Duke University

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT Plasticity mechanisms endow the brain with immense capacity to adapt to a wide range of experience and exposures. Protein synthesis, including locally at synapses, is a requirement for many forms of synaptic plasticity. The Integrated Stress Response, ISR, is a highly conserved biochemical pathway that regulates protein synthesis. The ISR markedly shifts which proteins are made by phosphorylating the initiation factor, eIF2alpha. The ISR was named for its effects body-wide - in which it provides a cell stress response mechanism. However, in the brain, the ISR has also been found to be a potent modifier of synaptic plasticity, learning and memory. At a high level of summary, ISR inhibition in the normal brain has been shown to lower the thresholds of experience needed to instantiate long-lasting memory and in some disease settings, such as traumatic brain injury, it rescues cognitive behavioral deficits. Mechanistically, ISR-inhibiting manipulations have been associated with long-lasting potentiation (LTP); while ISR activation is necessary for forms of synaptic depression (LTD). While trying to understand how the ISR contributes to diseases like dementia and dystonia, we recognized major gaps in the basic understanding of brain ISR actions, including when and where it was normally activated. We therefore developed a brain-wide viral reporter, SPOTlight, that gives a two-color readout for ISR activation state. Using SPOTlight, we uncovered a wholly non-canonical modality for the ISR in the brain – involving its constitutive activation in a class of neurons (striatal cholinergic interneurons) where it influences dopaminergic modulation of their firing response. Additionally, cell autonomous ISR inhibition in these cells recapitulated previous “learning enhancement” effects observed with systemic manipulations. These findings either upend, or at least substantially add to, working models for the ISR in plasticity, learning and memory. Here, we propose to advance understanding of how the ISR acts in the brain for neuromodulation, synaptic plasticity, learning and memory. We will focus on 3 knowledge gaps: (1) To what extent does ISR action in neuromodulatory cells, instead of at local synapses undergoing plasticity, explain ISR effects on synaptic plasticity and behavior? (2) What are the molecular mechanisms by which the ISR changes dopamine (D2R) signal transduction outcomes in cholinergic neurons? and (3) Can new ISR reporters be developed with the spatiotemporal resolution needed to resolve when and where the ISR activates protein synthesis under synaptic plasticity conditions? We expect the outcomes of this work to impact both general cell biological principles and specific striatal mechanisms and to have translational relevance for ISR-targeting therapeutic efforts that are underway.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT In children, asthma is a leading cause of respiratory symptoms and missed school. By mechanisms that are unclear, obesity in children with asthma is a strong risk factor for asthma that is more symptomatic and difficult to manage. Among asthmatic children, obesity is associated with a reduced response to conventional anti-inflammatory asthma drugs. Therefore, a better understanding of the underlying mechanisms of obesity-related asthma in children is needed. Discovering the mechanisms of `obese asthma' in order to foster more appropriate phenotype-specific therapies is a pressing public health need. Obesity, regardless of underlying asthma, is known to impair respiratory system compliance and inspiratory muscle efficiency leading to increases in the effort of respiration. Our lab found in asthmatic children that obesity is associated with more frequent asthma symptoms and a higher likelihood of having dyspnea. Inspiratory training and other forms of rehabilitation are common in adults but is understudied in pediatrics. Inspiratory `rehabilitation' training (IT) recently has been shown in both children and adults to reduce dyspnea and asthma symptoms. Our research team found IT over 6 weeks was well-tolerated, and led to improved inspiratory function and consistently improved trends in clinical measures, among children with asthma. The central objective of our proposed MICA (Mechanistic Study of Inspiratory Training in Childhood Asthma) study is to measure the effects of 8-weeks of IT on inspiratory function (strength and endurance) and small airway dysfunction measured by oscillometry in 6-17 year olds with asthma - with and without obesity. Our central hypothesis is that obesity promotes inspiratory muscle fatigue and small airway dysfunction, and that IT will mitigate these mechanisms. In a masked parallel arm trial, 75 youths with asthma (half with obesity) will be randomized to either IT (low or high dose) or SHAM control for 8-weeks. Inspiratory strength and endurance, small airway dysfunction (SAD), and exercise capacity will be assessed at baseline and after IT. Aim 1 will describe the contributions of inspiratory fatigue and SAD to obesity-related asthma. Aim 2 will determine the effects of IT on inspiratory muscle fatigue. Aim 3 will determine the effects of IT on small airway dysfunction. The MICA study will provide mechanistic knowledge about pediatric `obese asthma' by comparing treatment responses by obesity status. Measuring the changes in inspiratory muscle function and small airways dysfunction following a short regimen IT in children with symptomatic obesity-related asthma will provide much needed understanding about mechanisms underlying how obesity worsens asthma in children; and will uncover mechanisms and optimal dosing intensity involved in a safe non- drug intervention that will pave the way for larger phase III trials.

NIH Research Projects · FY 2025 · 2023-07

Summary Dry age-related macular degeneration (AMD) is the leading cause of vision loss in the Western World with a complex etiology. The fundamental abnormalities occurring in retinal pigment epithelial (RPE) resulting in their progressive dysfunction and subsequent atrophy in AMD, are still not known. However, candidate pathogenic pathways linked to the development of disease have emerged from the convergence of a sundry of epidemiological, genetic, morphological, and biochemical studies, including inflammation, lipid dysregulation, apoptosis, and RPE barrier dysfunction among others. Currently there are no drugs available to treat dry AMD. In this proposal we concentrate on investigating the biology and function of osteopontin (OPN), a matricellular glycoprotein known for its involvement in the pathogenesis of a variety of neurodegenerative and systemic diseases, in which inflammation is key, in part through its role as a macrophage recruitment and retention factor. However, OPN reportedly plays a two-sided role having both pro- and anti-inflammatory properties. Furthermore, OPN has also been shown to regulate cellular homeostasis effecting cell differentiation, metabolism, autophagy, phagocytosis, and apoptosis to name a few. This potential paradox in the role of OPN may in part be due to the fact that (1) OPN function is cell and tissue specific, and (2) most studies have not delineated the roles of the different OPN isoforms, which include intracellular versus secreted OPN and splice variants OPNa, b, and c. With this in mind, we propose to systematically investigate the role of OPN in AMD vulnerable cells with a focus on RPE biology and test the therapeutic potential of modifying OPN levels in animal models that present with dry ‘AMD-like’ pathologies. Published studies and key preliminary observations that precipitated pursuing this study include (1) circulating soluble OPN (sOPN) levels increase in a subpopulation of individuals with age; (2) the role of intracellular OPN, is less known but associated with amelioration of inflammation; (3) intracellular OPN expression in human RPE cells is decreased in early dry AMD donor tissue; (4) secreted OPN accumulates extracellularly in drusen and basal deposits of human AMD donor tissue; (5) plasma OPN is elevated in late dry AMD patients; and (6) human RPE cells secrete OPN following oxidant injury and are a potential local source. Based on our preliminary data and the known paradoxical anti- and pro-inflammatory roles of OPN in neurodegenerative and systemic diseases that share common pathogenic pathways with AMD, we will test the hypotheses that OPN regulates inflammation, aberrant RPE barrier function, and cell homeostatic mechanisms, in an isoform dependent manner.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Learning and performing complex skills such as speech or music requires precise control of motor variability. While elevated motor variability can spur the learning of new behaviors, excessive variability can impair performance of learned skills. How the brain controls motor variability during learning and in expert performance remains unclear. Intriguingly, the basal ganglia (BG) is an important source of motor variability in both health and disease, and is a key site where dopamine (DA) reinforces more successful behaviors. Indeed, the BG’s ability to regulate motor variability is especially critical for complex sequential skills such as speech, where variability can arise at both the level of elementary motor “syllables” and the sequential “syntax” in which these syllables are organized. How DA signaling in the BG influences motor variability during the learning of complex sequential skills akin to speech or music is poorly understood. Moreover, rather than acting alone, an emerging view is that DA signaling is strongly modulated by other signaling molecules, such as adenosine (Ado), which may track the metabolic costs associated with extensive motor practice. Here I will characterize how Ado and DA release in the BG are related to each other, to motor variability, and to the learning of vocal motor sequences. In direct service of BRAIN initiative goals, I will combine cutting-edge computational and optical tools along with an innovative molecular-genetic approach to dissect both neuromodulator and cell- type specific contributions to motor variability and learning. My Specific Aims are: 1) To image Ado and DA in the sBG during juvenile vocal learning. 2) To establish the necessity of sBG Ado to regulate variability and test for a direct link between Ado and DA release. 3) To genetically tag “indirect” and “direct” spiny neuron types and assess how Ado modulates their activity to influence song variability. Individually, each aim will move beyond a single-neuromodulator model of BG skill learning, and collectively they will help reveal fundamental mechanisms that control motor variability across learning and performance. I will conduct this research under the supervision of Drs. Richard Mooney, Josh Huang, and John Pearson, an interdisciplinary team of accomplished mentors that provides me with complementary expertise in behavioral, systems neuroscience, computational, and cutting-edge genetic techniques. In addition to my deep interest in understanding natural forms of behavioral learning, I bring my own expertise in analyzing behavior in concert with optical methods. This proposal will allow me to both deepen and broaden my expertise, and will provide significant training in novel behavioral, computational, genetic, and imaging techniques. This integrative approach to systems neuroscience and natural behavior will enhance my capabilities as an independent researcher while addressing BRAIN Initiative goals.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Congenital heart defects (CHDs) affect nearly 1% of live births in the United States. CHDs are associated with high morbidity and are the most common birth defect-related cause of death. Rapid advancement in the early diagnosis, medical management, and treatment of CHD have led to tremendous gains in survival; however, heart failure (HF) is the leading cause of death in adults with CHD. This makes identifying the risk factors of HF across the lifespan critical so that individuals at the highest risk can be identified and managed. We have brought together a multi-disciplinary team with significant experience in population-based CHD outcomes, social determinants of health, and cardiovascular genetics to fill these critical knowledge gaps. Since 2008, our team has led the North Carolina Congenital Heart Disease Surveillance Network (NC-CHD) which links the 5 major academic centers in North Carolina in a surveillance network for the vast majority of patients with CHD in the state. NC-CHD links clinical records to a variety of robust, state, and national databases to conduct outcomes-based research on survivors of CHD across the lifespan. This puts our team in a unique position to establish a prospectively enrolled cohort of CHD survivors with rich clinical phenotyping for comprehensive genetic and outcomes-based research to determine the causes of HF in this population. The goal of our study is to develop a large, well- curated, population-based cohort of individuals with CHD in the state of North Carolina (NC-DEFINE) to identify social determinants of health and genetic factors which influence CHD outcomes, specifically HF. We hypothesize that social determinants of health and rare genetic variants localizing to sarcomeric genes are associated with HF development among survivors of CHD. To test this hypothesis, we propose 3 specific aims: 1) To identify the social determinants of health associated with the development of HF in cohort of 600 patients with CHD which will comprise NC-DEFINE. 2) To determine the coding genetic variants associated with the development of HF among patients with CHD in NC-DEFINE. 3) To determine the functional impact of candidate variants associated with HF in CHD using patient-derived cardiac myocytes. If we are successful, this project will allow for identification of CHD patients at heightened risk of HF based on either social or genetic risk, allowing for informed counseling at the time of surgical repair and early intervention to control reversible risk factors associated with HF. Further, NC-DEFINE will lay the foundation for a genomic medicine-based approach to predicting CHD prognosis and outcomes.

NIH Research Projects · FY 2025 · 2023-07

Summary The PI3K/Akt signaling is one of most important oncogenic events in human cancers. It regulates many aspects of biological functions including cell proliferation, survival, and metabolism important for cancer initiation and progression. While extensive efforts have been made in the last three decades to understand the downstream effectors responsible for the biological and oncogenic processes regulated by PI3K/Akt signaling, the upstream signals mediating PI3K/Akt signaling activation upon diverse growth factor stimulation is not well understood. Understanding and defining the upstream mechanisms responsible for PI3K/Akt signaling activation will not only provide new insight into how PI3K/Akt signaling activation is orchestrated, but also offer novel paradigms and therapeutic targets for cancer intervention. Although it has been well established that PIP3 is critical for the membrane recruitment and subsequent activation of Akt, our recent studies provide the evidence that Akt undergoes methylation and subsequent non-proteolytic K63-linked ubiquitination, which are crucial for Akt membrane recruitment and subsequent phosphorylation and activation upon stimulation with diverse growth factors, opening up a new frontier for Akt signaling regulation. Of note, we identified SETDB1 as a methyltransferase for Akt K64 methylation and TRAF6 ligase as an upstream E3 ligase triggering K63-linked ubiquitination and activation of Akt, and these events are required for cancer progression. However, the outstanding questions remained to be addressed are how SETDB1 and TRAF6 are activated or recruited to the Akt complex upon growth factor treatment to trigger Akt methylation and subsequent Akt ubiquitination and activation, thus promoting oncogenic processes. The goal of this study is to dissect the upstream regulatory mechanisms by which growth factors activate and recruit SETDB1 and TRAF6 ligase to Akt complex to elicit Akt methylation and subsequent Akt ubiquitination, define the mechanism by which Akt ubiquitination facilitates Akt membrane localization and activation, and finally explore the role of these regulatory modes in cancer development and develop small molecule inhibitors targeting these regulatory mechanisms. Our preliminary results revealed that SETDB1 and TRAF6 undergo novel posttranslational modifications, which are crucial for methylation, ubiquitination and activation of Akt by growth factors and oncogenic activity. We hypothesized that SETDB1 and TRAF6 undergo the novel posttranslational modification upon growth factor treatment, which recruits SETDB1 to the Akt complex and activates TRAF6 E3 ligase to facilitate Akt methylation and subsequent Akt ubiquitination and activation, thus leading to tumorigenesis. Our innovative hypothesis has been formulated based on our preliminary results and prior research. We proposed three specific aims to validate this provocative and paradigm-shifting concept using cutting-edge technologies including xenograft, organoids from genetic mouse tumor models and patient derived tumors, patient-derived models (PDX), knockin mouse models, genetic mouse tumor models and pharmacological approaches. This application is significant, therefore, because it is expected to provide the knowledge needed to develop pharmacologic strategies that will allow concurring cancers with aberrant PI3K/Akt activation. Our study will open up a new avenue for PI3K/Akt signaling regulation, but also offer new concepts and strategies for cancer targeting.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Having a sense of belonging is vital to children’s and adults’ psychological health. In fact, lack of belonging is detrimental to children’s psychological health, causing acute and chronic distress and increasing risk for later anxiety/depressive symptoms and drug use. This fundamental need to belong can be undermined or strengthened by social experiences, such as gossiping (i.e., negative or positive talk about others). The effects of gossiping are well-studied in both adults and adolescents; members of both age groups who are associated with negative gossip feel less connected in their friend groups than those who are associated with positive gossip. Yet, no study to date has investigated the immediate and long-term effects of gossip on feelings of belonging at younger ages. The present proposal aims to address this gap. To understand the link between early childhood gossip and children’s social and psychological well-being, we aim to identify how spreading gossip or being the target of gossip impacts children’s feelings of belonging. The overall objectives will be achieved through two lab studies (Studies 1 and 2) and one naturalistic study (Study 3). Study 1 will use a novel experimental paradigm to identify the immediate effects of spreading gossip on children’s belonging and distress by testing the hypotheses that 1a) Children will feel closer to their conversation partners and feel less distressed if they gossip with them, and 1b) Children will feel closer to the peers who were the targets of positive gossip than peers who were the targets of negative gossip. Study 2 will use a similar experimental design to identify the immediate effects of being the target of gossip on children’s belonging and distress, by testing the hypothesis that 2) Children who are the targets of negative gossip will experience lower belonging and feel more distressed than the children who are the targets of positive gossip. In a novel analysis of an already collected observational social network dataset from classrooms, Study 3 will map long-term social network implications of children’s gossip over the course of an entire school year. Study 3 will test the hypotheses that 3a) Children who have higher connectedness (i.e., belonging) among friends will be less involved in spreading negative gossip and have lower likelihood of being a target of negative gossip, and 3b) Children who are more centrally connected to the friend group will be more involved in spreading positive gossip and have higher likelihood of being a target of positive gossip. The proposed research and training aims will identify the functions and impact of gossip on children’s feelings of belonging and distress and help the Candidate in furthering their career goals to lead their own lab as an independent PI. The findings can contribute to programs and policies to support healthy social development by increasing children’s feelings of belonging.

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT Angiogenesis is a central event in nervous system development that requires further elucidation within the context of the retina. In normal development, retinal angiogenesis initiates from the optic nerve and proceeds along the innermost layer of retina consisting of retinal nerve fibers and the developing astrocytic network. Astrocytic development occurs prior to angiogenesis and is critical for guiding the developing vasculature. Specialized endothelial cells called tip cells crawl along the astrocytes as the angiogenic wavefront advances, so that the vasculature matches the pattern of astrocyte arbors. It is well understood that astrocytes secrete VEGF-A in hypoxic zones to initiate angiogenesis, but the signals that advance the wavefront, and guide tip cells along the astrocyte template, are unknown. The objective of this proposed study is to identify novel astrocyte- derived molecular cues that guide retinal vascularization. The central hypothesis is that astrocytes provide molecular cues that promote angiogenesis by 1) enhancing tip cell production; and 2) guiding tip cell growth so that vasculature adopts the pattern of the astrocyte network. The rationale for this work is to provide a deeper understanding of glial-mediated regulation of angiogenesis that may be applicable to the entire nervous system while also providing candidate molecules to target in retinal pathologies such as retinopathy of prematurity (ROP). The specific aims are: 1) Identify astrocyte-derived cues driving progression of the angiogenic wavefront. Adrenomedullin (ADM) is a secreted peptide that was previously found to mediate angiogenesis via its receptor, calcitonin receptor-like receptor (CLR) in conjunction with receptor activity modifying protein, RAMP2, which are expressed by endothelial cells. Preliminary single-cell RNA-seq data shows that the adrenomedullin gene is highly expressed by immature astrocytes. To test this, its angiogenic properties will be tested utilizing a functional assay that consists of analyzing vasculature wavefront progression in cultured mouse retinal explants that shall be co-cultured with ADM-expressing HEK293 cells. 2) Identify astrocyte-derived cues that pattern growing vessels. Genes selectively expressed by astrocytes are candidates to guide tip cell growth and angiogenesis. To identify such molecules, each cell type in the nerve fiber layer will be purified for scRNA-seq. Astrocyte-specific genes encoding cell-surface or secreted molecules will be identified bioinformatically. As in Aim 1, a functional assay using retinal explants will be used to test angiogenic capabilities of putative tip cell-guiding astrocyte-specific genes. Completion of this work will be significant because it will reveal novel components and drivers of angiogenesis expressed preferentially by astrocytes in the retina. Such information will elucidate basic mechanisms of retinal angiogenesis, while also pinpointing important molecular cues that may be involved in angiogenesis during pathogenic conditions such as ROP.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract Patients with lethal castration-resistant prostate cancer (CRPC) are currently treated with agents targeting androgen receptor (AR) signaling. However, AR inhibition has not dramatically improved CRPC patient survival, underscoring the need to discover novel oncogenic mechanisms in CRPC and develop new therapies targeting these mechanisms. Cancer cells are addicted to aberrant RNA polymerase II (Pol II) transcription, which includes initiation, elongation, and termination phases, as well as a recycling step critical to repeated Pol II transcription of the same gene after the initial transcription cycle. While studies have already indicated that uncontrolled transcriptional initiation and elongation have oncogenic roles, it is unknown whether other Pol II transcription processes contribute to cancer-relevant transcriptional outcomes and cancer growth. In preliminary studies, our newly developed in vitro and cell-based transcription recycling assays have found that Pol II recycling is a key yet overlooked transcription process with relevance to prostate cancer. We have found that Mediator complex subunit 31 (MED31) drives Pol II recycling in CRPC cells, enhancing mRNA output during the recycling process. Importantly, high expression of MED31 is both sufficient and necessary for prostate cancer castration-resistant growth and is associated with poor prognosis of CRPC patients. While these findings identify the oncogenic MED31 as a new therapeutic target for CRPC, transcription regulators such as MED31 are generally considered untargetable by traditional, small molecule-based drug design. We have developed a safe lipid nanoparticle (LNP) system for targeted delivery of the CRISPR/Cas13d system to efficiently and specifically knock down oncogenic transcription regulators at the mRNA level. In preliminary studies, we have demonstrated that the LNP-Cas13d system effectively and safely knocks down MED31 mRNA and decreases CRPC cell growth in vivo, establishing the proof of the concept that the therapeutic window exists for targeting MED31 in CRPC. Together, our preliminary findings support the hypothesis that MED31-governed transcription recycling is a novel oncogenic driver for CRPC progression and that an LNP-Cas13d-based RNA targeting system can counteract oncogenic transcription driven by MED31 in CRPC with safety, specificity, and efficacy. In Aim 1, we will delineate the molecular mechanism, biological function, and clinical relevance of MED31-mediated transcription recycling. In Aim 2, we will target MED31-mediated transcription recycling using a CRISPR/Cas13d-based nanoparticle system. The successful completion of these aims will significantly elucidate the critical role of Pol II recycling in lethal prostate cancer and will provide an experimental basis for future clinical trials testing the utility of an LNP- Cas13d RNA targeting system to target this novel oncogenic mechanism in CRPC patients.

NIH Research Projects · FY 2024 · 2023-06

Abstract: Could fusion between two disparate cell types generate an unconventional memory of infection? Would this hybrid cell protect against tomorrow’s pandemics or promote neurodegeneration and cognitive decline? In virally infected mice, we serendipitously discovered the presence of long-lived cellular fusions between astrocytes, a major glial cell type in the central nervous system (CNS), and antiviral CD8+ T lymphocytes (T cells), which infiltrate the CNS following an upper airway infection. Remarkably, these T cell/astrocyte hybrids retain the cellular structure of an astrocyte but maintain gene expression only present in the T cell genome. While tissue- resident memory T cells are known to remain in the CNS after infection, T cell/astrocyte fusions represent an entirely novel cell fate that we consistently observe. This suggests that the transfer of genetic material from T cells could represent a widespread phenomenon associated with astrocyte reactivity. Histological studies of human brains have described hematopoietic fusions within the CNS, particularly in inflammatory settings, but the driving forces, mechanisms, and implications are unknown. Human pathogens, including those that cause upper respiratory infections (URI), directly infect or impact the CNS with symptoms ranging from mild inflammation to overt encephalitis and have been associated with cognitive changes and neurodegeneration, including “brain fog” associated with Influenza and Covid-19. These varying impacts on brain function are likely multifactorial but regional alterations in CNS gene expression are readily observed in animal models. We hypothesize that T cell/astrocyte hybrids represent a novel cell type possessing an engrained memory of inflammation that contributes to inflammatory and neurodegenerative processes. We will establish parameters that govern astrocyte and T cell hybrid formation and identify a core signature and biological consequences of infection-driven cell hybridization on inflammatory and immune processes.

NIH Research Projects · FY 2026 · 2023-06

Project Summary Malaria is an ongoing global health burden, and the spread of drug resistance threatens progress made to eradicate the disease. The liver stage of the Plasmodium lifecycle is a promising target for drug and vaccine development as its inhibition would prevent disease manifestation and transmission. Previously, we mapped transcriptional changes throughout Plasmodium infection of hepatocytes and identified host processes critical to parasite viability. The chemical inhibition and genetic disruption of specific host proteins, such as aquaporin 3 (AQP3), were found to hinder liver stage parasite development. In mammalian cells, AQP3 transports water, glycerol, and other small solutes across cell membranes. Interestingly, we demonstrated that AQP3 localizes to the parasitophorous vacuolar membrane (PVM), the interface between the host and pathogen, in multiple Plasmodium species and stages. Our goal is to better understand host-parasite dynamics that support infection by determining how and why Plasmodium repurposes AQP3. In Aim 1, we plan to elucidate the recruitment of AQP3 to the host-pathogen interface using live-cell imaging with chemical and genetic tools. We will delineate the dynamics of AQP3 interactions with the tubulovesicular network, a membranous system that extends from the PVM. We will further probe the role of known trafficking motifs and the host endomembrane system to understand how AQP3 associates to the PVM and observe this association at an ultrastructural level with immuno-electron microscopy. In Aim 2, we will investigate AQP3 function during Plasmodium infection using a suite of imaging tools combined with AQP3 mutants to identify molecules affected by the host protein. In Aim 3, we will develop AQP3-targeting chemical probes to explore protein dynamics in the Plasmodium liver stages, including human-infective P. vivax and P. falciparum, where genetic approaches are currently unavailable. Fragment-based probe discovery will be used to identify covalent AQP3-binding molecules to label and study AQP3 in cells. Together, this work will provide insights into AQP3 recruitment and function during Plasmodium infection, thereby uncovering mechanisms that may be ubiquitously used by Plasmodium parasites to hijack host proteins. Our small molecule approach offers a route to complete fundamental biological studies probing host- parasite dynamics throughout different stages of the Plasmodium lifecycle and lays a foundation for future host- targeting compounds to address malaria infections.

NIH Research Projects · FY 2025 · 2023-06

Project Summary Many of our most impressive skills, such as those supporting extreme athletic talent or precise musical expression, are learned by imitating the skilled performance of a tutor. To successfully imitate a tutor, a pupil must generate a range of behaviors, evaluate them relative to an example of the tutor, and then reinforce those that are similar to that example. The generated behaviors are often highly elaborate and produced without any source of comparison other than the pupil's internal template. As such, imitative learning depends intimately on the pupil's ability to evaluate and reinforce its own performance in the absence of any extrinsic reward or instruction. The brain mechanisms that support imitative learning remain poorly understood, although it is well known that the basal ganglia (BG) play a central role in classical forms of reinforcement learning. How the BG evaluates and reinforces behavioral variants over the course of imitative learning remains uncertain. In my research I will characterize the relationship between neural activity in the BG, behavioral exploration, and reinforcement during imitative learning. My Specific Aims are: 1) To model the imitative learning process by which songbirds explore subsyllabic structure within song. 2) To jointly model vocal variability and BG circuit activity during song learning. Aim 1 will advance our understanding of the behavioral mechanisms of vocal learning and develop computational frameworks for understanding complex learning processes, while Aim 2 will relate neural variability in the BG to vocal variability during these learning processes. The analyses and models I create in this proposal will both provide insight into the song learning process in zebra finches and create a more general framework for studying complex skill learning. I will conduct this research under the supervision of Drs. John Pearson and Richard Mooney, a team of accomplished, interdisciplinary mentors with complementary skillsets. Their collaboration has already proven to be fruitful. I will work closely with members of the Mooney lab to hone our scientific questions, refine our experimental design, and develop our analyses. In doing so I will build a balanced set of theoretical and experimental skills. I bring a deep passion for understanding complex behavior on both behavioral and neural scales, in addition to expertise in behavioral and computational methods. The experience I gain from this proposal will make me a competitive and independent investigator, accelerating me towards my long-term goal of obtaining a faculty position at a research institute.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY/ABSTRACT Traumatic brain injury (TBI) is a major public health concern, affecting more than 1.5 million individuals annually in the United States. Extracranial multi-organ dysfunction (MODS) occurs in approximately 60% of severe TBI patients and contributes to secondary brain injuries, increased risk for mortality, and poor functional outcomes over the year following TBI. Our prior research has demonstrated that the prevalence of early autonomic nervous system dysfunction is high following severe TBI and associated with cerebral metabolic crisis and MODS development. Although treatments for autonomic dysfunction following severe TBI are readily available, a complete characterization of the course of autonomic dysfunction following severe TBI that would be adequate to guide therapy is lacking. Therefore, a comprehensive characterization of autonomic dysfunction after TBI and an understanding of how early autonomic dysfunction contributes to episodes of cerebral metabolic crisis and extracranial organ injury are urgently needed to guide the development of therapies to improve patient outcomes following TBI. To address this, we propose the following specific aims using unique and granular waveform data from the BOOST-3 (Brain Oxygen Optimization in Severe TBI Phase 3 trial, U01 NS099946) clinical trial: 1) Determine the burden of early autonomic dysfunction and its relationship to cerebral metabolic crisis following severe TBI, 2) Determine the relationship of early autonomic dysfunction with extracranial MODS and functional neurologic outcomes following severe TBI, and 3) Assess the uniqueness and added value of cardiac waveform data in predicting risk for MODS and functional neurologic outcomes following severe TBI. Successful completion of this study will solidify our understanding of the effects of autonomic dysfunction after severe TBI, and inform trials to determine the impact of modulating autonomic dysfunction on the development of MODS and poor outcomes following severe TBI. Our long-term goal is to develop strategies to personalize critical care management in order to improve clinical outcomes after severe TBI in adults.

NIH Research Projects · FY 2026 · 2023-06

Project Summary Despite modern efforts to eradicate malaria, the disease continues to threaten the lives of over half the world's population. The urgency to uncover novel drug targets in the blood and liver stages of Plasmodium parasites, the causative agents of malaria, is exacerbated by strains resistant to frontline antimalarials. To address this need, a better understanding of pathogen biology is imperative, particularly relating to essential, druggable proteins. The Plasmodium falciparum heat shock proteins 90 and 70-1 (PfHsp90 and PfHsp70-1) are molecular chaperones critical to multiple stages of the parasite's lifecycle. These proteins have N-terminal nucleotide binding domains (NBD) that hydrolyze ATP and C-terminal domains that are critical for protein binding and functional dimer formation. We previously identified several molecules that bind to the PfHsp90 NBD that potently inhibit Plasmodium, but the role of the C-terminus for protein function has not been fully explored. We also discovered that the PfHsp70-1 C-terminus has a critical role in lipid signaling under heat stress, but we lacked chemical probes to resolve the function of the NBD in this process. Thus, we need domain-specific small molecule probes to unravel the role of these protein domains in Plasmodium proteostasis and signaling. Our goal is to understand mechanisms that support Plasmodium protein homeostasis and signaling by investigating domain-specific PfHsp90 and PfHsp70-1 functions and to uncover their interactomes. In Aim 1, we plan to develop domain-specific chemical probes that target PfHsp90 and PfHsp70-1. Candidates discovered through target-based screening will be assessed for their affinity with biochemical methods and the ligand binding site will be established with the mass spectrometry approach stability of proteins from rates of oxidation (SPROX). Our preliminary efforts to date have yielded putative novel N-terminal and C-terminal binders for both chaperones that we will optimize and employ in biological studies. In Aim 2, we will investigate the PfHsp90 and PfHsp70-1 interactomes using our chemical probes and genetic tools, respectively, in thermal proteome profiling and limited proteolysis strategies. Prioritized chaperone interactors will be validated with biochemical methods. In Aim 3, we will leverage our PfHsp90 and PfHsp70-1 chemical probes to link chaperone domains to the transcriptional stress response, global protein regulation, and digestive vacuole stability in parasites. We will use RNA-sequencing to assess the transcriptome and develop bioorthogonal noncanonical amino acid tagging (BONCAn to label and track nascent proteins in Plasmodium after chemical probe addition. Digestive vacuole stabilization assays will be completed after chaperone inhibition using live-cell microscopy. Together, this work will identify and characterize parasitic molecular networks involving PfHsp90 and PfHsp70-1 to gain insight into their biological function. Our chemical biology approach offers a route to complete fundamental biological studies throughout different stages of the Plasmodium lifecycle and lays a foundation to disrupt pathways in drug resistant parasites to address malaria.

NIH Research Projects · FY 2025 · 2023-05

Despite therapeutic advances, ovarian cancer (OC) remains the most lethal gynecological malignancy with a 51% five-year survival rate. Advanced-stage disease presentation is a critical determinant of poor outcomes, yet the biological mechanisms underlying heterogeneity in OC outcomes remain poorly understood. Emerging evidence suggests that psychosocial stress (PSS) may contribute to ovarian tumorigenesis and progression through inflammatory pathways. Recent research indicates that PSS may lead to vaginal microbiome dysregulation (dysbiosis), which has been associated with increased OC risk and advanced-stage disease. However, no studies have examined the full pathway between PSS, vaginal dysbiosis, and advanced-stage OC. This research will test the central hypothesis that multi-dimensional PSS leads to vaginal dysbiosis, contributing to advanced-stage OC and poorer survival outcomes. We will utilize the NIH-supported ORCHiD study, comprising newly diagnosed OC patients across nine US states with comprehensive psychosocial measures and vaginal microbiome data. We will develop an Integrated Psychosocial Stress Index (IPSI) that incorporates individual stress measures and neighborhood-level stressors. Our preliminary findings from 132 participants demonstrate distinct vaginal microbiome profiles associated with clinical cancer stage and independent associations between PSS measures and advanced-stage OC. Aim 1 will develop and validate the IPSI and evaluate its relationship with vaginal dysbiosis. Aim 2 will examine whether vaginal dysbiosis mediates the relationship between multi-dimensional PSS and advanced-stage OC. Aim 3 will determine the association between multi-dimensional PSS and OC survival, evaluating dysbiosis as a potential mediator. This integrative framework will elucidate novel biological mechanisms underlying OC progression and survival, informing targeted clinical interventions. By identifying modifiable biological and psychosocial risk factors, this research will advance understanding of the stress-microbiome-cancer axis and identify potential therapeutic targets. Training will include cancer epidemiology, biostatistics/bioinformatics, and microbiome methods, preparing me for independent translational molecular epidemiology research focused on improving cancer outcomes through mechanistic understanding.

NIH Research Projects · FY 2026 · 2023-05

Patients with TBI (age 18 and older) with mild-to-severe traumatic brain injury (TBI) face high incidence and hospitalization rates, and poor cognitive, physical, behavioral, and emotional impairments <12 months post-discharge. These impairments affect patients’ abilities to independently manage their health, wellness, and activities of daily living, resulting in dependence on family. The complexity of needs combined with the fragmentation of healthcare services creates the perfect storm for low patient quality of life (QOL), mismanaged symptoms, rehospitalizations, and increased caregiver strain. Lack of insurance or access to care aggravate these ongoing issues. Despite complex health needs, there are no U.S. standards for transitional care for patients with TBI. Transitional care is defined as actions in the clinical encounter designed to ensure the coordination and continuity of healthcare for patients transferring between different locations or levels of care (e.g., acute hospital care to home). In other patient groups with acute events (e.g., stroke, myocardial infarction), transitional care interventions have led to improved patient QOL and health outcomes. Yet, few TBI transitional care interventions exist. The paucity of theory-driven, evidence-based TBI transitional care interventions led our team to develop an intervention named BETTER (Brain Injury, Education, Training, and Therapy to Enhance Recovery). Based on the Individual and Family Self-Management Theory (IFSMT), BETTER is a patient- and family-centered, behavioral intervention for adults with TBI discharged home from acute hospital care and families. The goal is to improve patients’ QOL (change in SF-36 total score, primary outcome) by 16-weeks post-discharge, as this timeframe includes high rates of unmet patient/family needs and preventable clinical events. Skilled clinical interventionists follow a manualized intervention protocol to address patient/family needs; establish goals; coordinate post-hospital care, services, and resources; and provide patient/family education and training on self- and family-management and coping skills <16 weeks post-discharge. Findings from our NIH R03 pilot study showed BETTER significantly improved patients’ physical QOL by 31.36 points (p = 0.006) and that the intervention was feasible and acceptable with adults with TBI and families. Thus, the purpose of this study is to examine the efficacy of BETTER (vs. usual care) among adults with TBI who are discharged home from acute hospital care and families. Findings will guide our team in designing a future, multi-site trial to disseminate and implement BETTER into clinical practice to ultimately drive advancements to enhance the standard of care for adults with TBI and families.

NIH Research Projects · FY 2024 · 2023-05

Project Summary This proposal focuses on the fact that 400 children and young adults develop rhabdomyosarcoma (RMS) each year, and the vast majority of those with high-risk disease will not be long term, disease-free survivors. One of the major drivers of high-risk disease is the presence of PAX3-FOXO1 fusion protein. In what is commonly referred to as fusion-positive (FP) RMS, next generation DNA and RNA sequencing tools and molecular and cell biological approaches have yet to uncover targetable cancer drivers. As such, the treatment for these children and young adults has not fundamentally changed for several decades! Major challenges to unraveling FP-RMS and the biology of PAX3-FOXO1 are at least two-fold: First, we know much about the biology of the PAX3-FOXO1 fusion protein, including the fact that cooperating genetic or epigenetic changes are needed for it to drive RMS formation and progression. But our knowledge is not sophisticated enough to focus on the subset of cooperating genetic/epigenetic changes that can be leveraged as therapeutic vulnerabilities. Second, though some elegant, experimental models exist for FP-RMS, pure isogenic systems in which PAX3-FOXO1 expression can be quickly and completely turned “on” and “off” are not available. We are convinced that solving both of these challenges will provide a foundational step toward identifying actionable targets that are driven by the oncogenic fusion protein in FP-RMS. Over the next two years, we can accomplish this by completing two complementary but independent aims. First, we apply an innovative computational pipeline to nominate oncogenic drivers and tumor suppressors based on genetic and epigenetic changes in FP-RMS, and functionally validate them in a CRISPR/Cas9-based “mini-pool” assay using both FP and fusion-negative RMS models. Second, we will develop and validate a new degron- based system in which human PAX3-FOXO1 can be controlled in a dynamic and reversible fashion in native RMS cells and PDX models. Among other things, this system created in Aim 2 will be utilized to identify how the key drivers and suppressors from Aim 1 are controlled by PAX3-FOXO1. Our success will lay the foundation for future, hypothesis-directed studies of FP-RMS, generate sharable data and tools for the scientific community, and illustrate a general approach to tackling other translocation-driven cancers that pose challenges similar to FP-RMS.

NIH Research Projects · FY 2026 · 2023-05

Alzheimer’s Disease [AD] is a progressive degenerative disorder of unclear etiology and disease-modifying treatments remain elusive. Abnormal neurovascular regulation can lead to reduced substrate supply to brain, including capillary and small vessel pericyte regulation with neuronal activity, conduction from small vessels to larger scale vessels, and hemodynamic responses to neuronal activity. Neurovascular regulation mechanisms in the context of the aging brain can be differentiated from the premature aging and progressive neurodegeneration associated with AD and dementia syndrome. Early pathological neurovascular and metabolic alterations can reduce substrate delivery to the AD brain. Though brain metabolism is altered during aging, AD demonstrates more severe and premature metabolic insufficiency in comparison to age-matched controls, attributable to neurovascular dysregulation at multiple levels. We will analyze mechanisms of neurovascular regulation occurring in age-matched control genotypes (both wildtype C57Bl/6 and mNOS2-/-) compared to the progressive degeneration noted in the CVN-AD animal model of Alzheimer’s disease (APPSwDI +/+ mNos2−/−). This unique mouse model closely mirrors human phenotypic changes, particularly amyloid plaques around blood vessels, phosphorylated tau, and severe neurodegeneration. Our hypothesis is that degeneration, as noted in both human AD and the relevant CVN-AD animal model, is worsened by premature aging changes in substrate supply at the capillary, pericyte, conduction, and hemodynamic levels. Metabolic insufficiency can arise particularly from abnormal neurovascular coupling and conduction from small to larger vessels, blunting the hemodynamic response to dynamic neuronal activity. The CVN-AD model mirrors human AD phenotypes with a predictable time course of behavioral, vascular and circuit degeneration in relation to aging changes hence provides an appropriate pre- clinical, translational model for analyzing these concepts. We will study novel approaches to evaluating mechanisms of neurovascular regulation including chemogenetic approaches at the pericyte, mural wall cell level, assessing activity and conduction to larger capacity cerebral vessels, neurovascular coupling and hemodynamic responses, to understand dynamic mechanisms of hypoperfusion at critical times of substrate need, in both hippocampus and neocortex as a function of genotype, gender, and age.

NIH Research Projects · FY 2026 · 2023-05

Evidence for the roles of lipids in brain aging and Alzheimer (AD) and its related dementias (ADRD) is building. Lipidomics is providing new insights related to altered lipid turnover and metabolism in AD and their roles in brain aging. Our AD Metabolomics Consortium (ADMC) led by MPI Kaddurah-Daouk is part of the Accelerating Medicines Partnership-AD (AMP-AD) with centers of excellence in AD research, metabolomics/lipidomics, informatics, machine learning, and modeling. Over the last eight years we invested major effort exploring the AD metabolome with high-quality metabolomics/lipidomics datasets across different cohorts with rapid and broad data sharing and transparent reporting of methods, to maximize rigor and reproducibility. We defined metabolic failures across the trajectory of disease, connecting peripheral and central changes, delineating genetic modulation of metabolic changes in AD effort that lead to novel targets for drug development. MPI Arnold led the construction of the first molecular atlas for AD, a data integration resource for investigating AD and its biomarkers in a multi-omics context. MPI Meikle, a world-renowned expert in lipidomics, has created over 50,000 plasma lipidomic profiles from landmark studies, including AD cohorts (ADNI, AIBL, NSHDS) and the most advanced lipidomic profiling of human brain samples from the ROS/MAP cohorts. Our recent work, incorporated lipidomic GWAS with lipidomic profiling in AD cohorts and identified peripheral ether lipids associated with the ApoE risk and resilience variants. Lipid metabolism changes with age, potentially mediating the effects of age, the strongest risk factor for LOAD, on AD. However, it is unclear how age and lipid metabolism interact to affect the aging brain and AD susceptibility. An improved understanding of these relationships will open up new opportunities for early interventions to modify lipid metabolism pathways that modulate the immune system and preserve brain health. We will use state-of-the-art lipidomics to enable three complementary and one exploratory aim. Aim 1 derive reproducible peripheral and central lipidomic signatures for metabolic resilience and vulnerability to cognitive decline and calculate metabolic risk scores (MRS) that inform on AD risk and brain aging. Aim 2 catalogue the lipid-mediated effects of AD risk genotypes linked to vulnerability and resilience. Building on our methods to characterize the lipidome associated with APOE alleles, we will use GWAS, mediation analysis and Mendelian randomization analyses to uncover genetically modulated lipid alterations causally linked to AD and brain aging. Aim 3: evaluate the effects of lifestyle interventions on our derived lipidomic signatures to identify those interventions that can ameliorate lipid dysregulation to sustain brain health and prevent cognitive decline. Exploratory aim: perform lipidomic profiling of peripheral (immune) cells to capture a cellular lipidome and relate this to brain aging and AD pathogenesis. The outcome of our research will provide deeper understanding of role of lipids in brain aging and in AD and will lead to novel therapeutic approaches.

NIH Research Projects · FY 2026 · 2023-05

ABSTRACT Talaromycosis caused by the dimorphic fungus Talaromyces marneffei (Tm) is an invasive mycosis endemic in Southeast Asia. Tm causes a multi-organ disseminated infection that kills one in three affected persons with an immunocompromised condition despite antifungal therapy. Persons with advanced HIV disease (AHD, CD4<200 cells/L) have the greatest risk and account for 90% of diagnosed cases globally. In highly-endemic countries of Vietnam, Thailand, and China, Tm accounts for 18% of HIV-related hospital admissions and surpasses tuberculosis and cryptococcosis as the leading cause of AIDS deaths. An impediment to reducing talaromycosis mortality is our reliance on century-old diagnostic methods of culture which lack sensitivity and can take up to 28 days, leading to treatment delay and higher mortality. The scientific premise of this proposal is that early diagnosis of talaromycosis (up to 16 weeks before culture diagnosis) using a novel point-of-care test (POCT) is achievable, thus would enable early treatment and improve patient outcomes. We will engage multidisciplinary team science to translate a first-in-kind POC technology from bench to bedside to policy, and we will pave a way for an active screen-and-treat strategy to reduce disease burden and HIV mortality in Southeast Asia. Our objective is to develop, optimize, and validate a novel POCT for early diagnosis and for screening of talaromycosis, and to gather inputs from stakeholders to inform the development of a talaromycosis screening program in people with AHD. Contact MPI Le and colleagues have developed a sensitive and specific enzyme immunoassay (EIA) targeting a Tm-specific mannoprotein Mp1p abundantly secreted in patient blood and urine during infection. She has shown that Mp1p is a more sensitive biomarker than blood culture for diagnosis and can be detected up to 16 weeks before blood culture turns positive. MPI Chilkoti has pioneered the D4 POCT— a self-contained immunoassay that can quantify Mp1p levels in <30 minutes. Herein, we propose the translation of the Mp1p EIA onto the Mp1p D4 POCT to provide a rapid, cheap, and ultra-sensitive test for talaromycosis. AIM 1: Develop and optimize a Mp1p D4 point-of-care test (POCT) for early diagnosis of talaromycosis in human blood, serum, and urine. AIM 2: Determine the clinical utility of the Mp1p D4 POCT as a rapid diagnostic tool for symptomatic talaromycosis and as a screening tool for pre-clinical disease in two cohorts of patients with AHD. AIM 3: Determine the cost-effectiveness, feasibility, and acceptability of implementing a public health talaromycosis screening program. Impact: This proposal will translate a novel POC technology from ‘bench to bedside to policy’, forging a paradigm shift from passive treatment to an active screen-and-treat approach that will improve the individual patient outcome and reduce disease burden at the population level. It will build important partnerships with the health system and community and facilitate the implementation of a talaromycosis screening program in Vietnam.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY/ABSTRACT Prediabetes (PD) and type 2 diabetes (T2D) affect 122 million Americans. Although diabetes is the most costly chronic condition in the US—with an annual economic burden of $327 billion—it is severely underdiagnosed, with 84% of those with PD and 21% of those with T2D unaware of their condition. Screening guidelines for PD and T2D include coarse self-reports with low positive predictive value, and therefore have been unable to mitigate this severe diagnostic gap. The ultimate goal of this research is to develop and implement an innovative, practical, and scalable PD and T2D detection strategy by leveraging digital data obtained using personal consumer smart devices (smartphones and smartwatches). Smartphones and smartwatches are now prevalent in the general population, and the technology developed here will be directly translatable for immediate deployment to improve the detection of PD and T2D. Toward this goal, we recently developed wearable-based models using a research-grade wearable wristband to detect personalized glucose deviations, predict interstitial glucose values, and estimate the level of glycated hemoglobin (A1c), which are all key metrics for detecting and monitoring PD and T2D. The wearable-based models currently function on people with a limited A1c range (prediabetic and elevated normal). To translate this work and expand the reach and yield of current screening methods, we propose the following two Specific Aims: (1) Validate and extend the wearable models to distinguish between T2D, PD, and normoglycemia; and (2) Determine how we can leverage smartphones and smartwatches to improve the yield and reach of present screening methods for PD and T2D. In the first Aim, we will validate and extend our preliminary work developing the wearable models to function across a wider range of glycemic variability, interstitial glucose, and A1c values and to move from research-grade wearables to consumer-grade smartwatches. In the second Aim, we will expand the reach of current guideline-based screening using text message delivery of the American Diabetes Association (ADA) “60-second Risk Test” to directly assess and inform patients about their risk for PD and T2D with the goal of increasing A1c testing in patients that meet the risk criteria. We will increase the yield of true positives by adding objective smartwatch and/or smartphone measures (e.g., physical activity, sedentary habits, glycemic health parameter estimations) to the existing ADA 60-second Risk Test model. The innovations from this proposed research could transform PD and T2D detection for the 81 million Americans with undiagnosed PD and T2D through novel methods for real-time, continuous mobile screening. Successful completion of this project could ultimately revolutionize diabetes management by improving early detection and by enabling proactive intervention to prevent or reverse T2D progression.

NIH Research Projects · FY 2026 · 2023-05

ABSTRACT Nuclear structure-chromatin interactions underlie spatial genome organization and gene regulation during development and disease. However, the mechanisms by which distinct nuclear structures control these processes remain poorly defined. Understanding these mechanisms is critical, especially given that the changes in the nuclear structure can propagate to alterations in cell signaling, cell division and genome stability. The nuclear structure is in part composed of the nuclear pore complex (NPC) and lamina. Proteins that are the building blocks of the NPCs, called Nucleoporins (Nups), have been implicated in transcription and chromatin regulation by directly binding to chromatin. The NPC provides a nuclear compartment that accommodates subnuclear organization of genes, transcription factors and chromatin regulatory proteins. However, we still have very limited understanding on the molecular determinants and mechanisms of Nup- mediated transcription and chromatin structure in mammalian cells, and how these processes are governed during early development. Work to date suggests functional roles for Nups in cell type-specific gene regulation. Findings of this proposal will fill remaining gaps in knowledge regarding the exact mechanisms of how the NPCs influence binding of chromatin regulatory proteins at different genes, and how this mechanism influences transcription, peripheral chromatin organization and spatial positioning of genes. We recently provided new evidence that a particular Nup, NUP153, influences transcription and chromatin structure of developmentally regulated genes by mediating POL II pausing and binding of chromatin architectural proteins, CTCF and cohesin, at cis-regulatory elements. Towards dissecting the molecular basis of NUP153- mediated CTCF binding, we identified the catalytic subunit of the SWI/SNF chromatin remodeling complex, BRG1, as an NUP153 interacting protein. We hypothesize that the NPC-chromatin interactions through NUP153 mediate transcription, chromatin structure and peripheral chromatin organization by controlling BRG1 and CTCF binding. We propose that this mechanism in coordination with POL II pausing is necessary for cell type-specific gene regulation during early development. We will test this hypothesis by utilizing human HCT116 cells and mouse embryonic stem (ES) cells and performing genome-wide and genic assays. In Aim 1, we will determine the functional significance of NUP153-BRG1 interactions in NPC-mediated chromatin structure and transcription. In Aim 2, we will define regulatory function of NPC in spatial chromatin organization across the lamina. In Aim 3, we will elucidate the functional relevance of NPC-chromatin interactions in early development. Collectively, findings of this study will provide critical insights into the functional role for the NPC in integrating transcriptional regulation with chromatin remodeling, and spatial organization of chromatin, and how POL II pausing and activity participate in these processes during cell type-specific gene regulation in early development.

NIH Research Projects · FY 2026 · 2023-05

ABSTRACT Recombinant adeno-associated viruses (AAV) have emerged at the forefront of gene therapy as promising vectors for treating a wide spectrum of diseases. Despite the approval of 3 different AAV gene therapy products for ocular (Luxturna), neuromuscular (Zolgensma) and metabolic (Glybera) disorders, several challenges remain – most notably, the need for high doses of AAV to achieve therapeutic efficacy. This drawback imposes a significant burden on manufacturing processes and also the risk of dose dependent clinical toxicity. To this end, it is important to study key aspects of AAV biology that can profoundly influence manufacturing processes, vector yield and quality, which in turn impacts clinical outcomes. The current proposal is centered around one key question – how does AAV exit the host cell? Upon co-infection with a helper virus such as Adenovirus or Herpesvirus, wild type AAV undergoes a transition from a latent to lytic life cycle, hijacking the host cell machinery to lyse the cell. However, it is well known that during rAAV vector production, a significant fraction is secreted into media supernatant (as free or extracellular vesicle (EV)-associated particles), while a fraction is still retained within the producer cell. Despite this knowledge, the urgent need for process optimization and scale up in AAV manufacturing has resulted in adoption of upstream process/harvest steps in recombinant AAV production that involve detergent lysis of producer cells. This process step generates large quantities of cell lysate that is then subject to heavily burdened downstream processing steps that can result in compromised vector yield and quality. Recent work has revealed a novel +1 frameshifted open reading frame (ORF) in the VP1 region of the AAV cap gene that mediates expression of the membrane-associated accessory protein (MAAP). In the current proposal, we highlight exciting new findings from our lab that assign a novel function to MAAP in promoting AAV egress from host cells. Our overall scientific premise is based on strong supportive evidence that MAAP promotes AAV egress by hijacking host cell secretory pathways. Thus, the current proposal is focused on further dissecting the mechanism of MAAP-mediated AAV extracellular secretion. Specific goals of the proposal are to (1) dissect the role of MAAP as an egress factor for different AAV clades, (2) determine the molecular mechanisms underlying MAAP function and AAV secretion and (3) engineer novel MAAPs and stable MAAP producer cell lines for enhanced AAV secretion. Our overarching goal is to study and engineer AAV secretion to streamline process development and improve the clinical safety profile as determined by AAV vector quality.

NIH Research Projects · FY 2026 · 2023-04

Per- and polyfluoroalkyl substances (PFAS) exposure is widespread. Drinking water is considered a primary source of exposure, and high levels of PFAS are found in many communities, sparking concerns about health impacts on these populations. We have demonstrated high PFAS levels in drinking water in Pittsboro, NC. The residents in this city are currently experiencing disparate exposures of PFAS. However, the potential health risks associated with these exposures are not well understood. In addition, very little is known about the toxicity of “emerging” PFAS, including perfluorobutane sulfonic acid (PFBS), which are increasingly detected in the environmment and within humans. We have demonstrated the reproductive toxicity of PFBS. Epidemiological studies, supported by findings from toxicological studies, provide strong evidence that humans exposed to the PFAS legacy compounds are at risk for immunosuppression, including reduced antibody response to vaccination in children. Notably, there are lack of relevant data assessing the effects of exposure to emerging PFAS chemicals or PFAS mixtures and immunotoxicity in early life such as during pregnancy and lactation. In this proposal, we will test our hypothesis that maternal PFAS exposure results in reduced immune response to vaccination, during pregnancy in dams and in offspring after birth through altered cellular immunity and gut microbiota; decreased antibody transfer from dams to offspring through placenta and breast milk by disrupting endocrine signaling and antibobdy transfer receptors. Our specific aims are to: 1) Investigate maternal PFAS exposure and its effects on immune response to vaccination in dams and placental transfer of IgG from maternal to fetal compartment; 2) Examine the effects of maternal PFAS exposure on offspring through breastfeeding and antibody transfer and identify underlying mechanisms; 3) Determine the impact of maternal PFAS exposure on the establishment of gut microbiota and immune response to vaccination in offspring. This study is novel because we will address health impacts of PFAS mixtures mimicking highly contaminated community drinking water and an emerging PFAS compound, whereas most previous studies focused on legacy compounds; Although rodents are a commonly used model for immunotoxicity studies, rabbits are a more suitable animal model for investigating both maternal transfer of antibodies to the offspring and the development of the immune system during early life, including establishment of gut microbiota and immune response to vaccination. This study will provide new insights into the impact of perinatal PFAS exposure from breast-feeding and the subsequent health effects in offspring. Feasibility: The combination of expertise and preliminary studies provide a strong foundation for this proposal. Dr. Feng’s lab has established the perinatal PFAS exposure rabbit model; this proposal is an extension of her K01 project. Drs. Staats and Landon have extensive experience working with rabbits, such as immune responses to a variety of vaccinations in rabbits. Dr. Fenton has three decades of experience with mammary gland development, lactation, and toxicity in animal models, most of which pertains to PFAS exposure. Dr. Ji is an expert in analyzing single-cell sequencing and metagenomics data. Our study will make significant contributions to our understanding of the health impacts of PFAS and provide evidence to support regulations.

NIH Research Projects · FY 2026 · 2023-04

ABSTRACT Diffuse large B cell lymphoma (DLBCL) is the most common form of blood cancer and is characterized by a striking degree of genetic heterogeneity. It has been known for some time that African American patients have poorer outcomes than Caucasian patients. After correcting for potential differences in economics and access to care, our data indicate that the poorer outcomes in African Americans arise primarily from distinct genetics of their tumors. The underlying genetic causes of these poorer outcomes have not been studied thoroughly. Here, we propose to investigate tumor and germline genetics of African Americans to comprehensively understand the genetic basis of their poorer outcomes. Preliminary genomic analysis of African American DLBCL patients has revealed more frequent mutations in histone methyltransferase genes and other genes. In this proposal, we seek to comprehensively define the genetic origins of poor outcomes in African Americans with DLBCL and perform functional characterization of selected genomic alterations.