: Determine the effectiveness of KUPAA vs. standard of care for an outpatient population (n=432) by reducing disability and improving quality of life (primary outcomes); and reducing hospitalizations and reducing perceived family burden for caregivers (secondary outcomes) among matched patient-caregiver dyads. Aim #2: Examine the mechanisms of change underlying KUPAA as a group-based, family-involved intervention. Aim #3: Evaluate implementation outcomes guided by the Consolidated Framework for Implementation Research (CFIR) via mixed methods (n=122 semi-structured interviews, observational data, cost data). This project is a collaboration between the University of North Carolina at Chapel Hill, Duke University and four Tanzanian institutions: Muhimbili University of Health and Allied Sciences in Dar es Salaam, Mbeya Zonal Referral Hospital in Mbeya, Mirembe National Mental Health Hospital in Dodoma, and Kilimanjaro Christian Medical University College in Moshi. This proposal aligns with the NIMH cross-cutting global mental health theme and will contribute to NIMH’s goal of advancing mental health services to strengthen public health by providing insights on an adapted evidence-based practice designed for lower-resource settings that could also inform domestic implementation strategies for scaling family psychoeducation in the U.S.

NIH Research Projects · FY 2026 · 2024-06

Black adults in the U.S. have a disproportionately higher risk of Alzheimer’s disease and related dementias (ADRD) compared with non-Hispanic White adults. Differences in cardiometabolic risk factors and diseases and social determinants of health (including increased exposure to psychosocial stress, lower levels of education, and higher poverty levels, which increase ADRD risk) likely contribute, but exact mechanisms are unknown. We hypothesize that inflammation may be a key feature linking cardiometabolic and social determinants of health risk factors with the risk of incident cognitive impairment and dementia. We propose to test this hypothesis through systemic analysis of the circulating proteome with risk of incident cognitive impairment and trajectories of cognitive function in a large biracial population from the REasons for Geographic and Racial Differences in Stroke (REGARDS) study. REGARDS is a longitudinal cohort of 30,239 non-Hispanic Black and White adults across the contiguous U.S. with pre-existing genome-wide genotyping data and rich cardiovascular and neurocognitive phenotyping. We will use a case-cohort design including a 1100-person cohort random sample and 930 additional ICI cases (n=981 total cases) to assess 3,072 circulating plasma proteins (including >700 inflammation related proteins) for associations with risk factors for cognitive impairment, including age, social determinants of health, and cardiometabolic risk factors, and with incident cognitive impairment and trajectories of cognitive function over >17 years of follow-up. Differences in levels of identified proteomic biomarkers by race and sex will be assessed. Finally, we will integrate available genome wide genotyping data and the plasma protein data newly generated in this study to determine if the protein data can elucidate biological mechanisms underlying associations of previously identified genetic variants with dementia-related phenotypes. We hypothesize this work will identify non-invasive protein biomarkers of cognitive impairment and ADRD. The proteomic data generated in this study will be made widely available to the scientific community through appropriate public repositories (such as dbGaP). Results from this study may improve ADRD risk prediction, elucidate biological mechanisms of ADRD, and improve understanding of factors that contribute to ADRD health across the US.

NIH Research Projects · FY 2025 · 2024-06

Complicated skin and skin structure infections (cSSSIs) including abscesses, burn infections, cellulitis and diabetic foot infections represent an enormous burden on the healthcare industry. Treatment options are limited, and treatment failure is common, frequently leading to chronic wound infection. Staphylococcus aureus is the most common cause of these infections, followed by Enterococcus faecalis. Vancomycin, a lipid II targeting antibiotic that inhibits cell wall synthesis, is essential in the treatment of cSSSI. However, vancomycin-intermediate resistant S. aureus (VISA) and vancomycin-resistant Enterococci (VRE) are a growing problem. Additionally, even in susceptible populations, antibiotic tolerant persister cells can survive vancomycin exposure, making eradication of infection difficult to achieve. Identifying ways to increase the efficacy of vancomycin, to target persister cells and overcome resistance would greatly improve the treatment of cSSSIs. We find that palmitoleic acid, a non-toxic unsaturated fatty acid found in human serum potentiates vancomycin efficacy against S. aureus and E. faecalis. Strikingly, palmitoleic acid also rapidly sensitizes persister cells and resistant populations to vancomycin. Vancomycin causes accumulation of the hydrophobic lipid II molecule at the septum. Our preliminary data suggests that palmitoleic acid is recruited to these hydrophobic regions which destabilize the membrane and cause cell death. Vancomycin-resistant isolates induce expression of resistance genes when they encounter vancomycin. We hypothesize that the rapidity of palmitoleic acid- vancomycin killing may outpace the induction of resistance gene expression, resulting in death of the resistant strains. In this proposal, we will 1) determine how palmitoleic acid/vancomycin kills persister cells and overcomes vancomycin-resistance and 2) evaluate the efficacy of this therapeutic combination in a pre-clinical diabetic wound infection model. Utilizing host-produced unsaturated fatty acids to potentiate vancomycin killing against resistant organisms is conceptually innovative. If successful, this proposal will represent the first steps toward the development of a powerful therapeutic combination for the eradication of recalcitrant gram-positive wound infection.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY/ABSTRACT The prevalence of suicidal thoughts and behaviors (STBs) in late childhood and adolescence is alarmingly high, yet scientific understanding of STBs in youth is surprisingly limited. The NIMH has recently highlighted an urgent need for research examining mechanisms of complex health risk behaviors, including suicide, and a prioritization of child health research using existing datasets. The proposed study addresses these and additional scientific priorities by capitalizing on the unprecedented methodological advantages of the Adolescent Brain Cognitive Development (ABCD) Study to examine patterns of neurobiological, behavioral, and psychological processes, measured across multiple units of analysis, that define distinct subtypes of youth experiencing STBs in late childhood, as well as longitudinal associations between subtypes and STBs over adolescent development. This proposal addresses several critical limitations of prior research on suicide. First, despite epidemiological data that STBs frequently onset and escalate in late childhood and adolescence, suicide research has focused primarily on adults. Second, studies of suicide are largely cross-sectional and consider a narrow set of self-report variables. Third, most research takes a variable-centered approach and assumes a single set of risk-relevant processes apply similarly to all individuals, despite theoretical and emerging empirical evidence supporting the existence of multiple ‘subtypes’ of suicidal individuals defined by distinct profiles of risk-relevant vulnerabilities. The current study addresses these limitations in multiple ways. First, the proposed work adopts a primarily person-centered approach to examine patterns across neurobiological, behavioral, and psychological domains that distinguish distinct subtypes of youth who experience STBs by late childhood (ages 9-10)—the age at which suicide becomes a leading cause of death. Second, this work will test prospective associations of suicide subtypes with trajectories of STBs into adolescence, including potentially differential risk for persistence of suicidal ideation and/or onset of suicidal behavior. The proposal also offers opportunity to examine similar questions in a Supplemental Aim among initially nonsuicidal children, to examine risk for new onset of STBs in the adolescent transition. Finally, the proposal is not reliant solely on self-report and uses multimodal measures across units of analysis, including some that are relatively novel for suicide research (e.g., neuroimaging). In line with NIMH strategic objectives, the study will pursue these aims in the ABCD Study dataset. This dataset offers novel opportunity to address the aforementioned limitations of suicide research with longitudinal data on a large, heterogeneous, national representative youth sample from late childhood into adolescence. The research has potential to reduce heterogeneity of suicide by revealing distinct suicide subtypes earlier in development than has previously been examined. Findings can inform developmentally-salient risk models of youth suicide, and facilitate more precise interventions and treatments based on unique subtype profiles.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY Nonribosomal peptides are an important class of natural products which display great structural and functional diversity. As many nonribosomal peptides act as antimicrobials, the identification of novel bioactive NRPs can lead to the development of new therapeutics for the treatment of antimicrobial resistant microorganisms. Dehydrated nonribosomal peptides, peptides derived from or containing one or more α,β-dehydroamino acids (dhAAs), are key targets for further therapeutic development due to their structural rigidity, resistance to proteolytic degradation, and enhanced chemical reactivity. The Li Lab has focused on a specific class of dehydrated nonribosomal peptides whose biosynthesis stems from biosynthetic gene clusters (BGCs) containing nonribosomal-peptide synthetases (NRPSs) with C domains which modify amino acids (CmodAA). We have recently characterized a class of these CmodAA domains which dehydrates β-hydroxy amino acids to dhAAs in nonribosomal peptide biosynthesis, and work from our lab and others has demonstrated that these CmodAAderived nonribosomal peptides display a diverse array of bioactivities. However, the identity of many CmodAA derived nonribosomal peptides is undiscovered, and their biosynthetic pathways and biological roles remain uncharacterized. Through genome mining we have identified nearly 4,500 nonidentical CmodAA domains, many of which are distributed among soil bacteria including Streptomyces. The research described herein is aimed to elucidate the unknown products of several of these BGCs from Streptomyces and characterize the role of the CmodAA domain in their biosynthesis. I will use a combination of in silico analysis of the biosynthetic gene clusters, comparative metabolomics, genetic manipulation, and NMR analysis to elucidate the cryptic products of these BGCs. Once the nonribosomal peptide structure has been determined, I will perform a battery of in vitro enzyme assays to elucidate the core enzymes involved in nonribosomal peptide biosynthesis, and the biosynthetic role of the CmodAA domains within each BGC. Completion of the proposed research will lead to the discovery of novel CmodAA derived dhAA nonribosomal peptides with potential therapeutic applications. Furthermore, the findings of this research will provide a base of knowledge that will extend to the discovery of other CmodAA containing BGCs and characterization of their dhAA containing products.

NIH Research Projects · FY 2024 · 2024-06

A Phenomics-First Resource (PFR) for interpretation of variants Genomics is key to precision medicine; however, despite the ease of sequencing, clinical interpretation is still thwarted because relevant data (disease, phenotype, and variant) is complex, heterogeneous, and disaggregated across sources. Moreover, this evidence is sometimes incomplete, conflicting, and erroneous. Consequently, clinicians face long lists of candidate diseases, genes, and countless variants of unknown significance. This situation will not improve without capturing and harmonizing the underlying phenotypic information; computability of this information is the bedrock for the emerging field of ​phenomics​. From basic science to clinical care, communities need structured ways to represent and exchange phenotypes and disease definitions. Addressing these fundamental phenomics needs makes it possible to computationally assess and reveal links between diseases and variants. We have previously shown how the addition of phenotypic information using the Human Phenotype Ontology (HPO) can improve the diagnostic yield for hard-to-diagnose patients, and HPO is therefore now a global standard for “deep phenotyping”. We have demonstrated the applicability of deep phenotyping in the evaluation of rare diseases which have overlapping mechanistic underpinnings with common/complex diseases as well as evolutionarily conserved mechanisms in model organisms. Having coordinated the community and prototyped the underlying computational platforms, we will now align both phenotype ontologies and clinical terminologies, enabling better comparison and inference of phenotypes for improved diagnostic efficacy. We propose to develop a Phenomics-First Resource (PFR). ​Specifically we will: 1. Create a community-driven framework of interoperable phenotype definitions across species​ (uPheno) 2. Harmonize human disease definitions with the ​MONDO​ disease alignment resource 3. Create a community-wide exchange standard for clinical and model-organism phenotypes (​Phenopackets​) 4. Develop an integrated phenomics platform ​to provide the research ​(e.g. BioLink) and clinical (​FHIR​) communities with programmatic access to phenomics ontologies, data, and algorithms The dynamic suite of interlinked technologies will together leverage community-developed knowledge in order to make variant interpretation more reliable, better provenanced, and more clinically actionable.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY Continued access and adherence to antiretroviral therapy (ART) is crucial for pediatric populations living with HIV to prevent AIDS-related comorbidities. However, there are several unique challenges to treatment adherence in pediatric populations, including caregiver factors, and medication palatability and tolerability. Little work has been done in characterizing patterns and factors related to the timing of ART nonadherence since initiation of therapy in children. Additionally, new ART formulations available for adults have been slower to obtain approval for use in pediatric populations due to difficulty in assessing efficacy in trials. Bringing modern causal inference methods to the analyses of pediatric trials through rigorous estimation of per-protocol effects can help overcome some of these difficulties, and help bring analyses of pediatric trials into the modern age. The objectives of the proposal are to (1) characterize the barriers to pediatric dolutegravir combination therapy adherence, and (2) estimate per-protocol effects of treatment failure by 96 weeks in the pediatric HIV trial, taking into consideration the unique barriers to medication adherence in pediatric populations. Data from the ODYSSEY trial, a phase II/II open-label non-inferiority trial assessing dolutegravir-based ART compared to standard of care in an international sample of children aged 2 to 18, will be used to characterize baseline and time varying factors influencing time to protocol deviation, and then estimate per-protocol effects impacting 96-week treatment failure correcting for such protocol deviations through implementation of modern causal inference methods and machine learning. The completion of the proposed aims will provide evidence on characteristics contributing to timing of protocol deviation and estimates of per-protocol effects of treatment failure by 96 weeks corrected for such protocol deviations in the ODYSSEY trial for first or second line dolutegravir use compared to standard of care in a pediatric population. These results will (1) provide substantial information on factors related to timing of eventual ART nonadherence so that appropriate clinical intervention can be developed to prevent nonadherence related to these factors, and (2) provide an example of modern per-protocol analyses in pediatric trials which will serve as guidelines for future per-protocol analyses in pediatric trials. The training plan outlined in this F30 proposal will provide the applicant with necessary skills in modern causal inference methods and clinical medicine to become a productive and successful physician scientist at the intersection of pediatrics and application of modern causal inference methods for pediatric trials.

NIH Research Projects · FY 2025 · 2024-06

Somatic variants that arise during embryonic corticogenesis and result in brain mosaicism are increasingly recognized as significant contributors to the genetic risk of neurodevelopmental and neuropsychiatric conditions. The discovery of disease-causing somatic variants in the brain has not only contributed to the identification of novel genes and refined our understanding of the underlying genetic architecture of a range of neuropsychiatric diseases, but they also provide powerful research tools that can be used to understand how cell-type-specific changes contribute to disease pathophysiology. In this study, we seek to use single-cell genomic approaches in mosaic human brain tissue to establish which cell types harbor the disease-causing somatic variants and to determine cell-type-specific transcriptomic changes associated with the variant. While single-cell genomics approaches have been used to inform many novel aspects of pathophysiology across a range of diseases, no studies to our knowledge consider genotype and use deep single-cell RNA sequencing to study variant- associated transcriptomic changes in specific cell types from mosaic human brain tissue. Here, we propose to perform a proof-of-concept study to establish the extent to which genotype-informed comprehensive single-cell RNA sequencing of mosaic human brain tissue is reproducible and can inform novel aspects of disease pathophysiology. We will accomplish this goal using hemimegalencephaly, a severe brain malformation causing overgrowth of one cerebral hemisphere that is caused by a single highly penetrant, brain tissue-specific somatic variant in either AKT3, MTOR, or PIK3CA, as our prototype. We will isolate both DNA and RNA from single nuclei from therapeutically resected mosaic brain tissue of each individual, genotype the DNA to classify each isolated cell as variant-positive or variant-negative, and then perform RNA sequencing to determine cell-type-specific variant burden and define the transcriptomic changes associated with the variant in specific cell types. We will analyze three individuals with genetically diagnosed hemimegalencephaly caused by the same pathogenic somatic variant for each of the three known disease genes to allow for comparisons across individuals and genes. Comparisons of variant-positive to variant-negative cells in hemimegalencephaly cases, along with variant-negative cells from age-matched neurotypical control brain tissue, within like cell types, will allow us to identify cell type-specific cell-autonomous and non-cell-autonomous transcriptomic changes associated with the variant. We hypothesize that these studies will reveal reproducible convergent and divergent cell-type-specific disease mechanisms across individuals with the same pathogenic variants and across genes implicated in the same brain malformation. If successful, we will establish a powerful research approach that leverages somatic mosaicism to inform cell types involved in disease, identify pathophysiologically relevant specific cell-type specific transcriptomic changes involved in neurological and neuropsychiatric diseases, and illuminate novel therapeutic strategies in hemimegalencephaly and related conditions.

NIH Research Projects · FY 2024 · 2024-06

PROJECT SUMMARY CAR-T cell therapy has revolutionized the treatment of liquid tumors, including leukemia and lymphoma, and hold enormous promise for treatment of solid cancers as well. However, despite their unprecedented clinical success, widespread utilization of this therapy is hampered by the lengthy and labor-intensive manufacturing procedures. CAR-T cell manufacturing takes weeks, results in very high costs of therapy (~$500,000). The long manufacturing time creates delays of weeks or months to infuse CAR-T cells to patients with rapidly progressing disease. Finally, the extensive ex vivo manipulation creates cell products with heterogeneous composition and terminal differentiation that limit CAR-T cell engraftment and persistence. Effort to overcome these limitations have focused on closed and automatic manufacturing devices to contain the labor needed to manufacture CAR- T cells ex vivo, and allogeneic off-the-shelf CAR-T cells have been proposed to overcome the need of CAR-T cell manufacturing for each single patient. These technologies are promising, but reducing the time, costs and regulatory burden of manufacturing or eliminating ex vivo procedures entirely remains a critical unmet need. In vivo generation of CAR-T cells would eliminate the need for ex vivo procedures, prevent the terminal differenti- ation of ex vivo expanded CAR-T cells and ensure the potency and longevity of autologous T cells as compared to allogeneic CAR-T cell products that are extensively manipulated to prevent rejection and graft-versus-host disease. This proposal outlines the first steps in a highly innovative high-risk/high-reward effort to develop bioin- structive biomaterials scaffolds that generate CAR-T cells entirely within the patient and produce CAR-T cells with improved efficacy and persistence. Our endeavor is built on significant published and preliminary data demonstrating that our biomaterial scaffolds already efficiently activate and mediate CAR-T cell transduction in vitro and efficiently recruit and release CAR-T cells in vivo and reduce CAR-T manufacturing times from weeks to a single day. We propose that the biocompatible alginate biomaterial scaffolds can be modified to encapsulate T cell-attracting chemokines to recruit T cells to the scaffold. After recruitment, the biomaterial scaffolds will provide αCD3/CD28 signaling to activate the T cells. After activation, T cell-specific viral particles either already present in the biomaterial or administered to the biomaterial as a separate step will transduce the T cells, gen- erating tumor specific CAR-T cells in situ in manner compatible with irradiative lymphodepletion. Finally, inter- leukin signaling in the scaffold will expand and promote release of formed CAR-T cells for systemic function. If successful, this approach could have enormous clinical impact by significantly reducing therapy costs and dra- matically expanding the patient population benefiting from CAR-T-cell therapy. We expect that these studies will provide a foundational technology for CAR-T cells manufacturing and promote widespread patient access. In addition to the clear application in cancer, however, this rational, materials-based approach for cellular manu- facturing could be adopted to program therapeutic lymphocytes in solid tumors and for other diseases.

NIH Research Projects · FY 2025 · 2024-05

Project Summary Acute respiratory distress syndrome (ARDS) causes COVID-19 fatalities and is linked to uncontrolled release of pro-inflammatory cytokines, including interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF). Blockade of IL-1/IL-6 receptor signaling has shown success in treating severe COVID-19, but the molecular cues initiating excessive production of these cytokines remains unclear. A major source of IL-1 is the inflammasome, a supramolecular protein complex formed in response to two innate immune stimuli that executes IL-1β release. Meta-analysis of patient single-cell RNA sequencing data reveals exacerbated expression of IL1B and inflammasome-related genes in severe, but not mild, COVID-19. Co-culture of primary human airway epithelia (HAE) and primary human leukocytes during SARS-CoV-2 infection promotes IL-1β release, while infection in either cell type alone does not. These conditions also promote IL-6 release in an IL-1-dependent manner, highlighting how inflammasome activation and cell-cell communication amplify this inflammatory response. Furthermore, infected HAE undergo lytic cell death and secrete inflammasome-activating damage-associated molecular patterns (DAMPs). Therefore, the scientific goal of this proposal is to understand how SARS-CoV-2 infection promotes pathologic inflammasome responses with the central hypothesis that damage from dying, infected airway epithelial cells potentiate detrimental IL-1β release. Aim 1 will determine which inflammasome(s) mediate harmful IL-1β responses during SARS-CoV-2 infection in vivo and identify the tissue(s) from which this response is derived. Aim 2 will define the mechanism of SARS-CoV-2-mediated cell death in airway epithelial cells and its relationship with inflammasome-mediated cell death in myeloid cells using an established primary human co-culture system. Aim 3 will identify DAMPs released by infected primary human airway epithelial cells that stimulate inflammasome response via an unbiased multi-omics approach. These studies will define how damage caused by viral infection propagates inflammation and its role in disease, opening avenues for therapeutic targeting and identifying biomarkers related to pathologic inflammation in COVID-19. My goal is to become a tenured faculty member at a major research university with a research program studying fatal and emerging viral pathogens. My group will focus its efforts on understanding the innate immune response to viruses that regularly threaten human health and pinpoint pattern recognition receptor (PRR) ligands that lead to detrimental patient outcomes. Using a combined approach of primary human cell culture models, organismal murine models, and mechanistic molecular biology, I will identify readily translatable molecular targets to treat human viral diseases. Herein, I outline both a scientific vision and a plan acquire new skills through collaboration with local faculty, the support of my community and my advisory committee, and formal coursework to enhance my abilities further, creating a framework for my success as an independent investigator.

NIH Research Projects · FY 2025 · 2024-05

Neuroimaging technology such as magnetic resonance imaging (MRI) and positron emission tomography (PET) is critical to Alzheimer's disease (AD) and Alzheimer's disease-related dementias (ADRD) studies. In spite of the wide consensus that the integration of neuroimaging and data science approaches offers a powerful tool for understanding the underlying disease mechanisms and developing personalized treatment strategies for AD/ADRD, we are facing the urgent challenge of a capable workforce with computational neuroimaging skill-sets, due to the lack of early exposure to high-quality, hands-on research education experiences in the imaging-based AD/ADRD studies. To address this challenge, we form an inter-disciplinary team of data scientists and AD/ADRD experts to develop a summer research education program on computational neuroimaging to support the NIA’s priority on expanding research in AD/ADRD (ERA). Specifically, our education program consists of four inter-connected components. First, we will design a hybrid curriculum of computational neuroimaging for high school students that includes a set of synchronous/asynchronous short courses covering research ethics, imaging physics, biomedical image processing, statistical inference, machine learning, and AD/ADRD clinical applications. Second, we will develop a collection of hands-on tutorials to train the students how to formulate the AD/ADRD question into a well-posed computational problem, how to analyze neuroimaging data, and how to translate the computational tools into clinical applications. Third, we will build a cloud-based neuroimage processing platform with the integrative functions of visualization, analysis, and data management/sharing, which allows us to deliver the computational power to every student in this research education program. Lastly, and primarily, we will manage a project-based mentoring plan to offer each student the opportunity for a one-to-one mentored research experience with AD/ADRD experts at UNC, Duke, and Wake Forest University during the summer program. We will work with the academic mentors to continue the training for motivated high school students by tracking the project progress, providing career advice, and evaluating training performance. Under the umbrella of AD/ADRD research, we will partner with two NIH-funded Alzheimer's Disease Research Centers (ADRCs) at Duke/UNC and Wake Forest University. We will also align our education program with the existing renewed STEM programs at North Carolina School of Science and Mathematics and North Carolina Science & Engineering Fair to engage high school students with diverse scientific and cultural backgrounds. Our outreach strategy will promote broad participation in Alzheimer’s and related dementia (AD/ADRD) research education, ensuring that opportunities are accessible to qualified students. Participant eligibility will be determined through a merit-based selection process. The success of the program is built upon the unique neuroimaging, data science, and education expertise of the PIs (Drs. Wu and Kaur) and their collaborative relationships with experts in computer science, biostatistics, psychiatry, neurology, and radiology research faculties. The synergistic integration of research, mentoring, and outreach will enthusiastically engage more prospective students in AD/ADRD research, contributing to a collaborative research environment in the nation.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY Congenital deficiency of plasma clotting factor (F)XI is an autosomal disorder. Whereas some individuals are asymptomatic (non-bleeders), others have excessive bleeding after injury, primarily at sites with high fibrinolytic activity (mouth, nose, genitourinary tract) (bleeders). People with similarly reduced FXI have variable bleeding even within families sharing the same FXI mutation. Clinical assays cannot predict bleeding risk in FXI-deficient people, leading to under- or over-treatment to prevent bleeds. Moreover, FXI inhibition strategies are in clinical trials to reduce thrombosis; however, observations of individuals with congenital FXI deficiency suggest these therapies will incur bleeding risk in some patients, especially in prophylactic use. Uncovering mechanisms that determine bleeding risk in FXI deficiency and developing methods to predict bleeding will improve treatment for both bleeding and thrombosis. Our long-term goals are to characterize mechanisms that promote hemostasis in FXI deficiency, and translate these findings into clinically-accessible methods to predict bleeding. Using plasmas from two independent cohorts of well-phenotyped people with FXI deficiency, we developed and validated specialized plasma assays that differentiate FXI-deficient bleeders from non-bleeders, and discovered that inhibiting the contact pathway in these assays enhances the ability to identify bleeders. We also integrated computational modeling and in vitro assays to uncover synergy between FXIa and tissue factor that enhances coagulation. We built on these discoveries with new analyses that revealed plasma proteins whose levels differed significantly between non-bleeders and bleeders, and a novel computational workflow for advancing a prediction model. These findings and advances provide important clues to mechanisms that modify bleeding risk in FXI deficiency, and position us with innovative tools to identify these mechanisms. The objective of this application is to characterize the determinants and functional impact of differently present plasma proteins in non-bleeders and bleeders, and use computational methods to differentiate bleeding risk in FXI deficiency. The central hypothesis of this application is that in FXI deficiency, differences in plasma composition modify thrombin generation and clot formation, structure, and stability and determine the bleeding risk. Specific aims of this application are to: 1) Determine the functional impact and mechanisms differentiating differently present proteins in FXI-deficient non-bleeders and bleeders, 2) Use computational modeling and machine learning to identify predictive features that differentiate FXI-deficient non- bleeders and bleeders, and 3) Use multi-omic methods to define FXI deficiency and the bleeding phenotype. This proposed research is significant because the experiments will reveal molecular mechanisms that modify hemostatic potential in a predictive functional assay and in individuals with reduced FXI. Successful completion of this work will reveal new biology and lead to novel methods for predicting bleeding risk in individuals with congenital FXI deficiency and pharmacologically-reduced FXI for thrombosis prevention.