Univ Of North Carolina Chapel Hill

NIH Research Projects · FY 2026 · 2021-02

Tissue factor (TF) is a transmembrane protein that functions as a high-affinity receptor for factor (F)VII and FVIIa. The TF-FVIIa complex is the primary initiator of coagulation and plays an essential role in hemostasis. However, aberrant TF expression underlies most forms of thrombosis. TF expression is also induced in response to bacterial and viral infections as part of the innate immune response. This expression can be either protective by limiting the spread of the pathogen or pathologic by triggering disseminated intravascular coagulation (DIC). TF-dependent generation of coagulation proteases also leads to activation of protease-activated receptors (PARs). My lab has made major contributions to understanding the roles of TF, coagulation proteases and PARs in hemostasis, thrombosis, endotoxemia, ischemia-reperfusion injury, atherosclerosis, and viral infections. This R35 OIA application is an extension of two NHLBI funded R01 grants: Mechanism of venous thrombosis in pancreatic cancer; Role of the thrombin PAR1 pathway in viral infection. We have shown that levels of circulating tumor-derived, TF+ extracellular vesicles (EVs) are associated with increased venous thromboembolism in pancreatic cancer patients. In addition, we found that TF+ EVs enhance venous thrombosis in mice bearing human pancreatic tumors. We have also shown that TF-dependent activation of coagulation and PAR1 signaling is protective in response to Coxsackievirus B3 by boosting the antiviral IFNβ pathway in the heart. In contrast, PAR1 suppresses the pathologic NF-κB response in the lung in response to influenza A H1N1 infection. The OIA funding mechanism would provide stable funding and increased time for our group to pursue higher risk-higher reward projects, such as performing proteomic analysis of plasma and EVs, establish new technologies, such as the ExoView system, and following up on exciting discoveries. Our long-term goals are to further understand the protective and pathologic roles of TF, coagulation proteases and PARs in cancer and infections. There are two hypotheses for this proposal: 1/ TF enhances venous thrombosis and tumor growth in pancreatic cancer, and 2/ TF-dependent activation of coagulation is both protective and pathologic in response to viral infection. We will continue our studies on the identification of plasma biomarkers of thrombotic risk in cancer patients using clinical samples. We will identify prothrombotic pathways that contribute to cancer- associated thrombosis using mouse models. In addition, we will determine the roles of tumor and host derived TF in the growth of pancreatic tumors in mice. We will identify the cellular sources of pathologic TF in mouse models of viral infection that may lead to new treatments to prevent DIC. We will elucidate how PAR1 is protective by both enhancing the IFNβ antiviral pathway in the heart and suppressing the pathologic NF-κB pathway in the lung in response to viral infection. This knowledge may lead to the identification of new biomarkers of thrombotic risk, treatments for pancreatic cancer and protection from viral infection.

NIH Research Projects · FY 2025 · 2021-02

Project Summary/Abstract Neurons are capable of activating pathways that induce either the degeneration of the entire cell by apoptosis or to selectively degenerate only the axons. Physiological axon-specific degeneration, known as axon pruning, is important as it allows neurons to remove excessive or misguided axons and permit plasticity in neuronal connections. Aberrant pruning is observed in several neurodegenerative diseases, including Alzheimer’s Disease (AD). However, exactly how neurons activate this pathway to degenerate axons in physiological or pathological situations of AD is unclear. We have investigated the apoptosis and axon pruning pathways in a microfluidic chamber-based model utilizing sympathetic neurons. Upon nerve growth factor (NGF) deprivation, these neurons can induce either apoptosis (when NGF is deprived from both soma and axon compartments) or axon pruning (when NGF is deprived from only the axon compartment). Our research has identified substantial overlap but also distinct differences between the apoptosis and axon pruning pathways. For example, while caspase-9 (Casp9) and caspase-3 (Casp3) are required for both pathways, their activation is dependent on the apoptosome during apoptosis but is surprisingly independent of the apoptosome during axon pruning. While investigating the mechanism by which caspases are activated during pruning, we unexpectedly found that the inflammasome pathway plays an important function in axon pruning. Inflammasomes have been studied primarily in immune cells in the context of pathogen signaling. These are multi-protein complexes formed in response to pathogenic or danger stimuli, which result in activation of the proinflammatory caspase, caspase-1 (Casp1). Strikingly, we found that Casp1 and NLRP1 (a key component of a particular inflammasome) are both essential for axon pruning. These results are surprising as axon pruning does not involve pathogen exposure. In this proposal, we will identify the specific inflammasome pathway components that are critical for axon pruning and conduct mechanistic experiments to define this novel function of NLRP1 in neurons. In Aim 1, we will define the specific inflammasome proteins that are essential for axon pruning, and determine where they act in the known pruning pathway. In Aim 2, we will define the role of IL-1β/IL-18 in axon pruning. Our focus in Aim 3 will be to conduct mechanistic experiments to examine how NLRP1 is activated in the context of axon pruning. Importantly, in Aim 4 we will focus on AD and investigate whether the pathological degeneration of synapses and axons in AD are mediated by NLRP1. We will examine this in a microfluidic model of Aβ-induced axon degeneration in vitro as well as in a mouse model of AD in vivo where we will examine if NLRP1 deficiency reduces AD pathology and behavioral defects. This project opens an exciting new avenue of research into this unexpected function of the NLRP1 inflammasome in neurons.

NIH Research Projects · FY 2026 · 2021-02

PROJECT SUMMARY Reducing morbidity and mortality due to invasive cervical cancer (ICC) is a global health priority, which can be accomplished in part by improving the timely detection and management of cervical precancer. Precancerous cervical lesions characteristically progress from low-grade to high-grade cervical intraepithelial neoplasia (from CIN-1 to CIN-2/3). Low-grade CIN-1 can spontaneously regress to normal, persist over time, or progress to CIN- 2/3, but there is insufficient evidence to predict which CIN-1 cases will progress. Epigenetic alterations of DNA sequences, such as methylation at cytosine-p-guanine (CpG) sites, have been observed in cervical precancer and cancer. As such, methylation patterns have been proposed as potential biomarkers for the detection of high- grade cervical lesions. However, their utility as predictors of cervical disease progression is limited, as few studies have investigated the effect of methylation on the risk of progression from low-grade CIN-1 to high-grade CIN-2/3. In addition, while high-risk human papillomavirus (HPV) is known to cause ICC, little is known about the relationship between HPV infection and the host methylation patterns seen in cervical carcinogenesis. Epigenetic studies incorporating longitudinal clinical data are needed to understand the effect of DNA methylation on the risk of progression of HPV-associated precancerous cervical lesions. This study will assess relationships between early-stage DNA methylation, high-risk HPV infection, and the progression or persistence of CIN-1. The proposed research will utilize collected data from women in the Cervical Intraepithelial Neoplasia Cohort Study (CINCS) with CIN-1 at enrollment, baseline methylation at 450,000 GpGs, pertinent clinical and behavioral exposures, and one year of clinical follow-up data. Using this unique and robust dataset, the Specific Aims of this proposal are to 1.) assess the effect of baseline DNA methylation on CIN- 1 progression/persistence in 151 women at one-year follow-up and 2.) assess the association between baseline HPV infection status and DNA methylation in 151 women with CIN-1. Study findings have the potential identify novel methylation markers of cervical disease progression, improve the clinical management of low-grade cervical precancer, and contribute to our knowledge of HPV-related carcinogenesis. Through the completion of these research aims, the applicant will gain a unique set of skills in advanced epidemiologic methods and clinical research, including the analysis and interpretation of complex epigenetic and longitudinal clinical data. Expert mentors in cancer and genetic epidemiology, methylation analysis, statistics, and gynecologic oncology will support the applicant’s successful completion of the proposed research, associated training plan, and MD-PhD degree at the University of North Carolina at Chapel Hill. This F30 fellowship will critically aid the applicant’s development as a future interdisciplinary physician-scientist practicing at the intersection of cancer care, epigenetics, and women’s health.

NIH Research Projects · FY 2025 · 2021-01

Summary/Abstract Pertussis (aka whooping cough) is re-emerging in developed countries despite high vaccine coverage. Resurgence is due primarily to waning immunity to the causal bacterium Bordetella pertussis (Bp) in adolescents and young adults immunized with acellular (aP) pertussis vaccines. Moreover, while aP vaccination protects against disease, at least initially, it does not protect against colonization or prevent transmission, which puts infants, who are most vulnerable to serious and sometimes fatal disease, at greater risk. New vaccines that protect against both colonization and disease are needed. Their development requires a better understanding of the molecular mechanisms underlying Bp virulence. A strictly human-adapted pathogen, Bp is extremely closely related to Bordetella bronchiseptica (Bb), which infects nearly all mammals. Bp and Bb produce a nearly identical set of virulence factors, some of which, including filamentous hemagglutinin (FHA) and adenylate cyclase toxin (ACT), are functionally interchangeable. FHA is a critical adhesin, a component of acellular vaccines, and the prototypical member of the Two Partner Secretion (TPS) family. Using Bb and its natural hosts (rats and mice), we showed that in addition to mediating adherence to host cells, FHA plays important roles in controlling the initial inflammatory response to infection and in mediating defense of the bacteria against clearance by phagocytic cells, which contributes to persistence in the lower respiratory tract (LRT). Our work on the mechanism of secretion and processing of the “precursor” FhaB protein to FHA has led to major advances in the mechanism of two partner secretion, and we showed that FhaB itself, rather than FHA, is critical for bacterial persistence in the LRT. ACT also contributes to bacterial persistence in the LRT by mediating defense against phagocytic cell clearance. We and others have shown that ACT binds to FhaB/FHA on the bacterial surface. We propose a model in which ACT, while bound to FhaB on the bacterial surface, binds to CR3 on phagocytic cells, triggering degradation of the C-terminal, periplasmically-located, FhaB prodomain, resulting in efficient delivery of ACT to phagocytic cells and not to epithelial cells. We propose to: 1) Investigate the roles of DegP, CtpA, EnvC and LbcA in regulated degradation of the FhaB prodomain, 2) Investigate the relationship between ACT binding to CR3, regulated degradation of the FhaB prodomain, and delivery of ACT specifically to phagocytic cells, and 3) Investigate the consequences of dysregulated FhaB prodomain degradation on the establishment and maintenance of respiratory infection.

NIH Research Projects · FY 2025 · 2021-01

Project Summary: Multiple sclerosis (MS) is a debilitating autoimmune neuroinflammation disease inflicting millions of people worldwide. It is caused by dysregulated adaptive and innate immunity. CD4 T cells critically contribute to MS development in humans and EAE development in mice. Particularly, Th17 cells are central to autoimmune neuroinflammation. To understand the etiology of and develop treatment for MS, one of the main goals of MS research is to understand how T cell and Th17 cell function are controlled. Transforming growth factor–β (TGF-β) is instrumental in Th17 cell differentiation and function. Yet it is unclear whether and how TGF-β superfamily member other than TGF-β controls Th17 cell differentiation in MS/EAE. Our recent findings revealed novel TGF-β superfamily and related signaling to regulate Th17 cell function and the development of autoimmune neuroinflammation: (1) SKI protein, a TGF-β signaling suppressor that is degraded upon TGF-β stimulation, suppresses Th17 cell differentiation in vitro and in vivo, (2) SKI expression in T cells completely protected mice from EAE, (3) Activin-A, a TGF-β superfamily member that is closely related to TGF-β, was upregulated during EAE and in activated T cells in inflamed tissues and in the presence of proinflammatory cytokines, and (4) Activin-A+IL6 induced SKI degradation and the differentiation of Th17 cells that phenotypically resemble pathogenic- rather than non-pathogenic-Th17 cells to promote EAE. We therefore hypothesize that TGF-β superfamily member Activin-A is a novel factor distinct from TGF-β to promote pathogenic Th17 function and autoimmune neuroinflammation through SKI. In this study, we proposed to reach the following three Aims. AIM 1: Address whether Activin-A is required for Th17 cell function and EAE. AIM 2: Reveal the molecular program of Activin-A induced Th17 cell generation and function; AIM 3: Investigate the mechanisms underlying SKI controlled Th17 cell function and EAE. There is a great and yet unmet need in the understanding of how Th17 cell function during autoimmune neuroinflammation. This study aims to reveal previous unappreciated cellular and molecular mechanisms underlying TGF-β superfamily signaling in controlling Th17 cell differentiation and function for autoimmunity. The success of this study will gain critical mechanistic insights into T cell mediated autoimmune neuroinflammation and shed new light on how to mitigate related disease by targeting TGF-β superfamily signaling pathways.

NIH Research Projects · FY 2025 · 2021-01

Alzheimer’s Disease and Related Dementias (ADRD) are projected to affect 14 million Americans by 2050. To date, though, research on the signs and symptoms of ADRD has been sparse in early midlife populations, especially at the national level. It is crucial to conduct such research because early changes in cognitive functioning and the accumulation of risk factors for ADRD can begin decades before concrete signs and symptoms emerge. The challenging search for the causes of ADRD has made it clear that prospective and comprehensive data—including detailed social, biological, and health measurements across the life course— are needed to identify key predictors of ADRD. As such, the National Longitudinal Study of Adolescent to Adult Health (Add Health) provides an extraordinary opportunity to study the early origins of cognitive functioning/change and ADRD risk in a nationally representative cohort that has been followed since adolescence and will be in their mid-40s in the next wave of data collection (Wave VI). The overall goal of this project is to collect and disseminate critical data related to cognitive, physical, and sensory functioning in conjunction with the Add Health Wave VI Core Project to facilitate identification of early risk factors for later life ADRD. Adding such rich measures to Wave VI of Add Health will make possible tracking of cognitive, sensory, and physical functioning across the life course; coupled with the testing of biological risk markers, it will also lay the foundation for detecting signs of cognitive impairment and ADRD risk in early midlife. These new data, when combined with Add Health’s existing 25-year collection of extraordinarily rich multi-level and longitudinal measures and its new Wave VI data, will also aid in the scientific community’s understanding of the interplay of social, behavioral, and biological factors leading to ADRD in later life. Moreover, because Add Health is a nationally representative sample, adding these data to Wave VI will greatly increase understanding of disparities in cognitive, physical, and sensory functioning. The project’s specific aims are to: 1) Collect new in- depth, in-person assessments of cognitive functioning in early midlife for a nationally representative subsample of participants in Wave VI; 2) Collect automated, (largely) web-based measures of cognition in early midlife for all participants in Wave VI and compare them with our in-person measures of cognition to assess their feasibility and value; 3) Include assessments of physical and sensory functioning in early midlife for Wave VI participants of Add Health; 4) Test for biological markers of ADRD risk and cognitive function in early midlife; 5) Clean, document, disseminate, promote, and support the data collected in this project for the scientific community. All told, this project will collect and disseminate innovative data to thousands of researchers that will facilitate the rigorous study of cognition and risk factors for later life ADRD among a nationally representative sample of early midlife Americans.

NIH Research Projects · FY 2026 · 2021-01

Abstract Direct cardiac reprogramming holds great promise as a novel therapy for heart failure, a common and morbid disease that is usually caused by irreversible loss of massive functional cardiomyocytes. By leveraging the knowledge in developmental and stem cell biology gained during my PhD and postdoc training, in 2012 I demonstrated that in a murine acute myocardial infarction model, delivery of three transcription factors, Gata4, Mef2c and Tbx5 (GMT) converted cardiac fibroblasts (CFs) into functional induced cardiomyocytes (iCMs) that integrated electrically and mechanically with surrounding myocardium, resulting in functional improvement and scar size reduction. These findings suggest that iCM reprogramming is an effective means of regenerating heart tissue in vivo for human patients with heart disease. However, because relatively little was known about the factors that allow CFs to be reprogrammed, the applicability of cardiac reprogramming was limited to the context in which it had been attempted at that time. Since my independence, my own laboratory has established robust murine and human iCM reprogramming systems. By using these systems, we obtained novel insights into the transcriptional, post-transcriptional and epigenetic regulation of both murine iCM (supported by R01HL128331 as ESI) and human iCM reprogramming (supported by R01HL144551), and concomitantly improved the quality and yield of iCMs. This R35 EIA application is an extension to these two currently funded NHLBI R01 grants to further unravel the molecular mechanisms underlying iCM conversion, to test in vivo iCM reprogramming in non- acutely injured hearts and to exploit the latest single cell multi-omics and mathematical modeling for optimized and individualized reprogramming. Successful completion of this proposal will help to move direct cardiac reprogramming closer to its clinical application, provide new insights into molecular mechanisms underlying cardiac cell fate determination, and open new opportunities for the field to leverage the models and platforms we will develop here to study other cardiovascular physiological and pathological processes.

NIH Research Projects · FY 2025 · 2021-01

PROJECT SUMMARY Title: Biased PAR1 Agonism in Sickle Cell Disease Sickle cell disease (SCD) is caused by a single nucleotide mutation in the β-globin gene, resulting in altered red cell physiology that drives chronic hemolytic anemia and painful vaso-occlusive crisis (VOC) triggered by microvascular occlusion/stasis. VOC is the leading cause of hospitalizations of sickle cell patients. Activation of the main thrombin receptor, protease activated receptor 1 (PAR1), enhances the interactions between endothelial cells and sickle RBCs. In my recently published study, I demonstrated that PAR1 deficiency on nonhematopoietic cells or inhibition of PAR1 with vorapaxar attenuates microvascular stasis in a mouse model of SCD. In addition to thrombin, PAR1 is also activated by activated protein C (APC). The APC-mediated activation of PAR1 is referred to as “biased agonism” because it activates a different signaling pathway than thrombin and ultimately induces cytoprotective and anti-inflammatory effects. My central hypothesis is that canonical thrombin/PAR1 signaling contributes to microvascular stasis whereas non-canonical APC/PAR1 signaling reduces microvascular stasis and thromboinflammation. I hypothesize that inducing beneficial PAR1 biased signaling will be advantageous compared to complete PAR1 inhibition, which blocks the deleterious thrombin- dependent signaling as well as beneficial APC signaling. In the first aim I will compare the roles of thrombin/PAR1 and APC/PAR1 signaling on coagulation, inflammation, and microvascular stasis in sickle cell mice using mice with PAR1 point mutations that select for activation by either thrombin or APC. In the second aim, I will compare the therapeutic potential of a signaling-selective form of APC, 3K3A-APC, to two inhibitors of PAR1, parmodulin 2 and vorapaxar, on inflammation, microvascular stasis, and acute chest syndrome. Finally, in the third aim, I will investigate the effects of biased PAR1 agonism on mortality and end-organ damage in sickle mice. These studies will investigate the role of biased PAR1 signaling in the pathology of SCD.

NIH Research Projects · FY 2026 · 2021-01

DNA damage, particularly DNA double strand break (DSB), has detrimental effects on cell survival and genomic stability. In response to DNA damage, the cell activates several evolutionarily-conserved mechanisms to repair DNA damage, halt cell proliferation, or induce cell death. These surveillance mechanisms, collectively defined as the DNA damage response (DDR), constitute an important etiological factor for many human diseases, especially cancer. Moreover, the DDR is a key determinant for the therapeutic outcome of cancer treatment using radiation and other DNA damaging agents. A long-term goal of our laboratory is to delineate new DDR factors and mechanisms using comprehensive experimental tools, and thereby, revealing new insights into cancer progression and treatment. In a recent effort to systematically identify new components of the DSB “repairosome”, we identified Kif2C and several other MT regulators as potential DSB-associated proteins. Kif2C is rapidly recruited to DNA damage sites and plays an essential role in DSB repair. Strikingly, Kif2C mediates the spatial movement of DSBs, and controls DNA damage-induced chromatin remodeling. These functions of Kif2C are largely dependent on its MT depolymerase activity, and are likely to be achieved via coordination with other MT regulators. On the other hand, DNA damage modulates Kif2C phosphorylation and MT stabilization. These findings suggest a novel inter-organelle crosstalk between MT components and the DDR machinery that differs from the conventional perception that MT functions exclusively as a cytoplasmic structure. These findings also reveal new mechanistic insights into the clinical combinations of DNA damaging agents with anti-MT poisons in cancer therapy. In this project, we will further reveal detailed mechanisms via which Kif2C modulates the mobility of DSBs; we will uncover how Kif2C acts in concert with chromatin remodelers and other MT regulators to govern the dynamic chromatin compaction at damage chromatin; we will functionally characterize ATM-mediated Kif2C phosphorylation, and regulation of MT stabilization after DNA damage. Together, the project will potentially provide paradigm shifting additions to both the DDR and MT biology, and improve our understanding of how the cell coordinates various cellular components and mechanisms to maintain genomic stability and cell homeostasis after DNA damage.

NIH Research Projects · FY 2025 · 2021-01

Abstract Chimeric antigen receptors (CAR) expressed by T cells recognize tumor cells via single chains antibodies and activate T cell cytotoxic machinery and costimulation. In clinical studies, costimulation mediated by CD28 and 4-1BB endodomains integrated into the CD19-specific CAR has been shown to be equally effective in causing tumor regression. However, CD28 and 4-1BB costimulation differentially modulates the kinetic, metabolism and persistence of CAR-T cells, and the mechanisms governing these differences are not fully understood. In this study, we have identified that LCK recruited by co-receptors into the synapse of the CAR encoding CD28 leads to antigen-independent CAR-CD3ζ endodomain phosphorylation and imprints T cell activation upon antigen engagement. In contrast, the synapse formed by the CAR encoding 4-1BB recruits the THEMIS-SHP1 phosphatase complex that attenuates CAR-CD3ζ endodomain phosphorylation and T cell activation. We have also proved that the CAR synapse can be engineered to tune down the activity of CD28 costimulation or to tune up the activity of the 4-1BB costimulation. This discovery has been recently published in Cancer Cell. Remarkably, we observed that LCK mediated constitutive phosphorylation of CAR-CD3ζ in 4-1BB- costimulated CAR-Ts does not lead to premature exhaustion of CAR-Ts in xenotransplant models. Therefore, we hypothesize that the LCK-mediated imprinting in 4-1BB costimulated CAR-Ts leads to unique and critical signaling pathways in CAR-Ts. Furthermore, in addition to proximal signaling, CARs profoundly affect downstream T cell signaling. We found that NF-κB activity is influenced by the type of CAR costimulation. Precisely, 4-1BB induces more pronounced NF-κB activity than CD28 in CAR-Ts. NF-κB hyperactivity in 4-1BB is not caused by NF-κB overexpression, but rather by reduced A20 activity. Therefore, we hypothesize that 4-1BB sequesters A20 reducing its inhibitory effects on NF-κB. Furthermore, since NF-κB/A20 interplay is critical in controlling T cell function at multiple levels, we hypothesize that regulating NF-κB/A20 may enhance efficacy, persistence and safety of CAR-Ts. We will develop two specific aims: Aim 1: To mechanistically assess how LCK-mediated imprinting of 4-1BB costimulated CAR-Ts promotes rapid antitumor activity without causing T cell exhaustion. We will assess whether LCK overexpression in the 4-1BB CAR activates unique phosphorylation, transcriptome and metabolic pathways. Aim 2: To mechanistically assess how 4-1BB affect and how the NF-κB/A20 interplay can be manipulated to modulate CAR-T cell functions. Since A20/NF-κB interplay is differentially regulated in 4-1BB vs. CD28 costimulated CAR-Ts, we propose to understand how this interplay functions and to develop pharmacologic and genetic interventions to transiently or permanently modulate NF-κB activity to enhance safety, persistence and efficacy of CAR-Ts.

NIH Research Projects · FY 2025 · 2020-12

ABSTRACT Adeno-associated virus (AAV) vectors occupy a prominent role in recent CNS clinical trials particularly with respect to the use of AAV serotype 9 (AAV9) for single gene genetic disorders, such as spinal muscular atrophy and giant axon neuropathy (Mendell et al., 2018; Bailey et al., 2018). Given the need for selective cellular transduction, capsid modification, cell specific promoters and cell specific enhancers all have demonstrated success in achieving specific vector properties (Asokan et al., 2012; Dimidschstein et al., 2016; Grimm and Buning, 2017). In a recently accepted manuscript, we established a previously unknown interaction between the AAV9 capsid and different constitutive promoters, namely the ability to directly influence cell specific gene expression in the CNS. Using identical transgenes and the AAV9 capsid, CBA promoter driven gene expression exhibited a dominant neuronal gene expression in the rat striatum, but when gene expression was driven by the truncated Cbh promoter, gene expression was significantly shifted to striatal oligodendrocytes. Moreover, an AAV9 chimera containing six glutamate insertions after amino acid 139 in VP1/2 exhibited oligodendrocyte gene expression for both CBA and Cbh driven gene expression while a six alanine insertion in the same site reversed the Cbh driven gene expression back to neurons. Recently, preliminary findings revealed a similar AAV9 capsid interaction with the JetI synthetic promoter that influenced cellular gene expression in vivo. Given the highly novel nature of this capsid-promoter interaction, in vitro studies will define the mechanisms that underlie the glutamate and alanine shifts in cellular gene expression, including capsid conformation, intracellular trafficking, RNA splicing and VP1,2,3 interactions. In vivo studies will assess those promoter elements that contribute to the interactions, and specific AAV9 capsid elements that influence changes in in vivo cellular gene expression. Given the numerous applications of AAV9 vectors, the findings should significantly advance our understanding of basic capsid-promoter interactions and prove crucial to future design of AAV9 based gene therapies.

NIH Research Projects · FY 2025 · 2020-12

Idiopathic pulmonary fibrosis (IPF) is a progressive and end-stage lung disease of unknown etiology and no cure. It is likely that genetic changes increase a person's risk of developing IPF, and then exposure to certain environmental factors and/or aging trigger the onset of the disease. The MUC5B promoter variant rs35705950 is present in ∼50% of individuals with IPF and is recognized as the strongest known risk factor (genetic and otherwise) for the development of IPF. This variant leads to overexpression of MUC5B mRNA and protein in both distal airway epithelia and honeycomb cysts in the peripheral lung. These observations raise the question of why excessive MUC5B expression in distal airways is associated with IPF? Recently, our laboratory identified for the first time that the ER stress transcription factor XBP1S is highly expressed in the epithelium lining the distal airways and honeycomb cysts of IPF lung, activates MUC5B gene expression by direct binding to its promoter. Further, XBP1S differentially regulating the MUC5B promoter variant. These data connected activation of ER stress with excessive MUC5B expression in a promoter variant-dependent model likely impairs mucociliary clearance and function of distal airway stem cells that increase susceptibility of development of pulmonary fibrosis. We hypothesize that activation of XBP1S induces MUC5B expression in the distal airways that promotes pulmonary fibrosis. To test this central hypothesis, we propose the following aims: 1) XBP1S- mediated MUC5B secretion in distal airway epithelium enhances susceptibility to development of pulmonary fibrosis in vivo. We will assess the role of XBP1S by exposing Xbp1 airway epithelium-specific overexpression or deletion mice to bleomycin induced respiratory epithelial injury. 2) XBP1S-mediated MUC5B secretion impairs distal airway stem cell function to repair peripheral lung epithelia after injury. We will utilize mouse airway epithelium-lineage tracing system and in vitro 2-D and 3-D differentiation assays to evaluate the role of XBP1S and mucin in maintenance of the airway stem cell homeostasis in response to injury. 3) Activation of XBP1S and presence of MUC5B promoter variant cause abnormal mucus secretion, impaired mucociliary clearance and activation of myofibroblast differentiation. We will analyze the biochemical and biophysical properties of the secreted mucus and electrophysiology of the DAE carrying the MUC5B promoter variant rs35705950 at baseline and after activation of ER stress. We will also test whether XBP1S-expressing DAE carrying the MUC5B promoter variant and/or hypoxia-induced epithelial injury directly promotes myofibroblast differentiation. Completing the aims proposed in this application will provide novel mechanisms underlying ER stress-mediated excessive mucin secretion by DAE promotes pulmonary fibrosis. These mechanistic studies will likely identify novel biomarkers for early diagnosis as well as therapeutic targets (e.g., suppression of XBP1S activation as a promising approach to resolve MUC5B hypersecretion, even for those carrying the MUC5B promoter variant) in the distal lung to prevent or reverse fibrotic disease progression.

NIH Research Projects · FY 2025 · 2020-12

Project Summary There is a paucity of information known about how the lung recovers from acute respiratory distress syndrome (ARDS) and pneumonia, and this knowledge gap has contributed to the continued high morbidity and mortality of these diseases. A subset of immune cells, Foxp3+ regulatory T lymphocytes (Tregs), is thought to be important in resolution in several experimental models of acute lung injury (ALI). Furthermore, Tregs are present in the lung of patients with ARDS, suggesting that they contribute to ARDS recovery. Tregs can arise from either thymic or extra-thymic origins. Extra-thymic, peripherally-induced Tregs (iTregs) are converted from naïve T cells and serve distinct and essential functions in controlling adaptive immunity to restrain immune cell infiltrates in the bronchial and bronchiolar walls. Furthermore, our published studies show that during the resolving phase of lung injury, Tregs expand in number and change their gene expression profiles compared to Tregs in uninjured control lungs. Treg transcriptome profiling identifies several genes that shine a light on novel functions of Tregs during ALI resolution. One transcript that is markedly upregulated 23-fold in Tregs isolated from resolving lung tissue is matrix metalloproteinase 12 (Mmp12). Importantly, mice lacking endogenous Tregs and repleted with Mmp12-/- Tregs by adoptive transfer have elevated inflammatory cells and less epithelial proliferation during resolution than mice repleted with Mmp12+/+ Tregs. The Treg transcriptome also suggests that downregulated expression of Treg transcripts Kdm6b and Sik1, both of which act to regulate gene transcription, may function to regulate Treg homing, retention, stability, and survival. The Aims seek to test the central hypothesis that Tregs are critical to resolution and that by optimizing Treg responses, ALI severity can be reduced, and resolution hastened. Aim 1 investigates the role of iTregs during resolution of ALI resolution. The hypothesis is that iTregs are required for optimal resolution of inflammation and lung repair after ALI induced by LPS, S. pneumoniae, and influenza A. The direct effects of Tregs and iTregs will be determined using in vitro co-cultures of Tregs and AT2 cells. Aim 2 determines the impact of Treg-expressed MMP12 during the resolution of ALI. The hypothesis is that Treg expression of MMP12 exerts multiple roles in orchestrating and facilitating the resolution of ALI. Substrates of Treg MMP12 will be identified in BAL fluid and lung tissue. Aim 3 determines if the differentially regulated transcripts, Kdm6b and Sik1, regulate Treg-promoted resolution of ALI. We will test the hypothesis that Tregs deficient in Kdm6b have decreased Foxp3 expression levels and delayed, inadequate resolution and that Tregs deficient in Sik1 have improved quality of resolution. We anticipate that the proposed studies will determine the roles iTregs play in ALI resolution and identify mechanisms by which Tregs mediate resolution and repair. Identifying and elucidating these mechanisms will allow the development of therapeutic approaches to minimize collateral tissue damage without adversely altering the beneficial response to injury.

NIH Research Projects · FY 2025 · 2020-12

Project Summary In this study we seek to understand how rare genetic variation in all protein coding genes (the exome) influences the risk of developing obsessive-compulsive disorder (OCD). OCD is of major public health importance owing to its profound personal and societal costs. Little is known for certain about its etiology, and treatment, detection and prevention strategies are not optimal or directed by knowledge of pathophysiology. In other psychiatric disorders (e.g., autism, intellectual disability, schizophrenia, ADHD), whole exome sequencing (WES) in large numbers of subjects has begun to deliver fundamental knowledge about genetic architecture, identify specific loci for biological follow-up and localize pathways altered in disease. We intend to realize these same advances for OCD by markedly increasing the worldwide number of OCD subjects with WES data, in a first step toward elucidating the fundamental biology of this condition. Three overlapping areas will be investigated in this project. First, we will produce WES data from 5,100 OCD subjects and 3,000 ancestry-matched controls, all from Sweden and Norway. Sequencing individuals from these countries provides the substantial advantage of knowing about co-morbid conditions. We will call rare genetic variation from the sequencing data. Second, we will combine these new data with existing WES data for ~1,400 OCD cases and ~8,000 controls. This will increase power to identify OCD risk genes, which we will do using a combination of existing and novel analytical methods. Third, we will further refine our understanding of the genetic architecture of OCD, focusing on the relationship of OCD risk to risk for other neurodevelopmental disorders, including tic disorders, autism, ADHD, schizophrenia and bipolar disorder. Combining WES data from multiple large studies will enhance power to identify shared loci and begin to identify loci with greater specificity for OCD. Overall, we believe this study will improve our understanding of genetic risk factors for OCD, with a view towards improving clinical outcomes and reducing chronicity and societal costs.

NIH Research Projects · FY 2025 · 2020-12

PROJECT SUMMARY/ABSTRACT In contemporary practice for people with HIV, type 2 diabetes mellitus (T2DM) has become an important comorbidity. T2DM is 1.5 times more common in people with HIV than the general population. Among those with T2DM, people with HIV have greater risk for weight gain, lower diet quality, and higher hemoglobin A1c. All of this puts people with HIV and T2DM at substantial risk for complications, including chronic kidney disease, cardiovascular disease, and premature mortality. Food insecurity, “lack of access to enough food for an active, healthy life”, is a major contributor to this risk. Food insecurity is 2 to 3 times more common among people with HIV than the general population. Food insecurity is associated with both worse T2DM control and more T2DM complications. Medically tailored meal home delivery programs relieve food insecurity for people with HIV. Medically tailored meals emerged to treat food insecurity among those with AIDS in the 1990's. Medically tailored meal programs deliver fully prepared meals, tailored by a registered dietitian to an individual's medical needs. Although HIV care has changed, medically tailored meal interventions for people with HIV have not kept pace. Most medically tailored meal programs do not provide the intensive lifestyle intervention needed to counter the health threats seen in modern HIV care. These threats include the metabolic effects of anti-retroviral medications, chronic inflammation, aging, and obesogenic environments. For these reasons, it is critical to test new models of medically tailored meal for people with HIV. Our research team has developed a medically tailored meal intervention that combines meal delivery with an evidence-based lifestyle intervention designed to improve weight loss and diabetes self-management. The goal for this project is to test whether this medically tailored meal intervention can lead to improvements in hemoglobin A1c, weight, and in patient-reported outcomes such as food insecurity, quality of life, and diabetes distress, compared with a standard medically tailored meal intervention. Thus, we propose a randomized comparative effectiveness trial to assess a community-based medically tailored meals intervention (n=200). It will be conducted among diverse participants with HIV and T2DM, referred for medically tailored meals. Adults with hemoglobin A1c between 7.0% and 12.0%, and BMI ≥ 25 kg/m2 (≥ 23 kg/m2 for those with Asian ancestry) will be enrolled and randomly assigned to intervention or standard medically tailored meals. The intervention group will receive meal delivery and intensive lifestyle intervention for 12 months, while the comparison group will receive meal delivery along with standard nutrition education for 12 months. Outcomes will be assessed at 6, 12, and 18 months. The primary outcome is hemoglobin A1c at 6 months. Secondary outcomes include weight, food security, quality of life, and diabetes distress.

NIH Research Projects · FY 2024 · 2020-09

Project Summary/Abstract The ability to seek out situations that elicit pleasure and avoid those that lead to aversion or discomfort is a fundamental ability we all share, yet dysfunctional hedonic processing is prevalent in numerous psychiatric illnesses including substance use disorders (SUDs), alcohol abuse, and depression. As such, it is critical to understand the basic neural mechanisms underlying aversive affective states to ultimately apply treatment strategies to restore normal hedonic processing and aid in the recovery of maladaptive behaviors in mental illness. Here, we seek to understand the role of ‘top-down’ medial prefrontal cortex (mPFC) to nucleus accumbens (NAc) processing of innate and conditioned negative affective processing, incorporating electrophysiology, optogenetics, and a rat transcranial alternating current stimulation (tACS) method we developed. We focus on one type of behavior (taste reactivity, TR), measured in innate (unconditioned) situations and during conditioned taste aversion (CTA) and its extinction (restoration of positive affect). Notably, TR has exceptional translational value since it is preserved across species with similar behaviors present in rats, nonhuman primates and humans. Using electrophysiology, we will first determine how oscillatory, coordinated rhythms in mPFC and NAc circuitry are linked to affective processing in real time in the naïve state, how this signaling shifts when the sweet becomes devalued through CTA, and when it is restored in extinction. Since human and animal studies indicate that strengthening the mPFC can reduce negative affect we will then determine if strengthening these circuits using two distinct approaches can restore positive affect and associated neural function. We will use optogenetics (channelrhodopsin) to determine if targeted optical strengthening of infralimbic (IL)-NAc shell and/or prelimbic (PrL)-NAc core is sufficient to reduce negative affect and enhance CTA extinction. We will also examine if our novel rat transcranial alternating current (tACS) system, a relatively noninvasive approach with great translational value, can also modulate disrupted cortical oscillations in CTA, strengthen overall mPFC-NAc circuit coherence, and determine if this approach can increase positive affect. In both optogenetics and tACS studies, we will focus on 20 Hz (beta) frequency given its role in ‘top-down’ cognitive control as well as 80 Hz (gamma) frequency given studies that implicate this signaling in reward processing. Collectively, this multi-faceted approach will provide important insight into how mPFC-NAc systems modulate normal hedonic processing, how these systems are disrupted as negative affective states emerge and provide the foundation for the ultimate goal of developing methods to restore aberrant circuit function and hasten recovery from negative emotional states.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT More than 40 million Americans seek emergency care after traumatic stress exposure (TSE) each year. A substantial proportion of these individuals are at risk for chronic pain development and comorbid opioid use/misuse. Those at high risk of developing chronic pain can easily be identified (e.g., by severity of acute pain), creating tens of millions of opportunities each year for the prevention of chronic pain and associated opioid use/misuse. However, no interventions exist that prevent chronic pain development in those at high risk. In fact, current medications either have no effect on risk or actually increase risk. Therapeutic targets for post-TSE pain have historically focused on tissue injury-related pain generators, but increasing evidence suggests that physiologic systems involved in the stress response play a critical role in chronic pain development after TSE, opening up an exciting new landscape of potential therapeutic targets. Within this landscape, no target appears more promising than FK506-binding protein 51 (FKBP51), an intracellular protein known to affect glucocorticoid negative feedback inhibition. The investigators’ data demonstrate that FKBP51 inhibition reverses hyperalgesia after TSE, and suggest that FKBP51 inhibition after TSE can prevent enduring stress-induced hyperalgesia (ESIH). The investigators’ data further demonstrate that the effects of FKBP51 inhibition on ESIH after TSE are time, dosing, and duration-dependent. Available literature indicate that increased FKBP51 levels are associated not only with chronic pain after TSE, but also with other post-traumatic neuropsychiatric disorders often comorbid with chronic pain and opioid use/abuse, including posttraumatic stress, depression, and anxiety. Importantly, preliminary data from the investigative team indicate that FKBP51 inhibition does not have adverse cardiac effects or other adverse health or behavioral effects. Building on these data, the investigative team will perform the next critical steps in evaluating FKBP51 as a therapeutic target, including (1) evaluating the influence of dose, timing, and duration of FKBP51 inhibition after TSE on ESIH development, (2) assessing candidate mechanisms mediating the preventive effect of FKBP51 inhibition on chronic pain development, and (3) performing extensive testing of safety and addiction liability. All experiments will be performed across laboratories, animal models, pain measures, animal species, and animal sex and age, by a multidisciplinary team of experts in human and animal ESIH, animal behavior and addiction studies, FKBP51 biology, and FKBP51 inhibition. Completion of these experiments will substantively advance understanding of a target with exciting potential to prevent chronic pain and opioid use/misuse in millions of Americans who seek care in the early aftermath of traumatic stress exposures such as motor vehicle collision, sexual assault, and physical assault each year.

NIH Research Projects · FY 2024 · 2020-09

Abstract. The UNC Center for Excellence in SARS-CoV2 Serologic Research uses basic and applied research strategies to improve our understanding of the molecular and cellular mechanisms driving serological and humoral immune responses after SARS-CoV2 infection. Our overall goals are to 1) characterize the immune responses elicited to SARS-CoV2 infection, 2) understand the mechanisms driving the serological, humoral and cellular immune responses, 3) determine modifiers of the serologic memory and 4) determine the serological correlates of disease pathogenesis, and protection against future infection. The program includes three Research Projects led by internationally renowned exerts in coronavirus emergence, pathogenesis and immunity (Project 1: Baric), clinical and translational mucosal and systemic immune correlates of disease (Project 2: Bartelt & Margolis) and host-pathogen interactions driving innate and serological immunity (Project 3: Wallet & Maile). Program-wide support is provided by an Administrative Core A and two Shared Resource Cores B and C. Core A includes a robust infrastructure for programmatic oversight as well as participant recruitment, sample collection, tracking and sharing (Core A: Baric & Wallet). Core B is led by world renowned experts in characterization of human antibodies in protection and pathogenesis of disease (Core B: de Silva & Lakshmanane) and will provide recombinant spike protein antigens from SARS-CoV-2 as well as antigen- specific serological assays required for accomplishing the aims of all three Research Projects. Core C is led by serological experts (Core C: Ippolitto, Georgiou & Lavinder) who have revolutionized techniques to comprehensively analyze the molecular composition of the serological antibody repertoire (IgG and IgA) and the cellular antibody repertoire (i.e. B cell receptor) and thus will delineate these repertoires in and isolate human monoclonal antibodies from SARS-CoV-2+ individuals in cohorts defined in each Research Project. All three Research Projects are integrated, and each require the support of all three Cores. To this end, Project 1 will characterize the breadth and potency of polyclonal neutralizing antibody responses as well as determine the kinetics, magnitude and durability of the type-specific and cross neutralizing responses in both the systemic and mucosal compartments. Project 2 will determine the durability and the breadth of anti-SARS-CoV-2 serum antibodies and memory B-cells generated among convalescent plasma donors as well as determine the effect of convalescent plasma on the innate, adaptive and antibody repertoire in recipients. Project 3 will reveal innate immune signatures as a function of serology across the span of natural disease, as well as identify signatures which promote development of protective vs. pathogenic antibody repertoires, while delineating mechanisms of antibody mediated activation and suppression of innate immune function which drives severe vs. mild disease respectively. The integrated expertise of our Team is necessary and sufficient to address the novel cross-cutting hypotheses put forth which will improve our understanding of SARS-CoV2 serological and humoral immunity.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY The biracial population is the second-fastest growing demographic group in the US. Biracial adolescents also have higher risks of substance use as well as violent behavior, school problems, and poor physical and mental health than many of their monoracial peers. However, little is known about substance use prevention and interventions in this population. Historically, biracial youth have been either ignored in research or their many subgroups have been combined into a single “multiracial” category, potentially obscuring clinically relevant patterns. Moreover, no accepted model explains the factors that increase or decrease the risk of substance use among biracial youth. Discoveries made during the course of the proposed research will help accelerate the refinement of existing prevention and intervention programs for biracial adolescents and emerging adults, and will speed translation of its findings into public health practice. We propose to study the 4 subgroups of biracial youth that our prior research has shown to have the highest risk of substance use, namely biracial White-American Indian, White-Asian, White-Black, and White- Hispanic youth. In doing so, we will also test a newly developed model, the Double Jeopardy Hypothesis, that we propose to explain biracial substance use patterns. According to this model, biracial individuals experience not only the common risk factors for substance use, which are also experienced by monoracial youth but also a second set of risks unique to being biracial in America. This study takes advantage of existing data from two large, longitudinal and nationally representative databases that include adequate numbers of biracial persons to allow the sample to be divisible into subgroups, as well as multiple measures of social determinants of health (e.g., perceived discrimination, racial socialization), substance use, and other behavioral and physical outcomes. The first, Monitoring the Future, followed students from middle-/high-school through age 55 years. The second, the National Longitudinal Study of Adolescent and Adult Health, followed students from age 11 to 42 years. After integrating the two datasets using integrative data analysis to study adolescents and emerging adults ages 13-25 years old (Aim 1), we will determine the onset, prevalence, and developmental trajectories of substance use (i.e., cigarette, alcohol, marijuana, and polydrug use) from adolescence to emerging adulthood (ages 13-25) (Aim 2). Last, we will explore the relationships of common and unique risk and protective factors (in the individual, family, peer, school, and community domains) for substance use among biracial adolescents and emerging adults, examining evidence for the proposed Double Jeopardy Hypothesis (Aim 3). Findings will inform more effective and inclusive prevention approaches for an understudied but rapidly growing sector. If accurate, the Double Jeopardy Hypothesis will provide insight into the lived experience of biracial adolescents and emerging adults, forming a framework for future research on a range of outcomes.

NIH Research Projects · FY 2024 · 2020-09

Project Abstract Per- and polyfluoroalkyl substances (PFAS) are a family of over 5000 man-made chemicals that are ubiquitous in the environment, due to their chemical stability and bioaccumulative properties. Many of these “forever chemicals” have been linked with health concerns, including strong evidence of developmental health and harm to hormone-sensitive tissues. Manufacturers continue to substitute new PFAS for which exposure- based health risks are unknown. There is an urgent public health need to determine the effects of PFAS in use on both mammary gland development and increased breast cancer incidence. Current exposure studies use rodent models that require cumbersome end-point analyses as well as large monetary and time investments. Our proposal is aimed at developing an in vitro to in vivo extrapolation (IVIVE) pipeline of mammary gland development and maintenance to identify and prioritize potentially toxic PFAS, to ultimately mitigate number of animals needed for environmental exposure studies. Our approach is to develop in vitro models of the mammary gland of increasing complexity but decreasing throughput, identifying links between high-throughput and high- complexity model endpoint readouts to best prioritize large chemical libraries. A key technology to establish links across multiple in vitro culture platforms is optical coherence tomography-based structural-functional imaging (OCT-SFI), developed by MPI Oldenburg, which non-invasively visualizes label-free cells, their intracellular motility, and morphology of formed spheroids, within optically turbid tissue models. Our first specific aim advances a high-throughput paper-based culture system, developed by MPI Lockett, to study mammary epithelial cell invasion in physiologically relevant tissue microenvironments. The platform will evaluate 96 different exposure conditions in parallel. Our second specific aim employs 3D co-culture models that include fibroblasts to model stromal signaling known to affect mammary gland development. OCT-SFI will provide cellular motility and morphology of the organotypic spheroids that form in these cultures. Finally, our third aim will screen a library of 40 PFAS, with a particular focus on the perfluoroethercarboxylic acids (PFECAs) currently used in industrial coatings. In addition, 12 PFAS will be screened for which there is existing in vivo rodent model data available, and comparisons between in vitro assay outputs and in vivo gland remodeling will be used to refine the assay models and establish initial thresholds for screening. The models developed as part of this proposal will thus be predictive of biology, enabling the high-throughput capability needed for future screening of all PFAS as well as other emerging endocrine disruptors. The project’s risk is balanced by the known imaging capabilities of OCT-SFI to probe responses in 3D spheroid and paper- based co-cultures. The high-throughput nature of this IVIVE pipeline makes it ideal for screening libraries of potential toxicants, providing information-rich datasets of spatially and temporally resolved morphological and molecular changes across the tissue-like structures.

NIH Research Projects · FY 2024 · 2020-09

Abstract: The 2019 nCoV (SARS-CoV2 or nCoV2) is currently causing a global pandemic, and is on track to cause millions of infections, hundreds of thousands of deaths, and significantly disrupt healthcare systems and economies globally. nCoV2 is a group 2B coronavirus that is 75% identical to Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), which emerged in 2003. Approximately 10% of nCoV2 infections result in COVID-19 pneumonia that progresses to acute respiratory distress syndrome (ARDS), while a significant fraction of other individuals are asymptomatic or develop mild disease. While age, gender, and underlying health conditions predispose individuals to severe disease/death, we have a poor understanding of the factors that drive disease outcome. This knowledge is essential for understanding the pathogenesis of COVID-19, and for developing and testing safe and effective nCoV vaccines and therapies. However, while patient studies can provide insights into the disease risk factors, mechanistic analysis of these factors will require robust animal models of COVID-19 disease. Unfortunately, nCoV does not replicate in standard laboratory mice, and a significant need exists for new animal models that reproduce human-like COVID-19 disease, including ARDS. Collaborative Cross (CC) mice vary significantly in their response to SARS-CoV, and we were able to take advantage of this variation both to develop new models SARS-CoV-induced disease, while also identifying host genetic factors that regulate disease outcome. Based on this experience, we propose take advantage of a new mouse adapted SARS-CoV2 virus (maCoV2), which was recently developed in the Baric laboratory, to screen a panel of CC mouse strains for susceptibility to maCoV2-induced disease. This work will accomplish two critical research objectives by: 1) developing critically needed mouse models of nCoV2-induced disease, and 2) identifying polymorphic host genes/pathways that regulate resistance or susceptibility to nCoV2-disease.

NIH Research Projects · FY 2024 · 2020-09

SUMMARY In 2019, ~100 million Americans were obese, fueling increases in obesity-related morbidity, mortality, and health care costs, largely from cardiometabolic diseases (CMD). Large scale genetic studies have laid the foundation for many downstream investigations into the pathogenesis of disease and the translation of this information into public health applications. Over the last decade genome-wide association studies (GWAS) have substantially improved our understanding of the genetic architecture of obesity related traits. The potential of these study findings cannot be overstated for elucidating the biological or pathophysiological underpinnings of obesity and its costly morbidities. Although GWAS on common variants have made strides in identifying > 1,000 signals for obesity related traits, these studies are inherently limited without further translation into more actionable findings. In this proposal, we will narrow association signals and map causal genes and pathways underlying known obesity risk loci by applying innovative methods to integrate multiple OMICs (genOMICs, epigenOMICs, transcriptOMICs and metabolOMICs). Additionally, we will explore the clinical relevance of obesity susceptibility variants, genes, and pathways in a large BioBank linked to electronic health records (EHR) to validate expected phenotypic associations and reveal novel phenotypic associations. Finally, we will conduct in vitro functional studies of key variants and genes in physiologically relevant cells to reveal putative regulatory mechanisms of variants and effects on metabolites and thus the underlying mechanisms critical to obesity pathogenesis. Thus, in this proposal we leverage collaborations in the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE), TransOMICs for Precision Medicine (TOPMed) Program, the Genome Sequencing Project (GSP), and the EHR database from the Geisinger MyCODE Community Health Initiative study (MyCode) to narrow in on genes underlying GWAS signals, perform clinical characterization, and conduct in vitro functional studies to characterize the molecular underpinnings and biological mechanisms of obesity-risk loci. Our approach will substantially move the field away from tag variants and loci to causal variants, genes, and mechanisms. We anticipate that this work will generate fundamental and important insights into the underlying etiology of obesity and ultimately point the way forward towards prevention and treatment.

NIH Research Projects · FY 2026 · 2020-09

ABSTRACT The goal of this competing renewal of R01AI157758 is to develop and disseminate principled approaches to answering important questions in HIV research by combining (or fusing) data from multiple HIV trials and/or observational studies. While meta-analyses combine similar studies to improve precision, fusion analyses combine dissimilar studies to improve accuracy (validity and precision) or address questions that single data sources cannot. The scientific premise of this research is that results from both randomized trials and observational studies in HIV can be made more accurate and robust by combining multiple data sources and leveraging cutting-edge methods in epidemiology, statistics, and causal inference. The specific aims span the leveraging of enriched internal data, the combination of external data sources, and the answering of questions unanswerable with single data sources. Specifically, the aims are as follows. 1) Develop fusion study designs and analytic methods that improve accuracy by leveraging enriched internal study data on subsets of participants in HIV trials and observational studies. 2) Develop fusion study designs and analytic methods to improve accuracy by combining external study data from multiple HIV trials and observational studies. 3) Develop fusion study designs and analytic methods that answer new questions by combining data from multiple HIV trials and observational studies. For each aim, important examples from HIV have been selected to illustrate the novel analytic approaches. Estimators will be developed using modern statistical theory, tested with extensive simulation experiments, and illustrated by addressing timely questions in HIV research. We will explore the use of nonparametric ensemble machine learning for complex nuisance functions, which relax assumptions about model form, making results more robust to model specification. The assembled team features field-leading expertise in epidemiology, statistics, and HIV medicine. Products will include a series of scientific papers, presentations, and reproducible open-source software in multiple programming languages to facilitate dissemination (e.g., R, SAS, Python). The impact of the proposed work will be more accurate and robust inferences in HIV research with broad applicability to many other fields.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Giardia lamblia belongs to a group of gut pathogens associated with impaired child development and gut function, especially in children with inadequate nutrition. Although most Giardia infections in these children are clinically silent, Giardia may lead to longterm detriments. For reasons that are unknown, many of these children cannot clear the parasite. Further, infected children may be at risk for gut dysfunction as a result of Giardia infection, even if they are asymptomatic. We hypothesize that susceptibility to Giardia and subsequent disease result from a change in the resident intestinal microbial community as a consequence of low dietary protein. Animal models provide an opportunity to dissect how an individual pathogen like Giardia impacts early life intestinal health, and a model to understand mechanisms whereby specific nutrients support host defenses and physiology. The objective of this proposal is to use our novel mouse models of Giardia infection in a state-of-the-art environment where we can define and control for all microbial exposures in the gut (gnotobiotics). We will combine our gnotobiotics expertise with expertise in microbial metagenomics and metabolomics for a rigorous examination of how dietary influences exert a functional change in the complex community or resident intestinal microbes. In Aim 1, we will determine how resident intestinal microbes normally protect against persistent Giardia infection by transferring intestinal microbes from a well-nourished animal into susceptible hosts, and vice versa transferring permissive microbes from hosts with chronic infection into nourished mice. We will specifically examine whether the ability of microbes to metabolize bile acids are key to protection against Giardia infection, and whether microbes are necessary for effective immunity. In Aim 2, we will determine how Giardia and protein deficiency synergize to cause intestinal barrier dysfunction. Our model has a clinically relevant outcome of growth restriction and loss of intestinal barrier function during the combined insult of limited protein intake and Giardia infection. We will use our gnotobiotic model to generate metagenomic and metabolomic data that will identify pathogenic shifts in microbial communities during Giardia infection. We will specifically elucidate the role of aromatic amino acid metabolites and bile acids on gut function in the protein deficient state, as well as whether Giardia promotes pathogenic bacterial functions to cause enteropathy. Our gnotobiotic approach is innovative and will allow us to characterize critical interactions between resident intestinal bacteria and infection with Giardia that have not previously been elucidated. The proposed research will address longstanding questions related to the role of Giardia on gut function, and specifically whether Giardia exerts pathogenesis by altering microbial metabolism. Beyond Giardia pathogenesis, these results are expected to lead to new considerations for how healthy bacteria provide protection against gut infection, and how chronic enteropathogen exposures cause gut dysfunction absent diarrhea. We hope to discover new pathways that might leverage emerging microbial or molecular-based therapeutics to improve health of children vulnerable to malnutrition and intestinal infections.

NIH Research Projects · FY 2024 · 2020-09

Project Summary Early toxic stress can lead to enduring long-term effects on neurodevelopment and behavioral outcomes in children. One mechanism that may mediate these associations is the gut microbiome. There is robust evidence that patterns of gut microbiota may influence neurodevelopment and anxiety-related behaviors in rodents, but there is sparse literature on this association in humans. Recent findings from our research team are the first to reveal that the gut microbiome significantly predicts fear behavior in 1-year-old children. Thus, the proposed study will examine the influence of psychosocial stress on the development of gut microbiota, mediated by chronic HPA axis activation, as well as the bidirectional relationship between the developing microbiome and behavioral inhibition across the first four years of life. We will measure alterations in brain development across this time as a mediator of the relationship between the microbiome and behavioral inhibition. During this sensitive period the microbiome and brain are rapidly developing and may be most susceptible to environmental input. Participants (n = 200) will be drawn from a prospective longitudinal cohort study funded by NICHD (Brain and Early Experience Study (BEE); R01 HD091148-01A1). Assessments will be conducted during lab visits at 36 and 54 months and home visits at 6 and 24 months of age. Psychosocial stress will be assessed via observational assessments of negative parenting behavior and household chaos at 6 and 24 months, and HPA axis activation will be measured via hair cortisol at these visits. At all ages, fecal samples will be collected to assess microbial diversity and maturity and behavioral inhibition will be assessed via maternal report and observational measurement. Neuroimaging using high resolution magnetic resonance imaging, diffusion tensor imaging, and resting state fMRI will provide measures of volumetric growth of the mPFC and hippocampus from 15 months to 54 months, and structural and functional connectivity between the amygdala and mPFC. Finally, at 54 mos a brief structured diagnostic interview for major pediatric psychiatric disorders will be conducted (Mini International Neuropsychiatric Interview). This study will be the first to investigate the influence of early psychosocial stress on the gut microbiome, neurodevelopment and anxiety related behavior. Our long-term goal is to determine how colonization of the gut microbiome impacts human brain development and later risk for psychopathology in order to prevent the onset of psychiatric illness or reduce its severity. Findings from this study, during a sensitive period of early childhood, will provide important information that could inform intervention and prevention efforts soon after birth.