University Of Washington

NIH Research Projects · FY 2025 · 2022-08

Abstract: There are almost 500,000 deaths worldwide each year caused by rupture of intracranial aneurysms (IAs) with half the victims younger than age 50. Unruptured intracranial aneurysms (UIAs) can be treated by endovascular and microsurgical interventions to prevent rupture, however, the treatment carries a non- negligible risk of morbidity (5%–7%) and mortality (1%–2%). Current guidelines recommend intervention for UIAs larger than 7mm, when their rupture risk is higher than the intervention risk. However, more than 50% of ruptured IAs are smaller than 7mm. Identifying small aneurysms that are prone to rupture and performing selective intervention can potentially prevent rupture of these small aneurysms. A recent meta-analysis including more than 4000 UIAs with an average of 4 years’ follow-up showed aneurysms that grew during the follow-up were 30 times more likely to rupture than the non-growing aneurysms (3.1% vs. 0.1%). Identifying the factors that predict aneurysm growth can help select these high risk UIAs for treatment. Aneurysm wall enhancement (a surrogate marker of inflammation, identified by contrast-enhanced vessel wall MRI) and aneurysm geometric factors (such as shape or size ratio as identified on imaging) are two promising markers that may predict aneurysm growth. However, the current evaluation of these factors is limited by non-optimized imaging techniques that have flow artifacts, long scan time and subjective, qualitative image analysis. This project will develop optimally accelerated and blood suppressed imaging methods and quantitative image analysis methods for the evaluation of UIA wall enhancement and geometric characteristics, and investigate the parameters associated with aneurysm volume growth by longitudinal UIA evaluation using MRI. First, we will develop and optimize blood suppression and imaging acceleration techniques using in vitro phantoms and in vivo testing in patients. Second, we will develop automatic segmentation and quantification methods (Radiomics) for evaluating UIA wall enhancement and geometry. Finally, we will follow 200 patients with >3mm UIAs using MRI each year for up to 4 years, and investigate which clinical and quantitative imaging parameters are predictive of UIA volume growth.

NIH Research Projects · FY 2024 · 2022-08

PROJECT SUMMARY/ABSTRACT Social risk factors, or adverse conditions in which people are born, grow, live, and age1, contribute to greater mental health problems2,3 and lower treatment engagement worldwide.4–7 The prevalence and impact of social risk factors (SRFs) are particularly pronounced in low-to-middle income countries, where too few individuals— and particularly children—with mental health needs receive care. To our knowledge, in global mental health research, we have not systematically developed and evaluated strategies to address SRFs alongside mental health treatments. This study builds on an NIMH-funded trial in Kenya, “Building and Sustaining Interventions for Children (BASIC): Task-Shifting Mental Health Care in Low-Resource Settings” (R01MH112633). The study goal is to develop strategies to address and mitigate SRFs alongside evidence-based psychotherapies (EBPs) using decolonizing methodologies.8 Lay counselors and guardians involved in the BASIC study report that children enrolled in the culturally-adapted EBP (Trauma-focused Cognitive Behavioral Therapy)9 experienced high rates of SRFs (food insecurity, lack of support system), with strong calls for improving outcomes by addressing SRFs in therapy.10–12 Thus, this project responds directly to community needs and addresses a documented challenge to the effective implementation of EBPs given the negative effect of SRFs on mental health trajectory and treatment engagement. The study aims to: 1) Examine the experience of SRFs and the barriers and facilitators to addressing SRFs in therapy through qualitative interviews with PT counselors and guardians who received therapy in BASIC. 2) Collaboratively design impactful and practical strategies to address SRFs in therapy during a 2-day workshop with supervisors and counselors. 3) Investigate whether a 2-day training in the developed strategies and post-training supervision impact counselor-level outcomes (e.g., perceived self-efficacy/self-control, effectiveness, feasibility, and acceptability of the SRF strategies). As many scholars have highlighted problematic influences of coloniality in current and past global research,13,14 the study team will actively work against colonial attitudes by privileging the voice of community stakeholders during all research activities and decision-making throughout the study. Strategies developed through this project have the potential to improve engagement in and effectiveness of the EBP in BASIC and ideally will be generalizable to other EBPs globally. Our ultimate goal is to improve outcomes for individuals with mental health needs in low-to-middle income countries.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Covalent attachment of ubiquitin (Ub) to other proteins is among the most widespread and diverse modes of eukaryotic cellular regulation. The modification occurs on practically every protein in a cell at some point in its lifetime and is itself highly diverse. The type of ubiquitylation determines a product’s fate and a protein may undergo different modes of ubiquitylation depending on cellular circumstances. The origins of this diversity stem from the protein machinery responsible for Ub attachment. A trio of enzymes, E1, E2, and E3 coordinate the process, with several E1s, dozens of E2s, and many hundreds of E3s encoded in the human genome. Over the past 20 years, we have asked fundamental questions about how E2s and E3s work and have contributed to the structural, biochemical, and mechanistic understandings of the field. Our work began with the breast cancer tumor suppressor, BRCA1/BARD1 that was among the earliest RING-type E3 ligases to be identified. Over the years, we have expanded to study numerous E3s and E2s, making many unexpected discoveries along the way. The wide reach of protein ubiquitylation in cellular function means that dysfunction of components is associated with myriad human diseases and developmental issues. Such associations make the Ub system attractive for therapeutic targeting. Direct targeting of the ubiquitylation machinery as well as efforts to re-engineer protein ubiquitylation machinery to selectively target a specific cellular protein are both proving to be powerful strategies. Such translational efforts rely implicitly on mechanistic understanding and reveal the power of well- grounded structure/function research. Despite the apparent maturity of the field, there is still much we do not understand at a fundamental level. We do not know the full range of biochemical reactions carried out by the ~30 human E2s as fully one-quarter of these are uncharacterized. Existing data reveal that not all E2s carry out the presumed reaction that attaches Ub to lysine sidechains, implying the existence of ubiquitylated species that have yet to be investigated in cells. Second, understanding of how E2/E3s carry out mono-ubiquitylation is lacking. Unlike poly-ubiquitylation, attachment of a single Ub (mono-Ub) tends to occur in a site- selective manner implying that substrates to be mono-ubiquitylated are handled differently from those destined to have chains built upon them. Third, lack of knowledge regarding how mono- Ub attachment affects the structure and function of proteins limits understanding of how the modification regulates critical cellular processes including transcription, translation, and DNA damage response, among others.

NIH Research Projects · FY 2024 · 2022-08

PROJECT SUMMARY Our long-term goal is to develop safe and effective drugs to treat obesity. We propose to validate ALDH1A1 as a new drug target for obesity treatment by testing the anti-obesity efficacy of a novel inhibitor of this enzyme, N42, that was recently developed by our group. Retinoic acid (RA), a metabolite of vitamin A, is synthesized by three aldehyde dehydrogenases, ALDH1A1, ALDH1A2, and ALDH1A3, that are expressed in temporally and spatially distinct yet sometimes overlapping patterns in a tissue- and developmental stage-dependent manner. ALDH1A1 is the major RA synthesis enzyme in two metabolically important tissues, liver and adipose, where RA is known to regulate expression of genes involved in glucose and lipid metabolism and adipose tissue differentiation. Aldh1a1-/- mice develop normally, are healthy and fertile, and are also protected from developing diet-induced obesity. In agreement with observations in Aldh1a1-/- mice, we demonstrated that mice treated with WIN 18,446, a pan-inhibitor of ALDH1A enzymes, had lower weight gain due to a decrease in adipose tissue mass, demonstrating the feasibility of using pharmacological inhibitors of ALDH1A to treat obesity. Although WIN 18,446 treatment is promising for weight suppression, it inhibits other RA synthesis enzymes as well as ALDH2, causing unwanted side effects such as reversible spermatogenesis inhibition and alcohol intolerance. Therefore, we developed compounds that specifically inhibit ALDH1A1 and now propose to evaluate the efficacy and potential toxicity of a novel ALDH1A1-specific inhibitor, N42, for treatment of obesity and to determine mechanisms by which ALDH1A1 regulates weight gain by using comprehensive studies of gene expression and metabolite changes in response to ALDH1A1 loss. In Aim 1, we will determine if N42 treatment can (1) suppress weight gain in obese mice when they are continuously fed a high fat diet and (2) accelerate weight loss in obese mice when they are provided reduced calorie diet. We will also investigate potential organ toxicity of N42 treatment using standard, well-established protocols of clinical and toxicologic pathology. Metabolic changes associated with ALDH1A1 loss (N42 treatment or Aldh1a1-/- mice) will be comprehensively investigated using global metabolomics and gene expression studies. Phenotypic parameters including metabolites, gene expression, retinoid levels etc. will be associated with N42-mediated efficacy to identify potential biomarkers for future clinical use of this compound. In Aim 2, the role of ALDH1A1 in liver and adipose tissues will be further explored using tissue-specific Aldh1a1-/- mice. We will also determine how ALDH1A1 loss in adults alters adipogenesis and adipocyte hypertrophy using an adipocyte linage mouse model. Finally, we will identify tissue-autonomous functions of ALDH1A1 by combining a novel perifusion method, metabolomics, and systems-biology approaches. Successful completion of the proposed studies will validate ALDH1A1 as a novel target to treat obesity and provide a potential prototype drug.

NIH Research Projects · FY 2024 · 2022-08

ABSTRACT Women account for nearly 20% of new HIV cases in the United States but experience significant barriers to accessing HIV prevention, addiction, and sexual health services. Women who inject drugs (WWID) are particularly vulnerable to HIV due to a combination of social, biologic, and structural risk factors, and women comprise many of the new HIV diagnoses in recent outbreaks among people who inject drugs (PWID). Similarly, WWID are disproportionately impacted by major medical issues requiring hospitalization and leading to premature death, especially when injection drug use is compounded by transactional sex and unstable housing. HIV pre-exposure prophylaxis (PrEP), medications for opiate use disorder (MOUD), and treatment of sexually transmitted infections (STIs) are proven strategies for HIV prevention and addiction treatment among WWID; however, uptake remains low. Our preliminary data suggests that a co-located neighborhood clinic offeringwalk- in appointments dramatically improves uptake of HIV prevention and addiction treatment but does not result in sustained use of these evidenced-based interventions. Globally, delivery of HIV prevention and sexual healthcare at venues for exchange sex is an effectivetool for engaging marginalized populations, such as female sex workers; however, this strategy has not yet been implemented in the U.S. In direct contrast to traditional models of healthcare, an evening, drop-in clinic, co-located at a venue for exchange sex and drug use provides increased opportunity to access care in a client-centered environment. Leveraging a global to local approach, we propose a pilot venue-based pop-up primary and preventative care clinic for WWID. Formative, qualitative research and a bi-directional community engagement plan will inform the adoption of global models for HIV prevention and addiction services (e.g. PrEP, MOUD, STI screening, etc.), with the goal of supporting sustained us of evidence-based interventions. We hypothesize that venue-based care will be acceptable to WWID and improve uptake of preventive care. We also hypothesize that implementation of participant designed support strategies will improve adherence to PrEP, MOUD, and STI treatment. Aim 1 will assess barriers and facilitators to uptake and sustained use of PrEP and MOUD and inform conversations with our community advisory board to adapt a global to local venue-based care model, tested in Aim 2. In Aim 2, we will pilot a venue-based model of care for 50 WWID. Using novel rigorous laboratory science methods, we will measure the impact of venue- based care on sustained use of PrEP and MOUD (Aim 2a), and we will similarly measure the acceptability and feasibility associated with venue-based care for WWID. If successful, our study will provide data on a feasible model of care for reducing HIV acquisition among WWID, which can be assessed for cost-effectiveness and further tested at scale.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT The purpose of this project is to develop a novel image-guided approach for modulating the tumor microenvironment (TME) of HCC with combined mechanotherapeutic drugs (MechTx) and ultrasound cavitation treatment (USCTx), and evaluate its therapeutic efficacy. We plan to implement USCTx and the targeting and monitoring of the combined (with MechTx) treatment on a clinical scanner in order to make it widely available for future preclinical and clinical studies. Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide with an estimated 750,000 new cases per year. Most patients with HCC who are not candidates for surgical removal or ablation are treated with either transarterial chemoembolization or systemic chemotherapy. Yet these treatments result in only limited improvements in patient survival at the expense of considerable toxicities. Our main hypothesis is that the combined USCTx and MechTx will lead to tumor pressure, stiffness and vascular changes that promote increased local tumor uptake of systemic or transarterial chemotherapeutics, and will result in better therapy outcomes. While the discovery of new chemotherapeutic agents and interventional procedures will continue to evolve, our approach to modulate the TME with combined MechTx and USCTx aims to dramatically improve chemotherapy outcomes with both the existing and future chemotherapy agents. A strong interdisciplinary team (bioengineers, scientists, clinicians) from academia and industry will collaborate in the following aims: (Aim 1) Evaluate the ability of MechTx to modulate the tumor microenvironment and enhance drug delivery; (Aim 2) Evaluate the ability of USCTx to modulate the tumor microenvironment and enhance drug delivery; (Aim 3) Evaluate the ability of combined MechTx and USCTx to modulate the tumor microenvironment and enhance drug delivery; and (Aim 4) Demonstrate the preclinical efficacy of chemotherapeutics when combined with MechTx and USCTx in survival studies using two in vivo models of HCC. The innovation of the project is in: (a) the use of MechTx as a novel therapeutic strategy to modulate tumor pressure, microvascular flow, and stiffness of the TME; (b) implementing image-guided USCTx on a clinical ultrasound scanner leading to translatable precision medicine for HCC; (c) utilizing the synergy of MechTx and USCTx as a novel and innovative approach for modulating the TME to maximize chemotherapy outcomes; and (d) combining super resolution and nonlinear Doppler processing to spatially and temporarily super-resolve the vasculature of tumors undergoing treatments that target the TME. The proposed project will take advantage of the distinct, yet synergistic mechanisms of MechTx and USCTx as a novel paradigm of sensitization of the TME to chemotherapeutics, leading to better treatment outcomes and overall survival for HCC patients initially and patients of other malignancies and diseases in the future.

NIH Research Projects · FY 2024 · 2022-08

ABSTRACT Next-generation sequencing (NGS) has become increasingly integral to the practice of clinical oncology, where its ability to scalably examine hundreds to thousands of targets now routinely enables identification of prognostic and therapeutically actionable markers that support the practice of precision medicine. There are many applications for which it would be useful to detect and quantitate cancer-associated genotypes at ultra- low levels (<1 in 10,000 or more), such as identifying drug-resistance mutations in tumors, detecting residual cancer cells after therapy, or early cancer detection. Nevertheless, standard NGS technologies are hampered by a relatively high error rate (~1 in 100bp), below which true biological variation cannot be distinguished from noise. Various methods have been proposed to bypass this issue by allowing error correction of NGS sequence reads, but such techniques require redundantly sequencing individual template molecules at high depth such that an error-corrected consensus sequence can be produced. As a result, those methods require a large amount of sequencing power, are costly, and are limited in the number of specimens and genomic targets that can be examined. They have consequently seen little uptake in clinical use. There remains an unmet need for highly accurate sequencing methods that are cost-effective, scalable, and allow interrogation of enough gene targets for meaningful use in clinical practice. We have recently developed a new experimental paradigm, termed “Linked Duplex Sequencing”, that addresses these limitations. In our approach, we join the two strands of DNA from an initial template fragment into a single, covalently linked molecule. Error correction of the duplex can be performed by comparing separate reads from the two linked strands, thereby eliminating the need for redundant sequencing of template molecules. This innovative technology provides robust error correction with scalability, cost-effectiveness, efficiency, and quantitative precision, and is compatible with low-to-mid output short read sequencing platforms (ie, Illumina) that are already in widespread clinical use. In our first Aim, we will develop workflows to support Linked Duplex Sequencing, will develop supportive bioinformatic analysis pipelines, and will characterize the cardinal performance metrics of the approach using fresh and formalin-fixed reference material. In our second Aim, we will develop protocols for the targeted enrichment of genes or variants of interest for Linked Duplex Sequencing, and will evaluate performance using a variety of clinical materials. This work will provide information and deliverables with immediate, direct, and transformative benefit to cancer patients by substantially improving the quality of oncology sequencing assays while imbuing them with enhanced diagnostic capabilities for the ultrasensitive detection of cancer associated mutations relevant to disease emergence, relapse, and therapy resistance in routine clinical practice. Our goal is to make ultrasensitive, error corrected sequencing so inexpensive and straightforward that it will be used as standard operating procedure for NGS clinical oncology assays and cancer research studies.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT Lung transplantation improves survival and quality-of-life for patients with end-stage lung disease. Primary graft dysfunction (PGD) and acute lung allograft dysfunction (ALAD) are early and intermediate events, respectively, that threaten the long-term benefits of transplantation and increase the chances of chronic lung allograft dysfunction (CLAD)—the primary cause of long-term mortality among lung transplant recipients. Efforts to improve patient outcomes have relied, in part, on the use of risk-stratification to guide clinical decisions in lung transplantation. Specifically, the Lung Allocation Score (LAS) ranks transplant candidates based on the risk of death within one year of being listed and the probability of survival one year after transplantation. The estimated risk of PGD and ALAD may present additional opportunities for stratification. However, in order to predict PGD or ALAD accurately, recipient and donor risk factors exhibiting a strong association with these outcomes must be identified. Most clinical risk factors do not have sufficiently strong associations with PGD or ALAD to facilitate improvements in prediction outcomes. Biomarkers with a mechanistic role in the pathogenesis of PGD or ALD are likely to be the strongest predictors of these outcomes. Notably, a growing body of evidence shows that exosomes—30-150nm diameter lipid bound extracellular vesicles—released from immune and non-immune cells—modulate the immune response to antigens in a variety of diseases. Our team recently proposed a conceptual framework for the role of exosomes in innate and adaptive immunity that predicts the development of PGD, ALAD, and CLAD. We recently demonstrated the feasibility of measuring recipient-derived exosomes in patients with end-stage lung diseases and preliminary data suggest an association between exosome phenotypes and CLAD. However, it remains to be determined whether recipient-derived exosome phenotypes are associated with PGD or ALAD, whether changes in exosome phenotype occur post-transplant and if so, whether these changes increase the risk of ALAD. To address these knowledge gaps, we propose a three-year prospective cohort study with one-year follow-up of lung transplant recipients with the following aims: 1) Determine if recipient-derived exosome phenotypes are associated with PGD, 2) Determine if recipient-derived exosome phenotypes are associated with ALAD, and 3) Determine if PGD alters exosome phenotype post- transplant and/or the risk of ALAD. The ultimate goal of our research is to improve patient outcomes by increasing knowledge of biomarkers that predict PGD and ALAD. Evidence of an association between recipient- derived exosome phenotypes and PGD and ALAD accomplishes the first step of developing a risk-stratification tool to better inform transplant recipient selection and donor matching, and to further guide immunosuppression and other post-transplant management protocols. This line of investigation is also expected to enhance our knowledge of exosome-mediated immunoregulation, providing novel insights into the role of exosomes in the pathogenesis of PGD and ALAD and novel therapeutic targets for future investigation.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Worldwide, serious mental illnesses (SMI) such as schizophrenia or bipolar disorder are major causes of impairment and disability. In West Africa, the hardships of SMI are compounded by pervasive societal stigma, scarce treatment options, systematic exclusion, neglect, and abuse. People with SMI in West Africa describe the experience as akin to “receiving a death sentence.” West African mental healthcare systems have severely constrained resources that contribute to a large treatment gap. Most people with SMI receive services from traditional and faith healers who do not provide high quality care. Healers often use practices such as chaining, seclusion, and forced fasting that often worsen the negative impacts of SMI. Given the shortage of skilled mental health providers and the prevalence of healers, coupled with the established infrastructure healers have in place (e.g., referral networks, “prayer camps”, community ties) global health leaders have argued that healers and prayer camp settings may be leveraged as conduits for treatment, provided they receive appropriate training and support to provide higher quality care. Our multinational research team has developed M&M: a dual-pronged intervention package comprised of a mobile health program designed to train healers to deliver evidence-based psychosocial interventions while maintaining safety and patient dignity in practice (M- Healer) combined with pharmacotherapy delivered directly to the patients at their prayer camps via visiting nurse (Mobile Nurse). We have successfully completed usability, acceptability, feasibility and preliminary clinical testing of the modular elements of the intervention at prayer camps, with very promising results. We now propose to evaluate the effectiveness of the integrated M&M intervention in a fully-powered trial and share it widely through a new West African Digital Mental Health Alliance (WADMA), a mission centered network designed to jumpstart digital mental health research and cross institutional collaboration though grant funded studies, education, and integration of digital mental health tools in clinical practice. We aim to: 1. Evaluate the effectiveness of the M&M intervention using a stepped-wedge cluster randomized trial design; 2. Examine mediators and moderators of M&M intervention effects; and 3. Use qualitative methods to inform M&M intervention optimization and future implementation. Although focused on West Africa, the dual-pronged model we are testing to address quality of care needs, the outcomes we will measure, and the methodological lessons we will learn will all have translational implications for development and implementation of integrated technology-assisted treatment support packages for paraprofessionals caring for people with SMI in the United States.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY/ABSTRACT Patients with chronic life-limiting illness who develop acute respiratory failure are at high risk of death and disability. Because some such patients would choose to forego life-sustaining treatments in certain conditions, it is important that ICU clinicians assess patients’ values, goals, and treatment preferences, and incorporate these findings into shared decision-making to provide goal-concordant care. Unfortunately, my preliminary data as well as qualitative studies of other hospitalized populations suggest that ICU clinicians often miss opportunities to assess or incorporate patients’ goals of care into decision-making. This may lead to the delivery of unwanted intensive care and life-sustaining treatments, which is harmful to both patients and families, and results in the delivery of high-cost, low-value care. The proposed research in this application will use multimethod and mixed-methods approaches to: (1) examine current implementation of goals-of-care discussions for patients with acute respiratory failure and chronic life- limiting illness; (2) identify potentially modifiable clinician behavioral determinants that underlie deficiencies in goals-of-care communication, and iteratively redesign an existing outpatient communication-priming intervention toward the goal of promoting high-quality goals-of-care discussions in the ICU; and, (3) conduct a pilot randomized trial of the redesigned communication-priming intervention to improve the occurrence and quality of documented goals-of-care discussions for patients with acute respiratory failure and chronic life- limiting illness. Through a combination of mentored research activities and formal research training, I will receive valuable research training in analytical methods to enhance the validity of studies that use “real-world” data, application of mixed-methods research to inform health intervention design, and clinical trials. The proposed research and training activities will prepare me for a career as an independent clinical investigator, and will form the foundation for a body of research designed to improve outcomes for patients with acute respiratory failure. These research and training activities will be facilitated by the academically rich research environments of the University of Washington Schools of Medicine, Nursing, and Public Health, as well as the Cambia Palliative Care Center of Excellence at UW Medicine.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract In this project, we will test the overall hypothesis that PIEZO channels mediate mechanosensation in the cardiac pacemaker and that they are essential players on the heart rate acceleration evoked by mechanical stretch. We will leverage our expertise in the study of the cardiac pacemaker to enter into two new research fields for our laboratory: mechanosensation and mechano-electrical coupling. The heart is one of the most mechanically active organs in the body. Besides being a remarkably effective pump, the heart senses its mechanical environment and adjusts its performance to match the physiological demands. In a mechanism known as the “Bainbridge Reflex”, the cardiac pacemaker responds to the stretch induced by the increase in venous return with an acceleration of its pace to empty the heart effectively. To sense these constant changes in stretch, the pacemaker is equipped with stretch-activated channels, however, their molecular identity remains elusive. PIEZO channels mediate mechanotransduction in every cell type where its expression has been detected so far. Despite being expressed in the pacemaker and being considered the candidate to mediate pacemaker mechanotransduction, the role of PIEZO channels in this tissue has not been explored yet. This proposal will directly test the role of PIEZO channels in the stretch-activated response of the cardiac pacemaker. Our innovative approach includes the development of pacemaker-specific mouse lines to test the effect of PIEZO knockdown and overexpression in the pacemaker activity at the cellular, tissue, and animal level. We will combine immunodetection, in-situ hybridization, electrophysiology, high-resolution imaging, calcium imaging, and telemetry to: (Aim 1) Characterize the abundance, isoform relative expression, cell-expression specificity, and subcellular localization of Piezo1 and Piezo2 channels in the pacemaker tissue and isolated pacemaker cells. (Aim 2) Evaluate the role of PIEZO channels in the pacemaker stretch-activated current, the stretch- activated increase in intrinsic firing rate, the automaticity of pacemaker cells, and in their subthreshold calcium activity. (Aim 3). Determine the role of PIEZO channels in the stretch-activated electrical and calcium responses in the intact pacemaker tissue and their role in normal heart function in vivo. Completing the aims listed above will provide new insight into the molecular mechanisms of mechanotransduction in pacemaker cells and will help to identify novel targets for detecting and treating associated arrhythmias. Our results will also provide a diverse toolkit to identify multiple important mechanisms behind the translation of pacemaker stretch into heart rate acceleration, opening new avenues for our lab to study the downstream signaling pathways that are regulated by the mechanical activation of PIEZO channels in this tissue.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY/ABSTRACT: For many seriously ill patients, such as those with underlying life-limiting chronic conditions, deterioration in health often results in acute respiratory failure (ARF), a common critical illness that is associated with prolonged disability and high risk of death. Critical illness hospitalizations are resource intensive and expensive, and associated with significant family caregiver burden. As a result, these patients and their family caregivers are particularly vulnerable to significant financial hardship. We have previously shown that financial hardship is a major source of stress for patients with ARF and their family members. Importantly, it is persistent, worsens after hospital discharge, and is likely to be an important mediator of psychological distress and poorer quality of life in this population, yet few studies have sought to address the role of financial hardship in the context of patient- and family-centered outcomes over time. Financial hardship encompasses material (e.g. out-of-pocket expenses, unemployment), psychological (e.g. feelings of distress), and behavioral aspects (e.g. coping mechanisms). Although financial hardship has been established as an important and modifiable problem among outpatients with cancer, little work has been done with patients with ARF and their families. In order to develop context-specific interventions that are likely to be effective in meeting the unique needs of these patients and their family caregivers, we need to understand who is at highest risk for developing financial hardship during and after critical illness, which of these risk factors are modifiable, and the effect of financial hardship on patient- and family-centered outcomes. We will address this knowledge gap by using a multiple methods approach to advance our understanding of financial hardship for seriously ill adults with ARF and their family caregivers. In Aim 1, we will identify patients with ARF and family caregivers who are at highest risk for subsequent financial hardship by examining both non-modifiable baseline characteristics that identify those at risk, and modifiable factors that may be the focus for context-specific interventions. In Aim 2, we will make the important connection between financial hardship and patient- and family-centered outcomes (psychological distress, health-related quality of life and goal-concordant care) over time, something which has not been done for patients with ARF and their family but is a necessary next step for the development of interventions that will successfully disrupt the long-term consequences of financial hardship following critical illness. In Aim 3, using qualitative methods, we will explore drivers of material hardship and factors that influence psychological responses and coping behaviors from the perspective of key stakeholders. Our multi-disciplinary team has significant experience assessing patient- and family-centered outcomes, performing electronic health records-based data collection, and conducting qualitative research. Our findings will lay the groundwork for the development of interventions designed to reduce the burden of financial hardship on patients with ARF, family caregivers, and the healthcare system.

NIH Research Projects · FY 2025 · 2022-08

Dr. Lauren Cirrincione, an Assistant Professor at the University of Washington School of Pharmacy, is applying for a K23 award. Dr. Cirrincione’s career goals include making significant contributions to the field of hormone mediated drug interactions in transgender and gender diverse adults, specifically applying clinical pharmacology to transgender medicine. This grant will provide 1. expertise in probe substrate study methods and analysis and 2. longitudinal study design and 3. training to become an independent NIH-funded investigator with expertise in hormone mediated mechanisms of altered drug disposition and will address whether high-dose sex hormone therapy alters the disposition of other prescribed drugs. We will use a probe substrate-biomarker-protein activity framework to test differences in major drug handling proteins before and during estradiol treatment in vivo. Dr. Cirrincione will gain expertise in conducting mechanistic, clinical pharmacology studies in transgender adults. Data generated from this proposal will be used to advance clinical strategies to overcome changes in drug safety and efficacy in transgender adults and to increase available in vivo mechanistic data to establish the role of sex hormones on pathways of drug disposition.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY/ABSTRACT Clinical staging is gaining traction as a potentially powerful framework for understanding the pathogenesis and emergence of serious mental illnesses (SMI), such as schizophrenia (SCZ), bipolar disorder (BP), and severe depression (severe-DEP), across development, and for guiding early interventions that aim to alter disease trajectory. However, realizing the full potential of this approach requires overcoming a number of challenges. These challenges include the imprecise boundaries between psychiatric disorders; unclear validity and specificity of early clinical and neurobehavioral markers for predicting later illness onset; and growing recognition that current psychiatric nosology may not map onto differences in underlying etiology in an optimal way. Indeed, as large-scale genetic studies continue to unravel the genetic architecture of psychiatric disorders, it has become clear that genetic risk for each disorder is complex and highly polygenic; involves variants that span the allelic frequency range; and that individual genetic variants frequently confer risk for multiple disorders. Leveraging genetic risk profiles to define groups of at-risk individuals and map the progression of clinical phenotypes across development may therefore offer a more biologically valid approach for defining the nosology of psychiatric disorders, identifying biomarkers with the greatest predictive validity and specificity for different clinical outcomes, and optimizing early intervention. Towards this end, the current project will investigate the relationships between genetic risk profiles and early markers of psychopathology in the “Paisa,” a genetically and culturally homogenous population that predominates in the Andean Mountains of Colombia. Specifically, we will build upon our existing infrastructure for large-scale studies of SMI in the region to establish a new cohort of 3,000 children and early adolescents at elevated (n = 2,700) or low risk (n = 300) for SMI. We will obtain DNA samples and comprehensive clinical and neurobehavioral phenotyping and will generate common-variant based polygenic risk scores (PRS) for major psychiatric disorders, as well as rare variant scores summarizing burden of rare damaging variants and copy number deletions. We will characterize relationships between clinical syndromes and neurobehavioral traits in childhood (Aim 1). We will then map relationships between common variant-based genetic risk for SMI, rare damaging variant burden, and psychiatric diagnoses in childhood (Aim 2), as well as cognitive, motor, sensory, and psychological markers of functioning (Aim 3). Finally, using existing state-of-the-art psychiatric electronic medical record (EMR) databases to obtain longitudinal outcomes, we will explore genetic and clinical and neurobehavioral characteristics associated with poor clinical outcome within 2 years. Study findings will clarify trans-diagnostic vs. disorder-specific neurobehavioral profiles in childhood, identify clinical syndromes and neurobehavioral traits in childhood associated with genetic liability for SMI, and quantify the relative power of genetic versus clinical and neurobehavioral characteristics for predicting proximal psychiatric outcomes.

NIH Research Projects · FY 2025 · 2022-08

Project Summary P-glycoprotein pumps drugs, xenobiotics and nutrients out of cells via a partially characterized ATP-dependent mechanism. Due to the extreme substrate promiscuity of P-gp, it contributes to the disposition of nearly all small molecule drugs and to drug-drug interactions. P-gp may be particularly important in cancer cell drug resistance due to its over expression in several cancers. The aims of this proposal are to fill knowledge gaps in three distinct aspects of P-gp mechanism. Each aim shares the common mechanistic element of conformational dynamics. The first aim is to define the P-gp conformations at low ATP occupancy in order to understand how they control downstream conformational changes that are essential for substrate efflux, and how they are different in the presence vs. the absence of substrates. These conformational differences are hypothesized to be essential for minimizing wasteful expenditure of ATP in the absence of transportable substrate and to control proper conformational access in downstream steps. These differences will be identified by rapid, pre-equilibrium, kinetic methods with state- of-the-art kinetic modeling and Hydrogen/Deuterium Exchange Mass Spectrometry (H/DX) with P-gp in lipid nanodiscs. The second aim is to understand the extreme sensitivity of P-gp to its lipid environment. The lipid nanodisc platform provides fine control of the lipid bilayer in which P-gp resides. P-gp will be incorporated in nanodiscs with varying ratios of lipids with different head groups or acyl chains that provide different surface charge of bilayer fluidity. The basal- and substrate dependent ATPase activity in the different lipid environments will be correlated with changes in conformation determined by H/DX. The third aim is to determine the mechanism by which P-gp is able to interact with such a wide range of substrates and inhibitors. Substrate promiscuity is an increasingly appreciated behavior for many proteins throughout biology but the molecular basis is poorly defined. Here, pre equilibrium binding kinetics and single molecule fluorescence methods will be used to compare the distribution of binding parameters in P- gp ensembles vs. single P-gp nanodiscs in order to determine whether P-gp exploits interconverting conformations or persistent distinct conformations to recruit and retain drugs of varying structure.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY/ABSTRACT Acute kidney injury (AKI) is the most common form of organ failure in sepsis and sepsis-induced AKI is associated with prolonged hospitalization, need for acute dialysis, persistent AKI, and death. Despite this public health impact, no effective pharmacotherapy exists for the treatment of sepsis -induced AKI. One reason may be that heterogeneity is present within AKI, thereby concealing unique pathophysiologic processes specific to certain AKI populations. The applicant is an early career investigator who has generated preliminary data demonstrating that two novel AKI sub-phenotypes are present within the larger heterogeneous AKI clinical population. Our group demonstrated that these AKI sub-phenotypes have differing risk for clinical outcomes and response to vasopressin therapy, independent of traditional criteria to risk stratify AKI. We also identified a 3-variable model that included plasma markers of endothelial dysfunction and inflammation to identify the two AKI sub-phenotypes. These findings were subsequently replicated by an independent research group. Through this body of work, we have consistently shown in sepsis-induced AKI that these two AKI sub-phenotypes are reproducible, prognostic and predictive. Important next steps for translating these findings to the bedside include: 1) expanding the pool of candidate biomarkers to identify these or alternative AKI sub-phenotypes in the emergency room (ER), an earlier and critical time to implement strategies to prevent or treat AKI; 2) evaluating response of AKI sub-phenotypes to volume of intravenous fluids, a therapy given to almost all patients with sepsis-induced AKI; and 3) probing whether the AKI sub-phenotypes different in terms of pathophysiologic mechanisms. We will complete the proposed Aims in two ongoing NIH-funded studies, 1) Crystalloid Liberal or Vasopressor Early Resuscitation in Sepsis (CLOVERS) and 2) Kidney Precision Medicine Project (KPMP). In Aim 1, we will identify sepsis-induced AKI sub-phenotypes using biospecimens collected in the ER and determine associations with clinical outcomes in CLOVERS. We will apply two innovative approaches to identify AKI sub-phenotypes (an unsupervised clustering and a previously developed 3-variable model). In Aim 2, we will determine whether sepsis-induced AKI sub-phenotypes respond differently to a restrictive fluid/early vasopressor versus a liberal fluid/late vasopressor resuscitation strategy. In Aim 3, we will identify histological lesions from kidney biopsy tissue and urinary proteins associated with sepsis-induced AKI sub-phenotypes in biospecimens collected during AKI in KPMP. The outstanding qualifications of our team in the field of AKI, sepsis, molecular epidemiology, and interventional clinical trials uniquely position us to align clinical care with underlying molecular mechanisms to inform a ‘precision’ approach to the study and care of patients with sepsis-induced AKI.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Obesity is a major risk factor for cardiovascular (CV) disease. Various CV risk factors like insulin resistance, dyslipidemia, and inflammation are associated with mitochondrial dysfunction. Weight loss is the main recommendation to reduce cardiometabolic risk for people with obesity, but the protection is proportional to the magnitude of weight lost, and fades over time during weight maintenance with the reversal or worsened risk in those who regain weight. This proposal aims to investigate mitochondrial adaptations with weight loss that might lessen its capacity to protect against CV disease. We will do this by studying adaptation in the plateau phase, a time period when further weight loss becomes minimal despite efforts to continue losing weight, and weight regain often follows. The plateau phase is currently understudied and links the occurrence of a weight loss plateau with diminished protection against cardiometabolic risk. In AIM 1, I will determine functional differences in mitochondria at the weight loss plateau in immune cells isolated from people with obesity undergoing weight loss. In AIM 2, I will develop paradigms of the plateau- state in mice with diet-induced obesity to test whether reduced mitochondrial respiratory capacity in liver muscle and immune cells is an underlying mechanism for adaptation in energy expenditure (metabolic adaptation) at the plateau that promote weight regain. For AIM 3, I will determine in a mouse model of accelerated atherosclerosis whether mice the plateau state develop more severe diet-induced CMR, atherosclerosis, and immune cell redox stress. This proposal will be carried out in an institution with strong research programs in metabolism, obesity, and cardiovascular disease. I will receive state-of-the-art training in techniques and analytical tools necessary for completion of all aims, including clinical samples from NIH-funded obesity studies, indirect calorimetry in rodents, and assessment of atherogenic risk in mice. This proposal will generate data laying the foundation for my independent research career focused on mitochondrial mechanisms that influence cardiometabolic risk in obesity.

NIH Research Projects · FY 2024 · 2022-08

ABSTRACT This application proposes a pilot feasibility randomized trial of the USDA Healthy Mediterranean-style Food Pattern versus standardized guidance on fiber intake for patients with diverticulitis in response to PAS-20-160, Small R01s for Clinical Trials Targeting Diseases within the Mission of NIDDK. Diverticulitis is one of the most common gastrointestinal indications for inpatient hospital admission, outpatient clinic and emergency room visits, and colon surgery. At least 20% of individuals with an initial episode of diverticulitis will have one or more painful and unpredictable recurrences. Unfortunately, there is no proven pharmacologic means to decrease the risk of diverticulitis. Large, prospective, observational studies have identified diet and lifestyle risk factors for incident diverticulitis. In one study, men who adhered to a low-risk profile (< 51g/day red meat, >23g/day fiber, 2 hours exercise/week, normal BMI, and never smoked) were 75% less likely to develop incident diverticulitis. However, these modifiable risk factors have not been evaluated for secondary prevention. Studies of diet and plasma inflammatory markers suggest that chronic, systemic inflammation is a potential mechanism that underlies the dietary effects on diverticulitis development. The Mediterranean diet pattern is comparable to diets associated with primary prevention of diverticulitis, is more strongly associated with reduced inflammation, and is familiar to providers and many patients. Thus, we propose to conduct a randomized trial (n=75) of a USDA Healthy Med- style Food Pattern versus standardized guidance on fiber intake for patients with diverticulitis to evaluate the feasibility of this dietary intervention including willingness to randomize and adherence to a Med-style dietary pattern. We will employ state-of-the-art behavioral interventions in the form of electronic feedback to improve health-related behaviors and support dietary customization based on participant’s budget, dietary preferences, and restrictions. We will also examine plasma inflammatory biomarkers (interleukin-6, interleukin-10, and interleukin-1β) and fecal calprotectin at baseline, 6, and 12 months. The proposed feasibility trial will set the groundwork for a large multicenter RCT of a food pattern–behavioral intervention versus standardized guidance on fiber intake in patients with a history of diverticulitis. It will also expand our knowledge of inflammation and diverticulitis pathogenesis and gather prospectively collected samples for future biomarker study. Ultimately, given the prevalence and morbidity of diverticulitis and the lack of predictive and preventative measures, identifying a proven means of secondary prevention and a biomarker of risk would change treatment paradigms and improve the lives of millions of patients with diverticulitis.

NIH Research Projects · FY 2025 · 2022-07

Protein-mediated membrane fusion is essential for a multitude of fundamental biological processes. Despite intensive study, at present we have a limited mechanistic understanding of how fusion protein machinery manipulates lipid membranes in order to induce their fusion. This lack of knowledge is particularly acute regarding the structure of membrane intermediates, the extent to which their leaflets are bent or disrupted into nonbilayer structures, and how they are coordinated and remodeled by fusogens. Similarly, in terms of the structure of the fusion proteins themselves, very little structural information is available to describe how they change as they drive membrane fusion. These are processes that are targeted by therapeutics such as fusion inhibitors or neutralizing antibodies in the case of preventing virus infection, and they are processes that can go awry as a result of disease mutations for cellular fusogens. The proposed studies will expand our understanding of these fundamental processes and reveal general principles employed by divergent fusion machines. Cryo-electron microscopy and structural mass spectrometry provide powerful complementary methods to directly image and probe membrane fusion because they allow us to trigger a fusion reaction under native conditions then trap and then image or analyze intermediate states over the course of the reaction. Cryo-electron tomography in particular can resolve individual fusion machines and membrane leaflets captured in the process of fusing and can discern when the proteins and membranes have adopted non-canonical intermediate structures. Hydrogen/deuterium- exchange mass spectrometry complements cryo-EM by enabling us to monitor local backbone dynamics under native conditions. This approach is particularly effective for tracking conformational changes and for comparing protein structure in different states. Building on our work with influenza virus, we will apply these methods to investigate pathways of membrane fusion in two Class I viral fusion systems: the Env fusion protein used by HIV and the S spike protein used by SARS-CoV-2. These fusion machines employ sequential modes of activation and triggering involving receptor priming followed by either coreceptor binding (Env) or a proteolytic cleavage event (S). These systems thus offer the opportunity to analyze in detail the fusion system arrested at an intermediate, primed stage. For each of these systems, our goal is to image the architecture and progression of membrane remodeling leading to formation of fusion pores and to understand the means by which the protein machinery induces two separate membrane bilayers to join into one. By performing such an analysis, we will gain novel insight into general, obligatory events in Class I protein-mediated membrane fusion, while also revealing system-specific mechanisms. Our study should thus advance our structural and mechanistic understanding of the fundamental process of biological membrane fusion while also providing valuable insight into the mechanism of host invasion by two viruses that have ignited major pandemics impacting global health and society.

NIH Research Projects · FY 2025 · 2022-07

Project Summary Brain stimulation has shown great therapeutic promise for a wide range of neurological and psychiatric disorders. In addition to advanced engineering tools, successful implementation of brain stimulation requires a comprehensive un- derstanding of how this treatment drives changes in network dynamics and connectivity at a large scale and across multiple brain areas. It also requires the design of controllers that can relate stimulation effects to behavior and function. To achieve these goals, we will develop novel explainable machine learning models for psychiatric brain stimulation. To do so, we put forward three overarching goals. First, we aim to learn biologically plausible and flexible functional connectivity models from electrocorticography (ECoG) data. Then, we plan to develop a computational model based on a deep graph convolutional net to learn associations between ECoG data and network-scale connectivity. We will then design a machine learning based guide for psychiatric brain stimulation. Finally, we will use our tools to under- stand how the network evolves through time. To achieve these goals, the project brings together an interdisciplinary team of investigators with unique expertise in artificial intelligence and machine learning, computational and theoretical neuroscience, network science and biostatistics, bioengineering and brain stimulation experiments, and interventional psychiatric and neural engineering. The team will lead experimental and computational efforts that will produce ad- vanced explainable machine learning solutions informed by brain stimulation experiments and utilize these tools to design more efficient and effective brain stimulation therapies.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY: Microglia, the brain’s resident immune cells, are implicated in the pathophysiology of chronic ischemic cerebral small vessel disease (CSVD) and vascular cognitive impairment (VCI), a clinically important cause of dementia that preferentially affects white matter (WM). Colony stimulating factor 1 receptor (CSF1R) is a key regulator of myeloid lineage cells. Genetic loss of CSF1R blocks the normal population of resident microglia and systemic treatment with CSF1R antagonists such as PLX5622 results in a marked depletion of microglia in the CNS. Here we propose to evaluate the role of microglia and CSF1R signaling in a novel rodent model of CSVD/VCI developed in the laboratory of our colleague Dr. Edith Hamel (McGill University). Mice overexpressing transforming growth factor-beta1 (TGFOE) and fed a high cholesterol diet (HCD) display impaired cerebrovascular reactivity, diffuse leukoencephalopathy, and cognitive dysfunction that resembles human CSVD/VCI. We will first assess effects of TGFOE and HCD on regional and cell type specific gene expression in microglia and other myeloid cells using single cell RNA sequencing. Next we will determine if treatment with PLX5622 influences neurological, neuroimaging, and/or neurovascular outcomes in TGFOE mice (+/-HCD). We will assess longitudinal WM integrity outcomes using state-of- the-art 14 Tesla MRI with diffusion tensor imaging (DTI) and quantitative tractography. We will also use sophisticated assessments of resting-state functional connectivity (optical imaging of intrinsic signals) and neurocognition. Furthermore, we will perform longitudinal 2-photon brain imaging studies to track changes in the cerebrovasculature during critical periods of CSVD/ VCI disease initiation and progression. We will confirm the microglia-specific nature of our findings using an orthogonal genetic approach by crossing TGFOE mice with a Csf1rflox/flox : HexbCreERT2 bigenic line that exhibits stable, microglia-specific knockdown of Csf1r. We will then feed the trigenic TGFOE : Csf1rflox/flox : HexbCreERT2 line HCD and carry out the same comprehensive set of longitudinal parameters described above. We anticipate that both pharmacologic depletion of microglia and genetic deletion of CSF1R signaling specifically in microglia will attenuate WM inflammation and injury and result in improvement in multiple outcome parameters. In aim 3 we will collaborate with our colleagues in Radiology to use a novel CSF1R radioligand to carry out longitudinal positron emission tomography (PET) on TGFOE mice (+/-HCD) at multiple time points during disease initiation and progression. This will provide critical information on temporal and spatial in vivo expression patterns of CSF1R in the context of CSVD/VCI and generate foundational data for a novel biomarker for neuroinflammation that can be readily scaled up from mouse to humans. Overall, this microglia- and CSF1R signaling-targeted, WM-centric approach will provide important mechanistic information on the pathogenesis of ischemic WM injury in CSVD/VCI and elucidate new potential diagnostic and therapeutic approaches.

NIH Research Projects · FY 2025 · 2022-07

ABSTRACT Despite the significant public health impact of highly active antiretroviral therapy and pre-exposure prophylaxis on the HIV-1 pandemic, fundamental questions remain about the biological mechanisms that define the risk of sexually acquiring HIV-1. Notably, there is no explanation why, after accounting for genetic variants that directly modify HIV-1 entry and replication (foremost CCR5Δ-32), individuals with documented high exposure to HIV- 1 exhibit diverse outcomes ranging from rapid infection to prolonged natural resistance to HIV-1 infection. A better understanding of these phenotypes could lead to novel interventions to reduce HIV-1 acquisition. We previously identified functional variants (missense/nonsense, untranslated region, or splice site) in host genes that are strongly associated with altered risk of heterosexually acquired HIV-1 infection. Notably, genetic variants in CD101 had the strongest association with increased HIV-1 risk, while variants in AXL had substantially reduced risk. Both genes are thought to regulate host inflammatory responses. CD101 regulates T cell activation and is expressed on many immune cell types. We recently reported that circulating T cells and monocytes from donors with these CD101 variants, compared to donors without functional CD101 variants, show higher overall cellular activation, but with reduced expression of certain HIV-protective interferon stimulated genes. In contrast, AXL is a receptor tyrosine kinase thought to act through myeloid cells to inhibit cellular immune activation, possibly through inhibition of toll-like receptor-mediated innate immune responses. Our overarching hypothesis is that host variation in CD101 and AXL genotypes contributes to altered inflammatory homeostasis, thereby influencing the risk of HIV-1 infection. Specifically, we hypothesize that CD101 variants confer a higher risk of HIV-1 infection by increasing the abundance and activation of HIV-1 susceptible T cells, while also compromising their innate antiviral defenses. Conversely, the absence of these variants, possibly augmented by reduced inflammation conferred by AXL variants, lowers HIV-1 infection risk. We propose to evaluate our hypothesis in three aims: in Aim 1, we use genomic data from a cohort with well- characterized HIV-1 acquisition phenotypes to replicate in a second high HIV-risk African cohort that AXL variants balance the impact of CD101 variants on HIV-1 susceptibility. In Aim 2, we test the epidemiological and population generalizability of our hypothesis by quantifying the association of CD101 and AXL variation with susceptibility to HIV-1 infection in two US populations for which genomic data already exist: one that includes predominantly white men who have sex with men; and a second, ethnically diverse cohort recruited across the entire US. In Aim 3, we apply molecular techniques to blood and genital mucosal samples archived in a unique biospecimen repository to directly link the presence of these CD101 and AXL variants with host inflammatory mechanisms, and ex vivo HIV-1 susceptibility.

NIH Research Projects · FY 2025 · 2022-07

While Staphylococcus aureus (SA) commonly asymptomatically colonizes the skin and nose of healthy humans, severe disease can result from infection of the blood, bone, skin, and lungs, as well as sites of catheters and prosthetic devices. With currently approved therapy, about one-third of patients diagnosed with SA bacteremia succumb, accounting for more annual deaths than HIV, tuberculosis, and viral hepatitis combined. This R01 will develop an injectable vaccine depot comprising: (a) previously published cationic polymers to condense and charge neutralize anionic self-replicating mRNA (SR-mRNA) vaccines into nanometer-sized particles (i.e., “polyplexes”) that are then incorporated within (b) our recently reported injectable biodegradable gel of N-succinyl-chitosan (S-CS) and oxidized alginate (O-Alg). Ultimately, the temporary CS-Alg depots completely biodegrade into non-toxic by-products that are eliminated. This project will generate a self-immunizing biomaterials technology that is applied ONCE that is superior in immunization versus repeated systemic bolus injections.

NIH Research Projects · FY 2026 · 2022-07

The long-term goal of this research is a prosthetic socket for trans-tibial prosthesis users that continually adjusts size so as to both maintain a proper fit and stabilize limb volume. The socket relieves users of the burden of continually sensing if their socket fit has deteriorated, deciding what adjustment to make, and effecting a socket size change. Users may instead focus on other aspects of their life. The auto-adjusting socket should enhance independence, improve limb health, and enrich patient quality of life. The specific aims are to modify an auto-adjusting socket for Walking previously developed by our group to add a remote key fob interface and two new auto-adjustment algorithms. The compact key fob eliminates a barrier present in the prior design. A Low Activity auto-adjustment algorithm manages socket size to maintain fit during short bouts of walking mixed with standing, as would occur for example when moving about in a building. A Sitting algorithm facilitates limb fluid volume recovery during sitting. The algorithms are optimized through lab- based participant testing. Performance of the complete auto-adjusting socket system is tested in user free-living environments in three modes: auto-adjusting; manual-adjusting; and locked (sock changes only). The aims are accomplished by alternating between clinical studies and engineering design. In the first aim, prototype key fobs are fabricated and evaluated by participants with limb amputation and prosthetists in a qualitative study design. From the results a single design is specified for development. In the second aim, new auto-adjustment algorithms are created and added to the existing on-board control system to maintain fit during combined short bouts of walking and standing (Low Activity) and during sitting (Sitting). Fit is monitored using measurements from custom limb-socket distance sensors embedded in the socket. In a crossover randomized trial, socket comfort, residual limb health, variability in limb fluid volume, and control system error are assessed while participants wear the socket during a structured protocol with all three auto-adjustment functions enabled (Walking, Low Activity, Sitting) compared with only the Walking function enabled. Based on study results, the socket control system is updated accordingly. In the third aim, a crossover randomized trial is conducted in at- home testing where participants wear the socket in auto, manual, and locked modes. In the manual mode, participants adjust the socket using the key fob. Participant self-report scores, residual limb health, prosthesis use, activities, socket fit, number of manual socket adjustments and participant preference are compared. The health relatedness of this application is a reduction of the burden of socket fit monitoring and fit management for patients with limb loss. Rather than having to continually manage their disability, people with limb amputation may live more independent lives. The auto-adjusting socket should also reduce residual limb health complications from poor socket fit. The system may serve as a model for design of technologies that address other levels of amputation (e.g., transfemoral, upper-limb) where automatic adjustment to maintain fit is needed.

NIH Research Projects · FY 2025 · 2022-07

Preterm infants are at high risk for both transfusions and iron deficiency, both of which may independently contribute to poor neurodevelopmental outcomes. On average, infants less than 1000 gm require 4 to 5 blood transfusions during their initial hospitalization. Darbepoetin (Darbe) can increase the number of infants who remain transfusion-free, and for those who do require transfusions, can decrease the number of transfusions, cumulative volume of blood transfused, and unique donor exposures. However, the use of Darbe increases iron utilization, and treated infants may become progressively iron deficient with oral iron supplementation alone. When iron supply does not meet the iron demand of the rapidly expanding RBC mass, first iron stores in the liver and then non-storage iron in other tissues (including brain) will be compromised. This is of particular concern for preterm infants since iron is required for normal brain development, including such processes as myelination, dendritogenesis, production and degradation of neurotransmitters, and to sustain the brain’s high metabolic rate. Thus, iron deficiency during fetal and early postnatal months can result in irreversible neurodevelopmental abnormalities despite later iron repletion. Our overarching goal is to develop a therapeutic pathway to minimize transfusions while maintaining iron sufficiency, thereby optimizing developmental outcomes. We hypothesize that combined treatment of infants < 32 completed weeks of gestation with Darbe plus one of two slow-release intravenous (IV) iron preparations, ferumoxytol (FMX) or low molecular weight iron dextran (LMW-ID) will 1) be safe, 2) decrease or eliminate transfusions, 3) increase hematocrit, 4) maintain iron sufficiency, and 5) improve neurodevelopment. We further hypothesize that when compared to oral iron supplementation (standard care), IV iron will be better tolerated, with less effect on the gut microbiome. An advantage of using these slow-release IV iron preparations is that fewer, higher doses of iron are needed to prevent or treat iron deficiency. For example, an anemic iron deficient pregnant woman can be treated with a single dose of 1000 mg IV compared to 5 IV doses of iron sucrose. Because FMX and LMW-ID have not been tested in neonates, in Aim 1 we will compare these two drugs and evaluate total dosage needed to maintain iron sufficiency while being treated with Darbe. Starting doses will be 10 mg/kg and 20 mg/kg, repeated as needed to maintain ferritin >75 mcg/L. In Aim 2 we will compare the safety, efficacy and tolerance of the combined approach of using Darbe + IV iron (N=80) with standard care (oral iron supplements up to 12 mg/kg/day, N=40). Evaluation will include safety, gastrointestinal tolerance, and efficacy. The effect of oral compared to IV iron on the microbiome will be evaluated. The primary outcome will be hematocrit, transfusion burden and iron status at hospital discharge. In Aim 3, the long-term neurodevelopmental outcome of the 3 groups (Darbe + FMX, Darbe + LMW-ID, oral iron only) will be evaluated up to 2 years of age. We anticipate demonstrating the feasibility and potential benefit of Darbe plus slow-release IV iron to decrease transfusions, maintain iron sufficiency and improve neurodevelopmental outcomes.