Duke University

NIH Research Projects · FY 2026 · 2022-11

PROJECT SUMMARY/ABSTRACT Infectious insults are common during pregnancy; over the nine months of gestation ~60% of pregnant women self-report at least one illness, with viral upper respiratory tract (URT) infections being the most common. Although URT-trophic viruses replicate in the respiratory epithelium, the induced inflammatory cytokines like type I interferon (IFN) circulate systemically and can access the placenta. Recent work has shown that virally induced type I IFNs can be major drivers of adverse effects on fetal development. URT infections during pregnancy, however, are not typically linked to birth defects or miscarriage. It was therefore unclear why maternal infection with a pathogen like an influenza virus, which also induces to fetal IFN exposure, would not compromise fetal health. We hypothesized that an uncharacterized IFN regulatory pathway was the answer to this apparent discrepancy. By performing a genome-wide CRISPR/Cas screen, we identified a G-protein coupled estrogen receptor 1 (GPER1) dependent signaling pathway that protected fetal health from type I IFN signaling during maternal influenza A virus (IAV) infection. Disruption of this pathway led to fetal phenotypes as severe as those caused by direct congenital infections. Importantly, the activities of this pathway were restricted to reproductive and fetal tissues; alterations of its activity had no measurable effect on maternal health during IAV infection. The major goal of this application is to understand how GPER1-mediated signaling normally protects fetal health from inflammatory maternal cytokines such as type I IFN. In aim 1, we will define how GPER1-induced GPCR signaling suppresses IFN-induced JAK/STAT signaling and interferon-stimulated gene expression. These experiments will define a previously unknown mechanism for control of IFN signaling. In aim 2, we will characterize where and when GPER1 signaling is required to protect fetal health, as well as the effects of GPER1 dysregulation on cell physiology both in vivo and in primary human placental organoid cultures. These experiments will allow basic mechanistic insights into how maternal inflammation compromises fetal development. Finally, in aim 3, we will explore the consequences of IFN signaling on placental structure/function when GPER1 is absent and also evaluate the potential of hyper-activating GPER1 signaling under the inflammatory conditions that normally harm fetal development. Together, these studies will not only allow for a more complete understanding of IFN regulatory mechanisms and the fetal/maternal immune response but could also serve as the basis for an eventual first-in-class treatment designed to protect the fetus from inflammation without compromising maternal immunity.

NIH Research Projects · FY 2026 · 2022-09

ABSTRACT It remains considerably challenging to restore vision after developmental disturbances, such as congenital infantile nystagmus, and after injury or retinal degeneration. This is because the mechanisms establishing functional connectivity between retinal ganglion cells and their downstream targets in the brain remain poorly understood. This knowledge gap is partly because observing the functional emergence, stabilization, and maintenance of entire visual neural circuits is impossible in mammals. This project will leverage the strategic experimental advantages of the larval zebrafish, a vertebrate model system, to investigate the functional maturation of a conserved neural circuit underlying a visual orienting behavior, the optomotor response (OMR). This will form the basis for understanding how congenital disorders exert their effects and how new neurons added after initial circuit development can support healthy visual processing. Recently, we described the transformation of retinal visual motion signals into motor output and showed that it required many different types of neurons distributed across the brain. These neurons can be classified based on their diverse eye- and direction-specific response profiles, and they collaborate to compute how exactly visual scenes are moving. Fascinatingly, this collaboration supports stable behavior 5 days after fertilization, even though new neurons are added to the circuit throughout life. We will test the central hypothesis that after initial formation, the OMR circuit expands by adding new neurons in balanced response classes, permitting the continued execution of motion- guided behaviors. In Aim 1, we will test how the development of the behavioral repertoire and associated neural circuitry is affected by specific disruption of direction-selective retinal input. By training recurrent neural networks, we will generate predictive models of connectivity between direction-selective retinal ganglion cells and downstream targets. In Aim 2, we will investigate how the functional neural representations mature, and we will quantify the stability of individual neuronal responses over time. By computationally tracking all neurons, we will directly investigate the trajectory of new neuron functional integration into existing circuitry and determine how the balance of functional profiles varies over time and covaries with behavior. In Aim 3, will use holographic photostimulation to examine the role of activity in shaping ultimate circuit role for individual neurons. Together, these experiments will reveal how an entire motion-sensitive vertebrate circuit is functionally assembled, providing insight about the functional connectivity between retinal ganglion cells and their downstream partners and about the nature and utility of neurogenesis. These results will inform regenerative treatment strategies for developmental disorders or injuries to central visual processing areas.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Hypoglycemia is a dangerous complication of exogenous insulin therapy in Type 1 Diabetes (T1D) that is treated by exogenous glucagon secretion. However, the use of exogenous glucagon has its own side effects and can be complicated to administer in an emergency situation. As an alternative, stimuli that lead to robust endogenous glucagon secretion could be effective to counter severe hypoglycemia. Glucagon secretion can be stimulated by amino acids, like alanine and arginine, as well as by glucose-dependent insulinotropic peptide (GIP). Remarkably, we found that while alanine or GIP alone induce modest increases in glucagon secretion, the combination of alanine and GIP synergistically increase glucagon secretion in both isolated mouse and human islets, as well as mice in vivo. A better understanding of the physiology of this glucagon and the mechanism of its release is needed to determine if stimulating endogenous glucagon can treat hypoglycemia in T1D. We hypothesize that endogenous -cell stimuli, such as GIP + alanine, can counter insulin-induced hypoglycemia. The aims of this project are designed to elucidate whether -cell stimuli can mitigate severe hypoglycemia, how the effects of -cell stimulation are changed in T1D, and what is the mechanism that alanine stimulates the - cell to secrete glucagon. Successful completion of this project will enhance our understanding of the -cell and provide insight for the basis of therapeutics for hypoglycemia or insulin co-therapies. In addition, the aims will broaden the Candidate’s technical expertise and develop conceptual understanding of -cell physiology that will provide a foundation for a career as an independent investigator.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Glaucoma is a group of diseases, second leading cause of permanent blindness worldwide, characterized by the chronic degeneration of RGC axons and progressive loss of retinal ganglion cells (RGCs), which results in visual field defects and vision loss. Elevated intraocular pressure (IOP) is the best-well known factor contributing to the onset and progression of glaucoma. There are not therapeutic treatments to offer neuroprotection in glaucoma. Current glaucoma therapies are directed at lowering IOP, but cannot rescue RGCs. A better understanding of the exact molecular mechanisms triggering RGC death and axonal degeneration in glaucoma is essential for the development of neuroprotective treatments. Autophagy is a lysosomal degradative process, which plays a central role in cellular homeostasis by eliminating damage organelles and proteins. In addition to having a key role on maintaining cellular and tissue homeostasis, autophagy is regarded as a survival pathway, involved in stress-induced adaptation. Dysfunction of the autophagy pathway has been associated to a growing number of human diseases, in particular age-related diseases, as well as to several neurodegenerative disorders. Paradoxically, in the neural tissue, autophagy plays an important role in neuroprotection as well as neuronal injury and death depending on the circumstances. Although not extensively, autophagy within a context of glaucoma, has been investigated by independent laboratories using different experimental models. While all of the studies agree that autophagy is activated in RGC in response to injury or elevated IOP, there is no consensus on whether autophagy promotes survival or triggers cell death. Latest studies seem to suggest that a protective or pro-death role of autophagy depend on the initial injury (i.e traumatic insult vs IOP elevation). Moreover, autophagy seems to have a different role in RGC death and axonal degeneration. The purpose of this grant application is to investigate the independent contribution of autophagy to apoptotic RGC death and axonal degeneration in acute injury and chronic hypertensive experimental models of glaucoma. For this, we will use unique tools generated in our laboratory, including our unique DBA/2J transgenic mouse glaucoma models with upregulated and deficient basal autophagy. We anticipate that completion of this project will contribute to a further understanding of the role of autophagy in neurodegeneration in glaucoma. Most importantly, our studies have the potential of identifying a novel therapeutic target for the treatment of ocular hypertension and glaucoma.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Corneal nerves that mediate pain, blink reflexes and tear production are indispensable in the proper maintenance of ocular surface homeostasis. However, the complexity of these neurons, both at the axon level in the cornea, and cell bodies in the trigeminal ganglion, has made it increasingly difficult to grasp their full nature, resulting in key knowledge gaps in the field. Consistent with the call for the current U01, we will address this barrier via comprehensive analyses of corneal nerves at the morphologic, molecular, and functional level. We have assembled a multidisciplinary team with complementary expertise, enabling integrated analyses of spatial, electrophysiologic, genomic and behavioral profiles in mice; and AI-assisted structure-function studies in human subjects, to open new inroads in the field. In Aim 1, we will anatomically and functionally map corneal-projecting trigeminal afferents in mice. Genetic approaches will be applied to rigorously profile the spatial arrangement of nociceptors in the cornea and trigeminal ganglion using seven- color immunolabeling. This spatial information will be directly linked with the electrophysiological profiling of these respective populations. In Aim 2, genomic analyses of corneal-projecting trigeminal afferents will be conducted applying a new platform for spatial RNA-seq at cellular resolution. We will use mouse strains to parse out the transcriptomes and behavioral outputs at the genetic level. Moreover, we apply two novel approaches to selectively target corneal nerves involved in tear production versus blinking. In Aim 3, we will discover morphological patterns of corneal nerves that predict blinking, lacrimation, and nociception in humans. We will image corneal nerves with in vivo confocal microscopy of subjects with differential blink, tear, and nociceptive behavioral outputs, thereby capturing functional analogs of the mouse experiments in Aims 1 and 2. With the AI-based auto-segmentation algorithm that we are developing, we will be able to apply machine learning for multidimensional profiling of nerve patterns, and then compare these with respective behavioral outputs. These AI-guided efforts will provide critical clues for understanding corneal afferent structure-function in humans. In summary, our collective studies will lead to an unprecedented cartography of corneal afferents in blink, lacrimation, and nociception. The advancements from this work will be poised to facilitate a deeper understanding of related pathobiology including neuropathic ocular pain and dry eye disease that will lay the foundation for future translational and clinical research. All genomic, imaging and electrophysiologic datasets produced will be made publicly available, and all software products for corneal nerve image segmentation will be made freely available online as open-source and easy-to-use software packages.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Three-dimensional ultrasound imaging (3D-US) is an essential clinical tool for visualizing, navigating, and investigating patient anatomy and pathologies in real time in 3D. Owing to its moderate cost and lack of ionizing radiation, 3D-US plays an important role in many clinical applications for diagnosis and intervention. Despite the significant clinical value and potential, 3D-US is not a widely accessible and capable technology with its current implementations: existing 3D-US solutions are challenged by many limitations such as low imaging speed, low functionality, bulky devices that are inconvenient to use, and a high cost of designated equipment. For decades, there has been a long-standing quest for developing an accessible, functional, and user-friendly 3D-US technology. In this proposal, we will develop a new 3D-US solution (called FASTER) that uses a novel, fast-tilting microfabricated acoustic reflector to achieve high-speed and high-functionality 3D-US imaging. The acoustic reflector is water-immersible and enclosed in a clip-on device that is compact, lightweight, and low-cost. It can be easily attached to and removed from different types of ultrasound transducers to turn a conventional 2D ultrasound system into 3D. Unlike conventional 3D-US technologies (e.g., wobbler transducers and 2D matrix arrays), FASTER does not require the procurement of additional ultrasound transducers for different applications. Also, FASTER achieves a much higher imaging volume rate (up to 1000 Hz) than conventional 3D-US technologies. FASTER is compatible with most ultrasound systems on the market ranging from premium scanners to portable and handheld devices. In this proposal, we will conduct basic technology development research and carry out preliminary clinical studies to build a solid technical foundation for the FASTER 3D-US technology. In Aim 1 we will focus on developing the Phase-1 FASTER device that uses a double-axis reflector for extended range of imaging volume rate and field-of-view (FOV). We will also develop Phase-1 FASTER into a stand-alone device that does not need external equipment and communicates wirelessly with the ultrasound system. Aim 2 will focus on developing advanced imaging modes for FASTER, including 3D blood flow imaging (3D-BFI) and 3D shear wave elastography (3D-SWE). Pilot clinical studies will be conducted for both Aims 1 and 2 to facilitate the development and optimization of the FASTER device and imaging sequences. In Aim 3 we will conduct a clinical study to evaluate the performance of FASTER 3D-US in characterizing suspicious axillary lymph nodes (ALNs) for breast cancer patients undergoing clinically indicated biopsy of ALN. We will also evaluate the performance of FASTER in localizing clipped ALNs from patients undergoing neoadjuvant chemotherapy. The study aims will be carried out by a team of experts in ultrasound imaging, micro sensors and systems, medical device design, and breast cancer from the campuses of University of Illinois Urbana- Champaign, Texas A&M University, and Mayo Clinic.

NIH Research Projects · FY 2025 · 2022-09

Predicting how a particular patient's vascular system with respond to different treatment or stimuli and adapt over long periods of time remains a grand challenge in precision medicine. The lack of real-time turn around critically limits our ability to search a wide treatment space to identify optimal intervention plans based on long-term, personalized predictions. Moreover, it prevents real-time monitoring of a patient's hemodynamics based on streaming, dynamic data such as that acquired from wearables. By moving from simulations that can capture only several heartbeats to modeling months or even years, we shift the utilization of patient-specific digital twins to provide on-demand tracking of a patient's hemodynamic state. Such data would improve screening for cardiovascular disease, improved monitoring, and finally, inform treatment planning by enabling prediction of longterm flow effects currently not attainable. The major objective of this proposal is to develop and apply a methodology coupling physics-based simulations with machine learning that, combined with wearable sensors, provides real-time, personalized predictions of 3D, complex hemodynamic patterns over months to years. A better understanding of how a patient's circulatory system and underlying hemodynamics responds under different physiological states over time is of broad relevance to treating a wide range of vascular diseases.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY One out of every ten infants is born preterm. Preterm birth can cause retinopathy of prematurity (ROP), a leading cause of childhood blindness. Even in the absence of ROP or neurological disability, children born preterm exhibit subtle impairments in visual acuity and visual function, though the etiology of this suboptimal vision in preterm infants remains unclear. The fovea, an indentation in the central retina, is the most critical region determining visual acuity. The fovea is surrounded by anastomosis of three layers of capillaries, forming the foveal avascular zone. It is well-established that children and adults with history of prematurity have a small or absent foveal avascular zone, retained foveal inner retinal layers, and decreased photoreceptor function, and that these abnormalities are more severe in individuals with history of treated ROP. The development of human perifoveal vasculature, however, is difficult to study due to the absence of fovea in most easily accessible animal models and the rarity of human histopathological samples during late gestation. Our multidisciplinary team with complementary expertise will work together to use advanced bedside handheld optical coherence tomography (OCT) angiography imaging to fill the gap in our knowledge of perifoveal vascular development in infants. We propose to elucidate the human perifoveal vascular development through the following specific aims: 1) optimize bedside handheld infant OCT angiography to visualize perifoveal vascular development; 2) determine if perifoveal vascular development is delayed in preterm infants compared to term infants, further delayed in ROP, and associates with poor outer retinal maturation; and 3) determine the association of macular edema and decreased perifoveal deep vascular complex formation and outer retinal maturation in preterm infants. This research will expand our knowledge of human foveal development and inform the pathophysiology of diseases of macular and retinal vascular development.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT – Duke Human Vaccine Institute Duke University is pleased to respond to RFA-IP-22-004 entitled “US Platform to Measure the Effectiveness of Seasonal Influenza, COVID-19 and other Respiratory Virus Vaccines for the Prevention of Acute Illness in Ambulatory Settings” by submitting the application for Component B. Duke University will coordinate the activities of the Centers for Disease Control and Prevention’s Respiratory Virus Vaccine Effectiveness Network incorporating the collective breadth of scientific, program management, regulatory, data management, statistical, and information technology expertise of the Duke Human Vaccine Institute (DHVI). In particular, we will leverage our vast prior experience coordinating clinical investigations for both NIAID and the CDC to help facilitate the work of this project. The Duke Network Coordinating Center (NCC) will provide logistical and coordinating support by facilitating network communications through hosting video and in-person conferences, hosting a network SharePoint, providing reports and project updates and establishing a clear communication plan for network activities. As the NCC, The DHVI will help facilitate protocol development and establish standard operating procedures for network investigations. Studies will include evaluations of both influenza and SARS-CoV-2 vaccine effectiveness at preventing symptomatic respiratory infection in the community and household settings. As the NCC, the DHVI is also well poised to support network studies assessing vaccine immunogenicity in addition to studies using more complex virologic and immunologic influenza assays to detect influenza virus infection and the host immune response to infection. Working with the Duke University Health System IRB, the Duke NCC will provide the regulatory support to facilitate single IRB requirements. Through our established quality management programs, we will also assure that network studies are performed in a manner which adhere to good clinical practice. The Duke NCC will provide data management and statistical support for network studies. Duke will build and host project specific REDCap databases from which information can be readily exported to provide project updates through dashboards. Data exports will also be utilized to create reports, presentations and manuscripts to disseminate information regarding the current effectiveness of the respiratory virus vaccine being evaluated given the circulating virus strains or variants.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract Opioids are potent analgesics, and despite considerable side effects and risks for overdose and addiction, many patients continue long-term opioid therapy. Based on prior NIDA-funded research (DA040154), we have published initial data from brain functional MRI (fMRI) studies that demonstrate significantly altered brain response to reward in patients on long-term opioid therapy. Our preliminary data from innovative high- resolution fMRI of the cervical spinal cord revealed disrupted resting-state functional connectivity of the spinal cord dorsal horns in the same patients. Thus, long-term opioid therapy has neurobiological consequences on responses to stimuli and neural circuitry at both brain and spinal cord levels. Due to the opioid epidemic, there is an urgent need to understand neurobiological consequences of opioids, as stated in Goal 1 of the NIDA Strategic Plan, to aid patients and clinicians in opioid cessation strategies, and to inform novel ways to reverse neurobiological consequences of long-term opioid use. Our overall objective in this project is to characterize neurobiological consequences of long-term opioid therapy on brain reward systems and spinal cord circuitry, 2 interacting focal points in the central nervous system. Our central hypothesis is that in long-term opioid therapy patients, opioid use transiently improves responses to stimuli, while disrupting functional connectivity of neural circuits within the brain and spinal cord. To test this hypothesis, we will collect and analyze data from task-based and resting-state fMRI of the brain, and high-resolution fMRI of the spinal cord in long-term opioid users (ie, > 90 days duration, homogeneous sample of female patients with fibromyalgia, as included in our preliminary data). We will evaluate brain and spinal cord fMRI-based activity in opioid patients (N = 40) by using a novel within-subject design to compare activity in active vs non-active opioid states (timed to opioid administration and blood opioid level) to activity in opioid-naïve patients (N = 40) and healthy controls (N = 40). In Aim 1, we will characterize neurobiological consequences of long-term opioid therapy on brain fMRI-based response to reward probability, and on resting-state fMRI-based functional connectivity of a key brain reward circuit. In Aim 2, we will characterize neurobiological consequences of long-term opioid therapy on spinal cord fMRI-based response to noxious heat stimuli, and on resting-state fMRI-based functional connectivity between dorsal horns. To identify neurobiological targets related to clinical endpoints of opioid use/misuse and addiction behavior, exploratory analyses will be integrated across aims to assess relationships between brain and spinal cord fMRI-based endpoints, and cognitive-affective and clinical measures. Together, the proposed project will provide important and rigorous opioid dose-timed evidence of neurobiological consequences of long-term opioid therapy across the central nervous system.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT The cerebellum plays a key role in motor coordination and learning. Classic models posit that cerebellar learning is instructed by teaching signals from climbing fibers (CFs) that act according to the principles of supervised learning. While such models work well to describe CF activity and learning in some behaviors, they are not sufficient to explain CF activity in others. By developing an operant, reward-guided cerebellar-dependent task for the mouse, as well as a modified classical conditioning task, we used calcium imaging of CF input to Purkinje cell dendrites to demonstrate that CFs can be driven by reward-related task parameters. Our data suggested the possibility that CFs might engage in reinforcement learning to report predictions about expected rewards (reward prediction errors) in a similar manner as dopaminergic neurons of the ventral tegmental area (VTA). Importantly, however, our data also show significant differences from some predicitons of leading reinforcement learning models, and many other properties of cerebellar reward-based learning remain unclear. Thus, it remains largely unknown how the cerebellum operates in reward-based learning. Here will rigoursly test the hypothesis that CFs instruct cerbellar learning according to reinforcement learning rules: In the first aim, we will test whether CF activity obeys the many diverse requirements of reward prediction error signals, for example by scaling with both the probability and size of an expected reward. To do so, we will use two-photon calcium imaging to monitor CF input to Purkinje cell dendrites while manipulating reward contingencies during a classical conditioning paradigm. We will also determine the contribution of behavioral context, learning, and motor output to CF activity. In the second aim, we will test whether reward-predictive CF activity is generated by reward- responsive CF activity. This is a key property of reinforcement learning because it binds activity driven by an unconditioned simulus (US) to activity driven by a conditioned stimulus (CS). We will use classical blocking experiments and optogenetic manipulations to determine the neccessity and sufficiency of US-linked CF responses to generating CS-linked CF responses. Finally, in the third aim, we will determine whether CS-linked CF activity drives learned changes in behavior and cerebellar output. Thus, we will use a combination of optogenetics and extracellular electrophysioloigcal recordings test the function of reward-related CF activity. Together, these experiments will reveal the key principles that govern reward-based cerebellar learning, and how this learning alters cerebellar output and behavior.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Organ transplantation is the optimal treatment for end stage organ disease and results in improved patient survival and quality of life. Black patients have rates of end stage organ disease nearly three times that of white patients yet comprise only 20% of organ transplant recipients. The transplant selection process is not standardized across transplant centers and therefore health system and transplant center processes may influence access to transplant. Further, national transplant data collection and monitoring exclude the transplant selection process. The overall objective of this proposal is to identify, characterize, and develop interventions to improve access to organ transplant via consistent health system and transplant center processes of care. Our is that lack of standardization and inconsistent care delivery throughout each phase of the transplant selection process creates obstacles to transplant access. This project will quantify the extent of patient elimination in the screening, evaluation and committee decision phases of the transplant selection process using electronic health record data (aim 1), Characterize process vulnerabilities in screening, evaluation and committee decision phases of the transplant selection process that impede transplant access (aim 2) and design and pilot a toolkit for use by transplant centers to improve transplant access. This results of this project elucidate the contribution of inconsistent health system and transplant center processes of care to creating and exacerbating patient difficulty with accessing organ transplant. The research and additional knowledge and skill development will form a strong foundation for the candidate’s transition to an independent investigator independent investigator developing and testing interventions to improve access to organ transplant.

NIH Research Projects · FY 2025 · 2022-09

The Helping to End Addiction Long-term (HEAL) initiative was developed to identify scientific solutions to the national opioid public health crisis as quickly as possible. As part of this initiative, the NIH HEAL Pain Management Effectiveness Research Network (HEAL Pain ERN) will continue to use the infrastructure of the NCATS Trial Innovation Network to provide scientific guidance and coordination of the HEAL Pain ERN Trials. Specifically, the three Trial Innovation Centers (TICs) at Duke/Vanderbilt, University of Utah, and Johns Hopkins/Tufts will continue to provide integrated functions for the clinical resource coordinating center (CRCC), data coordination resource center (DCRC), and the statistical and safety resource center (SSRC) for the HEAL Pain ERN. Two additional HEAL Pain ERN trials will be selected which will require full support from the HEAL Pain ERN CCRC, DCRC, and SSRRC. In addition, five ongoing trials supported by the HEAL ERN will continue to receive dedicated support from the resource centers. The Duke/Vanderbilt Trial Innovation Center is poised to: Specific Aim 1. Provide clinical trial leadership and expertise, assistance in study and protocol design, study implementation, and management, in collaboration with the other HEAL ERN Resource Centers, for existing and new HEAL ERN trials. Specific Aim 2. Serve as the clinical coordinating resource center (CCRC) for existing and new HEAL ERN trials. Specific Aim 3. Provide single IRB support for HEAL ERN trials for existing and new HEAL ERN trials. Through completion of these specific aims, we will harmonize the HEAL studies to achieve major innovations in trial design and execution, including: combination and reuse of valuable data, efficiencies of implementation, shared pain expertise among investigative teams, and the integration of three TICs as a cohesive unit, poised for future trial implementation.

NIH Research Projects · FY 2025 · 2022-09

Breast cancer mortality differs significantly by several demographic factors, including socio-economic status, access to care, geographic residence and race/ethnicity. Despite extensive investigation, the known causes to date do not adequately explain this mortality gap. Largely missing in the literature is a rigorous examination of how exposure to chronic societal stress (CSS) might impact adverse breast cancer outcomes. Multiple lines of evidence, when considered together, indicate that this exposure merits investigation. CSS (e.g., interpersonal conflict, limited residential resources, etc) is associated with a range of adverse health effects, and chronic psychosocial stress due to CSS can become embodied via hyperactivation of the hypothalamic pituitary adrenal (HPA) axis, leading to elevated markers of multisystem allostatic load. We hypothesize that exposure to CSS contributes to—and helps explain—excess breast cancer mortality in certain demographic groups, however, no empirical study has directly tested this hypothesis in a single cohort. To address this gap, we will generate a new prospective cohort with 2,498 incident breast cancers and a 2,678 sub-cohort random sample from two parent cohorts -- the REasons for Geographic and Racial Differences in Stroke (REGARDS) and Southern Community Cohort Study (SCCS) cohorts. Both parent cohorts over participants representing various socio-economic, access and race/ethnic demographic groups, including from southern states with a history of CSS; moreover, they obtained extensive baseline epidemiologic and covariate data. We will newly assess measures of CSS exposure across multiple domains, multiple geographic levels (e.g., census tract, state level), and multiple time points across the lifecourse. Our study will provide the first thorough prospective evaluation of the distinct influence of CSS, above and beyond other population-level risk factors, on breast cancer outcomes in a large, broadly representative US cohort. By quantifying the distinct impact of CSS and exploring pathways that may mediate this association, our study will: i) illuminate specific social and biological drivers of breast cancer outcomes, ii) improve the poor accuracy of current breast cancer prognostic models across various demographic groups, and iii) inform primary prevention strategies to mitigate CSS exposure and reduce breast cancer mortality.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT The high-throughput sequencing technology allows us to query both common and rare variants for complex human diseases. When variants are rare, single variant association analyses suffer from low power. To increase power, existing whole-exome sequencing studies often aggregate the rare variants (RVs) across an entire gene to study their collective effect. Presumably, when a gene harbors many pathogenic RVs, the aggregation will increase the signal-to-noise ratio and thus the power. However, a gene often carries many mutations, while only a subset will lead to novel or altered activities. These mutations usually do not distribute uniformly across the entire gene or domain. For genes whose functional mutations are localized or concentrated to the specific subregions, aggregating all the RVs across the entire gene or domain will dilute the signal, resulting in a loss of power. Besides, even if the gene- or domain-based analysis can identify the pathogenic genes, they cannot pinpoint the pathogenic subregions. Pinpointing the pathogenic subregions is preferred because it is usually more unified in function and will be more informative to the downstream disease mechanism and translational studies. To address these concerns and needs, we propose a novel statistical and computational method for rare-variant association analysis with the three main features. First, it automatically searches the GVSs with different sizes for their disease associations to optimize power. Second, it can pinpoint the disease-associated GVSs with high resolution to facilitate the downstream disease mechanism studies. Third, it can be easily customized to fit the special needs, such as preserving data privacy, incorporating functional annotations, and adjusting for varying ancestry loadings for admixed populations. We will establish a rigorous mathematical and statistical foundation for the GVS analysis and develop the software to realize its implementation on high- throughput sequencing studies. We will apply our method to an ongoing whole-exome sequencing study of amyotrophic lateral sclerosis (ALS) to identify ALS-related genomic subregions.

NIH Research Projects · FY 2026 · 2022-09

Hypertension is a common chronic disease with a significant impact on public health, yet its basic pathogenesis is not fully understood, and new therapeutic targets are needed. A beneficial role for prostanoids in hypertension was suggested because non-steroidal anti-inflammatory drugs (NSAIDs), which block the production of all prostanoids, can cause sodium retention and exacerbate hypertension. Among prostanoids, PGE2 and its EP4 receptor (EP4R) have been implicated in blood pressure control, but these mechanisms are unknown. Our previous work showed that conditional deletion of EP4R from all tissues in adult mice dramatically exacerbated Ang II-dependent hypertension. However, the elimination of EP4R from vascular smooth muscle cells, endothelial cells, and macrophages had no impact on hypertension development. By contrast, specific removal of EP4R from whole renal epithelia recapitulated the phenotype of exacerbated hypertension, indicating that EP4R attenuates hypertension by direct actions in the renal epithelium. Recent single-cell sequencing studies demonstrated that EP4R expression in renal epithelia is enriched in the collecting duct (CD). CDs have pivotal roles in final urinary sodium excretion through the actions of the epithelial sodium channel (ENaC). Our preliminary studies showed that mice with EP4R deletion in renal epithelia throughout the nephron had increased responsiveness to ENaC inhibitor, and PGE2 inhibits the ENaC activity in isolated CDs. Thus, we hypothesize that EP4R resists the development of hypertension through actions in the CD to reduce sodium reabsorption via ENaC. The project’s objective is to identify mechanisms underlying the anti-hypertension effects of EP4R and to exploit them for new treatments of human hypertension. Our Aims are: 1) Identify cell specificity for EP4R actions in kidney epithelia to resist hypertension. We will generate mice with EP4R deleted from entire CDs, principal cells, or intercalated cells, respectively, to assess the consequences of these genetic alterations on blood pressure, sodium homeostasis, and ENaC function in hypertension; and 2) Determine the mechanisms of ENaC regulation by EP4R. We will perform patch-clamp electrophysiology in isolated CDs to characterize EP4R downstream signaling pathways that mediate its powerful effects on attenuating the development of hypertension. Successful completion of the proposed research is expected to identify the mechanisms underlying the antihypertensive actions of EP4R. The long-term goal is to identify novel therapeutic targets for essential hypertension.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT: Temporomandibular disorders (TMDs) are the most common form of chronic orofacial pain, affecting 5% of U.S. adults. Despite substantial clinical and research interest in this area, progress to identify and target pathophysiological mechanisms underlying TMDs has been slow. This lackluster progress is owed in large part to our relatively primitive understanding of the basic neuroanatomical, molecular, and physiological features of sensory afferents present within the temporomandibular joint (TMJ) tissues. The Restoring Joint Health and Function to Reduce Pain (RE-JOIN) Consortium seeks to address this knowledge gap through the formation of interdisciplinary teams which can define the innervation of articular and peri-articular tissues that collectively make up the jaw joint. To this end, project MPIs Donnelly (Duke University School of Medicine), Emrick (University of Michigan School of Dentistry), and Cai (University of Michigan Medical School) have partnered together to comprehensively map the peripheral neural architecture of the tissues of the temporomandibular joint (TMJ) in mice and humans. Using MRI-guided stereotactic approaches to deliver retrograde dyes and viral tracers with spatiotemporal precision, we will investigate the molecular properties of peripheral sensory neurons which innervate distinct tissues within the murine TMJ in both steady-state and TMD conditions, using this information to build new intersectional genetic mouse models to permit whole-TMJ mapping using lightsheet microscopy. In addition, using intersectional genetic approaches in conjunction with chemogenetics, in vivo Ca2+ imaging, and behavioral phenotyping, we will characterize the physiological/functional properties of TMJ-innervating sensory neurons, allowing us to identify neuronal subpopulations which contribute to chronic pain in TMD. To address the translational gap between mice and humans, we will establish a biobank of TMJ tissues from TMD-free healthy human donors and from a cohort of clinically-phenotyped patients pursuing elective TMJ surgeries to manage chronic intraarticular TMD conditions, followed by quantitative analysis of peripheral afferent subtypes across TMJ tissues in each cohort. Finally, we will build a free, user-friendly web- based platform to integrate the resulting transcriptomic, functional, and macroscopic imaging datasets to permit widespread dissemination of these data, which we anticipate will yield a working model of the sensory architecture of the temporomandibular joint tissues in mice and humans, including alterations in TMDs compared to steady-state conditions.

NIH Research Projects · FY 2025 · 2022-09

Chromosome double strand breaks (DSBs) that evade DNA damage checkpoints can persist into mitosis. These DSBs are in danger of forming highly detrimental structures connected to genome shattering and tumor progression, collectively referred to as micronuclei. To find mechanisms that prevent micronuclei, we discovered that Drosophila papillar cells naturally inactivate DNA damage checkpoints, and as a result frequently exhibit DNA fragments in mitosis. These fragments lack centromeres (acentric DNA), yet remarkably segregate during papillar mitosis. This process prevents micronuclei and tissue development defects. The distinctive dependence of papillar tissue development on acentric DNA segregation holds promise to reveal fundamental responses to DSBs that persist into mitosis. From our combination of in vivo genetic screens, live imaging, and complementary biochemistry approaches with collaborators, we are poised to make unique conceptual advances in this area. This proposal leverages our expertise, new findings, and a genetically amenable Drosophila model to uncover regulation of broken chromosome segregation. The significance of our proposed work is evident in the frequent contribution of micronuclei to genome instability and the evolutionary conservation of the molecules studied, including the Alternative End Joining (Alt-EJ) repair protein DNA Polymerase Theta, conserved monoubiquitination of the DNA repair scaffold FancD2, and the ubiquitin ligase CRL4CDT2. The innovation of our approach derives from our model system that is evolutionarily wired to solve the challenge of frequent persistent broken chromosomes, and the enhanced in vivo genetic screening capability of our system. These advantages led to the preliminary data presented in this proposal. In Aim1, we will define the pathway leading to poleward segregation of acentric DNA. In this Aim, we will identify Pol Theta domains that function in acentric DNA segregation, pinpoint the extent to which Alt-EJ occurs in papillar cells with DSBs, and assess the role of FancD2 in regulating Pol Theta after DSBs. In Aim2, we will define the signaling pathway that promotes the transition from lagging to segregating acentric DNA. In this Aim, we will determine if CRL4CDT2 functions together with Pol Theta/FancD2 to promote acentric DNA segregation, uncover whether critical regulation of CRL4CDT2 activity or in papillar cells with DSBs, and assess if inactivity of interphase checkpoints leads to a requirement for CRL4CDT2 in segregating acentric DNA in a non-papillar cell context (wing cells). In Aim 3, we will determine how regulation of mitotic chromosome condensation contributes to segregation of acentric DNA fragments. We will build on biochemical, genetic, and protein localization data connecting CRL4CDT2 to the mitotic complex Condensin I. We will assess the role of CRL4CDT2 in regulating Condensin I localization during acentric DNA segregation and determine the role of a conserved CRL4CDT2 recognition sequence in Condensin I subunits. Collectively, our approach will uncover fundamental regulation of broken mitotic DNA and can inform on disease-relevant biology.

NIH Research Projects · FY 2025 · 2022-09

5U24AT009676-09 Since 2012, the NIH Pragmatic Trials Collaboratory Coordinating Center (CC) has worked with the NIH to nurture >35 large-scale embedded pragmatic clinical trials (ePCTs) by providing leadership, resources, tools, training, and coordination of diverse elements. In 2019, the CC began working with a group of ePCTs supported by the Pragmatic and Implementation Studies for the Management of Pain to Reduce Opioid Prescribing (PRISM), a program of NIH’s Helping to End Addiction Long-term Initiative (NIH HEAL Initiative). The PRISM trials are determining the effectiveness of non-opioid interventions for treating pain and assessing the impact of implementing interventions or guidelines to improve pain management and reduce reliance on opioids. The CC also supports additional HEAL pain trials to address pain management in rural populations and coordinated pain care in the primary care setting. The CC maintains expert Core Working Groups and committees; supports dissemination and implementation efforts; and established the online Living Textbook of Pragmatic Clinical Trials to translate learnings for the research community. The CC has created and disseminated guidance so that investigators can work with health systems to embed their research in the delivery of health care and use routinely collected health care information as a core data source for the full spectrum of clinical research, including randomized trials. The CC is also providing essential guidance to funders, reviewers, institutional review boards (IRBs), and data safety monitoring boards regarding the ethical and regulatory aspects of ePCTs. In this next phase of work, we will build on lessons learned and new experiences gleaned from the next set of Demonstration Projects to continue to refine the pragmatic trials model (Aims 1-3).

NIH Research Projects · FY 2025 · 2022-09

Abstract – The identification of mutations in the isocitrate dehydrogenase 1 (mIDH1) gene has led to significant advances in understanding lower grade gliomas. Lower grade gliomas, WHO grade II-III, can have a longer median overall survival than WHO grade IV, yet they still develop significant neurological morbidity and ultimately mortality, as standard of care therapies are not curative. New therapeutics are needed to target lower grade gliomas to improve quality of life outcomes and survival for these patients. The mIDH1 mutation exists in over 70% of glioma subtypes and is the oncogenic driver in lower grade gliomas by leading to the extreme overproduction of the oncometabolite R-2-hydroxyglutarate (2HG). Because the mutation is specific to the tumor, it provides significant targets for the development of: 1) targeted therapeutics, such as mIDH1/2 inhibitors and immunotherapeutic vaccines (e.g., vorasidenib and PEPIDH1M, respectively) and 2) non-invasive tumor diagnosis and monitoring methods, via magnetic resonance spectroscopy (MRS). More recently, it has been reported that in addition to driving the oncogenesis of lower grade gliomas, 2HG has immunosuppressive actions. Its role as a driver of gliomagenesis and now induction of immunosuppression of tumor-fighting T-cells places 2HG as a critical target in fighting lower grade gliomas. Thus, we propose a clinical trial for a combined therapy of vorasidenib and PEPIDH1M. We hypothesize that early suppression of 2HG by vorasidenib would allow subsequent or concurrent administration of PEPIDH1M vaccine to generate a more robust T-cell response and lead to increased immune-mediated tumor cell kill (Specific Aim 1). In this phase I, single-site clinical trial, we will determine the safety of this combinatorial approach. Toxicity events will be evaluated and graded by CTCAE 5.0 criteria. The RANO criteria will be used to assess radiographic progression-free survival based on routine MRI imaging. As an exploratory goal, we will map 2HG across the brain longitudinally using existing high-speed magnetic resonance spectroscopic imaging (MRSI) and offline processing to monitor the effects of vorasidenib (Specific Aim 2). Finally, in parallel with the clinical trial, we will convert the validated 2HG mapping tool into a standardized works-in-progress (WIP) package for reporting 2HG levels on both GE and Siemens scanner DICOM workflow (Specific Aim 3). This project builds on 1) our previous experiences participating in the early clinical trials for PEPIDHM1 and vorasidenib, where we also characterized longitudinal 2HG levels, and 2) our decades-long experience with automating MRS methods and translating them into manufacturer supported WIP packages.

NIH Research Projects · FY 2025 · 2022-09

Abstract Electrotransfer (ET) or electroporation has been widely used to deliver molecular cargo into cells for genome and epigenome editing, cell engineering, and DNA vaccination in treatment and prevention of diseases (e.g., the COVID-19 pandemic). To enhance ET efficiency, different chemicals have been combined with electric pulses in treatment of cells. However, a challenge with this strategy is how to screen for chemicals. In most studies, the screening process is accomplished empirically in vitro, but this approach is impractical or prohibitive in vivo. Furthermore, a chemical treatment that works well for ET in cultured cells may not necessarily work for ET in the body. To this end, the proposed study will explore a new approach to chemical screening. Since mechanisms governing molecular transport are conserved across different cells and tissues, can chemical treatments that assist DNA transport be used to ubiquitously improve ET in vitro and in vivo? To answer this question, the Yuan lab will build upon their previous successes to deepen the understanding of transport mechanisms. One area of focus is to investigate cytoplasmic transport. Specifically, the study will determine mechanisms of vesicular transport and the escape of DNA from vesicles prior to nuclear entry. The second area of focus is to investigate the nuclear entry of DNA in non-dividing cells. The study will address key questions such as: How does DNA travel through the nuclear pore complex? How does the transport depend on the size and structures of DNA? How does the amount of nuclear localization signal or DNA nuclear targeting sequence per DNA molecule influence the transport? In addition to the mechanistic studies, the Yuan lab will develop new techniques to screen for nontoxic compounds and nanoparticles that can be used to improve cytoplasmic transport and nuclear entry. The third area of focus is to explore different combinations of strategies to simultaneously improve intracellular and extracellular transport of DNA in tissues. The study will demonstrate that the combination can synergistically enhance ET efficiency and prolong the transgene expression in vivo. The Yuan lab has extensive experiences in the analysis of molecular transport in cells and tissues, especially in the context of ET research. This R35 will give the lab the flexibility and power to advance the understanding of transport mechanisms, and open new avenues for research and applications. Findings from the studies mentioned above will lead to the development of common strategies to enhance DNA transport that in turn will improve ET in vitro and in vivo.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY/ABSTRACT DNA shed from tumors into the blood stream, termed circulating tumor DNA (ctDNA), is an easily obtained source of tumor material. As most ctDNA is identical to normal DNA, some distinguishing feature is needed to demark a cancer origin. In this regard, a fifth or more of all human cancers harbor a cancer-causing (oncogenic) point mutation in the gene KRAS. This raises the exciting possibility that sequencing for the presence of KRAS-mutant ctDNA could be used to detect many types of cancers from a simple blood draw. Indeed, the Guardant360® ctDNA-detection assay is used for just this purpose in the clinical care of cancer patients. The challenge to detecting ctDNA is that this form of DNA is found at vanishingly low levels in the blood. This limitation is borne out in our own clinical experience at Duke, where we find that the Guardant360® assay successfully detected KRAS-mutant ctDNA in only half the cases in which the patient's cancer was documented to be KRAS mutation- positive by direct sequencing of resected or biopsy tumor tissue. Thus, while Guardant360® is real-world proof that ctDNA can be used as a `liquid biopsy' in the clinic, there is clearly much room for improvement. In this regard, we adopted the Maximum Depth Sequencing (MDS) technology, originally developed in the microbiology field to detect rare antibiotic-resistance mutations in bacteria populations, to detect oncogenic mutations in KRAS. By barcoding the original KRAS template and making multiple first-strand replicates thereof, coupled with ultra-deep sequencing of these targeted DNA products, we were able to detect mutations engineered into KRAS templates at a limit of 5x10-7, which is 2,500 to 50,000 times more sensitive than the detection limit of 1x10-3 to 2x10-4 reported for the Guardant360® assay. Given this, we will combine the basic research of Dr. Counter into this KRAS-specific MDS (K-MDS) assay with the clinical and translational expertise of oncologist Dr. Abbruzzese to optimize the K-MDS assay for blood samples (aim 1) and then evaluate K-MDS to Guardant360® a prospective clinical comparison (aim 2). Completion of this study will thus provide an new technology to screen for KRAS-mutant ctDNA in the blood of cancer patients at a sensitivity orders of magnitude greater than current clinical assays, initially to monitor either recurrence of KRAS-mutant cancers or detect such cancers in high-risk patients, but more long term, in combination with screening for other hotspot mutations and using different sources of tissue, for the early detection of multiple cancer types.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT The burden of cancer disproportionally affects children in low- and middle-income countries (LMICs), which accounts for over 80% of global childhood cancer cases and deaths. Children in LMICs are four times more likely to die of cancer than children in high-income countries. One of the main reasons impacting poor outcomes for vast number of children with cancer in LMICs is the lack of strong health systems, which impacts timely access to care along the entire care continuum. In order to improve cancer outcomes for children in LMICs, interventions are needed to improve comprehensive care using a health system strengthening approach . My long-term goal is to improve outcomes for children with cancer in LMICs through support of locally-relevant programs to strengthen health systems. I am a global health epidemiologist with a passion for children’s health care in LMICs. Although my formal training and research experiences have been formative for the analysis of disease patterns and health systems, I will use this award to develop skills to improve health systems and to translate epidemiologic data into clinical practice. With my extensive epidemiologic experience, I have previously measured and identified delays in care for children with surgical needs in LMICs due to geographic, financial, and health system barriers. However, I lack the expertise to translate those findings into practice through real-world, evidence-based, sustainable solutions. Training in this gap between science and practice requires an integrated approach to health system strengthening. To achieve my career goals, I need additional training in health services research and implementation science. The skills acquired during the K01 training grant will equip me to become an independent investigator in low-income settings with my career goal of becoming researcher to improve global health systems for children. My mentorship team’s expertise in global care for children, health services research, implementation science, and intervention development will support my proposed training and research objectives. In addition, the proposed aims will gather preliminary data needed for the next step of a R01 proposal aimed at testing interventions to improve cancer care for children in Tanzania.

NIH Research Projects · FY 2025 · 2022-09

Project Summary / Abstract Autoimmune diseases (AD) are the third most prevalent disease in America, disproportionately impacting females 4x more than males. Despite the high prevalence and cost to society, there are few effective treatments and no definitive cure because the genetic factors and environmental triggers for autoimmunity remain poorly defined. Existing studies implicating hormonal differences to explain the sex differences in autoimmunity and immune response fail to reconcile the increased susceptibility to autoimmune diseases in Kleinfelter males, who have two X chromosomes (XXY) like females (XX) and unlike most males (XY). In XX individuals, the long noncoding RNA (lncRNA), XIST (Xist in mice), is required for silencing one of the X’s to achieve gene dosage compensation. This project aims to elucidate the XX-linked preponderance for autoimmune disease development through studying the female-specific lncRNA, XIST, and the proteins associating with XIST in the XIST ribonucleoprotein complex (RNP) as a potential immune complex trigger for autoimmunity. In this proposal, Dr. Dou proposes to test the novel hypothesis that Xist RNPs increase autoimmune risk using three multi-level aims: (1) In vitro, through controlled stimulation of immune cells with Xist RNPs, (2) In vivo, through autoimmune disease modeling in a transgenic Xist mouse wherein male mice viably express Xist RNPs, (3) diagnostically, using autoimmune disease patient serum to test reactivity against XIST RNP proteins. Completion of the project will build a comprehensive model of the pathways, genes, and specific immune cell types involved using powerful and high-resolution single-cell sequencing (Aims 1 and 2), disease models in mice (Aim 2), rigorous and specific clustered regularly interspaced short palindromic repeats (CRISPR) gene perturbation experiments (Aim 1), and a sensitive protein antigen array (Aim 3). This work will be performed in a world-class environment at Stanford University under the supervision of Dr. Howard Y. Chang, a lncRNA authority who excels at developing and applying sequencing techniques to study diseases, with co-mentorship from Dr. PJ Utz, a clinically trained rheumatologist with particular expertise in autoantibodies and autoimmune disease mouse models. An advisory committee and consisting of experts in single sequencing in immune cells (Dr. Satpathy), high throughput CRISPR screens (Dr. Bassik), proteomics (Dr. Lundberg) and autoimmunity (Dr. Fiorentino) will provide additional mentorship to Dr. Dou and the resources necessary to achieve her project goals. Completing the project and associated training plan will allow Dr. Dou to meet key milestones in her transition to independent investigator. The 3 Aims are designed for parallel investigations. The first half of each aim will be completed during the mentored K99 phase to provide the platform for the R00 independent phase. This project will be the springboard for Dr. Dou to launch an independent career and achieve her long-term goal of resolving the genetic and epigenetic factors underlying autoimmune diseases.

NIH Research Projects · FY 2025 · 2022-09

Excessive alcohol use is the 5th-leading risk factor for premature death and serious morbidity. The health and economic burden associated with alcohol use is concentrated in ~14 million adults with alcohol use disorder. Pharmacological and behavioral interventions, especially when combined, reduce alcohol use and related harms and assist long-term recovery. However, only ~5% adults with alcohol use disorder receive formal treatment in health care settings. Recent societal phenomena, namely the rapid rise of high-deductible health plans and reduced health care used due to the 2020 public health emergency, might contribute to delayed alcohol use disorder diagnosis and treatment. Deferred care might especially affect low-income and rural residents. In the last decade, high-deductible plans requiring potentially prohibitive out-of-pocket payments for alcohol use disorder services have expanded rapidly, now covering 57% of workers. The 2020 public health emergency led to stay-at-home orders and closure of nonessential businesses, dramatically reducing healthcare use. The overarching goal of this proposal is to examine major societal factors affecting alcohol use disorder treatment access among lower- versus higher-income populations, including modifiable high-deductible health plans and the 2020 public health emergency. The study will assess alcohol use disorder-related measures before and after 2 key change dates of interest: the date that employers mandate a switch to high-deductible health plans (using a rolling cohort accrual period), and March 2020 when restrictions due to the 2020 public health emergency began. More specifically, the project seeks to: (1) determine the effect of high-deductible health plans on alcohol use disorder diagnosis, treatment, and adverse outcomes; (2) assess whether high-deductible health plans differentially affect alcohol use disorder diagnosis, treatment, and adverse outcomes among key subpopulations; and (3) examine the 4-year impact of the 2020 public health emergency on alcohol use disorder diagnosis, treatment, and adverse outcomes among key subpopulations. The study will draw from an 18-year rolling sample (2007-2024) of ~50 million members aged 18-64 enrolled through a national health insurer. The study will apply rigorous, quasi-experimental interrupted time series designs with segmented regression and segmented survival analyses. We expect that findings will demonstrate the health insurance benefit designs that optimize access to AUD treatment, informing potential modifications to Internal Revenue Service regulations that exempt certain services from high out-of-pocket costs under high-deductible plans. Findings could also help inform policymaking by identifying subgroups at risk of delayed diagnosis and treatment during public health emergencies.