University Of Washington

NIH Research Projects · FY 2024 · 2022-09

Project Summary/Abstract Age-related macular degeneration (AMD) is a leading cause of visual impairment and blindness in adults over the age of 65 and is expected to affect ~288 million people worldwide by the year 2040. Recently, induced pluripotent stem cells (iPSC)-derived RPE generated from AMD patients and those with phenotypically similar monogenic diseases have been shown to approximate elements of AMD disease phenotype in culture, including the formation of sub-RPE deposits resembling drusen, dysregulated complement, and mitochondrial dysfunction. Our groups and others have measured metabolite usage, glycolysis, mitochondrial function, and lipid metabolism in a variety of iPSC RPE model systems. While in vitro RPE models show significant promise in the discovery of disease mechanisms and therapeutic targets, there is also increasing awareness of potential limitations, including reproducibility across model systems and fidelity to native conditions. A comprehensive review of recent iPSC RPE studies shows that the most used traditional culture media are highly diverse in nutrient and metabolite content which may significantly alter RPE metabolism. Moreover, multiple types of plating substrates used could contribute to the variability in nutrient environments. A lack of consensus on baseline nutrient environments and knowledge of their impact on RPE metabolism makes comparisons between findings challenging. The goal of this proposal is to characterize the metabolic and disease-relevant phenotypic profiles of AMD iPSC RPE cells in three distinct and commonly used traditional media and physiological medium closely approximating the composition of human blood. Two AMD iPSC RPE lines and their CRISPR-corrected isogenic controls will be used in this study. RPE will be differentiated from one NIH/NYSCF AREDS2 subject iPSC line with multiple known high-risk alleles, selected to gender and complotype-match RPE lines generated from an individual with early onset macular drusen (EOMD). A splicing mutation in the CFH gene results in this severe subtype of AMD, and our preliminary data show that EOMD iPSC RPE display AMD disease-relevant features, including complement dysregulation, sub-RPE deposit formation, and altered metabolism. iPSC RPE will be cultured on twp substrates (Matrigel®, vitronectin), and maintained in four media preparations (MEM-α based, DMEM/F-12 based, X-VIVO 10TM and PlasmaxTM). This project aims to determine the impact of culture microenvironment on AMD and EOMD iPSC RPE metabolism and disease phenotype. The outcome of this project will be a new and more comprehensive understanding of how traditional and physiologic media influence the metabolic profile and phenotypic characteristics of normal and diseased RPE cells. This new understanding will aid in the interpretation of metabolite studies across model systems and help to inform the design of more physiologic cell culture media for future studies.

NIH Research Projects · FY 2024 · 2022-09

ABSTRACT The COVID-19 pandemic has disrupted lives and affected behavioral health of many. Unhealthy changes in substance use are a great concern. Early data indicate substance use has changed during the pandemic, particularly for some sub-groups of the population. This project aims to examine changes in cannabis, alcohol, and tobacco use and related health risk behaviors (i.e. driving while—or riding with a driver—under the influence of cannabis, alcohol, and simultaneous effects of cannabis and alcohol) during the course of COVID- 19 pandemic among young adults in Washington State. Specifically, we will address the following questions: What are the patterns of young adult substance use during the pandemic and how do these relate to use before the pandemic both in terms of individual trajectories and normative patterns over the course of young adulthood? What are the predictors of escalation of use vs. desistance from use during the pandemic and what is the role of pandemic stressors in these processes? How do community-level differences in access to resources and access to substances relate to patterns of substance use during the pandemic? To answer these questions, we will use data from the WA Young Adult Health Survey (YAHS) that we collected over the past 7 years with funding from the WA State’s Division of Behavioral Health and Recovery. YAHS is an accelerated longitudinal cohort sequential study of young adults ages 18-25, with cohorts added annually and followed over time (2015-2021). Two cohorts were added after the onset of the pandemic, and five cohorts have longitudinal data spanning the time from before to during the pandemic. These data will be linked with community-level variables (e.g., neighborhood disadvantage, availability of substance use-related outlets and services) before and during the COVID-19 pandemic. We will assess changes in patterns (e.g., mode of use, sources, frequency, and amount) of cannabis, alcohol, and tobacco use, simultaneous cannabis and alcohol use, and SU-related risk behaviors (e.g., driving while intoxicated) from before to during the pandemic. The role of community-level factors and differences by socio-demographic characteristics (e.g., sex, sexual and gender minoritized status, race/ethnicity, college student status) in these changes will be examined. Moreover, we will examine within-person changes in risk factors such as norms and perceived harm of cannabis, tobacco, and alcohol use and COVID-19 pandemic related stressors by socio-demographic and community-level characteristics. Finally, we will assess within-person changes in substance use and related risk behaviors (e.g., driving while intoxicated), focusing specifically on initiation, escalation, and desistance and their predictors and potential explanatory mechanisms. Findings will inform planning of prevention and intervention efforts aimed at improving health and reducing problem behaviors.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT The Pacific Northwest Agricultural Safety and Health (PNASH) Center, established in 1996 at the University of Washington, conducts research and promotes best occupational health and safety practices for Northwest farming, fishing and forestry. One of nine regional centers, PNASH works throughout Washington, Idaho, Oregon, and Alaska integrating expertise from multiple disciplines, institutions and community partners. PNASH's Vision is, Research for healthy workers, strong communities & productive agriculture. The agricultural industries (farming, forestry, and fishing) are foundational to our rural communities and national productivity, yet rank among the most dangerous jobs. Fatality rates exceed the national average by 6-times for farming, 29-times for forestry, and 23-times for commercial fishing. Adding to the safety and health complexity of the worksite is the blended nature of work with community and family, and the dependency on seasonal and contract workers. The PNASH Center proposal is a collaborative effort, integrating research and education among University of Washington investigators and regional institutions and stakeholders. Our institutional partners for this new cycle include: Oregon State University; University of Idaho; and, Washington State University. Our faculty, staff, and students in these partnerships bring expertise in the fields of medicine, industrial hygiene, exposure science, epidemiology, animal and agricultural sciences, forestry, fisheries, engineering and education. Our proposed research includes: animal handling safety; forestry activity recognition; cannabis respiratory health; pesticide application technology, smoke and heat solutions, fishermen lifejacket program, and health indicator monitoring. Our research theme is, Growing agricultural safety and health with technology. Our research is rooted in partnerships with the workers and industries we serve, and builds on their strengths. We use best practices for community-engaged research to collaborate, enabling the Center's interdisciplinary capacity, while maintaining a deep respect for the communities’ cultures and lived experiences. As researchers, we have the capacity to enter and exit communities and therefore carry the responsibility of empowering communities beyond funding cycles. Exiting a research site is as important as entering it, particularly in those with marginalized workers. With this in mind, PNASH Outreach Core places particular emphasis on: (1) returning research progress, results, and workplace solutions in a timely manner with participants, partners, and the public, and (2) sustaining opportunities that empower organizations to adapt our safety and health resources for their own communities.

NIH Research Projects · FY 2025 · 2022-09

Abstract We aim to move the field of genetics as applied to juvenile idiopathic arthritis (JIA) away from the identification of genetic associations and toward a mechanistic understanding of how genetic variants exert risk-conferring effects. We will accomplish two major tasks now facing the field: (1) identification of the variants that exert the biological effects that confer risk (the “causal variants”); (2) identification of the genes whose expression levels are altered by those variants (the “target genes”). In accomplishing these aims, we will also elucidate mechanisms through which those variants alter gene expression and cellular functions. One of the striking findings from GWAS for many complex traits, including rheumatic diseases such as JIA, is the frequency with which disease-associated genetic variants appear in the non-coding genome. As in other complex traits, the JIA genetic risk loci are highly enriched for H3K4me1/H3K27histone marks, epigenetic signatures frequently associated with enhancer function. This finding has led to the hypothesis that genetic risk in JIA impinges on enhancer function, leading to transcriptional abnormalities that can be observed in peripheral blood cells. In this application, we focus on CD4+ T cells, which our preliminary data suggest are among the cells likely to be impacted by causal genetic variants in JIA. In Aim 1, we will identify causal variants based on distinct biological properties. We will identify histone quantitative trait loci (hQTLs) in CD4+ T cells of children with JIA, i.e., regions where genetic variants are associated with differences in read depth on H3K4me1/H3K27ac Cut-and-Run sequencing. We will use the same approach as that previously used by our co-investigator, Dr. Gaffney, in his investigations into the genetics of systemic lupus. We will then identify the variants within the hQTLs that alter DNA topology, a critical determinant of regulatory function. Finally, from variants that pass both screens, we will use a massively parallel reporter assay (MPRA) to identify those variants within the hQTLs that have a significant influence on gene expression. In Aim 2, we will identify the target genes within the JIA risk haplotypes. The underlying premise of these studies is that, although the causal variants may not impact the nearest gene, the majority of relevant interactions will occur within the same topologically associated domains (TADs). Using Cut-and-Run data that we generate in Aim 1 as well as H3K27ac HiChIP data and supplemented by our published CTCF ChIPseq/HiChIP data, we will identify interactions between H3K27ac-marked regions on the risk haplotypes and gene promoters, focusing on those within CTCF-anchored TADs. Knowledge of the 3D chromatin structure, patient genotype, and RNAseq data will then allow us to identify the likely target genes of variants on the risk haplotypes.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY First line radical treatments for localized prostate cancer are associated with significant morbidity and side effects impacting urinary, bowel, and sexual quality of life. As a result, there is strong interest in focal therapy in which the cancer tissue is eradicated while sparing normal healthy prostate and adjacent structures (such as urinary sphincter, rectum, and neurovascular bundles) in order to maintain oncologic outcomes while reducing side effects. To date, clinically available focal therapies rely on thermal ablation (heating or freezing) to induce coagulative necrosis and cell death. Presently, the most widely used thermal technique for focal therapy of prostate cancer is transrectal high intensity focused ultrasound (HIFU). While existing data suggest that thermal HIFU has less morbidity than radical treatments, its ability to effectively control cancer is still uncertain with series reporting positive biopsies in up to 30-40% of patients within one year of treatment. Limitations of thermal HIFU systems, including heat diffusion/sinking and minimal real-time treatment feedback, may explain efficacy concerns. Our team has developed a HIFU-based method termed boiling histotripsy (BH) that uses sequences of milliseconds long HIFU pulses with shock fronts to mechanically ablate targeted tissue to subcellular debris without thermal effects and with real-time ultrasound imaging feedback. We have developed the first prototype of a pre-clinical system and have demonstrated the feasibility of transrectal BH for non-thermal ablation of prostate tissue in a canine model. The specific aims of this proposal are built upon the previous work to refine BH technology into a clinically viable format for focal therapy of PCa. In Aim 1, a novel transrectal multi-element array transducer will be developed and built facilitating efficient volumetric BH prostate ablation using electronic steering and mechanical translation of the focus. The BH transducer will be combined with an imaging probe and both will be controlled by a Verasonics Flexible Ultrasound platform. Comprehensive acoustic characterization of the BH system will be performed, and BH treatment protocols will be designed accordingly and evaluated in tissue phantoms and ex vivo prostate tissue. In Aim 2, improved ultrasound-based imaging algorithms will be developed to enable pre-treatment tumor localization using shear wave elastography and quantitative tissue liquefaction feedback using plane wave Doppler imaging. In Aim 3, the resulting BH array system, exposure protocols, and imaging algorithms will be evaluated in a series of acute and chronic in vivo studies in a canine model to demonstrate the safety and efficacy of the device. At the conclusion of the project period, transrectal BH technology will be ready for submission to FDA for an Investigational Device Exemption in preparation for future clinical trials.

NIH Research Projects · FY 2024 · 2022-09

Project Summary We propose to establish a Hemophilia A Analytical Cohort Research Program (HARP) to support the development of an Intergenerational Precision Medicine Program for the study of factor VIII (FVIII) immunogenicity in severe hemophilia A. The first major aim of HARP is to form a new longitudinal antenatal/neonatal/pediatric severe hemophilia A cohort to study the development of FVIII inhibitors. The cohort will encompass at least 50 mother-baby pairs, following first genetic carrier mothers and then their babies with severe hemophilia A over at least the first two years of life. HARP will be responsible for the overall project coordination, administration, data and biospecimen management, and biostatistics/data analytical support for the program. The second major aim of HARP is to enable the development of the HARP Shareable Resource comprised of well annotated data and biological samples collected over the course of the program accessible through a portal, F8portal.org and a linked biorepository. These resources will be made available to community researchers. As part of aim 2, protocols will be developed by the HARP Protocols Expert Panel (PEP) in conjunction with the Consortium of Clinical Centers to support research in four HARP Scientific Emphasis Areas (SEAs): 1. maternal antenatal research, 2. perinatal research, 3. neonatal/pediatric multi-omics and immunology research, and 4. hemophilia-event driven neonatal/pediatric multi-omics and immunology research. The third major aim of HARP is to perform hypothesis-testing research in each of the four Scientific Emphasis Areas. For the maternal antenatal SEA, we will deeply characterize the mother's expression of FVIII and the antenatal environment to test our hypothesis that the mother's hemophilia carrier phenotype modulates inhibitor development by influencing exposure to FVIII tolerizing proteins and future bleeding risk of infant. For the perinatal SEA, we hypothesize that perinatal exposure to FVIII via expression of F8 gene products are critical in modulating the FVIII immune response and propose to investigate this via genetic methods in the neonate and maternal/cord blood. For the neonatal/pediatric -omics and immunology SEA, we hypothesize that environmental, genetic, and persistent maternal factors promote perturbations in the immune system leading to a FVIII immune response. We propose to rigorously investigate these factors using genetic, immunologic and - omics evaluation beginning in neonates and following through early life. Finally, we propose to investigate FVIII antibody affinity, epitope, and peptide presentation to test our hypothesis that immunologic and/or inflammatory events occurring around FVIII exposure influence production of FVIII-IgG1 and ultimately inhibitors for the hemophilia event-driven SEA. Together, this proposal seeks to provide rigorous scientific and clinical data with biospecimens and expert support for a communal resource to drive innovative investigations of FVIII immunogenicity.

NIH Research Projects · FY 2025 · 2022-09

Project Summary We propose to establish a Hemophilia A Analytical Cohort Research Program (HARP) to support the development of an Intergenerational Precision Medicine Program for the study of factor VIII (FVIII) immunogenicity in severe hemophilia A. The first major aim of HARP is to form a new longitudinal antenatal/neonatal/pediatric severe hemophilia A cohort to study the development of FVIII inhibitors. The cohort will encompass at least 50 mother-baby pairs, following first genetic carrier mothers and then their babies with severe hemophilia A over at least the first two years of life. HARP will be responsible for the overall project coordination, administration, data and biospecimen management, and biostatistics/data analytical support for the program. The second major aim of HARP is to enable the development of the HARP Shareable Resource comprised of well annotated data and biological samples collected over the course of the program accessible through a portal, F8portal.org and a linked biorepository. These resources will be made available to community researchers. As part of aim 2, protocols will be developed by the HARP Protocols Expert Panel (PEP) in conjunction with the Consortium of Clinical Centers to support research in four HARP Scientific Emphasis Areas (SEAs): 1. maternal antenatal research, 2. perinatal research, 3. neonatal/pediatric multi-omics and immunology research, and 4. hemophilia-event driven neonatal/pediatric multi-omics and immunology research. The third major aim of HARP is to perform hypothesis-testing research in each of the four Scientific Emphasis Areas. For the maternal antenatal SEA, we will deeply characterize the mother's expression of FVIII and the antenatal environment to test our hypothesis that the mother's hemophilia carrier phenotype modulates inhibitor development by influencing exposure to FVIII tolerizing proteins and future bleeding risk of infant. For the perinatal SEA, we hypothesize that perinatal exposure to FVIII via expression of F8 gene products are critical in modulating the FVIII immune response and propose to investigate this via genetic methods in the neonate and maternal/cord blood. For the neonatal/pediatric -omics and immunology SEA, we hypothesize that environmental, genetic, and persistent maternal factors promote perturbations in the immune system leading to a FVIII immune response. We propose to rigorously investigate these factors using genetic, immunologic and - omics evaluation beginning in neonates and following through early life. Finally, we propose to investigate FVIII antibody affinity, epitope, and peptide presentation to test our hypothesis that immunologic and/or inflammatory events occurring around FVIII exposure influence production of FVIII-IgG1 and ultimately inhibitors for the hemophilia event-driven SEA. Together, this proposal seeks to provide rigorous scientific and clinical data with biospecimens and expert support for a communal resource to drive innovative investigations of FVIII immunogenicity.

NIH Research Projects · FY 2025 · 2022-09

Quantitative model-based ESUS reclassification using cardiac and cerebral vessel wall MRI Stroke is a major cause of death and the leading cause of permanent disability worldwide. Ischemic stroke is the dominant stroke variety, representing approximately 80+% of strokes in the United States. Defining the specific underlying pathophysiology of ischemic strokes is critical for personalized secondary prevention treatments with the goal of minimizing the risk of recurrent events. However, even with extensive diagnostic workup in current clinical practice, a large portion of ischemic strokes are classified as embolic stroke of undetermined source (ESUS), leaving these patients without optimal treatment tailored to their specific pathophysiology. Recent literature has demonstrated that among subjects diagnosed with ESUS, there may be under-detected lesions of atherosclerosis in intra/extracranial arteries or cardiac pathology on a path towards atrial fibrillation, a so called “atrial cardiopathy”. This implies that there are opportunities to improve the sensitivity and accuracy of etiologic diagnosis to reduce ischemic strokes classified into the ESUS category, allowing for more targeted, personalized secondary prevention measures. New developments in magnetic resonance imaging (MRI) of intra/extracranial atherosclerosis and atrial cardiopathy may provide new opportunities to detect these currently under-detected lesions and allow reclassification of ESUS patients into large-artery atherosclerosis or cardioembolic categories leading to focused treatment strategies. However, there are still significant challenges to using these imaging methods in practice: 1) Specialized vessel wall and cardiac MRI (ESUS-imaging) and image analysis algorithms need to be integrated into the standard of care workflow of stroke patients; 2) A model-based analysis will be needed that combines new findings from ESUS-imaging and findings from existing clinical workup so that new “risk features (RFs)” can be defined for reclassification; and 3) The impact of using these RFs on stroke subtype reclassification needs to be studied prospectively. In this proposal, we plan to develop a model-based analysis focused on ESUS-imaging and test the hypothesis that among acute ischemic stroke subjects diagnosed as ESUS under current clinical workup, a new set of RFs drawn from ESUS-imaging will allow reclassification of a subset of ESUS into large-artery atherosclerosis or cardioembolic categories. The specific aims will: 1) establish new vessel wall and cardiac MRI (ESUS-imaging) and image analysis techniques; 2) develop a multiparametric statistical model that combines information from the standard stroke workup and new ESUS-imaging to identify a set of RFs that can reclassify ischemic stroke etiology; and 3) evaluate the impact of the model on ischemic stroke subtype re-classification. If successful, this proposal will help to establish a clinical workflow that includes ESUS-imaging in ischemic stroke workup and provide a model-based algorithm to assist in future stroke subtype classification.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT: Progesterone (P4) is a key sex hormone governing the physiological changes of the menstrual cycle. We and others have shown that it also has anti-inflammatory effects on the immune system locally in the female reproductive tract (FRT), but the mechanisms are not well understood. There are also indications that its immune effects reach beyond the FRT and affect immunity systemically. P4’s effects are likely modified by the more pro-inflammatory effects of estradiol (E2). A detailed understanding of the mechanisms behind P4’s immune activities at local and systemic sites, and its interactions with E2, may contribute to women’s health by explaining sex-based and menstrual cycle-related fluctuations in inflammatory diseases. Some inflammatory diseases fluctuate during the menstrual cycle, suggesting that P4 has systemic immune effects. For example, asthma and inflammatory bowel disease worsen in the P4-low follicular phase, while rheumatoid arthritis often improves in the P4-dominant luteal phase. On the other hand, P4-induced immunosuppression could limit the response to vaccination or resistance to infection. P4 surges during the luteal phase of the menstrual cycle, reaching >40 times above follicular phase levels. Our data show a clear immunological difference in the FRT between the phases: luteal inhibition of macrophage- tropic chemokines by P4. Monocytes/macrophages and dendritic cells (DC) are antigen-presenting cells (APC), which provide key signals for the localization and survival of resident memory T cells (TRM). We propose complementary human and mouse studies: (1) a human cohort with studies at the APC level, probing their connections to TRM in the FRT and other barrier sites and (2) mouse experiments to corroborate P4’s immunological properties in combination with E2 and to test the hypothesis that vaginal CCL2/CCL4 administration partially reverses P4-induced immune suppression. In Aim 1, we will recruit a clinical cohort to study the effects of the P4-dominant luteal phase in humans on APC and TRM activation and function in the FRT, the gastrointestinal tract, the upper respiratory tract, the skin, and the blood. We will use MSD-based immune factor profiling of secretions and flow cytometry and single cell RNA sequencing of mucosal cells to address three hypotheses: (1) inhibition of APC-tropic chemokine production is a hallmark of barrier site immunosuppression during the luteal phase; (2) mucosal APCs are fewer or less inflammatory during the luteal than the follicular phase; and (3) the APC and cytokine changes during the luteal phase associate with lower number and/or activation status of TRM. In Aim 2, we will use mouse models to establish a causal link between P4 treatment, chemokine production, and APC & TRM frequencies and activation state. We will address two hypotheses: (1) P4 inhibits production of CCL2 and CCL4 in major barrier tissues, reducing APC and TRM frequencies and/or functional potential; and (2) vaginal CCL2/4 administration prevents P4-induced suppression of APC and TRM.

NIH Research Projects · FY 2024 · 2022-09

SUMMARY We propose to develop the foundations of a platform for direct sequencing of native, full-length protein strands using unfoldase-coupled nanopore array technology. In principle, this technology could be used to identify protein primary sequence, in addition to certain post-translational modifications (PTMs) found in prokaryotic and eukaryotic cells, with single-molecule resolution. It is a foundational advance over existing and other next-gen proteomic technologies such as Edman degradation, mass spectrometry, fluorescent label approaches, and immunoaffinity-based methods that suffer from limitations in read length, throughput, sensitivity, labeling efficiency, and/or the availability of suitable affinity reagents. Nanopore sequencing of intact protein strands overcomes these limitations because the ~1 nanometer-long sensor directly interacts with the protein strand as it is linearly-driven through the pore by the unfoldase motor protein, manifesting sequence-specific ionic current signals. Thus, complete sequence analysis of native protein molecules can be achieved. This method is a natural technical extension of current nanopore sequencing platforms that use molecular motors to control movement of nucleic acid strands through nanopores in DNA/RNA sequencing. During the grant period, we will pursue three specific aims: 1) Establish baseline methods of controlled protein translocation through nanopore sensor arrays using unfoldase motors; 2) Develop computational and bioinformatic methods to translate raw nanopore signal data into protein sequence information (amino acid calling and PTM detection); and 3) Establish techniques for analysis of native proteins and proteomic samples. Our team of investigators is uniquely qualified to take on this project: i) We pioneered the analysis of full-length protein strands using unfoldase-coupled nanopore sensors and recently demonstrated that the Oxford Nanopore MinION nanopore array device can be used to directly detect peptide strands and resolve single amino acid substitutions (Nivala). ii) Co-investigators on this application have elucidated and exquisitely characterized the enzymatic mechanisms of unfoldase motor activity through in vitro biochemical, single-molecule, and structural studies (Martin), and have led the development of nanopore raw signal analyses for sequencing of nucleic acids, including direct RNA sequencing, genome and transcriptome-wide detection of modified bases, and assembly of a human genome using ultra-long DNA nanopore reads (Jain). iii) Collaborators will provide access to enabling nanopore technology platforms and expertise, including highly-parallel nanopore sensor arrays and customized nanopore proteins, and offer natural routes to technology transfer (Oxford Nanopore), contribute to characterization and comparison of project results to traditional analysis methods such as protein mass spectrometry (Guttman), and advise on compelling technological applications that will be enabled by successful execution of this project (Timp).

NIH Research Projects · FY 2025 · 2022-09

The overall goal of The CASCADE CLIMB (Cervical cancer prevention in women Living with HIV (WLWH) research Mobilization Base) is to generate critical evidence to inform optimization, implementation, and scale- up of effective cervical cancer prevention interventions for WLWH. Functioning as a scientific hub of the CASCADE Network, CLIMB will drive the network's scientific agenda through skilled and innovative scientific and statistical leadership, rigorous and robust operational oversight, and training and capacity-building opportunities to facilitate research, implementation, and scale-up of cervical cancer prevention interventions in intended-use settings. Our highly capable team of investigators has complimentary and integrated multidisciplinary research and clinical expertise in HPV/cervical cancer prevention and control, gynecologic oncology, women's health, and HIV/AIDS; methodological expertise in epidemiology, statistics, and implementation science; and a strong record of leading clinical trials and studies across the continuum of effectiveness to implementation research in both the US and in low- and middle-income countries (LMICs). The specific aims are to: 1) Develop and conduct rigorous pragmatic, multi-site clinical trials that address unmet needs in cervical cancer prevention for WLWH, and 2) Nurture emerging investigators in resource-limited settings by offering mentorship, capacity-building opportunities and training in the skills and disciplines needed to lead future research in cervical cancer prevention. We will develop trial concepts and protocols and lead implementation, operational oversight, statistical analysis, and manuscript preparation for three trials. We propose pragmatic trials to evaluate important clinical effectiveness and implementation outcomes in the US and in LMICs, with the following aims: 1) Compare strategies for offering HPV self-sampling vs. local standard of care screening approaches on rates of screening and precancer detection; 2) Compare molecular triage (e.g., p16/ki-67 dual stain, methylation) vs. immediate ablation after an HPV-positive result on follow-up rates of HPV/precancer detection; 3) Compare a task shifting strategy to nurses with electronic quality assurance for same day cervical cancer screening and treatment vs. usual care procedures that require referral for follow-up on rates of screening and treatment; and 4) Compare thermal ablation vs. LEEP for treating precancers in WLWH with type 2 transformation zones on rates of post-treatment HPV detection/precancer recurrence. We will leverage the University of Washington Department of Global Health's Treatment, Research, and Expert Education and E-Learning Programs to deliver training and capacity-building activities to CASCADE Clinical Sites, including synchronous online courses in research fundamentals, virtual and on-site trainings to build research capacity, and on-site clinical training in cervical cancer prevention. We will promote development of early-career investigators through a CASCADE Scholars program, including training in epidemiology and statistics and mentorship for manuscript preparation.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT BACKGROUND. The goal of this proposal is to improve outcomes for hospitalized patients with Alcohol Withdrawal Syndrome (AWS). AWS is common in hospitals and can be fatal without appropriate management; however, treatments for AWS also have dangerous side effects. Suboptimal treatment (persistent agitation or oversedation) leads to adverse events (e.g., use of physical restraints or mechanical ventilation), which are potentially avoidable but have never been studied. Moreover, the two types of medications used as first-line therapy for inpatient AWS—benzodiazepines and phenobarbital—have never been rigorously compared in hospitalized patients. Both treatments are recommended by guidelines at the discretion of clinicians, yet their comparative safety remains unknown. CANDIDATE. Dr. Tessa Steel is a critical care physician who co-led a national panel of experts to develop a comprehensive research agenda for improving the care of severe AWS. The proposed K23 builds on this foundation and her prior studies, showing wide variation in current treatments for inpatient AWS. RESEARCH. First, Dr. Steel will investigate how and why inpatient physicians choose to treat inpatient AWS with benzodiazepines versus phenobarbital, using interviews and an implementation science framework for understanding provider behavior. Second, she will evaluate the risk of adverse events (e.g., use of physical restraints) associated with benzodiazepines versus phenobarbital in diverse groups of hospitalized patients, using real-world data from electronic health records (EHRs). These studies will inform her third aim—to design and pilot a pragmatic randomized trial, embedded in hospital care, comparing benzodiazepines to phenobarbital for hospitalized patients with AWS. The pilot will serve as groundwork for a definitive multi-center randomized trial (Dr. Steel’s planned R01) to determine the safest first-line therapy for inpatient AWS. CAREER DEVELOPMENT. Dr. Steel’s goal is to be an innovative clinical researcher who improves outcomes for hospitalized patients with alcohol use disorder. To launch her career as an independent physician scientist, the proposed K23 award provides essential training in (1) qualitative research methods, (2) advanced analytic strategies for using EHR data, and (3) the design and conduct of embedded pragmatic clinical trials. Dr. Steel will conduct her research at the University of Washington (UW) under the primary mentorship of Dr. Katharine Bradley, who is an expert in mixed methods research using EHR data, including pragmatic trials. She will be co-mentored by Dr. Nicholas Johnson, providing expertise related to clinical trials conducted in hospital settings and Dr. Kevin Hallgren, a methodologic expert on the use of EHR data for clinical AUD research. PUBLIC HEALTH IMPACT. The proposed career development award will support an early-career investigator conducting research to improve management of AWS for the ~2 million patients hospitalized with this problem per year in the United States.

NIH Research Projects · FY 2026 · 2022-09

The long-term objective of this Pathway to Independence Award is to support candidate Dr. McCabe in building an independent research program and to facilitate his transition into an independent faculty research position. To date, Dr. McCabe’s research has focused on 1.) refining quantitative methods applied in addictions research, and 2.) understanding individual differences in stress, developing self-regulation, and their associations with alcohol use (AU) among sexual minority and non-minority communities. Dr. McCabe seeks to expand his training in AU development, minority stress theory, and applied quantitative methods to a new emphasis on intersectionality and sexual and gender minority (SGM) AU risk, machine learning and multilevel methodologies, and ecological factors influencing AU disparities. This long-term objective will be achieved through a five-year training plan involving a carefully selected mentorship team as well as targeted coursework and hands-on training experiences. The goals of the proposed research are to 1) distinguish SGM subgroups and intersections at heightened risk for AU (e.g., bisexuals and trans persons, SGM young women of color), 2) assess the role of state policies in moderating AU risk, and 3) delineate moderators and mechanisms of heightened AU across SGM populations within and beyond the coronavirus pandemic. The mentored phase (K99) will involve cross-sectional analysis of the All of Us Research Program (AURP), a large (N=331,360) and diverse national dataset. Aim 1 will identify heterogeneity in alcohol and other substance use behaviors among sexual (1a; n=38,820 non-heterosexual) and gender minority (1b; n=2,660 transgender or nonbinary) communities. It will then test race/ethnicity and age as intersectional moderators of SGM inequities (1c) and state-level policies impacting SGM communities (1d; e.g., hate crime laws enumerating SGM identity) that further differentiate AU risk among SGM groups. During the independent phase, findings will be extended to address mediators and moderators of AU in the monthly AURP COVID-19 Participant Experience Survey (Aim 2; n=100,340) as well as the longitudinal, biennial AURP data that extends beyond the pandemic into 2027 (Aim 3). Aim 2 will test pandemic stressors as mediators of between-person AU among SGM intersections (2a) and examine intersectional (2b) and multilevel moderators (2c) of within-person AU. Aim 3 will test differences in post-pandemic recovery in AU among SGM intersections (3a) and determine pandemic mediators (3b) and moderators (3c) of this change. Findings will serve as the foundation for an NIAAA R01 submission during the R00 phase focused on geocoded neighborhood-level factors influencing developing alcohol risk across adolescence and young adulthood across SGM intersections. Mentors (Drs. Rhew, Lee, Helm) and consultants (Drs. Grimm, Bauer, Raifman) are committed to the candidate’s training, each providing unique expertise to the research and training plan. This award will support the candidate’s development as an independent cross-disciplinary prevention scientist in AU disparities and quantitative methods.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Giardia lamblia, the causative agent of giardiasis, leads to gastrointestinal disorders, long-term growth defects and even death. Estimates place worldwide incidence at over 200 million symptomatic cases per year. Of concern, up to 20% of giardiasis cases are resistant to front-line clinical treatments, and resistance to all major antigiardial drugs has been reported. In addition to their increasing lack of effectiveness, front-line and second- line nitro drugs act intracellularly via non-specific free radical generation, and therefore have a high incidence of negative side effects. There is a critical need to better understand the basic biology of the parasite in order to ultimately design improved intervention and treatment strategies. Flagellated trophozoites proliferate to colonize the intestine where cues including cholesterol starvation and increased pH at the end of the intestinal tract promote terminal differentiation into cysts that detach for transmission. While current treatments target the flagellated trophozoites, encystation could be exploited to clear Giardia infections. The regulation of encystation is poorly understood across the diversity of encysting parasites; thus, studies focused on the encystation pathway are of fundamental cell and developmental biology interest, as well as profound clinical relevance. The aim of this proposal is to understand how encystation cues are perceived and transduced to initiate the encystation program. Our preliminary studies indicate that elevated cAMP is necessary to activate encystation. Our preliminary studies identified a role for Giardia’s sole Rho family GTPase, GlRac, in the regulation of cAMP. Protein-protein interaction studies with GlRac identified a previously uncharacterized seven- transmembrane PQ-loop protein that belongs to the TOG (transporter/opsin/GPCR) superfamily. Knockdown of this protein results in increased encystation indicating that it negatively regulates encystation. We named this protein EncystR for its role in encystation. EncystR localizes to the plasma membrane in actively growing cells, but exposure to encystation stimuli (high pH and cholesterol depletion), causes rapid internalization. EncystR internalization requires GlRac, supporting a functional relationship between these proteins. EncystR negatively regulates encystation through control of cAMP levels, but the mechanism remains to be determined. Related PQ loop proteins are multi-function amino acid transporter-receptors that are known to be pH responsive and can regulate development. EncystR is an exciting new entry point into uncovering the regulation of Giardia’s developmental program. The overall goal of this project is to delineate the roles of EncystR and GlRac in regulating cAMP and the role of cAMP in eliciting the encystation program. This work will establish a framework for understanding the regulation of encystation from signal detection to encystation. Ultimately, we aim to identify the means to short circuit the normal encystation program such that cell cycle arrest and cytoskeletal disassembly can be activated without producing a protective cyst wall. This would clear infections without the risk of transmission.

NIH Research Projects · FY 2024 · 2022-09

Project Summary/Abstract The goal of this research is to investigate the development of inflammatory macrophages and disease in Macrophage Activation Syndrome (MAS). MAS is a serious and potentially fatal complication of rheumatic disease or viral infection. MAS is characterized by the development of cytopenias, including anemia and thromobocytopenia, and accumulation of hemophagocytes— activated macrophages that phagocytose red blood cells (RBCs). I will investigate spontaneous MAS-like disease in a mouse model of Systemic Lupus Erythematosus. In the TLR7.1 model, the overexpression of and constitutively active signaling through the endosomal single-stranded RNA sensor Toll-like receptor 7 (TLR7) drives chronic inflammation and subsequent MAS disease. TLR7.1 mice develop thrombocytopenia, anemia, and a novel population of hemophagocytes, inflammatory hemophagocytes (iHPCs), that spontaneously differentiate from Ly6Chi monocytes. Further, in this model, the development of anemia is positively correlated with iHPC phagocytosis of RBCs indicating that iHPC activity may be driving disease. In humans, SNPs in the gene encoding the transcription factor interferon regulatory factor 5 (IRF5) are associated with MAS development. IRF5 signaling is critical for the development of inflammatory macrophages in the context several inflammatory diseases. Previous work in our lab showed that IRF5 ablation in vivo ameliorates iHPC differentiation downstream of acute TLR7 signaling. However, in the context of in vivo TLR7- driven MAS, the role of IRF5 signaling in iHPC production and in MAS disease is unclear. In preliminary data, I show in TLR7.1 mice that signaling through IRF5 is critical for iHPC development, iHPC RBC phagocytosis, and anemia and thrombocytopenia indicative of MAS disease development. Based on these findings, the goal of my proposal is to determine the role of IRF5 signaling, specifically in Ly6Chi monocytes, in TLR7- driven iHPC differentiation and MAS disease (AIM 1), and to determine what other signals synergize with TLR7 to drive iHPC differentiation and MAS disease, with a specific focus on heme and type I interferons (AIM 2). Overall, completion of the proposed research along with my additional training described here will allow me to pursue my goals of becoming an independent investigator and faculty member with a focus on education.

NIH Research Projects · FY 2024 · 2022-09

Project Summary Dilated cardiomyopathy (DCM) is a highly prevalent inherited cardiac disease, characterized by systolic dysfunction, eccentric hypertrophy, and left ventricle dilation. While pharmacologic and mechanical treatments have been shown to partially improve cardiac function, these results are often short lived and highly variable from patient to patient. Moreover, recent studies have demonstrated that epigenetic and matrix dysregulation can persist, even in patients with improved systolic function after treatment. Given that aberrant chromatin remodeling and extracellular matrix (ECM) deposition have been identified as drivers of dilated remodeling—and that chromatin and ECM remodeling can become irreversible over time—it is crucial to understand the time- dependent effects of DCM mutations on reversing maladaptive remodeling at the genome, myocyte, and matrix levels. The central hypothesis of this proposal is that the reversibility of the DCM phenotype is time-dependent due to the accumulation of permanent ECM and chromatin remodeling as the disease progresses. To complete this proposal, I will utilize a DCM mouse model created by the Davis lab which contains a mutation (I61Q) in cardiac troponin C (cTnC) that directly decreases the myofilament’s Ca2+ sensitivity, leading to eccentric hypertrophy, systolic dysfunction, and left ventricle dilation. Importantly, expression of this mutant can be temporally controlled with doxycycline to specifically test our central hypothesis. In this proposal I will 1) Determine the time-dependent effects of I61Q cTnC expression on myocyte, matrix, and chromatin accessibility, 2) Examine the reversibility of DCM remodeling in myocyte, matrix, and chromatin accessibility at different stages of the disease, and 3) Determine if myocytes retain epigenetic memories of the mechanical disequilibrium caused by DCM. Improving our understanding of the time-dependent reversibility of DCM remodeling will better inform therapeutic targets and treatment windows for patients with DCM. Moreover, completion of this project will enhance Bella’s training as an independent scientist and prepare her to one day become a professor in cardiovascular engineering. Receiving the NIH F31 predoctoral fellowship will facilitate important experiments and training that will aid in her pursuit of this career goal. In this project, Bella will gain expertise in multi-omic approaches—such as proteomics, RNAseq, and ATACseq—which are growing in popularity due to their unbiased screening capabilities. The UW Genomics Core will assist Bella in learning how to effectively use these tools, which will not only benefit this project but will also be an incredibly useful skillset for Bella’s future career. Given the clinical relevance of this project, we have engaged Farid Moussavi-Harami, MD, an acting physician- scientist who practices cardiology within the UW Department of Medicine. Dr. Moussavi-Harami’s input as a clinician will be crucial for ensuring our research questions and aims remain relevant to patients, and his mentorship throughout this project will enhance Bella’s training and career development as she aims to eventually run a lab with an emphasis in translational therapeutics and technologies for cardiovascular diseases.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY Sexual aggression, which refers to a continuum of sexual activities with a nonconsenting partner from unwanted sexual contact to forced penetration1, is an intractable public health problem. Despite a substantial investment in prevention and intervention efforts, rates of sexual aggression perpetration remain high, with 26 – 42% of men self-reporting past perpetration of sexual aggression2. One pathway to sexual aggression perpetration that has received considerable attention and empirical support is sexual misperception, or the erroneous perception of a potential partner’s sexual interest or consent3-6. Sexual misperception shares several key risk factors with sexual aggression perpetration, making it a valuable intermediary mechanism for examining perpetration constructs. Specifically, both sexual misperception and sexual aggression perpetration are associated with alcohol intoxication6-12 and a constellation of attitudes collectively referred to as the Confluence Model4,5,13-18. Although evidence suggests acute intoxication and Confluence Model constructs interact to predict sexual misperception, the nature of this interaction is unclear and the underlying mechanisms remain unexamined. This proposal aims to provide a comprehensive evaluation of the effects of alcohol and Confluence Model constructs on sexual misperception and directly assess this interaction through an alcohol myopia (AM) framework. AM theory posits that alcohol’s effects on behavior are driven by its ability to direct attention to impelling (“go”) cues and away from inhibiting (“stop”) cues19. Attention to these cues is also influenced by a priori knowledge structures20-22 such as those encapsulated in the Confluence Model and those activated by the environment. The proposed research will directly measure the impact of Confluence Model constructs on intoxicated men’s attention to impelling and inhibiting cues of sexual interest and the effects of attention to these cues on sexual misperception in a field setting. Conducting this research at drinking venues addresses several significant gaps in the literature, namely the generalizability of these processes and the effects of contextual cues, which have substantial potential to inform intervention design and evaluation. This proposal is commensurate with the candidate’s desire to develop a research program centered around understanding the mechanisms underpinning alcohol-involved sexual misperception and sexual aggression perpetration. Through the proposed research and training plan, the applicant will develop the skills and expertise needed to make a substantive contribution to alcohol and sexual aggression research as an independent clinical scientist.

NIH Research Projects · FY 2025 · 2022-09

PROJECT ABSTRACT/SUMMARY. Cardiomyocyte cell state is dramatically altered during postnatal development. During this period, cardiomyocytes terminally differentiate, fundamentally changing their energetics, functional machinery, and mechanism of cell growth. Although we have a good understanding of the factors controlling embryonic heart development, we still have a poor understanding of the mechanisms that establish cardiomyocyte terminal differentiation during postnatal development and maintain this cell state in the adult. Critically, cardiomyocyte terminal differentiation is integrally linked to cardiac remodeling and regeneration. While a terminally differentiated transcriptome is necessary for cardiomyocytes to accommodate adult circulatory demands, this transcriptional program also suppresses cardiomyocyte proliferation, underlying the inability of the mammalian heart to meaningfully regenerate after injury. Indeed, in model systems of cardiac regeneration, cardiomyocytes must first de-differentiate and return to a fetal-like transcriptomic state in order to proliferate. Few studies examine the factors responsible for maintaining cardiomyocyte terminal differentiation, but it stands to reason that targeted disruption of cardiomyocyte terminal differentiation could unmask latent pro-proliferative pathways in the adult heart and promote endogenous cardiac regeneration. In this study, we will examine the role of the RNA-binding protein Muscleblind-like protein 1 (MBNL1) in controlling cardiomyocyte terminal differentiation and cardiac regeneration. Although MBNL1 expression increases during cardiomyocyte terminal differentiation and MBNL1 is known to promote fetal-to-adult isoform switching of a number of developmentally regulated genes, it has never been examined directly for controlling cardiomyocyte terminal differentiation or integrated into regulatory mechanisms surrounding cardiac plasticity. Specifically, this proposal will use a variety of in vivo and in vitro genetic approaches to address the following aims: (1) to determine the role of MBNL1 in maintaining cardiomyocyte terminal differentiation in the adult mammalian heart and (2) to determine the role of MBNL1 in controlling cardiac regenerative potential. Insight gleaned from these aims will characterize MBNL1-dependent post-transcriptional regulatory mechanisms governing cardiomyocyte terminal differentiation and will determine if MBNL1 could be used as a novel therapeutic target to promote endogenous cardiac regeneration.

NIH Research Projects · FY 2026 · 2022-09

Project Summary/Abstract: Contribution of somatic mitochondrial DNA mutation to the transition from normal aging to Alzheimer’s disease. Candidate and Training: Dr. Sanchez-Contreras is an MD, PhD, Acting Instructor in the Department of Laboratory Medicine and Pathology, University of Washington (UW). Her research is directed towards understanding the effects that somatic mutations of the mitochondrial DNA (mtDNA) have on mitochondrial function during aging, and further differentiate these from pathogenic mtDNA mutations that cause mitochondrial dysfunction in Alzheimer’s disease (AD). To develop her area of research, Dr. Sanchez- Contreras will apply her expertise in duplex sequencing and in neurodegeneration, while she will acquire skills in procedures to measure mitochondrial physiology and data analysis. This training will be focused on the underlying mitochondrial biology of aging guided by four mentors that are experts in mitochondrial genetics and biology, neuropathology and in vivo models of aging and AD and immersed in a research group that is a leader in aging and in AD research in the country. Research: Somatic mutations of the mtDNA and mitochondrial dysfunction are found in the brain of AD patients. As these findings also accompany normal aging, it is unclear what determines the departure from normal to pathogenic in AD. The main hypothesis of this study is that somatic mtDNA mutations abnormally increase at preclinical and early stages of AD, and that they contribute to mitochondrial and synaptic dysfunction, and the worsening of AD pathology. This hypothesis will be tested in two aims. In Aim 1, pre- clinical AD patients will be studied to find somatic mutations and mitochondrial and synaptic abnormalities that associate with AD pathology. In Aim 2, a systematic evaluation of somatic mtDNA mutation and mitochondrial function will be performed in the mouse brain by increasing somatic mutagenesis at multiple ages using the mutator mito-APOBEC1 transgene. Lastly, the impact of somatic mutation in AD will be studied in two models of the main neuropathological components: amyloid pathology and tauopathy. These two approaches will contribute to understanding of how the entorhinal cortex and the hippocampus respond to increasing somatic mutations and mito-dysfunction early in the progression of AD. Career Development Plan: The execution of this K01 award is designed to ensure Dr. Sanchez-Contreras’ successful transition to an independent faculty position in her department. To this aim, a structured plan is presented that includes the commitment of her institution and her department to support her efforts, a strong mentorship committee, the consolidation of strategic collaborations and the performance of crucial experimental protocols that will result in significant advancements in the field of aging and that will be the foundation for R21 and R01 submissions at the conclusion of this K01.

NIH Research Projects · FY 2026 · 2022-09

Project Summary The paraventricular nucleus of the thalamus (PVT) is a critical node between ascending nociceptive information and goal directed behavior. Lesions, chemogenetic and optogenetic inhibition of the PVT reduces an animal’s ability to complete goal-directed behaviors. Studies have suggested NAc-projecting PVT neurons are necessary for mediating opioid dependence, or opioid use disorder. Chronic opiate use induces opioid dependence, a complex disease whose components including tolerance, drug seeking or craving, and physical dependence characterized by withdrawal avoidance behavior. The PVT is rich with all 4 opioid receptors, particularly the mu-opioid receptor (MOR) – the primary target of analgesic drugs like morphine and fentanyl. Interestingly, activity-driven expression of the immediate early gene c-fos indicates increased excitation of the PVT in response to opioid withdrawal. Given the growing body of evidence that the PVT is involved in the expression of opioid dependence, I will examine the effects of acute and chronic opioid administration on PVT neurons. Thus, Aim 1 is dedicated to characterizing the acute actions of opioids on MORs pre- and postsynaptic to the PVT and the role of phosphorylation in the process of acute desensitization in the PVT. Phosphorylation of sites on the C-terminus of MORs results in the induction of acute desensitization which is thought to be a precursor for the development of long-term tolerance. Tolerance, or a diminished responsiveness has been well described in hindbrain regions such as the locus coeruleus (LC) yet less is known about how tolerance shapes circuits that mediate goal directed behavior. Since phosphorylation of MOR is thought to be the initial step in the development of tolerance, I have characterized these mechanisms in the PVT. Sequential work will determine long-term adaptations that lead to withdrawal to multiple opioids including, morphine, fentynal, and bupinorphine in the PVT; as well as presynaptic adaptations of MOR (+) inputs to the PVT. The overall goal of this proposal is to characterize the adaptations of MORs in PVTs seen after chronic opioid treatment. Additionally, this work stands as the foundation of what I aim to achieve in science and bring to my post-doctoral fellowship. In Aim 2, I propose to identify a post-doctoral mentor with expertise in mood disorders, chronic stress, and/or addiction who can utilize my electrophysiology expertise and train me in rigorous experimental design and data analysis for in-vivo imaging and behavioral assays. The broad, long term goal is to build the skill set required to dissect a circuit molecularly and follow these adaptations to awake and moving animals. This proposal seeks to address the BRAIN Initiative’s Scientific Review and High Priority Research Area for using multimodal methods to monitor neural activity. My electrophysiological foundation will serve well to seize the challenge of recording dynamic neuronal activity from both complete neural networks and single cells; informing us about dynamic networks with molecular precision.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT The overall scope of the problem is that as the US population lives longer, kidney disease becomes more abundant. In particular, elderly patients face worse disease outcomes, and they are now the largest group to undergo first-time dialysis. The goal of this proposal is to prove that aged podocytes are central to the many glomerular changes with aging. Changes to and loss of podocytes remain the best predictors of age-related glomerulosclerosis and reduced GFR. Major unmet needs are understanding the mechanisms of podocyte aging and the crosstalk between aged podocytes and neighboring parietal epithelial cells (PECs). To close these knowledge gaps, we performed a transcriptome analysis comparing podocytes from aged vs. young mice. Much to our surprise, transcripts for immune response processes such as inflammasome components, inflammatory factors (e.g. TNFα, interferons, interleukins and chemokines) and SASPs were significantly enriched. Importantly, similar changes were confirmed in human kidney biopsies. Based on these preliminary data, we propose a novel paradigm that aged podocytes secrete inflammatory signals and SASPs that in autocrine loops directly impact podocytes themselves. Specific Aim #1 will prove that this newly discovered inflammatory aged podocyte phenotype directly shortens the podocyte's lifespan and reduces their health-span. We will test the hypotheses that in aged podocytes: (1) Inflammasome-induced de novo intracellular inflammation reduces podocyte lifespan; (2) The PD1 signaling pathway acts downstream of the NLRP3 inflammasome; (3) A specific subset of secreted inflammatory mediators accelerates the podocyte aging phenotype through autocrine loops. We also propose a second novel paradigm in which aged podocytes play a paracrine role in accelerating PEC aging. This is based on the facts that (i) podocyte aging temporally precedes PEC aging; (ii) PEC aging is typically only present in individual glomeruli in which podocytes exhibit an aged phenotype; (iii) inhibition of the inflammasome or PD1 pathways in aged podocytes reduces PEC aging. In Specific Aim #2 we propose that SASPs and inflammatory cytokines derived from aged podocytes accelerate the PEC aging phenotype through paracrine loops. We will test the hypotheses that: (1) The inflammatory podocyte phenotypes in aged mice precedes and accelerates PEC aging. (2) A distinct subset of SASPs and inflammatory cytokines derived from aged podocytes accelerates the PEC aging phenotype. These studies are based on many innovative experimental approaches including aging studies in transgenic mice, primary human podocytes and PECs, Design-of-Experiment methodology and novel co-culture models. Finally, the focus of our study is significant for its short-term translational impact by intersecting our mouse data with a large transcriptomic data set on aged human kidneys and its long-term impact in developing therapeutic strategies that will counter the age-dependent demise of kidney function.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Background: Worldwide, the majority of people with depression receive no treatment, despite the existence of evidence-based, low-cost treatments. In Zimbabwe, at least a third of people presenting to primary care screen positive for depression, but most cases go unrecognized and untreated. Research strategy: This study includes the development and pilot testing of an implementation strategy to improve uptake of clinical guidelines for depression diagnosis and treatment in primary care. It has three phases: 1) Assessment of current practice: In a sample of patients presenting to routine primary care (n=200), determine what percent of patients with probable depression are recognized by primary care nurses and offered treatment, 2) Adaptation of an implementation strategy: Using a human-centered design (HCD) approach, conduct qualitative interviews and hold focus groups with nurses to iteratively adapt a implementation strategy that combines in-person practice facilitation with mobile phone-based clinical supervision, 3) Pilot trial of the implementation strategy: In order to evaluate whether the implementation strategy is feasible and acceptable and to inform a future cluster randomized trial of the strategy, pilot test the strategy in two clinics (n=12 nurses, n=30 patients), comparing outcomes to two control clinics (n=12 nurses, n=30 patients). Outcomes include a) feasibility and acceptability of the implementation strategy (qualitative interviews, surveys, and program data), b) exploratory implementation outcomes (e.g. depression screening, recognition, treatment receipt), c) exploratory clinical outcomes: PHQ-9 score of patients at 6 and 12 weeks after initial visit, and d) feasibility of data collection strategy. Training plan: To conduct the proposed research and transition to being an independent investigator, Dr. Jack will receive mentorship and training in the following areas: 1) foundational understanding of methods for the design and analysis of clustered clinical trials, 2) implementation science with a focus on mixed methods, and 3) human-centered design. Mentorship: Dr. Jack’s primary mentor (Rao) brings expertise in implementation science and global mental health in primary care, which is complemented by her co-mentors who are experts in depression research in Zimbabwe (Chibanda) and mental health treatment guidelines and clinical trials in low and middle income-countries (Thornicroft). She also has a scientific advisory team (Dorsey, Hallgren, and Lyon) who will support her training and research in implementation science, clustered trial design, and HCD, respectively. Candidate: Dr. Jack combines clinical training in Internal Medicine with a research background that has focused on global mental health and the integration of behavioral health into primary care (42 publications, 15 as first author). This K23 builds on her eight years of prior research in Zimbabwe and will allow her the dedicated time for research and training required to become an independent, NIH-funded investigator in implementation science to address the behavioral health treatment gap worldwide.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract Type 2 diabetes affects over 30 million people in the US and carries a high burden of cardiovascular morbidity and mortality. Identifying new, modifiable mechanisms that may influence the development and macrovascular complications of this multifactorial disease will make a substantial public health impact. This application will investigate the associations of hydrogen sulfide (H2S) with risks of incident diabetes and diabetes-associated cardiovascular disease (CVD). H2S is a gasotransmitter that is crucial for cell signaling and cell function. In addition, H2S appears to play an important role in the development of diabetes and mitigating the related toxicities. In the pancreas, H2S regulates insulin secretion and protects beta cells from apoptosis. In the liver, the main organ for the synthesis and storage of glucose, H2S reduces insulin resistance and improves glucose uptake. In preclinical studies, compounds that release H2S, and therefore increase systemic concentrations, protect from endothelial dysfunction, cardiac hypertrophy, myocardial injury and atherosclerosis in rodent models of diabetes. Studies in humans are limited to cross-sectional studies showing a reduction in plasma H2S in patients with type 2 diabetes. Prospective studies are needed to bridge the gap between pre-clinical studies and future clinical trials. Therefore we propose to conduct efficient case-cohort and longitudinal studies of H2S, diabetes and CVD. We hypothesize that higher levels of H2S in plasma are associated with lower risks of incident diabetes and diabetes-associated CVD; and we hypothesize that cellular H2S levels protects human hepatic and cardiomyocyte from insulin resistance and the resulting cellular dysfunction. To test these hypotheses, we will measure circulating H2S levels in existing samples from two prospective cohorts and examine H2S associations with type 2 diabetes and diabetes-associated CVD (Aim 1). Importantly, in Aim 2, we supplement these observational studies with functional experimental studies in human derived cell lines to investigate the molecular mechanism of H2S hepatic and cardiac protection in insulin resistance, and to discover novel H2S-regulated pathways that may lead to future targets for the prevention of diabetes and CVD.

NIH Research Projects · FY 2025 · 2022-09

Candidate: Dr. Ellington is a pediatric pulmonologist at the University of Washington (UW) and K12 UW-Implementation Science Training Grant recipient (NHLBI). She completed a Master of Science in Epidemiology at the UW School of Public Health and has a long-term track record of successful global health research. Dr. Ellington’s long-term goal is to become an independent physician-investigator working to improve health outcomes of pediatric respiratory conditions in resource-constrained settings. Her career development plan includes coursework, experiential learning, and mentorship to achieve training goals in 1) complex qualitative and mixed methods, 2) pragmatic clinical trials and quasi- experimental studies, 3) advanced implementation science, and 4) global health leadership. Environment: The proposed training and research activities will be facilitated by existing strong international collaborations, resources, infrastructure, and academically rigorous environments of the UW Schools of Medicine and Public Health and the Makerere University Lung Institute and College of Computing & Information Science. Research: Acute lower respiratory illnesses (ALRI) are a leading cause of childhood mortality in children under 5 years, despite effective prevention and treatment strategies. The majority of deaths occur in sub-Saharan Africa. Major challenges in case management exist in low- and middle-income countries (LMICs), including poor adherence to ALRI guidelines, lack of health worker training, and lack of decision support to recognize wheezing illnesses (asthma and bronchiolitis), which represent up to 50% of ALRI in children. Acute Lower Respiratory Illness Treatment Evaluation (ALRITE) is a comprehensive guideline- and evidence-based ALRI clinical decision support mobile health tool Dr. Ellington developed with her team. ALRITE has demonstrated preliminary acceptability and usability in Ugandan health centers but requires optimization for health system integration and pilot-testing in clinical care. Dr. Ellington proposes a feasibility study in 4 Ugandan health centers to 1) identify promising implementation strategies for the ALRITE intervention to optimize implementation, 2) pilot-test study feasibility of the ALRITE tool and its effectiveness for diagnosis and management of wheezing illness and pneumonia in children 1-59 months using an interrupted time series design, and 3) assess the outcomes of implementation following ALRITE deployment and barriers/facilitators of ALRITE use. Successful completion of these aims will improve implementation of decision support tools in Uganda and other LMICs, provide critical preliminary data to support Dr. Ellington’s planned R01 for a cluster RCT evaluating clinical effectiveness of the ALRITE intervention in LMICs, and provide Dr. Ellington with the training and experience needed to transition to research independence.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Dental caries is a prevalent but preventable oral disease. In caries-active subjects, there is a shift towards a microbial community dominated by acidogenic and acid-tolerant bacteria. Streptococcus mutans is a major cariogenic pathogen. Commensal bacteria normally help to control S. mutans via bioactive products such as H2O2. However, high-sugar consumption, which is frequently found in populations with a high rate of caries development, could inhibit the generation of H2O2 through carbohydrate catabolite repression and thereby disrupt homeostasis. Several metal titanates have recently emerged as effective green chemistry solutions to produce reactive oxygen species (ROS), including H2O2, O2˙–, ˙OH, and 1O2. They trigger oxidation stress in certain bacteria and subsequently inhibit them. Metal titanates have the potential for broad dental applications due to high compatibility with various dental materials, including dental adhesive systems, resin composites, ceramics, and metals. Furthermore, as photocatalysts, metal titanates will not be consumed in the catalyzed reaction but can act continuously, thus offering long-lasting benefits. Our group has demonstrated that gold titanate could catalyze and produce H2O2, which could inactivate S. mutans while having limited impact on commensal oral bacterial S. gordonii and S. sanguinis. It is hypothesized that selective photoactivated semiconducting metal-titanates will produce extrinsic H2O2 from O2 reduction and other ROS to inhibit S. mutans, while giving an advantage to commensal bacteria and thereby maintain homeostasis in dental biofilms and thus prevent dental caries. To test our hypothesis, Aim 1 will optimize the application conditions of metal titanates to maximize the potentials of the antibacterial efficacy. We will measure different species of ROS production from gold titanate with in situ probe compounds. Then, we will synthesize semiconducting metal titanates to enhance the photocatalytic activities (i.e., activation by visible light, more ROS generation) and improve clinical performances (e.g., esthetics, compatibility, physical property, stability, and toxicity). Aim 2 will identify the gene expressions of S. mutans and commensals exposed to metal titanates. The transcriptional profiles of S. mutans with metal titanates will be revealed by mRNA sequencing (RNA-seq) and the oxidative stress relevant genes will be specifically monitored. Aim 3 will focus on diverse multi-species oral biofilms, especially in high sugar condition. First, we will examine the effect of metal titanates on the spatial organization and composition of the dual-species biofilms of S. mutans and commensal bacteria in flow cell system. Second, we will apply plaque-derived multispecies microbial biofilms and S. mutans-infected multispecies oral microbial community to understand the response of species within complex oral biofilms to metal titanates in the oral cavity, for which 16S rRNA gene sequencing will be employed to measure the composition shift. Collectively, the study will provide a comprehensive understanding of the effects of metal titanates on the formation and ecology of dental biofilms and guide the development of an effective dental application.