Univ Of North Carolina Chapel Hill

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY The molecular and cellular biology of metastasis is multifactorial, and this complexity contributes to the challenge of treating patients with metastatic disease. One of the challenges of metastasis biology is to determine why certain tumor subtypes have preferential sites of metastasis: e.g. TNBC/Basal-like breast cancers tend to metastasize to visceral organs, while ER-positive/Luminal breast cancers tend to metastasize to the bone. In addition, once metastases are established at a distant site, it is critical to understand how these tumors interact with the local microenvironment and identify the tumor-to-organ crosstalk might be occurring to sustain tumor growth. This proposal aims to address these questions by utilizing a comprehensive and carefully curated dataset of paired human primary tumors and metastasis samples, and a similar paired Genetically Engineered Mouse Mammary tumor models dataset. We seek to identify the molecular determinants of lung and liver organ-specific metastasis using supervised learning approaches and this extensive human and in vivo mouse models breast cancer data set. Lastly, using spatial transcriptomics, we will characterize unique organ microenvironments to identify critical crosstalk that contributes to sustained tumor growth, thus possibly offering a new means for therapeutic interventions.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT HIV remains persistently detectable within tissues during antiretroviral treatment (ART) and can rebound throughout the body following a treatment interruption. Low concentrations of antiretrovirals (ARVs) may be present within these tissue viral reservoirs, such as lymph nodes, which may provide localized sanctuaries for virus. Understanding the potential role of pharmacology in the persistence of virus during treatment and subsequent viral reactivation following treatment interruption is critical for targeting strategies to achieve a functional cure. This remains a knowledge gap because of existing limitations in measuring tissue ARV concentrations by standard LC-MS/MS methods. These methods cannot capture variability in drug penetration within the tissue microenvironment that may limit exposure to cells targeted by virus. We have developed a new approach for quantifying ARVs in tissues based on IR-MALDESI MSI, which offers the ability to map spatial distributions of drugs. Using this approach we have demonstrated heterogeneous ARV distributions, often detected as monotherapy, across a range of putative reservoir tissues. We hypothesize that progressive HIV pathogenesis can induce structural and morphological modifications in tissue (e.g. fibrosis) that may limit penetration of ART to some T cell populations within reservoir tissues. To investigate this question, we will combine multiple cutting-edge imaging techniques to simultaneously characterize and quantify distributions of drug, virus, and host response within tissues of a rhesus macaque (RM) animal model using a well-characterized SIV strain that provides a robust, predictable pathogenic response. With long-acting injections (LAI) emerging as an alternative to daily therapy, this effort will investigate differences in ARV tissue penetration through each of these two routes of administration. Three specific aims are proposed: 1) Characterize tissue PK using IR- MALDESI MSI in reservoir tissues following treatment initiation in acute or late chronic infection. 2) Use a novel multimodal spatial imaging suite (IR-MALDESI MSI, RNAscope in situ hybridization, spatial transcriptomics) to evaluate differences in tissue PK/PD based on timing of treatment initiation. 3) Identify lipidomic/metabolomic markers of immune responses in tissue by IR-MALDESI MSI and develop an innovative single-assay approach to tissue PK/PD assessment. When completed, this proposal will benchmark PK of long-acting and daily treatment combinations for the first time in healthy RM and assess how the extent of immunopathogenic damage prior to treatment initiation influences ARV tissue penetration. These data will provide insight into how delivery of new therapies LAI strategies can optimize clinical outcomes

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY The goal of this K01 application is to provide an individualized program of mentored research training for Dr. Deanna Saylor, an Assistant Professor in the Department of Neurology at the Johns Hopkins University School of Medicine. Dr. Saylor has been based full-time in Zambia since 2018 where, in collaboration with local stakeholders, she has launched the first neurology inpatient unit and first neurology post-graduate training program in the country. Dr. Saylor is applying for an International Research Scientist Development Award to gain skills and experience in implementation science research. Combined with her background in clinical and epidemiological research, this new skillset will uniquely position her to both generate new knowledge and help develop care systems that effectively implement new knowledge to improve patient outcomes. The research plan outlined in this application leverages the developing system of neurological care in Zambia to understand current stroke care practices at the University Teaching Hospital (UTH; Zambia's national referral hospital), develop locally relevant stroke clinical practice guidelines, and examine the effect of their implementation on stroke outcomes. Since stroke is the second leading cause of adult disability and mortality worldwide and stroke prevalence in sub-Saharan Africa is amongst the highest in the world, these data are urgently needed to address this national, regional, and global health problem. The proposed research includes a pre-intervention prospective cohort of patients with stroke at UTH to characterize current stroke care clinical practices and stroke outcomes (aim 1). A stroke working group including relevant stakeholders will then be convened to investigate community, patient, provider, and health systems factors leading to difficulty achieving stroke quality measures and optimal outcomes. The Adopt-Contextualize-Adapt framework will then be used to develop locally relevant clinical practice guidelines from international stroke guidelines for implementation (aim 2). These guidelines will then be implemented, and a post-intervention observational cohort study conducted to examine their impact on achieving stroke quality measures and improving stroke outcomes. This K01 will also support Dr. Saylor's training goals which include gaining additional training and mentorship in the following key areas: (1) implementation science frameworks and methodology; (2) qualitative data methods and analysis; (3) stroke clinical research methods, especially aspects unique to global settings; and (4) developing an international network of collaborators for future large-scale and generalizability studies. In order to achieve these goals, Dr. Saylor has established an international team of mentors with expertise in implementation science, qualitative studies, stroke research, neurology research in sub-Saharan Africa, and stroke center development in international settings. This award will provide Dr. Saylor with the mentorship, knowledge, and experience necessary to become an independent investigator in global neurology conducting clinical and implementation research to improve outcomes for patients with stroke and other neurologic disorders.

NIH Research Projects · FY 2025 · 2025-08

Viral infections and air pollution rank among the highest threats to lung health. People with higher melanin expression, bear a disproportionate burden of environmental lung diseases and respiratory infections due to higher exposure to air pollution, genetic predispositions, and other factors, such as nutrition, including increased prevalence of vitamin D deficiency. In addition to reduced nutritional intake of vitamin D-rich foods, high melanin levels in the skin of people with higher melanin expression prevent UVB absorption and biosynthesis of vitamin D. Lower vitamin D levels have been linked to higher incidence and severity of chronic lung disease, such as asthma and viral infections, such as influenza. However, oral vitamin D supplementation has shown mixed results in treating lung diseases, potentially due to the inability of reaching the lungs. Therefore, the objective of this proposal is to investigate inhalation of vitamin D as a route of direct delivery to the airways to protect against air pollution and respiratory infections. Preliminary studies I have conducted indicate that vitamin D may act as a membrane antioxidant, upregulate the expression of the antimicrobial peptide cathelicidin, and attenuate oxidative stress and inflammation induced by air pollutants. Based on these data, I hypothesize that inhaled vitamin D can reduce the severity of respiratory infections and abnormal immune responses to environmental pollutants in human bronchial epithelial cells (HBECs). First, I aim to understand how inhaled vitamin D protects the airway epithelium from lung inflammation caused by respiratory infections and environmental pollutants. Using a novel aerosol deliver system I have developed, HBECs will be pre-treated with aerosolized vitamin D before influenza infection or pollutant exposure, followed by characterization of markers of inflammation, oxidative stress, viral load, and immune dysfunction. I will then determine the potential mechanisms behind this through investigation into the membrane antioxidant properties of vitamin D using live cell imaging techniques. Second, I aim to investigate demographic differences in the pharmacokinetics and pharmacodynamics of vitamin D in the airway epithelium to determine the feasibility of vitamin D inhalation as a therapeutic strategy. HBECs from diverse donors will be treated with aerosolized or basolateral vitamin D, and concentrations of vitamin D metabolites and vitamin D-related signaling proteins will be measured over time. Despite established links between vitamin D status and respiratory health, no studies have investigated the use of inhaled vitamin D as a therapeutic or adjunct treatment for airway inflammation. This proposal is innovative as it uses a new in vitro aerosol delivery system to evaluate a novel route of delivery of vitamin D, investigates its membrane antioxidant and antimicrobial mechanisms as a prophylactic against lung inflammation, and determines demographic differences in the pharmacokinetics of this compound. This application is impactful as delivery of vitamin D could provide a potent, cost-effective solution for treating the pulmonary effects of vitamin D deficiency, potentially reducing health disparities associated with environmental lung diseases and respiratory infections.

NIH Research Projects · FY 2025 · 2025-08

Abstract: “Targeting Signaling Pathways with Small Molecules in Osteoarthritis” One of today’s greatest needs in managing osteoarthritis (OA) is an intervention that stops or delays joint tissue destruction and progression to “joint failure”. The overall goal of this exploratory/developmental project is to use a novel high throughput small molecule screening (HTS) technology combined with CRISPR screening and target prioritization tools to identify signaling pathways and therapeutic targets for disease modification in OA. The pathobiological processes that promote OA result from activation of cell signaling pathways and subsequent changes in gene transcription mediated by a host of factors. The complexity of OA explains why a single target approach for disease modification has met with limited success. We propose that use of small molecules to target pathways that regulate expression of multiple OA mediators will represent a viable and more promising approach. We have established a robust cell-based high throughput functional screening (HTS) assay using normal human chondrocytes treated with a matrikine relevant to OA progression. We found this system to be a valid in vitro model of the OA chondrocyte phenotype that allows us to investigate multiple pathways and targets, providing a distinct advantage over single target analysis. Our aims are: Aim 1: Identify OA signaling pathways and potential therapeutic targets through high throughput cell-based compound screening, network analysis of genetic and genomic datasets, and CRISPR screening. Aim 2: Determine the ability of compounds that target prioritized pathways to restore the catabolic and anabolic balance in joint tissues. At the completion of the project, we expect to: 1) identify one or more new cell signaling pathways contributing to dysregulated catabolic and anabolic activity in OA joint tissues; 2) identify specific therapeutic targets within those pathways; and 3) discover small molecules for further testing in preclinical models of OA. These outcomes would serve as the basis for lead optimization and further development of disease-modifying drugs for OA in humans.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT In Kenya, like most low- and middle-income countries (LMICs), rapid urbanization and changing food environments are leading to increased access to energy-dense nutrient-poor foods that are contributing to rising rates of obesity and the burden of nutrition-related noncommunicable diseases. Adolescence is a critical lifecycle period for the prevention of obesity, and an opportunity to establish lifelong habits for healthy eating. Adolescent girls are particularly at risk for overweight and obesity, and women in urban Kenya have more than double the rates of obesity compared to men. Although there is widespread agreement on the need to improve adolescent girls’ dietary practices in LMICs, effective obesity prevention interventions are lacking, particularly in sub-Saharan Africa. With the mentorship and training proposed in this K01 application, I will pilot test theory- based adolescent obesity prevention intervention components for girls in an urban informal settlement in Kenya. In Aim 1, I will follow a community-engaged process to refine school-based adolescent obesity prevention intervention components that were designed based on our previous formative research. In Aim 2, I will conduct a pilot multiphase optimization strategy (MOST) study to determine the feasibility of recruitment, randomization, intervention delivery, retention, and data collection procedures, and the acceptability of intervention components in preparation for a future fully-powered factorial trial. In the pilot MOST study, 8 secondary schools will be randomized to one of eight combinations of intervention components, and data will be collected from students at baseline; after the 3-month intervention; and after a subsequent 3-month follow-up period. My aims are linked to training objectives that will advance my conceptual knowledge and skills in obesity research, the design of adolescent obesity prevention interventions, and MOST. This research will provide preliminary results to inform a future trial testing a multicomponent adolescent obesity prevention intervention for girls in urban Kenya. My mentoring team has expertise in the design of adolescent obesity prevention interventions, global obesity research, and the design and analysis of MOST and factorial trials, as well as extensive experience mentoring early career investigators. This research will generate novel insights into the design of school-based adolescent obesity prevention interventions in urban LMIC settings. The K01 will help me achieve my long-term career goal of becoming a leader in adolescent obesity prevention intervention research. With the support from this award, I will transition from conducting intervention research focused on undernutrition to creating an independent research program that is building the evidence base for effective obesity prevention interventions. The proposed mentorship, training, and research activities will generate data needed to develop a competitive R01 application to identify effective obesity prevention interventions for adolescent girls in urban areas.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY / ABSTRACT People living with HIV (PWH) often exhibit a spectrum of cognitive, motor and mood symptoms, together referred to as HIV-associated neurocognitive impairment (HIV-NCI). HIV-NCI is estimated to affect over 40% of PWH and is more common as PWH age. HIV can enter the CNS within days of initial infection, and individuals with suppressed plasma viral loads might still have detectable HIV in their cerebrospinal fluid (CSF) for up to a decade, leading to inflammation within the CNS and subsequently HIV-NCI. Inflammation is believed to start very early in the disease process and precede overt clinical signs and symptoms of NCI. Identifying biomarkers of neuroinflammation related to presence of HIV virus in the CNS could help detect PWH who are now experiencing the neuroinflammatory effects of HIV. While incompletely understood, HIV-NCI is believed to be linked to chronic inflammatory processes in PWH, since immune responses may lead to both repair and damage in the CNS. Chronic inflammation may also contribute to changes in brain structure and altered connectivity. Our own prior work of 21 PWH ages≥50 shows that heightened segregation in the brain correlates with inflammatory biomarkers in PWH, while higher CSF NfL is related to higher markers of neuroinflammation or microglial activity. Our findings highlight the potential relationship between inflammation, neurodegeneration and functional connectivity (FC). Our overall goal in this proposal is to determine if the impacts of FC deficits, neuroinflammation, neurodegeneration or greater white matter hyperintensity (WMH) volume in HIV-NCI are mediated by CNS HIV viral persistence. We aim to (1) evaluate the specific contribution of HIV in the CNS to associations with inflammation, brain structure and function and cognition, and (2) examine whether detectable HIV RNA in the CSF mediates the associations between inflammation and neuronal injury, cerebrovascular disease and Alzheimer’s pathology in older PWH. Leveraging our team’s expertise in the fields of neuroHIV, biomedical imaging and virology, we leverage an existing research infrastructure and banked biospecimens from an existing cohort of older PWH at UNC. We will examine if the associations between fluid biomarkers of inflammation using next-generation proteomics and multimodal MRI markers of aging differ in PWH age≥50 with detectable vs. undetectable HIV RNA in CSF. We will also examine if the associations between biomarkers of inflammation and biomarkers of neuronal injury (NfL), cerebrovascular pathology (WMH volume) and AD pathology (NfL, phosphorylated tau217) are differentially impacted by presence of HIV within the CNS. The results of this study will provide an understanding of the mechanistic pathways by which viral presence in the CNS affects brain function and connectivity and neuroinflammation and provide potential future therapeutic targets to reduce the burden of HIV-NCI.

NIH Research Projects · FY 2025 · 2025-08

SUMMARY/ABSTRACT T follicular helper cells (Tfh) are recognized as critical regulators of antibody (Ab) responses. Consequently, Tfh in autoimmunity have primarily been studied for their role in regulating autoantibody production. However, Tfh are also linked to T cell-mediated autoimmune diseases such as type 1 diabetes (T1D). Evidence suggests a role for Tfh in T1D. For example, elevated levels of Tfh correlate with the development of diabetes in at-risk individuals, and NOD mice that lack expression of IL-21, the signature cytokine of Tfh, are protected from insulitis and diabetes. Despite these and other findings, whether and how Tfh regulate effector T cell (Teff)- mediated events leading to b cell destruction, and the properties of diabetogenic Tfh, remain poorly understood. It is also unclear if regulatory Tfh (Tfr) suppress the diabetogenic response. To address these fundamental questions, we propose three Specific Aims. Aim 1 tests the hypothesis that islet Tfh undergo both qualitative and quantitative changes as T1D progresses, leading to a Tfh pool with enhanced diabetogenic potential. Aim 2 focuses on defining the role and mechanisms of islet Tfh in regulating b cell autoimmunity. We hypothesize that islet Tfh mediate destructive insulitis during late-stage preclinical T1D by sustaining the pathogenic function of islet Teff. Conversely, Tfr limit islet Teff function indirectly by directly suppressing islet Tfh. Aim 3 will test the hypothesis that a hierarchy exists among islet Tfh subsets in their diabetogenic function, which is dependent on T-bet expression. This proposal is expected to provide novel insights into the properties, roles and mechanisms of Tfh and Tfr in regulating T cell-mediated b cell autoimmunity.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY High-grade serous ovarian carcinoma (HGSOC) manifests with peritoneal metastases in over 75% of patients. Despite aggressive treatment, including cytoreductive surgery and platinum-taxane chemotherapy, the remaining metastases almost inevitably give rise to chemoresistant recurrences for which no curative treatments exist. There is an urgent need for complementary strategies to address resistance in these residual tumors. Altering cell membrane lipid composition has emerged as one such strategy. This proposal aims to utilize photodynamic alterations of cell membrane lipids to promote chemotherapy penetration and efficacy in preclinical models of peritoneal metastases. This will be achieved by combining two clinically viable approaches: 1) Co- delivery of easily oxidizable polyunsaturated fatty acids (PUFAs) with lipophilic photoactivatable molecules (photosensitizers) and 2) Induction of lipid peroxidation within cancer cell membranes that occurs downstream of photochemically-generated reactive oxygen species (ROS)—photodynamic priming (PDP). This approach is based on a hypothesis that lipid pathology initiated by incorporating PUFAs into membrane-forming phospholipids, followed by exposure to photochemically-generated ROS, will synergistically “prime” cells for subsequent chemotherapeutic eradication. Intraperitoneal light delivery, proven feasible in University of Pennsylvania trials, allows integration of the proposed strategy within the current standard of care. In this approach, PUFA and photosensitizer would be administered prior to cytoreductive surgery, during which light- irradiation would photodynamically alter cell membranes to enhance response to postoperative chemotherapy. During the mentored K99 phase, Dr. Overchuk will investigate the effects of free PUFA, PDP, and combinations on carboplatin efficacy in orthogonal models of HGSOC. As a clear path to independence, Dr. Overchuk will develop a lipoprotein-mimetic nanoparticle to harness natural lipid trafficking mechanisms for codelivery of PUFAs alongside a photosensitizer. Upon her transition to the R00 phase, Dr. Overchuk will compare PDP using free versus nanoencapsulated PUFAs and photosensitizers with respect to tumor selectivity and carboplatin enhancement. A mentoring committee will guide Dr. Overchuk’s research and facilitate her transition to independence. As primary mentor, Dr. Imran Rizvi will train Dr. Overchuk in physiological models of HGSOC peritoneal metastasis. As co-mentor, Dr. Alexander Kabanov will provide specialized training in nanoformulation development. Drs. Robert Chapkin (PhD), Victoria Bae-Jump (MD/PhD) and Huang Chiao (Joe) Huang (PhD) will bring further expertise in membrane therapies, lipid metabolism, clinical management of HGSOC and translational photomedicine. To summarize, this proposal aims to address key barriers to treating peritoneal metastases with a mechanistically distinct approach and establish a cancer-agnostic platform translatable to a wider variety of malignancies. The mentorship provided by this K99/R00 Award will allow Dr. Overchuk to pursue this clinically relevant research and enable her transition to an independent career.

NIH Research Projects · FY 2025 · 2025-08

Abstract This proposal is for the purchase of a 300 keV cryogenic-transmission electron microscope (cryoTEM) to be housed in the CryoEM Core at the University of North Carolina at Chapel Hill (UNC-CH). This cryoTEM, the JEOL CRYO Arm 300 II (JEM-3300), will serve as a multi-functional instrument to support high resolution data collection for single particle (SP) cryoEM projects as well as for cryo-electron tomography (cryoET) and micro electron diffraction (microED). Currently, the UNC-CH CryoEM Core houses only a single 200 keV cryoTEM, yet the core still serves over 120 researchers representing 50 research groups. These researchers are located not only at UNC-CH, but also at several nearby universities. The UNC CryoEM Core therefore serves as a regional resource to those without cryoEM access and expertise. The addition of a JEM-3300 is crucial for our core to have the capacity needed to support the already high, and continually growing, demand from our NIH-funded user base. As the UNC-CH core has only a single cryoTEM, we struggle to service all users in a timely fashion, even though our instrument is maximizing its available usage time. Additionally, UNC-CH does not have a 300 keV instrument, a prerequisite for performing high-resolution cryoET. The proposed instrument will therefore both expand our throughput for collection of SP cryoEM data, while also expanding our capabilities to perform cutting-edge cryoET and microED experiments. The JEM-3300 will support scientists studying a wide range of research questions relevant to public health. Some of these questions are fundamental, and address regulation of genomic processes, cell division, and exocytosis. Still others address how the enzymes involved in lipid metabolism affect heart health, study the role of amyloid fibrils in Alzheimer's disease, and address viral exosomes and their causative role in cancer. These research questions are diverse but are united in their reliance on high-resolution structural biology to answer mechanistic questions about cell biology. CryoEM has revolutionized modern structural biology, and the new technology fueling this revolution allows researchers to obtain high-resolution structural insights with unprecedented speed and resolution. These insights will allow us to understand the etiology of multiple diseases and move towards therapeutic solutions.

NIH Research Projects · FY 2025 · 2025-07

Abstract Failure to detect and treat atherosclerosis in younger adults worsens cardiovascular morbidity and mortality. Current strategies of assigning preventive therapies are not based on screening for atherosclerosis, but rather on short-term risk scores that heavily weight age and leave most at-risk young and middle-aged adults untreated. A new paradigm of screening for and identifying subclinical atherosclerosis via coronary computed tomography angiography (CCTA) is emerging because subclinical atherosclerosis has prognostic superiority over traditional risk scores. However, it is still unknown how best to screen for and identify younger adults who have subclinical coronary atherosclerosis, and how best to treat them to reduce atherosclerotic burden. Both LDL-C and inflammation are causal in the pathogenesis of atherosclerosis, but it is unknown whether treating either or both processes in younger individuals can effectively and safely reduce atherosclerotic burden. The PREEMPT (Prospective RandomizEd trial of the Evaluation and Management of Premature aTherosclerosis) study will address this urgent public health need by determining optimal strategies to screen for, identify, and treat subclinical coronary atherosclerosis in young and middle-aged adults. Aim 1 will focus on screening younger adults for subclinical coronary atherosclerosis. We will evaluate the effectiveness of three strategies to detect coronary atherosclerosis in young and middle-aged adults (women aged 40−60 and men aged 30−50) at low 10-year, but high lifetime risk. These strategies will include an electronic health record search, outreach to family members of those with premature heart disease, and community outreach/social media. The outcome will be proportion screened with coronary artery calcium (CAC) score >0. An adaptive design will be utilized to modify or eliminate ineffective strategies. Aims 2 and 3 will focus on assessing the efficacy and safety/tolerability of 3 therapeutic strategies to reduce atherosclerotic burden. Individuals who meet clinical eligibility criteria through the screening study, or opportunistically through preexisting imaging evidence of coronary calcification, and have a CAC score of 1−99 will undergo CCTA to confirm presence of measurable non-calcified plaque (NCP) volume, and will then be randomized to a placebo-controlled, double-masked, 2x2 factorial randomized trial of rosuvastatin 20mg, colchicine 0.5mg, or the combination vs. placebo. All participants will also receive a state-of-the-art mHeath behavioral intervention to ensure lifestyle modification for all participants. The primary endpoint will be centrally adjudicated NCP volume on CCTA at 2 years, adjusting for baseline NCP volume. If successful, PREEMPT will reduce the unacceptably high morbidity and mortality of cardiovascular disease by providing randomized trial evidence supporting a paradigm shift away from a 10-year risk-based prevention strategy and towards earlier detection and treatment of subclinical atherosclerosis in younger adults.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY/ ABSTRACT Advancing understanding of the human virome is a cross-institutional national priority. The National Institute of Allergy and Infectious Diseases (NIAID) has a long-standing investment in U19 Genomic Centers for Infectious Diseases, renewed in April 2024 (RFA-AI-23-015). Meanwhile, the Centers for Disease Control and Prevention (CDC) recently funded 5 Pathogen Genomics Centers of Excellence to identify needs and opportunities for genomics in the U.S. public health system (CDC 2023). Simultaneously, NIH’s Common Fund Human Virome Program will support projects that will comprehensively capture the role of viruses in the human body (RFA- RM-23-019). This investment trio reflects the enormous clinical and public health potential of expanding our understanding of human viruses, providing unexpected insights into individual and population health. Inevitably, viromics research teams will confront complex ethical, legal, and social implications (ELSI) of this rapidly expanding area of science. In addition to standard concerns like risk, privacy, and broad consent, viromics might raise concerns distinctive to this area of science, including conceptions of the “human virome” and “viral colonization,” gaps in regulatory pathway for bacteriophage research, or public uncertainty generated by complex genetic interactions among viruses, hosts, and vectors. In this R21, we propose to focus on priority areas from NIAID and NHGRI: (1) support the integration of bioethics work with genomic research in infectious diseases, analyzing ethical implications in the planning and conduct of NIAID viromics research; and (2) significantly extend the focus of NHGRI’s ELSI and policy issues that arise with the design and conduct of genetic and genomic research. Our project consists of three interlocking aims with a planned expansion to an R01 proposal. (1) We will undertake a multidisciplinary horizon scan based on an NIH-framework for governance of emerging technologies. This approach includes an expert filtration of the scientific literature to select 5 high-priority topics in virome research and associated ELSI, comparing ELSI of human genomics, microbiome, and virome. (2) Findings from Aim 1 will be developed into vignettes of virome research including potential clinical and public health applications (such as use of bacteriophages in clinical care and phylogenetics in hospital surveillance). Data will be collected from interviews with experts from a variety of disciplines and professions, including responses to and expansions on the 5 vignettes. (3) Using results from Aims 1 and 2, we will produce and disseminate vignette-based resource guides to be used by viromics teams and trainees as they encounter ELSI. Resources will be disseminated through professional communities of practice and the ELSIHub, a platform that supports multidisciplinary resource sharing, including for lay audiences. In sum, through conceptual and empirical analysis, and vignette development and dissemination, this project will advance ELSI resources and illuminate ELSI and policy implications that arise in human virome research.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Developing neurons achieve a complex, elongated morphology, involving substantial increases in plasma membrane surface area that far exceed the accompanying growth in cytoplasmic volume. We have previously shown that during exocytosis, the fusion of secretory vesicles provides sufficient membrane material for developmental plasma membrane expansion. Similar to the synapse, the synaptic SNARE proteins, VAMP2, syntaxin1, and SNAP25 are present in developing neurons and mediate vesicle fusion. At the synapse, several synaptic vesicles are tethered to the presynaptic plasma membrane, representing the readily releasable pool. Their synchronous fusion and release of neurotransmitter is evoked by a Ca++ spike. In contrast, in developing neurons, our studies have demonstrated that fusion is not synchronous, and single vesicle fusion events occur in what has previously been considered a constitutive manner. However, we recently reported a surprising requirement for the activity of the nonreceptor tyrosine kinase focal adhesion kinase (FAK) in promoting vesicle fusion, in particularly during the transition between SNARE complex formation and vesicle fusion. The goal of this proposal is to investigate how FAK scaffolding function and/or FAK kinase activity regulates SNARE complexes and exocytosis. Our work utilizes neuronal cultures, multiple modes of light microscopy to visualize single vesicle fusion events at high spatial and temporal resolution, and quantitative mass spectrometry. Completion of this work will provide fundamental information about regulation of exocytosis during neuronal development.

NIH Research Projects · FY 2026 · 2025-07

Abstract The heart’s native pacemaker complex, the sinoatrial node (SAN), is a multicellular bioelectric signaling center responsible for rhythmically initiating the impulses that drive cardiac contraction. SAN dysfunction is extremely prevalent in humans and represents one of the leading causes for the surgical implantation of artificial pacing devices. SAN dysfunction is a progressive and noncurable. To date the molecular pathophysiology of SAN dysfunction remains poorly understood. Indeed, significant gaps remain in even our basic understanding of the biophysical processes that control electrical impulse generation in the heart. This is compounded by the fact that major discrepancies still exist between our theoretical descriptions of SAN electrical activation and experimental data. Therefore, we have used a combination of in vivo experimentation and computational modeling to reevaluate fundamental principles of cardiac pacemaking. More specifically, we have modeled how extracellular ion dynamics can influence the behavior of excitable cells arranged within fixed tissue geometries. This has revealed that robust entrained automaticity can emerge within cellular networks through repetitive elevation/depletion of extracellular potassium [K+]o. The stability of this automaticity is highest when [K+]o is elevated within diffusion limited extracellular nanodomains that are accessed by multiple cells. In addition, our model predicts that entrained automaticity can be achieved even when not all cells within a population are natively autorhythmic or connected via gap junctions. We have termed this form of “outside-in” regulation of pacemaking Extracellular Mediated Automaticity (EMA). In support of our computational simulations we have localized [K+]o within intact embryonic SAN tissue and identified that: i) [K+]o is indeed elevated in nanodomains between adjacent cardiac pacemaker cells (CPCs) in the SAN, ii) [K+]o oscillates within these nanodomains in a manner predicted by EMA, and iii) depletion of [K+]o from these nanodomains results in rapid loss of SAN automaticity and entrainment. Collectively these data have led to our overall working hypothesis that extracellular ion cycling serves as a baseline mechanism for imparting rhythmic, multicellular, impulse generation in the SAN. This hypothesis will be tested across two specific aims in which we will define [K+]o as the critical ionic species that underlies this form of entrained CPC automaticity (Aim1) and identify the mechanisms that control nonuniform extracellular ion distribution within the SAN (Aim2). Thus, successfully completion of the proposed studies will uncover an entirely novel set of paradigms for coupled electrical oscillation in the SAN and identify new extracellular regulators of SAN function. These data will fundamentally alter current theories of cardiac pacemaking, identify new factors to target as causative for SAN dysfunction, and directly inform future design criteria for clinical restoration of homologous biological pacemaking in patients.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY/ABSTRACT Half of all lifetime mental health disorders are present by adolescence, but the neural mechanisms underlying these disorders, particularly in the period leading up to adolescence, are still poorly understood. Early life and early adolescence are theorized to be sensitive periods characterized by large changes in structural (white matter-based) and functional (activity-based) brain networks and during which individuals are at increased vulnerability for later psychopathology. The correspondence between structural and functional connectivity, termed structure-function (S-F) coupling, also changes across development and, critically, relates to psychopathology. However, no studies have yet examined how S-F coupling changes across both sensitive periods in the same individuals, and it is unknown how S-F coupling relates to risk for psychopathology that transcends diagnostic boundaries (i.e., transdiagnostically). The proposed study will leverage the UNC Early Brain Development Study (EBDS), a unique dataset consisting of 10 timepoints of diffusion and resting-state functional neuroimaging data spanning two weeks to 16 years in the same individuals. This study will characterize trajectories of S-F coupling between birth and 16 years and will measure psychiatric outcomes at ages 6, 10, and 16 using the general psychopathology factor (p-factor), which captures transdiagnostic risk for psychopathology. Critically, 40% of infants in the EBDS dataset were at increased risk for future psychopathology at enrollment (e.g., due to premature birth, maternal mental illness). Therefore, this dataset is ideally suited for a longitudinal study examining transdiagnostic outcomes. With these unique data, this project will be the first to establish how S-F coupling changes across development within individuals and how these changes relate to transdiagnostic psychopathology. To accomplish these goals, this project will: 1) characterize longitudinal trajectories of S-F coupling from birth to adolescence, 2) determine what features of S-F coupling in early childhood, middle childhood, and adolescence relate to transdiagnostic psychopathology cross-sectionally, and 3) determine how trajectories of S-F coupling from birth to adolescence predict transdiagnostic psychopathology in adolescence. Through the results of these aims, this study will not only advance our knowledge of neurodevelopmental trajectories across childhood and adolescence, but it will also contribute to the identification of early neural markers of future transdiagnostic psychopathology. This essential early work may guide future clinical practice to improve outcomes for children of all ages. The expertise of an exceptional mentorship team combined with a unique dataset and top-tier resources available at the University of North Carolina at Chapel Hill will allow the trainee to use this research project and training plan to prepare for a productive career as a physician-scientist applying advanced neuroimaging analysis to study typical and atypical neurodevelopment.

NIH Research Projects · FY 2026 · 2025-07

Project Summary Oxidative stress is a byproduct of energy production necessary for all living organisms and caused by unregulated reactive oxygen/nitrogen/carbonyl species (ROS/RNS/RCS) among others. Nature has evolved the oxidative stress response (OSR) as a key component of metabolism that maintains cellular homeostasis by detoxifying and neutralizing aberrant reactive molecules. Spatiotemporally control of OSR is achieved through compartmentalization and redundancies that are coupled to create a redox balance to promote survival. Unbalanced OSR due to a defective or overactive capacity to resolve oxidative damage is associated with various human diseases. For example, chronic OSR is a hallmark of obesity, a global epidemic as well as a major risk factor for developing cardiovascular diseases, metabolic syndrome, and cancer. To better understand obesity, it is paramount that we elucidate the coordination of the OSR metabolon, defined here as the sequential antioxidant enzymes, biochemical reactions, and cellular compartments that maintain redox homeostasis. The proper regulation of and adaptive changes by OSR require rapid signaling taking place in the seconds-to- minute timeframe. Such dynamics must therefore require fast regulatory networks such as protein post- translational modifications (PTMs). Phosphorylation of serine (S) (~90%), threonine (T) (~9%), and tyrosine (Y) (~0.1-1%) residues are one of the many ways cells regulate pathways that maximize survival. Initial evaluation of the published phosphoproteome stratified by enzyme classification and pathway enrichment analysis indicates that, despite low intracellular stoichiometry, pY are enriched on antioxidant enzymes. However, the majority of pY sites on antioxidant enzymes are not functionally characterized. My overarching goal in this proposal is to gain network level insight into the pY directed regulation of antioxidant enzymes and the resulting dynamics of dysregulated OSR. I hypothesize that obesity-driven pY on multiple antioxidant enzymes modulates their catalytic activity to produce systemic changes in OSR. I will test this hypothesis by employing proteomics, metabolomics, structural analysis, and computational modeling. During the mentored phase of this application, I will predict the functional role of previously uncharacterized pY, validate predictions using in vivo as well as in vitro enzyme kinetic assays, and demonstrate pY-driven OSR dysregulation in an in vivo high-fat diet (HFD)- induced obesity mouse model. Through these interdisciplinary approaches, I aim to define systems of pY- modified enzymes that “tune” metabolic response to HFD, and evaluate differential regulation of OSR in a sex specific manner. Additionally, I will determine how altered dietary serine, glycine, or addition of small molecule antioxidants ameliorate HFD phenotypes, and the sex specific responses in the OSR metabolon that may be therapeutically relevant. This proposal and the outlined training plan will equip me with the technical skills, scientific knowledge, and professional training that will serve as the foundation to launch my research focused on OSR regulation as an independent investigator.

NIH Research Projects · FY 2025 · 2025-07

ABSTRACT The goal of this proposal is to generate an enriched mechanistic understanding of post-stroke motor recovery by examining corticomuscular circuit function in adults with stroke. The study of specific neural circuits in stroke aligns with our long-term goal of developing targeted and personalized stroke treatments based on an individual’s neural circuitry. Towards this precision rehabilitation approach, we have previously assessed neural oscillatory activity in 30 persons with stroke using electroencephalography to measure functional connectivity between brain and muscle (corticomuscular coherence, CMC). Findings from this work suggest that CMC in a motor-relevant frequency band (beta, 13-30 Hz) is both a marker of post-stroke motor function and recovery. The cortical and muscular sources that comprise this corticomuscular circuit may thus serve as a potential therapeutic target for neuromodulation. This proposal will determine the modulatory effects of beta-burst repetitive transcranial magnetic stimulation (rTMS) on corticomuscular circuit function as measured by CMC. We hypothesize that the enhancement of neural oscillatory rhythms that are consistent with corticomuscular circuit function using beta - burst rTMS with strengthen the corticomuscular circuit. We will test this hypothesis in 20 persons with chronic (≥ 6 months) stroke during a single research visit using a randomized cross-over study design. During this visit, participants will receive brief bouts of stimulation just prior to the execution of a precision grip task performed with their affected upper extremity. Aim 1a focuses on target specificity by comparing CMC change following beta-burst stimulation to an active site within the corticomuscular circuit vs. a control/decoy site outside of the immediate circuit. Aim 1b responsibly addresses stroke-related injury by determining how downstream damage to the ipsilesional corticomuscular circuit, based on corticospinal excitability evaluation with TMS, impacts beta-burst rTMS efficacy. This aim was informed by our prior work along with others showing that injury to the corticospinal tract significantly predicts participants’ response to treatment. Aim 1b will begin to identify neurophysiological characteristics of potential responders and non-responders to beta-burst rTMS. Independent of Aims 1a and 1b, Aim 2 will determine if beta-burst rTMS impacts motor task performance (reaction time and grip precision). This aim will provide preliminary evidence that corticomuscular circuit enhancement has motor behavioral relevance. This proposal introduces an innovative strategy by evaluating corticomuscular circuit function during goal- directed movement in stroke using a frequency-specific probe (rTMS). Findings generated from this work will provide proof-of-concept and causal support that dysregulated coupling between brain and muscle represents a treatable target for enhancing post-stroke function.

NIH Research Projects · FY 2026 · 2025-07

Project Summary Inflammatory bowel disease (IBD), which includes Crohn's disease and ulcerative colitis, are chronic, relapsing- remitting conditions that affect more than 1.6 million adults and 80,000 children in the US alone. Patients suffer from severe abdominal pain with bloody diarrhea caused by chronic inflammation and ulcer formation in the gastrointestinal tract. Current therapies provide only symptomatic relief, with 25% of IBD patients requiring hospitalization and 45% relapsing. Many of the drugs administered cause severe side-effects. We hypothesize that an orally administered nanobiologic secreted by a bioengineered yeast therapeutic, capable of (a) reducing the inflammatory response and (b) modulating the gut microbiome, will offer a safer and more effective treatment for IBD. To test this hypothesis, we will design, characterize and optimize fibronectin-targeting, nanobody drug- secreting S. boulardii as a function of S. boulardii proliferation and fibronectin density through a stable and dynamic ligand expression system (Aim 1). In Aim 2 we will determine the pharmacokinetics, colon retention, immunogenicity, and biodistribution of the dynamic and stable ulcer-targeting, nanobody drug-secreting S. boulardii. We will use physiologically-based computational modeling to guide dosing regimens. Aim 3 of the study involves evaluating the therapeutic effectiveness of S. boulardii and its potential to modulate the host microbiome in mouse models of ulcerative colitis. Specifically, one of the sub-tasks will involve the use of a human microbiota-associated IBD mouse model. This newly developed approach will be utilized to assess how engineered S. boulardii may alter a human IBD-associated microbiome. Our studies will advance targeted microbial therapeutics and oral nanobiologics towards the clinic to improve outcomes for ulcerative colitis patients. Here we combine pharmacoengineering, computational modeling, pharmacokinetics studies, and microbiome analysis to develop highly integrated, microbe-based therapeutics. We expect that the outcome of these studies will yield new strategies for IBD therapy that will be relevant to other diseases that affect the gastrointestinal tract or other ulcerative conditions.

NIH Research Projects · FY 2025 · 2025-07

Title: Mechanistic dissection and inhibitor targeting of autophagy in RAS driven cancers PI: Kirsten L. Bryant, PhD Description/Abstract Autophagy is a self-degradation process whereby cells can orderly clear defective organelles and recycle macromolecules as a nutrient source. Autophagy is elevated and essential for the tumorigenic growth of KRAS- mutant pancreatic ductal adenocarcinoma (PDAC), providing the rationale for clinical evaluation of the autophagy inhibitor hydroxychloroquine (HCQ) for PDAC. Disappointingly, when used as monotherapy or in combination with standard of care, HCQ has shown limited to no clinical efficacy for PDAC. We recently determined that the treatment of PDAC with inhibitors of the key KRAS effector pathway, the RAF-MEK-ERK mitogenic activated protein kinase (MAPK) cascade, unexpectedly caused further elevation of autophagy, rendering PDAC acutely dependent on this process, and hypersensitive to autophagy inhibition. We determined that ERK inhibition impaired other critical processes that then led to compensatory upregulation of autophagy. Our findings, together with essentially identical conclusions by another independent co-published study, have led to the initiation of clinical trials evaluating either MEK (trametinib, binimetinib) or ERK (LY3214996) inhibitor in combination with HCQ for metastatic KRAS-mutant PDAC. While early observations from compassionate use of this combination support a significant clinical impact, our preliminary studies support our premise that we can improve upon this therapy. We propose two aims to further advance autophagy inhibition as an anti-RAS therapeutic approach. HCQ is a lysosome inhibitor and consequently not selective for autophagy. We hypothesize that inhibitors of the proteins upstream in the autophagy pathway, will synergize with HCQ and more potently and durably inhibit the autophagy pathway. Additionally, a comprehensive evaluation of the effect of inhibition of each individual node of the autophagy pathway on flux in the context of an autophagy-addicted cancer such as PDAC has not been performed. Thus, we will determine the effect of individual and combined inhibition of different nodes of the autophagic pathway on autophagic flux (Aim 1). Our Aim 2 studies are based on our recent observation that PDAC cells upregulate macropinocytosis in response to sustained inhibition of the RAS ERK MAPK pathway. We hypothesize that the upregulation of macropinocytosis facilitates resistance to both RAS- and autophagy- targeted therapeutics. We will characterize the mechanistic signaling underlying macropinocytic upregulation as well as evaluate RAS inhibitor resistant cells to determine whether upregulated macropinocytic activity is a driver of resistance. In summary, our studies will enhance our understanding of regulation of nutrient scavenging pathways in PDAC and aid in the development of novel combination therapies to target autophagy and macropinocytosis for the treatment of KRAS-mutant cancers.

NIH Research Projects · FY 2025 · 2025-07

Classical Klebsiella pneumoniae (cKp) isolates are typically associated with hospital-acquired infections such as pneumonia, bacteremia, and urinary tract infections (UTIs) in immunocompromised patients. Over the past thirty years, a distinct pathotype known as hypervirulent Klebsiella pneumoniae (hvKp) has emerged, recognized for causing severe infections, including liver abscesses, in otherwise healthy individuals. Despite the availability of targeted antibiotic therapies, hvKp infections can escalate into severe complications such as endophthalmitis and meningitis. Previous mouse infection models have demonstrated that hvKp evades clearance by Kupffer cells in the liver sinusoids, highlighting a critical gap in our understanding of hvKp virulence mechanisms. However, due to rapid mortality in experimental models, the bacterial factors required for liver abscess formation remain underexplored. We have established a novel mouse hvKp wound infection model which progresses to systemic infection, including the formation of macroabscesses in the liver. Initial findings indicate that a mutant deficient in both the type 1 and type 3 fimbriae is unable to form liver abscesses but retains virulence in the wound and the kidney. Fimbriae are surface expressed protein appendages that play a crucial role in the infection process. Type 1 fimbriae in cKp strains bind mannose residues on uroepithelial cells to mediate urinary tract infections (UTIs). Type 3 fimbriae mediate biofilm formation and facilitate attachment to abiotic catheters in a UTI model. However, Klebsiella fimbriae are dispensable for gut and lung colonization and their contribution to liver abscesses is unknown. In this study, we will investigate the specific contributions of type 1 and type 3 fimbriae to the pathogenesis process and evaluate their potential as targets for therapeutic intervention. Our methods include constructing and evaluating the ability of hvKp strains lacking type 1 and/or type 3 fimbriae to infect mouse wounds, disseminate from the wound and establish liver abscesses. We anticipate that strains deficient in these fimbriae will demonstrate significantly reduced liver colonization, underscoring their importance in hvKp pathogenicity. This investigation will significantly advance our understanding of hvKp virulence strategies in liver abscess formation and may lead to the development of novel anti-virulence therapies.

NIH Research Projects · FY 2026 · 2025-07

The increasing availability of large-scale lifespan brain MRI data, from Fetal Tissue Annotation (FeTA), Developing Human Connectome Project (dHCP), National Database for Autism Research (NDAR), Baby Connectome Project (BCP), Multi-visit Advanced Pediatric (MAP) Brain Imaging Study, Autism Brain Imaging Data Exchange (ABIDE), Human Connectome Project (HCP), Adolescent Brain Cognitive Development (ABCD), Open Access Series of Imaging Studies (OASIS), Chinese Color Nest Project (CCNP), UK Biobank (UKB), Alzheimer’s Disease Neuroimaging Initiative (ADNI), and Australian Imaging Biomarkers and Lifestyle Study of Ageing (AIBL), affords unprecedented opportunities for precise charting of lifespan brain changes from the fetus to the elderly, providing important insights into the origins and aberrant evolving patterns of neurodevelopmental, psychiatric, and neurodegenerative disorders. However, a major barrier is the critical lack of lifespan-dedicated computational tools/pipelines for accurate and consistent processing and analysis of challenging lifespan brain MRIs, which suffer from age-dependent imaging artifacts and low and dynamic tissue contrast—especially for infant and elderly brain MRIs—as well as large inter- site data heterogeneity inherent to different imaging protocols and scanners across sites. Most existing tools/pipelines are designed for age-specific groups, and directly applying these to lifespan data results in inconsistent and misleading outcomes. Therefore, the overarching goal of this project is to create and disseminate the first knowledge-empowered deep learning pipeline for accurate and consistent atlas construction, skull stripping, artifact correction, super resolution, harmonization, tissue segmentation, topological correction, and brain labeling of lifespan brain MRIs. Specifically, we will develop an anatomy- guided atlas learning neural network to construct the first set of lifespan atlases with temporally consistent and spatially detailed patterns, and subject-specific atlas with personalized prior for subsequent aims (Aim 1). Guided by prior knowledge from Aim 1, we will develop a novel lifespan skull stripping framework (Aim 2). After skull stripping, we will then propose a novel deep learning framework for joint artifact correction, super resolution, harmonization, and tissue segmentation (Aim 3). We will propose an anatomically constrained topological correction network to ensure the topological correctness of cortex and further a transformer brain labeling to divide the cortex into anatomical/functional regions of interest (Aim 4). Finally, we will integrate our lifespan- dedicated tools and atlases into a comprehensive software package that is conveniently executable in both local and cloud-based environments (Aim 5). We will freely release our software, atlases, and processed lifespan data (150,000+ scans from 50,000+ subjects) to the public.

NIH Research Projects · FY 2025 · 2025-07

Abstract Investigations of embryonic development are critical for understanding congenital anomalies that arise from altered development. Additionally, the derivation of tissues in vitro from pluripotent cells relies on directed differentiations along developmental pathways. However, this has proven challenging for organs such as the mammalian kidney which proceeds through three different phases of development along the anterior to posterior axis before the third phase induces development of the final, metanephric kidney. Questions remain about whether cellular programs of the metanephric kidney are actually induced in vitro or whether they represent earlier phases of development. The intermediate mesoderm, which is thought to derive independently of the adjacent paraxial and lateral plate mesoderm from the primitive streak, generates the kidney tissues, gonads, and adrenals. The metanephric kidney is induced to form caudally at the hindlimb level. However, despite over a century of research into the intermediate mesoderm and metanephric kidney development, the origins remain clouded by studies focusing on anterior intermediate mesoderm, conflicting results, and building evidence that may suggest alternate mesodermal origins. Our own imaging of early embryos as well as a thorough consideration of previous findings has led us to hypothesize that the intermediate mesoderm which gives rise to the metanephric kidney is derived from paraxial mesoderm. Therefore, it is imperative that we reexamine the intermediate mesoderm and kidney origins considering these findings. Advances in tools and methodology available will enable us to interrogate intermediate mesoderm and metanephric kidney development and shed new light onto the cellular origins. In Aim 1 we will perform novel lineage traces of paraxial mesoderm and utilize whole embryo light-sheet and live imaging to examine the paraxial, intermediate, and kidney cell populations at critical developmental timepoints to ascertain their identity and origins. In Aim 2, we will utilize directed differentiations from induced pluripotent stem cells (iPSCs) to test whether in vitro derived paraxial mesoderm is competent to generate intermediate mesoderm and kidney tissue. Together, we predict that these novel and innovative studies will redefine and refine the cellular origins of intermediate mesoderm and its derivatives. These findings will contribute essential foundational knowledge to the field of developmental biology and aid the development of effective tissue replacement and regenerative strategies, as well as further our understanding of developmental anomalies which significantly affect human health.

NIH Research Projects · FY 2025 · 2025-07

Abstract Bone morphogenetic protein as a bone regeneration biologic has experienced steady decline in use given its side effects. Our published work has shown, for the first time, that a natural compound, dietary hesperidin (HE), can control BMP side effects likely via multiple mechanisms. These include control of inflammation, osteogenesis and osteocyte function in regenerated and native mandible bone. Due to the complexities of the multifactorial effect of phytochemicals such as HE, a multi-cellular system mimicking the bone environment would be an ideal ex-vivo setting for undertaking mechanistic studies to understand its role on BMP function and craniofacial bone. Such system could be targeted to simulate the mechanics of the bone environment given specific shear stresses. Based on preliminary data, it is our hypothesis that HE limits inflammation induced by exogenous BMP2 via TLR receptors, and that it can promote new high-quality bone formation via its modulation of osteoblast-osteocyte interaction, promoting BMP signaling, favoring fast matrix remodeling and Ca2+ deposition. Further, this grant proposes the utilization of an innovative 3D co-culture platform which will facilitate the understanding of the effect of the bone exposome containing HE +/- BMP on bone cells and allow data collection in a bone network set to mimic the microenvironment. This proposal is in response to the NIH RFA DE-025-002 which will support the validation of novel dental, oral and craniofacial (DOC) organ-on-a-chip (OoC). The proposal offers high reward since, if successful, will provide further understanding of BMP2 function in bone regeneration, a novel approach on modulation of BMP2 via phytochemicals and ultimately, provide validation of a new 3D bone culture system mimicking the bone cell interactions under mandible-like mechanics (frequent shear stresses) (the OoC). The results can be compared to our previous pre-clinical studies and ultimately the proposed bone exposome-on-a-chip may offer validation, equivalence and perhaps outperform animal testing.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Glioblastoma is the most common primary brain malignancy in adults yet has no cure. Patients typically only live 12-15 months after treatment, with a five-year survival rate of less than 10%. Despite the poor patient outcomes, only three new treatments have been approved for glioblastoma since 2005: temozolomide, bevacizumab, and tumor-treating fields. The most common treatment method is maximal surgical resection followed by radiotherapy or chemotherapy, but the highly progressive and recurrent nature of the disease causes treatment to be ineffective. Chimeric antigen receptor (CAR) T cell therapy has emerged as a successful anti-cancer immunotherapy with significant impact in treating blood cancers. There have been six FDA approved CAR T cells therapies since 2017, demonstrating its clinical relevance. Unfortunately, typical CAR T cell production is a complex and expensive process involving harvesting the patient’s blood, isolating the T cells, transferring the CAR gene, extensive ex vivo expansion, and finally infusion of the expanded CAR T cells back into the patient. This process costs around $500,000 per dose and can take over 20 days to complete. It is often used as a last resort, causing many patients to succumb to their disease before receiving treatment. In addition, the lengthy ex vivo expansion deteriorates T cell quality as it encourages T cell differentiation. It has been clinically shown that having less-differentiated naïve/stem-like and central memory phenotypes after infusion is directly related to patient outcomes due to the enhanced persistence of these phenotypes compared to effector phenotypes. The goal of this project is to reduce the CAR T cell manufacturing time to three days and improve the quality and potency of CAR T cells by using biomaterials to manufacture CAR T cells in vivo. These scaffolds will be used to manufacture CAR T cells against GD2, a tumor-associated antigen overexpressed on glioma cells but limitedly expressed on adjacent healthy cells. Limiting ex vivo manufacturing will reduce CAR T cell differentiation for enhanced CAR T cell quality and persistence to improve therapeutic efficacy. For Aim 1, in vitro studies will determine how CAR T cells are released from the scaffold over time and the phenotype of released cells. In vitro coculture experiments with scaffold-produced CAR T cells and glioma cells will be performed to evaluate tumor killing ability and secretion of proinflammatory cytokines. For Aim 2, an in vivo patient-derived orthotopic xenograft model will be used to determine anti-tumor efficacy and survival in mice treated with biomaterial- generated CAR T cells. In vivo CAR T cell phenotype and persistence will be evaluated in the blood, brain, bone marrow, and spleen. This project will have a profound impact on glioblastoma treatment by demonstrating the potential for biomaterials to provide sustained local delivery of less-differentiated CAR T cells, show enhanced efficacy, and prevent disease relapse, which are highly important factors for clinical success in GBM patients.

NIH Research Projects · FY 2025 · 2025-07

Project Summary The screening of biologically active substrates’ analogs and derivatives represents an eƯective and straightforward strategy in new drug discoveries, as such strategy provides Structure Activity Relationship (SAR) study data that can be useful in identifying the most promising drug structures. The screening could also be incorporated into High Throughput Experiments (HTE) to facilitate rapid research process, and sometimes could generate unexpected findings from screening to inspire new drug designs. Radiofluorinated Positron Emission Tomography (PET) tracers are an important type of radiopharmaceuticals that are used in PET imaging, which could provide valuable diagnostic data in the early stages of disease progressions. Even though the benefits of screening large numbers of drug candidates’ analogs apply to the development of both nonradioactive pharmaceuticals as well as radiopharmaceuticals, the challenges in radiosynthesis can sometimes present obstacles in utilizing such strategy in novel PET tracer development. This is especially a concern in 18F-labeled radiopharmaceutical development, as the poor nucleophilicity of the [18F]fluoride ion exacerbates these radiosynthetic challenges. To promote the utilization of quick candidates screening strategy in 18F-labeled PET tracer development, the fluorine-18 nuclide must be incorporated at late-stages of the synthesis in a manner that is fast (to avoid decay loss) and selective (to allow for the construction of PET tracer analogs with diƯerent functional groups for SAR studies). This can be done either via 1) radiofluorination methods that are functional group compatible, or via 2) the utilization of robust and selective 18F-labeled prosthetic groups. Progress in both categories have been reported by many groups in recent years. My proposal focuses on the further developments of both new labeling methods and prosthetic groups, and to construct large numbers of 18F-labeled PET tracer analogs in a quick and iterative manner via these new methods/prosthetic groups for biodistribution evaluation in order to support radiopharmaceutical development via quick screening strategy. To be specific, I propose to develop late-stage direct radiofluorination method to synthesize [18F]fluorohydrin substrates via organophotoredox-mediated reactions. I also propose to develop new prosthetic groups such as radiolabeled isocyanate prosthetic groups and new strained alkynes for Strain- Promoted Azide Akyne Cyclizations (SPAAC). These proposed method/synthon developments will promote quick and iterative constructions of new PET tracer analogs for biodistribution evaluations, facilitate SAR studies, and have the potential to promote rapid development process of radiopharmaceuticals.