University Of California, San Diego

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY/ABSTRACT This resubmitted project evaluates the role of the positive valence system (PVS) in the interpersonal processes of disclosure, help-seeking, and safety planning in a transdiagnostic sample at risk for suicide. Interpersonal factors are central to contemporary models of the transition from suicide ideation to behavior, and social resources are key to effective suicide safety planning. Yet, interventions to promote informal help-seeking have been ineffective to date, and almost half of people do not disclose suicidal thoughts to anyone prior to attempt. Basic research on the mechanisms underlying social approach or avoidance behaviors in suicide could help identify new targets for more effective suicide prevention interventions. Here, we focus on investigating how the reward system (i.e., valuation, responsiveness and/or learning) is involved in engaging in social outreach behaviors before and during crises. In this proposal, we will recruit participants with either affective or psychotic disorders, stratified by current active suicidal ideation. Purposive sampling of both affective and psychotic diagnoses enables inclusion of a group with a similar high background risk of suicide, yet varying PVS and negative valence system (NVS) profiles. We will use a measurement burst longitudinal design which integrates lab-based tasks with bouts of ecological momentary assessment and passive sensing. Participants will be followed longitudinally for 12 months. Among lab-based tasks, we will administer our validated dyadic paradigm that simultaneously evaluates PVS and NVS components in a simulated social affiliation and disclosure context, and enables facial emotion coding and natural language processing of speech. In Aim 1, we will administer lab-based tasks and focus on evaluating the impact of PVS on suicide- related social affiliation, including social elements of the standard safety plan (e.g., people to contact in crisis) and help seeking opportunity within each individual’s social network. In Aim 2, we will use mobile assessments to model short-term dynamics of suicidal ideation and whether and to whom ideation is disclosed. In Aim 3, we will integrate lab-based and ecological momentary assessment data streams to build and test integrated predictive models of suicidal behavior over 12 months, evaluating stability of effects across affective or psychotic syndromes. We will evaluate whether social affiliation dynamics mediate the relationship between PVS components and suicidal ideation and behavior. The intended products of this research include an integrated dataset informative for translational interventions to improve long-term and acute suicide prevention. Innovative aspects of the proposed study include the lab-based dyadic social affiliation task, use of mobile health and computational methods to model dynamic aspects of help seeking, and the focal comparison of psychotic and affective syndromes. This proposal aligns with RDoC by probing connections across domains (PVS, NVS and Social Processes) in a transdiagnostic sample, and is also responsive to the NIMH Strategic Plan (Aim 2.2, Aim 3.2) and the NIMH Notice of Special Interest in Digital Mental Health.

NIH Research Projects · FY 2025 · 2023-05

Project Summary/Abstract Prostate cancer affects nearly 1 in 7 men in the United States yet the diagnosis of early aggressive cancer remains elusive at the expense of many unnecessary biopsies, delay in diagnosis from biopsy sampling error, or the diagnosis of indolent disease. In order make a diagnose clinically significant prostate cancer, there is an urgent need find an imaging modality that can be implemented uniformly, yet also provide clinical utility. MRI- guided prostate biopsy has rapidly become a common modality to perform prostate biopsy; however, limited emphasis has been placed on the quality and accuracy of the image acquisition. Our proposal supports an academic-industry partnership to develop and imaging biomarker that could revolutionize how prostate cancer is diagnosed and monitored by using restriction spectrum imaging (RSI)-MRI. We seek to improve the operating characteristics of prostate MRI with a novel, non-invasive method of a short- duration, targeted magnetic RSI-MRI sequences then undergo FDA-cleared, class II post-processing software (OnQ Prostate) to attribute values to suspicious areas of the prostate. RSI-MRI employs multiple b-value acquisitions with multiple diffusion gradient directions at each b-value to acquire raw data; images are post- processed to isolate the signal from isotropic, restricted water compartments typically found in cancer cells. A resultant “restricted signal map” corresponding to tumor location is derived and quantified functioning as an in vivo biomarker of tumor grade and enhances tumor conspicuity for reader detection. In Aim 1, weseek to validate our previous findings regarding the operating characteristics of the RSM values and its association with clinically significant prostate cancer (grade group 2 or higher). We will use a non-randomized, single arm clinical trial to investigate both PI-RADS scores with and without the RSM values. In Aim 2, we see to calibrate the RSM map values across manufacturers(Siemens, GE, and Phillips MRI scanners). We will use a two-scan approach which will invite a sample of men with a range of PI-RADS values to undergo a research reference scan after they have undergone their standard of care scan within one of three locations housing a specific MRI vendor. The two scans will be compared and used to calibrate the RSM values using several calibration and normalization techniques with targeted biopsy results. In some men that do undergo prostatectomy, we will further make a mold of the prostate using the preoperative MRI then perform whole-mount sectioning, scanning, and alignment with RSI-MRI to obtain accurate reads of normal and cancer areas. In Aim 3, we will compare RSM to currently available FDA-approved biomarkers and clinical risk to determine clinical utility. In the proposed project, we seek to improve specificity of prostate MRI using RSI. Specific deliverables will include the ability to amend the standard radiological reporting system (PI-RADS) withspecific RSM output vales. We seek to validate the restricted signal maps between vendors and provide clinical assessment tools demonstrating a measurement of clinical utility.

NIH Research Projects · FY 2026 · 2023-05

Project Summary/Abstract The Microbiota-Diet-Host interactions are critical to the balance of health and disease. Understanding this axis will be important to the development of nutritional precision medicine. Although there are some important discoveries where microbes make molecules that dramatically affect health, most of the molecules produced by microbiota still remain structurally uncharacterized. In addition, even when a molecule is known and the activity is known, this knowledge tends to be buried in papers and there is not a systematic organization of this knowledge that can be readily leveraged by data scientists. This prevents a deep mechanistic understanding of the microbiome. The goal of PAR-21-253 to which this application is applying to and the accompanying RFA (RFA-DK-21-014) is to build a microbial metabolite knowledgebase that can be used by the larger scientific community. In order to build the knowledgebase, the R01’s funded under PAR-21- 253 are discovery grants that will provide the knowledge of new microbial metabolites and their bioactivities. This proposal will 1) obtain MS/MS signatures for up to 5,000,000 molecules synthesized, using combinatorial/diversity driven synthesis, using diet and host precursors that are accessible to human microbiota. The synthesis is biased towards common bio-transformations. This will be the largest metabolomics reference data set ever assembled, even when all public and non-public libraries are combined; 2) we will use our big data mining strategies, especially our mass spectrometry search tools, to not only discover what molecules are made by microbes, but also understand phenotype, disease, organ/tissue/biofluid, food associations; 3) the newly discovered microbial molecules that are up or down regulated in inflammatory bowel disease (and other gut metabolic disorders) will be further screened in cell-based assays and colitis animal studies to understand the biological effects of the newly discovered microbial metabolites; 4) we will transfer all the knowledge obtained by this R01 to the knowledgebase that will be created by the accompanying RFA. The PIs and Co-Is are well suited to help build the proposed knowledge base, due to their expertise in microbial metabolomics (Dorrestein), synthesis (Dorrestein, Siegel), microbiome (Dorrestein, Knight, Raffatellu), data science (Dorrestein, Knight) and having a track record of doing very large projects and share newly discovered knowledge publicly for the benefit of the community – generally made available years before any publication (Dorrestein, Knight).

NIH Research Projects · FY 2026 · 2023-05

The field of ASD screening is at a crossroads: the sensitivity of the most popular screening tool is only 33%- 38%1,3, and pediatricians consistently refer only about a third of children who fail a screening tool for an evaluation4,5 - citing a lack of confidence in screening results as the primary reason for non-referral5. Within this context, it is not entirely surprising that the mean age of ASD diagnosis and eventual treatment remains at ~52 months6 - years beyond the disorder’s prenatal origins7, and beyond the age when it can be reliably diagnosed in many cases8. Clearly, new approaches need to be tested. Eye-tracking, which generates biologically-relevant, objective, and quantifiable metrics of social and non-social visual attention patterns, is a technology that holds considerable promise as a tool to dramatically change how screening is implemented. With the help of NIH funding, we developed 6 novel eye tracking tests that tap into key challenge areas for children with ASD including visual social attention, gaze shifting, and auditory social attention. Leveraging our large legacy eye tracking dataset collected from >2,000 toddlers spanning multiple diagnostic groups including ASD, non-ASD delay, and TD we determined optimal eye tracking metrics and cut-off values across for each test that result in very high specificity and PPV (~97% & ~90%) but modest sensitivity (~20% per test). Combining across all 6-tests however, dramatically improves sensitivity (~90%) and results in high classification accuracy (AUC .95). These findings, however, were demonstrated in a laboratory setting with utility in real-world clinical settings unknown. Thus, in AIM 1, we take the bold step of testing whether eye-tracking administered across 8,000 12, 18, & 24 month well-baby check-ups (from ~5,2000 unique toddlers) serving families from a wide range of racial, ethnic, and SES backrounds can improve ASD early screening when implemented by medical staff in pediatric offices. Toddlers who fail eye-tracking using resarcher defined criteria, and a percentage who pass, will be evaluated by a licensed psychologist blind to eye tracking scores, and diagnostic classification accuracy of eye-tracking computed. Relationships between eye tracking profiles and clinical phenotype will also be examined. In order to fully understand the accuracy of eye tracking as a screening tool, diagnostic outcomes of the entire screened cohort needs to be determined. Thus, in AIM 2 electronic health records (EHRs) will be leveraged to allow us to not only determine the true sensitivity, specificity, PPV and NPV of eye tracking for detecting ASD, but to also compare results to rates of ASD detection using the CSBS, a parent report screening tool used as standard of care in San Diego as part of our Get SET Early model9. State of-the-art bioinformatics will allow us to further determine if combining eye-tracking with parent report is superior relative to either approach alone. Statistical modeling will reveal whether or not factors such as age at screening, sex, race, ethnicity or SES impacts eye tracking scores. Finally in AIM 3, pediatricians and parents will rate their satisfaction with eye tracking.

NIH Research Projects · FY 2026 · 2023-05

Project Summary/Abstract The exact mechanisms by which light stroking and deep pressure – components of massage therapy – induce pleasantness and pain modulation are not understood. Given the frequent use of massage therapy for relaxation and pain relief, and the need for complementary tools for chronic pain, there is a critical need to study the mechanisms of specific forms of affective touch. Our long-term goal is to elucidate the mechanisms by which manual therapies induce pleasant relaxation and pain relief. Our overall objectives in this application are to (1) Determine the extent to which A- and C-fibers contribute to affective effects of gentle stroking, 2) Determine the extent to which A- and C-fibers contribute to affective effects of deep pressure, and 3) Determine the contribution of C-fibers to neural mechanisms of touch-induced pleasantness and pain modulation. We will also (Exploratory) use machine learning to identify individual contributions of C-tactile (CT) fibers to neural mechanisms of touch pleasantness and pain modulation, and the association of interoceptive sensibility with CT effects. Our central hypothesis is that A-fibers are necessary but not sufficient for the pleasantness of gentle stroking and unnecessary for its pain reduction, but are necessary and sufficient for the pleasantness and pain reduction of deep pressure. Further, we hypothesize that left anterior insula and anterior cingulate cortex activation will predict CT contributions to modulation of pain by touch. We will conduct a two session within-subject study in healthy adult volunteers to test the effects of A- and C-fiber blockade on the pleasantness and pain modulation induced by slow stroking (N = 24) and deep pressure (N = 24). We will then test effects of A-fiber blockade on brain mechanisms of pain modulation during fMRI, and relationships between CT contributions and touch pleasantness, pain modulation, and interoceptive sensibility (N = 36). Effects of CT inputs on heart rate variability will comprise a secondary outcome. Upon successful completion of the proposed research, we expect to have identified the role of A- and C-fibers in the pathways and major effects of affective touch. This contribution is expected to be significant because it will define the pathways for two major forms of affective touch and their effects on pain, providing reliable information about non-invasive measures for pain control and potential targets for noninvasive neuromodulation of pain. Further, this project is innovative because it explores a novel affective touch pathway, applies a novel method to causally test afferent pathways for affective touch, and uses machine learning to explore individual differences in the contributions of CTs to neural mechanisms for affective touch. Our proposed project seeks to elucidate pathways for two major forms of affective touch commonly engaged by massage. This research will have a positive impact by opening new horizons for mechanistic research on effects of touch in health and disease, and may provide targets for neuromodulation in the treatment of pain.

NIH Research Projects · FY 2025 · 2023-05

Project Summary/Abstract Iron is an essential trace mineral involved in vital cellular and systemic functions. Humans and other vertebrates require a sufficient iron supply to carry out essential metabolic functions and oxygen transport while maintaining homeostatic iron levels to prevent iron toxicity. Organismal iron content is controlled by dietary absorption, iron partitioning in erythrocytes, iron recycling by macrophages, and iron storage in hepatocytes. Precise machinery has evolved to conserve iron stores and regulate iron delivery to tissues. A critical member of this system is hepcidin, a liver-derived peptide hormone. Hepcidin is a master regulator of systemic iron homeostasis through its ability to negatively regulate ferroportin, the sole known mammalian iron-exporter in the cell. Hepcidin synthesis is activated physiologically by elevated serum iron level and pathologically by inflammation and infection. Hepcidin expression is repressed by increased erythropoietic demand. Heparan sulfate (HS), a prominent glycan part of the cell glycocalyx, was recently established as a critical component of hepcidin regulation. Inhibition of HS biosynthesis in hepatoma cells, primary human hepatocytes, and mice reduced baseline and stimulated hepcidin expression and worsened the pathophysiology characteristic of anemia of inflammation. Preliminary studies have identified Syndecan-1 (SDC-1), the primary heparan sulfate proteoglycan (HSPG) in the liver, as a novel cell surface receptor involved in hepcidin regulation. Genetic and pharmacological inactivation of SDC-1 demonstrated that hepcidin expression is SDC-1-dependent in human hepatoma models. However, its precise mode of action remains to be elucidated. The proposed study will (i) validate SDC-1 as the key HSPG that regulates basal and inducible hepcidin expression, identifying a novel arm of iron homeostasis modulation, (ii) elucidate the mechanism by which SDC-1 regulates hepcidin in vitro which can provide novel insights into the control of iron homeostasis and (iii) use in vivo liver-specific SDC-1 inactivation to study the impact of SDC-1 depletion and structural alteration on systemic iron homeostasis. The overarching goal of this proposal is to evaluate the relationship of Syndecan-1 structure to iron metabolism, with the long-range goal of defining new potential targets to reduce the risk of iron-loading disorders, such as hemochromatosis and anemia of inflammation.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY Activating Transcription Factor 4 (ATF4), also known as cAMP-Response Element Binding Protein 2 (CREB2), belongs to the ATF/cAMP responsive element-binding (CREB) protein family of transcription factors. As a well- characterized stress-response transcription factor, ATF4 is constitutively and ubiquitously expressed at low levels but can be rapidly induced under a variety of cell-stress conditions. Previous studies have shown that ATF4 functions in diverse cell types and tissues under various physiological and pathological conditions, including cardiac diseases. However, little is known as to the specific role of ATF4 and its target genes in mammalian cardiac development. To address this gap in knowledge and to determine the role of ATF4 in cardiomyocytes (CMs), we have generated novel Atf4 CM-specific constitutive knockout (cKO) and tamoxifen- inducible CM-specific knockout (icKO) mouse lines. Our preliminary studies revealed that Atf4 cKO mice exhibited perinatal death and cardiac morphological defects, associated with reduced CM proliferation. RNA- seq and ChIP-seq analyses of embryonic ventricular tissues revealed upregulation of a series of cell cycle arrest-associated genes known to be downstream of p53, and downregulation of a series of p53-independent cardiac development/function and/or cell cycle progression associated genes, most of which harbored ATF4 binding regions, in Atf4 cKO mice. Loss of ATF4 in developing CMs also resulted in increased p53 protein levels but not Trp53 mRNA levels. Moreover, p53 ablation in Atf4 cKO mice partially restored ventricular wall thickness and ameliorated upregulation of p53 target cell cycle arrest genes at E17.5, but failed to rescue lethality beyond postnatal day 1. Conversely, inducible ablation of Atf4 in adult CMs had no effect on cardiac function or left ventricular dimension, suggesting distinct roles for ATF4 at specific stages of CM development. Taken together, the foregoing evidence leads us to the hypothesis that ATF4 plays an essential role in CM proliferation and function via p53-dependent and -independent mechanisms at specific stages of cardiac development. Accordingly, our Specific Aims are: 1. To determine the role of ATF4 in cardiomyocyte proliferation and cardiac development by analyzing Atf4 cardiomyocyte-specific knockout (Atf4 cKO) mice, and to elucidate mechanisms by which ATF4 regulates target gene pathways in a p53-dependent and/or -independent manner; and 2. To determine times at which ATF4 is required for embryonic and neonatal cardiomyocyte proliferation and function (from E7.5 to P30) by analysis of Atf4 inducible cardiomyocyte- specific knockout (icKO) mice. Our proposed studies will help us to understand the specific roles of ATF4 and p53, as well as other novel ATF4 targets in CMs at critical developing stages in vivo, as well as to determine a safe therapeutic window for the potential application of ATF4 and/or p53 inhibitors in cardiac diseases.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY While transformative, all-oral treatment regimens have recently been endorsed by World Health Organization (WHO) for the treatment of drug resistant TB (DR -TB), the efficacy of these new regimens is dependent on prior knowledge of the susceptibility profile of key drugs before treatment initiation. Companion molecular diagnostics for drug susceptibility testing (DST) of the new and repurposed anti-tuberculosis drugs is the most likely path to achieving practical and timely DST for these new regimens. However, there are currently no commercially available molecular diagnostics to detect mutations conferring resistance to bedaquiline, pretomanid, linezolid or delamanid, nor is there an easy developmental path forward using existing nucleic acid amplification test (NAAT) approaches given the complexity of the molecular targets for these drugs. Culture- free, next generation sequencing (NGS) has the greatest potential for delivering a comprehensive diagnostic solution, but existing workflows are highly complex, expensive, and rely on highly skilled staff to run them. To achieve broad adoption of NGS approaches, particularly in low resource settings, these workflows must be simplified substantially to reduce both their complexity and cost. Our goals for the proposed study are to bring culture-free NGS closer to patient care by simplifying NGS workflows for DR-TB diagnosis to the point that NGS can be run mostly hands-free by any laboratorian who can run a NAAT and to reduce costs by removing several enzymatic processes. Our objective is to demonstrate that automated, single amplification sequencing (ASAS) can be used to accurately diagnose resistance to all legacy and new/repurposed drugs for which there are recognized molecular targets (i.e., isoniazid, rifampicin, pyrazinamide, amikacin, moxifloxacin, bedaquiline, clofazimine, linezolid, and delamanid). We will achieve this objective by completing the following three aims; Aim 1) Expand with additional gene targets an existing single amplification targeted NGS assay and describe the assay performance; Aim 2) Integrate an existing Akonni sputum extraction/PCR workstation with our novel single amplification sequencing assay and sequence on Illumina iSeq100; and Aim 3) Evaluate the accuracy of the ASAS solution for detection of drug resistance against a phenotypic and genotypic reference standard in sputum samples from patients at risk for DR-TB under field conditions at clinical laboratories in India, South Africa, and Moldova. The results of this project will demonstrate key characteristics of the ASAS workflow and will provide a solid developmental foundation for the application of this tool in clinical settings, reducing cost and time-to-result for comprehensive DST without culturing.

NIH Research Projects · FY 2025 · 2023-04

PROJECT SUMMARY: The objective of this proposal is to test cerebroprotective interventions for the SPAN program by utilizing an intraluminal middle cerebral artery occlusion (MCAO) mouse model or an animal blood clot embolic model. Our lab has been using innovative techniques in producing highly consistent mouse MCAO and clinically relevant animal blood clot embolic stroke models. Our animal surgeons have more than 15 years of experience in various animal ischemic stroke models and have performed surgeries on thousands of animals of various species (e.g., mice, rats, and rabbits). We established an easy-to-use technique to monitor the middle cerebral artery (MCA) blood flow throughout the peri-MCAO periods. Our animal operating and behavioral exam rooms have about 900 square feet of space and were completely renovated in 2019. Our three animal surgical stations with matching monitoring instruments are state-of-the- art. Our Bruker 9.4 T MRI Scanners are advanced and allow for high-resolution quantitative assessment of CNS structures and functions. Our state-of-the-art infrastructure, together with our highly skilled personnel, allows us to run multiple studies in parallel. We have about a decade of experience in performing interactive multi-institutional projects funded by the NIH. We have published more than ten studies about stroke-related comorbidities. Death and disability/dependency are always key primary outcome measures in stroke clinical trials. However, many functional tests in animal stroke studies are not designed to assess animal “natural (i.e., minimal investigator interaction)” disability; rather, animals are required or forced to perform tasks. These tasks are highly useful to test specific deficits but may differ from “natural” disability/dependency that presents in stroke clinical trials. Therefore, we recently established two novel tests to reflect animal long-term “natural” disability (unable to work, eat, and drink by themselves): (i) nest building activity measuring ability to work, and (ii) PhenoTyper monitoring the “total” activity, food and drink intake activities, and time of death. These long-term “natural” behavioral tests are objective, easy-to-use, measurable, and sensitive, and may mimic the clinically relevant disability/dependency benchmarks used in stroke clinical trials. In the first year, we will: (i) set up the required infrastructure and animal models; (ii) sign agreements and participate in all SPAN meetings; (iii) share data with the Coordinating Center (CC); and (iv) execute the animal studies following the assignments and protocols set forth by SPAN. In the second year, we will further: (i) execute the animal studies; (ii) present our results to SPAN for recommendations of go/no-go, and (iii) participate in all SPAN meetings and share resources, infrastructure, and protocols. In the third year, we will continue to execute the animal studies and participate in all scheduled SPAN meetings. We will consult the CC: (i) to get a consensus of the best intervention(s) and, if agreed by the CC, (ii) validate the clinical benefits of the best intervention(s) with a clinically relevant animal blood clot embolic stroke model.

NIH Research Projects · FY 2026 · 2023-04

THE NETWORK DATA EXCHANGE – A NETWORK COMMONS FOR BIOLOGISTS PROJECT SUMMARY Knowledge of biological networks has been critical to the analysis of big biomedical datasets, including interpretation of the molecular heterogeneity seen within and across tumors. NDEx, the Network Data Exchange (ndexbio.org), is an online commons where scientists find, share, and publicly distribute biological networks. With a previous grant from the Informatics Technology for Cancer Research program (ITCR U24 CA184427), we created the NDEx open-source database platform along with its public website and query system. Further, we enabled interoperability with the Python and R data science platforms and built NDEx seamlessly into the Cytoscape network analysis and visualization ecosystem. NDEx presently serves a large base of users and network content, with approximately 3285 accounts linked to >180,000 networks, of which approximately 6500 are publicly accessible. NDEx is the approved network repository for articles published in the Springer Nature and Public Library of Science (PLoS) families of journals. We are requesting sustained ITCR funding for the NDEx project to maintain, scale, and enhance the NDEx system, meeting the evolving needs of the biomedical research community via the following five Specific Aims: 1. Maintain and scale NDEx and its services to support a large and growing community of users. Regular software releases of NDEx will continue to extend its capabilities. Targeted server hardware upgrades will increase capacity and performance to match the growing use of NDEx. 2. Extend the NDEx corpus of cancer pathway networks. We will continue to maintain and extend the NDEx Cancer Collection (NCC) corpus of networks via outreach, collaborations, and automated text and image analysis methods to capture cancer networks from literature. 3. Enable the use of networks in next-generation cancer genome analysis. We will enhance the use of networks in existing 'omics analysis tools used by the cancer research community and develop and maintain analysis tools that leverage NDEx functionality and data. 4. Enable publishing of interactive networks. Building on our success in enabling publications to link to NDEx networks, we will add new capabilities for authors and readers to enhance the dissemination of interactive networks and their use in publications. 5. Provide support and training, track usage, and gather feedback. The NDEx Project is committed to user engagement and development driven by the needs of cancer researchers and their user feedback. Through these aims, the NDEx project will transition from system-building to sustaining and enabling network biology for the NCI-supported cancer community.

NIH Research Projects · FY 2026 · 2023-04

Abstract Sensorineural hearing loss is a debilitating condition with no cure that directly impacts >30 million people. A common cause of inherited hearing loss is the disruption of cochlear hair cell stereocilia, which are essential for transducing sound vibrations to the brain. For proper function, stereocilia must be tall enough to reach the tectorial membrane (TM) and form stereocilia-TM junctions (STJs). Stereocilia elongation greatly depends on actin regulation; as such, disruption of actin regulatory proteins and other cytoskeletal elements cause profound hearing loss in humans and model organisms. Thus, it is critical to elucidate the dynamic regulation of stereocilia lengths to understand normal hearing and determine how to rescue stereocilia-related hearing loss. Previous work demonstrated that stereocilia actin filament elongation depends on epidermal growth factor pathway substrate 8 (Eps8), an actin-regulatory protein that is critical for hearing in both mice and humans. Eps8 paradoxically contains domains with both actin filament capping and bundling activities, which are typically associated with shortening or elongation of actin filaments, respectively. Moreover, it was found that two other deafness-associated proteins essential for stereocilia elongation, Myosin-XVa (MyoXVa) and whirlin, bind to Eps8 at stereocilia tips in a tripartite complex. Preliminary data show that stereocilin, another deafness-associated protein which links stereocilia to the TM at STJs, is not properly targeted to stereocilia tips in Eps8, MyoXVa, or whirlin knockout (KO) mice, and that all these mice lack normal STJs. Moreover, early (≤postnatal day 1) adeno-associated virus (AAV)-mediated delivery of Eps8 can rescue stereocilia elongation, stereocilin localization, and STJs in Eps8 KO mouse apical hair cells, suggesting that it may be possible to rescue hearing function. This proposal tests the hypothesis that Eps8 regulates stereocilia elongation and the proper formation of STJs by directly regulating actin bundle growth through its C-terminal actin capping and bundling regions and indirectly regulating stereocilia growth through interactions with MyoXVa and whirlin. Further, it is proposed that there is a critical window of hair cell maturation during which stereocilia plasticity is sufficient for full rescue. To test these hypotheses, an AAV-mediated delivery of Eps8 mutants lacking either or both capping and bundling domains (Aim 1) or Eps8 mutants lacking MyoXVa-, whirlin-, or actin-binding activity (Aim 2) in Eps8 KO models will be used. In addition, novel light- and chemically-inducible Eps8 mice will be employed to explore cochlear plasticity and define the critical window for restoring hair cell function in vivo (Aim 3A). The potential for partial reprogramming to expand or restore the critical window for rescuing hair cell function (Aim 3B) will be investigated. Thus, by combining advanced genetic tools, high-resolution imaging, and hearing assays, the basic cell biological mechanisms of stereocilia length regulation will be elucidated and innovative strategies for restoring hearing will be developed.

NIH Research Projects · FY 2025 · 2023-04

This patient safety learning laboratory (PSLL) will address the intersection of two major gaps in the science of patient safety: 1) psychosocial patient harm / avoidable patient suffering, and 2) patient safety for transgender individuals. It will leverage an interdisciplinary team of clinical and systems improvement experts to make an innovative, and sustainable contribution to AHRQ’s patient safety mission. It will also address AHRQ’s call for health services research to advance health equity. Transgender individuals are an AHRQ priority population. We will also address intersectional impacts of transgender status by targeted recruitment from additional AHRQ priority populations: low-income individuals, minority groups and people with disabilities. Psychosocial harm has been largely neglected in the science and practice of patient safety, despite evidence of its pervasive and consequential impact. The TRANS-SAFE PSLL will advance patient safety science by identifying and addressing the systemic causes of psychosocial harm in perhaps the most vulnerable patient population–transgender people. Because of widespread stigma, many transgender individuals live on the margins of society, facing discrimination, exclusion, and violence. Transgender and gender non-binary (TGNB) individuals also experience mistreatment in the healthcare system, leading to direct (psychosocial) harm and indirect physical harm. >30% of transgender individuals report delaying or not seeking care due to discrimination, which may lead to diagnostic delays and preventable disease progression. As a result, they experience poor health compared to “cisgender” (non-transgender) people. This PSLL will improve patient safety for the transgender population through 3 specific aims: Aim 1: Identify the contributing factors leading to avoidable patient suffering in transgender individuals [Problem analysis]. Apply human factors, improvement science, risk management, and biopsychosociotechnical systems approaches to conduct a human-centered problem analysis that identifies determinants of avoidable patient suffering in transgender individuals, including issues such as misgendering, disrespect, abuse, and “getting lost in the system.” Aim 2: Co-design human-centered solutions to prevent and mitigate avoidable patient suffering in transgender individuals [Design, development]. Engage with the full range of stakeholders in an iterative process of co-design and development, using both new and proven tools to produce interventions that address the systemic determinants of psychosocial harm in transgender individuals. Aim 3: Evaluate the effectiveness of proposed interventions in real and simulated and clinical environments [Implementation, evaluation]. Test the interventions designed in Aim 2 in both actual practice settings and in simulation to evaluate effectiveness, acceptability, usability, implementability, and sustainability. An innovative dissemination approach (a certification program in partnership with the World Professional Association for Transgender Health) will facilitate widespread and sustainable impact.

NIH Research Projects · FY 2025 · 2023-04

Project Summary/Abstract Land plants like flowering plants and liverworts produce an array of natural products with various biological functions. Polyketide meroterpenoids, comprising structures partially derived from terpenoid biosynthetic pathways and partially derived from polyketide synthase biosynthetic pathways, have attracted scientists for decades from their unique and diverse biological activity. Neuroactive plant meroterpenoids, like phytocannabinoids from Cannabis sativa, represent a particularly exciting suite of compounds with therapeutic promise due to their ability to cross the blood-brain barrier and engage GPCR targets. However, much of the pharmacological data present in the literature to-date has focused on the cannabinoids, Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), while minor constituents and unique analogs from other producers remain less well studied. Lack of pharmacological data for these compounds is partly due to low accumulation of more rare plant meroterpenoids in native producers, production in less widespread plants (i.e. specific liverwort species), and lack of convergent synthetic routes capable of producing several analogs from one common intermediate. Heterologous production of plant meroterpenoids has been accomplished in eukaryotic hosts (i.e. yeast) but suffers from a pathway bottleneck caused by low catalytic activity and poor expression of plant prenyl cyclization enzymes (e.g. THCA synthase). Bacterially derived cyclization enzymes that generate the same key intermediate, an ortho-quinone methide, provide an attractive alternative for biocatalyst generation toward production of plant-like meroterpenoids and their analogs. Here, I propose the development of new cyclization biocatalysts engineered from bacterial biosynthetic enzymes for chemoenzymatic production of rare and designer plant-like meroterpenoid products and their pharmacological evaluation to assess therapeutic promise. This proposal aims to address issues of supply present for rare meroterpenoids with low accumulation in native producers, generate novel, structurally diverse scaffolds using engineered biocatalysts, and test the pharmacology (i.e. therapeutic promise) of such compounds. In Aim 1, I will engineer biosynthetic pathway enzymes recently identified from the Moore lab, Clz9 and Tcz9, to chemoenzymatically produce meroterpenoids with alternative regioselectivity and steric modification. While neuroactive meroterpenoids are predominantly produced by flowering land plants, other species, like liverworts or marine bacteria, produce similar-looking natural products. In Aim 2, I will identify new prenyl cyclase enzymes from marine bacteria and liverwort sources, expanding the toolkit of biocatalysts for producing plant-like meroterpenoids, especially compounds with unique stereochemistry and larger steric modifications. In Aim 3, produced compounds will be subjected to pre- pharmacokinetics experiments to determine likely metabolic products, and both compounds and their metabolic products will be assessed for ability to activate GPCR targets from the human brain. This research proposal will produce rare and designer plant meroterpenoids using biocatalysts from bacterial hosts and identify which of the produced analogs possess noteworthy therapeutic potential.

NIH Research Projects · FY 2025 · 2023-04

Primary open angle glaucoma (POAG) is a leading cause of blindness in the United States and worldwide. It is estimated that over 2.2 million Americans suffer from POAG and that over 130,000 are legally blind from the disease. As the population ages, the number of people with POAG in the United States increased to over 3.3 million in 2020 and is expected to be >110 million worldwide by 2040. POAG is a progressive disease associated with characteristic functional and structural changes that clinicians use to diagnose and monitor the disease. Optical coherence tomography (OCT) and visual field (VF) testing are the clinical standard for measuring the structural (OCT) and functional (VF) changes associated with the development and progression of POAG. Combining data from these sources as well as information about patient demographics, medical history, and clinical measurements is critical to is critical in detecting glaucoma early, identifying signs of progression, and selecting appropriate treatment. Recent progress in AI and deep learning (DL) have provided tools to build predictive multimodal, models that incorporate multiple different types to make predictions. A central hypothesis of this updated research plan is that applying multimodal, longitudinal DL to clinical measurements, VF testing, and OCT imaging will improve the accuracy of predicting progressive structural and functional changes in glaucoma. This updated plan builds on the original research proposal by incorporating new methods and datasets. With this update, there is even greater potential to improve care and preserve vision by helping clinicians tailor glaucoma management to individual patients. This proposal also summarizes the research, training, and career development achievements made the K99 phase of the award. Working with my mentor, Dr. Linda Zangwill, I was able to conduct and publish impactful research during my K99 phase. I was also able to complete the training and career development objectives laid out in the original proposal. During the mentored phase, I helped developed infrastructure and secure access to real-world clinical datasets that will allow me to make immediate progress on the proposed research. This proposed R00 transition will put me in an ideal position to research, publish, mentor, secure funding, and advance my career as an independent investigator.

NIH Research Projects · FY 2025 · 2023-04

PROJECT SUMMARY/ABSTRACT Alzheimer’s Disease (AD) is the leading cause of dementia characterized by progressive cognitive decline. The underlying causes remain poorly understood after decades of efforts, and the disease still cannot be cured, prevented, or significantly delayed. Myelin impairment can disrupt axonal transport, integrity, and plasticity, leading to a massive reduction in signal transduction. Given its essential role in development and maintenance of higher cognitive functions, loss of myelin could play a key role in the pathogenesis of AD. A non-invasive MR imaging technique that can accurately evaluate myelin could therefore be of critical importance for precise diagnosis of AD. Synthetic MRI (SyMRI) has been proposed to indirectly map myelin by assessing brain parenchymal fraction (BPF) and myelin parenchymal fraction (MyPF). However, myelin has a very short T2 (<<1 ms) and is invisible with conventional sequences such as those used with SyMRI. As a result, SyMRI only indirectly evaluates myelin via measuring water signals, and cannot evaluate myelin quality using the T1 and T2* relaxation times. Ultrashort echo time (UTE) sequences with echo times (TEs) of ~8µs, which are 100-1000 times shorter than the TEs of conventional sequences, make it possible to directly detect signal from myelin protons and so circumvent problems associated with SyMRI. The major challenge with UTE approach is the concurrent detection of high signal from various water components. The 3D short TR adiabatic inversion recovery UTE (STAIR-UTE) sequence employs an adiabatic inversion pulse for uniform inversion of long-T2 tissues, which together with a short TR and a high flip angle allow robust suppression of all water components with a wide range of T1 values, thereby selective myelin mapping. The first aim of this study is to validate and compare 3D STAIR-UTE and SyMRI sequences for myelin imaging in phantoms and brain specimens from donors without (n=20), and with AD (n=20) using histology as reference standard. The second aim is to evaluate STAIR-UTE and SyMRI sequences for myelin mapping in patients with mild cognitive impairment (MCI) (n=40) and AD (n=40) as well as healthy controls (n=40). The participants are part of Dr. Du’s newly funded R01 grant. We will correlate SyMRI metrics (BPF, MyPF) and STAIR-UTE metrics (myelin PD, T1, T2*) with cognitive assessments including Mini-Mental State Examination (MMSE), Consortium to Establish a Registry of Dementia (CERAD), and Clinical Dementia Rating (CDR) scales. Our central hypothesis is that 3D STAIR-UTE sequence will robustly detect changes in myelin, and that mapping of myelin quantity with myelin PD and quality with myelin T1 and T2* will provide more specific evaluation of myelin damage and correlate better with disability and disease progression in AD than metrics derived from the SyMRI approach. Dr. Athertya and her mentor, Dr Du, have designed a detailed training plan and assembled a strong research team to guide Dr. Athertya through her fellowship period towards preparing her for a productive career in translational research for AD.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY/ABSTRACT Cannabis has undergone widespread increases in recreational use and legalization in recent decades. Cannabis use, particularly when it begins in adolescence, is associated with impairments in multiple domains of cognition and mental health. Recently, genetic studies have found several variants associated with cannabis use and cannabis use disorder, as well as several phenotypes that are genetically correlated with cannabis use. However, it is unclear how genetic risk for cannabis use affects adolescent development and behavior, particularly in the period before substance use begins. In this project I plan to use advanced methods in statistical modeling to examine the effects of several substance use genetic risk profiles on behavior. Aim 1 of this project will test the influence of a cannabis use disorder genetic risk score, as well as several genetic risk scores known to be related to cannabis use, on multiple domains of mental health and behavior in two large samples of substance-naïve adolescents (The Adolescent Brain and Cognitive Development Study [ABCD], and the Norwegian Mother, Father and Child Cohort Study [MoBa]) totaling over 115,000 participants. By incorporating multiple related genetic risk scores in univariate and multivariate statistical models, this project will be able to tease apart the unique and overlapping effects of genetic risk for cannabis use disorder compared to other genetic risk profiles. Aim 2 will follow a similar analysis technique, applied to restriction spectrum imaging measures in the brain, to understand the influence of genetic risk for cannabis use on brain microstructure in substance-naïve adolescents from the ABCD study. Finally, Aim 3 will incorporate genetic risk for cannabis use, childhood mental health measures, and brain microstructure to predict age of onset of regular cannabis use. In line with the motivation of the ABCD Study, this project will use pre-exposure data to aid in prediction of substance use behavior during adolescence. The proposed research project will leverage existing population-based longitudinal datasets to tease apart the interplay between genetic variation, brain development, and behavior. The addition of several related genetic risk scores to our statistical models will allow us to understand the shared genetic variants that contribute to brain structure and behavior. Importantly, the examination of development both before and after the initiation of cannabis use will be instrumental in understanding the relationship between cannabis use and development, and will allow us to distinguish effects that follow from cannabis use versus those that may predispose adolescents to begin using.

NIH Research Projects · FY 2025 · 2023-04

Social relationships contribute enormously to our health and well-being. Social disconnection is a common and disabling feature of anxiety and depressive disorders that does not respond sufficiently to our best available treatments. These outcomes suggest first-line treatments do not adequately engage the mechanisms that support positive connections with others. Animal and human research suggests the dopamine system plays an important role in responding to social reward cues and opportunities that drive our motivation and behavior toward connecting with others. Diminished social reward anticipation is observed across anxiety and depressive disorders – pointing to a trans-diagnostic mechanism that may underpin social disconnection. Directly modulating dopaminergic functioning in a dose-dependent manner would provide a strong causal test of social reward-mediated disconnection pathways. This is important because first-line pharmacotherapies for anxiety and depression do not directly target this system, which may explain in part why social disconnection persists for many patients following treatment. The proposed two-phase, milestone-driven project will address this gap by testing the hypothesis that modulating the dopamine system pharmacologically will enhance social reward anticipation (the treatment target) and therefore improve social connectedness (primary outcome) in individuals with clinical levels of anxiety or depression. We will selectively engage this system using pramipexole – a D2/D3 dopamine receptor agonist shown to enhance dopamine signaling in the striatum – thereby providing a strong proof of mechanism test. The R61 project will evaluate dose-dependent effects of pramipexole on striatal activation during social reward anticipation (primary outcome) and opportunities to disclose to others. Secondary outcomes will be measured during dyadic affiliation and shared experiences tasks. Aim 1 will test the hypothesis that pramipexole increases social reward anticipation compared to placebo following 6 weeks of treatment. Aim 2 will determine which dose of pramipexole produces a greater effect on social reward anticipation. Pramipexole blood concentrations will be used to confirm dose-dependent target engagement. If pramipexole is superior to placebo in increasing striatal activation to social reward anticipation, the R33 project will attempt to replicate the R61 findings (Aim 1) and examine whether increases in social reward anticipation are associated with improvements in social connectedness (Aim 2) following a 6-week double-blind, randomized, placebo-controlled trial (dose informed by the R61). Secondary outcomes will be change in positive and negative valence symptoms (e.g., social anhedonia, anxiety, depression). An exploratory aim will examine treatment effects on negative valence processes (e.g., threat sensitivity). Positive findings would validate a new CNS target for remediating social disconnection that could be studied in larger confirmatory efficacy trials. Regardless of study outcomes, important new information will be gained about the role of dopamine-mediated processes that are believed to govern whether and how we connect with others.

NIH Research Projects · FY 2025 · 2023-04

PROJECT SUMMARY The overall vision of the proposed research is to gain a comprehensive understanding of how phosphorylation regulates the activity and function of a key regulator of immune signaling, the Ser/Thr protein kinase C (PKC) Theta (). This kinase is selectively expressed in hematopoietic cells where it transduces signals resulting in T cell and platelet activation.1,2 Its dysregulation is associated with a variety of pathophysiological conditions including blood cancers,3,4 inflammatory diseases,5 thrombosis,6 and hemostasis.7 Despite this, the regulation and function of PKC remains largely unknown and necessitates further investigation. Phosphorylation of PKC plays an essential role in regulating its maturation, catalytic activity, and subcellular localization,8 all of which are crucial for PKC function in T cells and platelets. This proposal aims to understand how phosphorylation at known conserved priming sites (activation loop, turn motif, hydrophobic motif),9 a bioinformatically-identified new potential priming site (Ser662), and an uncharacterized activation-induced site (Ser685), regulate the maturation, activity, and/or localization of PKC. Unbiased phosphoproteomics approaches have revealed that phosphorylation of Ser685 significantly increases in T cells10 and platelets11 in response to stimulation, however its function has not yet been determined due, in part, to a lack of available research tools. This site, and Ser662 are positioned on a key regulatory segment, the C-tail, and are evolutionarily conserved. The central hypothesis driving this proposal is that phosphorylation of S662 is involved in the maturation of PKC and that activation- induced phosphorylation of S685 promotes the re-autoinhibition of activated PKC to facilitate signal termination. To this end, I will investigate how nonphosphorylatable or phosphomimetic mutations at these residues impact PKC biochemical properties, cellular activity, subcellular localization, and downstream signaling (Aim 1). Additionally, I will examine the phosphoproteome of PKC in Jurkat cells and platelets and examine how phosphorylation at the agonist-induced site, Ser685, affects downstream signaling. I will also aim to identify the kinase(s) regulating PKC Ser685 phosphorylation using various phosphoproteomics approaches (Aim 2). These key studies will elucidate the functional impact of PKC phosphorylation at critical residues and how this influences downstream signaling. Moreover, this proposal will elucidate substrates and signaling networks regulated by PKC. Uncovering the regulation and function of PKC, a key regulator of T cells and platelets, is crucial to understanding T cell and platelet signaling in both normal and disease states.

NIH Research Projects · FY 2026 · 2023-03

Project Summary/Abstract All life forms on Earth use the same set of natural genetic alphabets: adenine (A), cytosine (C), guanine (G), thymine (T) (and uracil (U)) as the building blocks for storage and retrieval of their genetic information. Recently, a major breakthrough was made in developing the first semi-synthetic organism that is able to store and retrieve genetic information containing an unnatural base pair (UBP) in vivo. However, the molecular basis of transcription processing of UBPs is poorly understood. An important and long-standing question remains unanswered: How are these UBPs recognized by cellular transcription machinery? A lack of clear answers to this important question represents a major knowledge gap in the field. The long-term goal of this project is to tackle this important question. We hypothesize the transcription recognition of UBPs is governed by two layers of specific interactions: specific interactions between the unnatural nucleic acid template and substrates as well as their interplays with the active site of RNA polymerase. We will perform kinetic studies and compare the transcription processing of three classes of representative UBPs by different RNA polymerases, including single-subunit and multi-subunit RNA polymerases. We will determine the structural basis of transcription recognition of UBPs and gain the mechanistic insights into how transcription machineries recognize unnatural nucleotide substrate and catalyze the nucleotide addition reaction. We will utilize a combined approach that includes X-ray crystallography, cryoEM, biophysics, biochemistry, computational biology, and nucleic acid chemistry. The proposed research is significant and groundbreaking, because the novel knowledge and structures obtained from this proposed research will have a transformative impact on the fields of transcription, nucleic acid chemistry, as well as synthetic biology and vertically advance our understanding of the protein- nucleic acid interactions and how unnatural nucleic acids and nucleotides are recognized by different RNA polymerases. Ultimately, such knowledge will provide a framework for developing next generation of UBPs and would produce novel therapeutic nucleic acids and proteins containing new functional groups.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY There is growing evidence that viral infections of the central nervous system (CNS) contribute to chronic brain disease. During development, multiple viruses including cytomegalovirus, herpes simplex virus, rubella virus, human immunodeficiency virus, and Zika virus annually cause thousands of cases of microcephaly — small head size resulting from impaired neurogenesis within the cerebral cortex. The sequalae of these viruses later in neurodevelopment and adulthood are less understood, but here they also disrupt neurogenesis and have been implicated in disease. These viruses share a common ability to eliminate neural progenitor cells (NPCs) in the developing and adult brain. However, the complex biology of these viruses has precluded our ability to identify a precise mechanism by which these infectious agents ablate neurogenesis. We recently discovered that the widely used recombinant adeno-associated virus (rAAV) rapidly kills dividing NPCs and early post-mitotic neurons in the adult murine dentate gyrus (DG) in a dose-dependent manner. Unlike the other viruses described above, rAAV is replication defective and is not known to cause significant pathology. This has resulted in its wide use as a vector in both experimental biology and human gene therapy. However, evidence is mounting that rAAV-based gene therapies are not without significant risk, with at least 7 rAAV-related deaths and numerous adverse outcomes reported in pediatric rAAV trials during the past three years alone. While some of these adverse effects are thought to be caused by immune reactions to the capsid or transgene, increasing evidence indicates that the rAAV genome, which contains two 145-base pair DNA segments named inverted terminal repeats (ITRs), is a major source of rAAV toxicity. Our preliminary experiments indicate that rAAV ITRs bind to and deplete Parp1, a first responder in cellular DNA damage response (DDR) within the nucleus. Moreover, rAAV toxicity mimics pharmacological inhibition of Parp1, inducing cell cycle arrest and cell death, and can be partially reversed by Parp1 activation. We aim to capitalize on these findings to identify the cellular pathways that mediate ITR-induced toxicity and discern whether therapeutic targets within these pathways are shared among viruses that cause microcephaly.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY/ABSTRACT Genes and variants often act in combination to drive cellular and organismal phenotypes. Mapping these functional interactions advances our fundamental understanding of biological systems and has broad applicability to therapeutics development. Gene-gene interactions also likely constitute a considerable component of the undiscovered genetics underlying human diseases, due to the extensive buffering encoded in genomes which makes many individual genes appear dispensable. In this regard, we and others have shown that combinatorial screens, such as those based on CRISPR-Cas systems, are powerful platforms for mapping synergistic relationships among genes and variants. However, unlike screens based on single-gene perturbations which are broadly utilized, combinatorial screens have been significantly harder to deploy. Two fundamental challenges underlying combinatorial CRISPR screens are: 1) the requirement to physically link multiple perturbagens on the same library element which, in addition to complicating library generation, prevents different classes of genome and epigenome engineering toolsets from being readily combined; and 2) analysis of the resulting combinatorial screening data is highly complex, especially in the context of multi- dimensional phenotypic assays. Furthermore, because the perturbation space scales exponentially with the number of simultaneous perturbagens, it is critical to be able to computationally infer interactions beyond those measured experimentally. To address these challenges, we propose to engineer a new screening platform, CombinX, that auto-tethers individual library elements expressed at the RNA, instead of the DNA, level to enable massively multiplexed combinatorial screens. Scalability of this platform is thus limited only by cell culture and sequencing power. We propose to develop the system for both two-way and multi-way (>2 perturbagen) combinatorial screens via application to genetic interaction mapping and cellular reprogramming respectively. Resulting screening data will be interpreted via new advanced computational methods and machine learning approaches to systematically determine genetic interactions, as well as to predict interactions well beyond those that can be covered by direct experimental screens. We anticipate this experimental and computational platform will have broad applicability in basic science and therapeutics discovery, and that it will generate widely useful reagents and data.

NIH Research Projects · FY 2026 · 2023-03

Abstract Vulnerability to substance abuse and addiction has a heritable component. In particular, family and twin studies demonstrate that about 45-65% of the vulnerability to develop alcohol use disorder is determined by genetic factors. In the past decade, advances in genetics have provided critical clues in the search for the molecular basis of alcohol use and abuse. Human genome-wide association studies (GWAS) have expanded dramatically in size and sophistication, which has led to hundreds of loci being implicated in a range of alcohol- related traits. One major impediment to studies of alcohol use disorder is the complexity of the phenotype and the lack of control of environmental variables. We propose a complementary and multidisciplinary approach that combines next-generation sequencing with state-of-the-art behavioral screening in a unique, genetically diverse, nonhuman animal model. The primary goal of this proposal is to identify gene variants that are associated with increased vulnerability to compulsive alcohol use, tolerance and response to FDA approved medications by performing a GWAS in N/NIH heterogeneous stock rats. We will use the most relevant animal model of alcohol use disorder (i.e., escalation after chronic intermittent access to alcohol vapor) and highly standardized measures of compulsive alcohol self-administration combined with longitudinal assessment of withdrawal signs. To increase the impact of these findings and facilitate translational and basic research studies on the mechanisms underlying compulsive alcohol use, we will also establish a data/tissue repository from behaviorally and genetically characterized animals that will allow researchers to further investigate the cellular and molecular mechanisms underlying compulsive alcohol use and identify the biological changes associated with the expression of specific gene variants. This project is likely to have a sustained and powerful impact on the field because it will (1) characterize the transition from controlled to compulsive alcohol use in male and female outbred rats, (2) identify genes associated with compulsive alcohol use and the the response to the currently FDA approved medications , (3) create the Alcohol BioBank which will provide free access to brain, kidney, liver, spleen, ovary, testis, adrenal, and blood samples with a variety of tissue preservation protocols that will allow the generation of induced pluripotent stem cells as well as neuroanatomical, molecular, biochemical, and pharmacological studies on behaviorally/genetically characterized animals.

NIH Research Projects · FY 2026 · 2023-03

Summary/Abstract There are currently limited treatment options for improving endothelial dysfunction in vascular diseases such as sepsis, resulting in high morbidity and mortality. Endothelial dysfunction results in endothelial cell activation, disruption of endothelial barrier function and sensitivity to apoptosis. The long-term goal of this proposal is to delineate the pathways by which the endothelium can resist injury and disruption to facilitate the advancement of new targets for therapeutic development. Activated protein C (APC) is a promising therapeutic and exhibits multiple beneficial effects including stabilization of endothelial barriers and anti-apoptotic activities. Protease- activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR), is the central mediator of APC cellular signaling, which requires caveolin-1 (Cav1) and compartmentalization in caveolae. We discovered that APC- activated PAR1 signals primarily through b-arrestin-2 (b-arr2) to promote endothelial barrier protection, and not heterotrimeric G proteins like thrombin (Th)-activated PAR1. The overall objective of this proposal is to develop a mechanistic understanding of how APC/PAR1 generates b-arr2 transducer bias to promote endothelial cytoprotection. We hypothesize that distinct GRK5 determinants and co-receptors facilitate APC/PAR1-induced b-arr2 transducer bias to promote endothelial cytoprotection through pathways enabled by Cav1 phosphorylation. We propose three specific aims. Aim 1: To delineate the mechanisms that enable GRK5 to distinctly regulate APC- vs. Th-induced biased signaling. GRK5 is required for APC-stimulated signaling and desensitization of Th-induced signaling. However, the mechanisms that enable distinct GRK5 functions is not known. We will determine if distinct GRK5 functions are regulated by localization to discrete plasma membrane microdomains such as caveolae using human cultured endothelial cells, a native system that permits the study of endogenous PAR1 and GRK5 and HEK293 CRISPR/Cas9 knockout cells. Aim 2: To determine the mechanisms by which APC vs. thrombin control b-arrestin transducer bias. It is not known how b-arrestin transducer bias (signaling vs. desensitization) is induced by APC- vs. Th-activated PAR1 nor how APC/PAR1 promotes two distinct b-arr2-mediated cytoprotective signaling pathways: dishevelled2 (Dvl2)-Rac1 controls endothelial barrier protection whereas sphingosine kinase 1 (SphK1)-Akt regulates anti-apoptotic activities. We will determine if distinct determinants of b-arrestin and GPCR co-receptors control different b-arr2 binding modes and functions induced by APC vs. thrombin. Aim 3: To define the mechanisms by which APC/PAR1 regulates Cav1 function to promote cytoprotection. APC/PAR1 stimulates Cav1 phosphorylation but how this modulates Cav1 function and is integrated into the cytoprotective pathway is not known and will be determined. The proposed research is innovative because it will test novel hypotheses to explain how GRK5, b-arr2 and Cav1 drive APC/PAR1 endothelial cytoprotective responses in a physiologically relevant context and will help advance the status of new targets as a future therapeutics for the treatment of endothelial dysfunction.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY Errors associated with DNA replication, transcription, mRNA processing, and protein biogenesis result in the continuous production of potentially toxic defective proteins. The error-prone nature of these essential processes requires robust quality control (QC) systems to effectively triage and destroy defective translation products. Protein quality control is an essential component within the larger protein homeostasis (proteostasis) system and proteostasis dysfunction has been implicated in human aging-related pathologies. On one hand, elevated protein QC function is needed to enable neoplastic cell proliferation in cells with high mutational burdens or chromosomal abnormalities. Conversely, impaired proteostasis and defects in protein QC function result in the enhanced production of misfolded and toxic aggregation prone proteins that typify many neurodegenerative disorders. These observations suggest that developing molecular strategies to predictably alter QC function to either enhance, or limit QC capacity as needed can improve aging-associated disorders and extend human healthspan. However, there is a surprising and substantial gap in our understanding of not only how QC systems selectively engage their substrates, but also how substrates evade detection during proteostasis dysfunction. To make substantive progress toward the goal of leveraging QC systems to combat aging-associated disorders, it is necessary to identify and characterize cellular and molecular mechanisms that enable detection and degradation of diverse QC substrates. Recent research progress from my lab has identified a spatially restricted QC pathway that acts on stalled and collided ribosomal complexes both before and after translation initiation to target defective translation products for degradation and recycle ribosomal complexes. Further, we have developed a systematic pipeline for biochemical, structural, and cellular interrogation of enigmatic but critical QC ubiquitin ligases that have been implicated in targeting diverse substrates for degradation by unknown mechanisms. We have focused our initial studies on the ubiquitin ligase HUWE1. Our recently described HUWE1 structure represents the first full-length structure of a HECT-domain ligase. We have generated a unique and powerful set of genome-edited cell lines and HUWE1 variants that have and will enable molecular dissection of HUWE1 function, HUWE1 substrate identification, and identification of cellular stress conditions that require HUWE1 for cellular survival and proliferation. Research outcomes achieved by the proposed studies will mechanistically determine how terminally stalled ribosomes are sensed and resolved and how ribosome-associated QC pathways can be manipulated to alter proteostasis function. Further, we will establish mechanisms by which QC ligases engage substrates under normal and stressed conditions. Successful completion of the proposed research will provide substantial progress toward our long-term goal of combating aging-associated human pathology through the development of molecular strategies to modify cellular responses to chronic proteotoxic stress and improve cellular fitness following proteostasis insults.

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY The primary objective of this application is to investigate siderophore-based immunization and antibiotic delivery strategies designed to inhibit the growth of Escherichia coli and non-typhoidal Salmonella (NTS). These Gram-negative facultative anaerobic bacteria are major causes of infections in diverse patient populations. E. coli includes commensal organisms, pathogens, and pathobionts (organisms that are usually harmless but are pathogenic in some settings) and cause infections that include urinary tract infections (UTI), bacteremia, meningitis, and sepsis. Moreover, a pathovar known as adherent-invasive E. coli (AIEC) is commonly isolated from patients with Crohn’s disease, a form of inflammatory bowel disease. NTS, including Salmonella enterica serovar Typhimurium (STm), are major causes of inflammatory diarrhea. The primary site of E. coli and NTS colonization is the gastrointestinal tract, where these organisms thrive during colitis and disseminate to other body sites. Recent studies, including work from our laboratories, demonstrate that iron (Fe) availability is a key factor for the progression of E. coli and NTS colonization in the gut, motivating the research proposed in this grant application. Our central hypothesis is that targeting siderophores and their uptake machineries can limit enteric pathogen growth in vitro and in vivo. Both E. coli and NTS deploy the catecholate siderophores enterobactin (Ent) and salmochelin (DGE, diglucosylated enterobactin) in the gut to scavenge Fe3+ from the host. We propose that blocking Ent&DGE-mediated Fe3+ acquisition by bacterial pathogens or targeting Ent&DGE transport systems to deliver antibiotics will provide a means to inhibit the growth of STm and AIEC in the inflamed gut. In support of this notion, we developed a siderophore-based immunization based on Ent that inhibits STm and AIEC growth in the murine gut, and we synthesized and evaluated siderophore-antibiotic conjugates (SACs) based on the Ent&DGE scaffold that target E. coli and STm. In Aim 1, we will use mutants in Fe acquisition genes in STm and AIEC to test whether CTB-Ent immunization results in specific inhibition of pathogen growth and association with the gut mucosa when the pathogen produces Ent&DGE; determine the effect of CTB-Ent immunization on the mucosal-associated versus luminal microbiota; and ascertain whether neutralizing anti- Ent&DGE Ig mediate protection by limiting pathogen association with the mucosa. In Aim 2, we will evaluate the antimicrobial activity of three Ent&DGE-based SACs. Studies in vitro will largely focus on how key environmental variables that characterize diverse host environments affect the antimicrobial activity of SACs, whereas studies in vivo will evaluate the consequences of SAC administration on the gut microbiome composition as well as on inhibiting mucosal expansion of STm and AIEC during colitis. This work may lead to future development of siderophore-binding antibodies and SACs as therapeutics to limit colonization of enteric pathogens and pathobionts in the inflamed gut.