Washington University

NIH Research Projects · FY 2024 · 2022-09

Abstract Despite the development of dozens of drugs since the start of the HIV/AIDS epidemic, the emergence of drug resistance and lack of a vaccine or cure necessitate the development of new antiretroviral compounds. The HIV-1 capsid (CA) protein plays essential roles throughout the viral replication cycle. After an immature HIV-1 virion buds from a host cell, the structural Gag polyprotein undergoes proteolytic cleavage and rearrangement. The retroviral core is formed when rings of CA, held together by intra- and inter-subunit interactions, arrange into a conical lattice around the viral genomic RNA (gRNA) and enzymes. Since many CA-CA interactions are required to form a stable lattice, CA is genetically fragile and a favorable drug target. In fact, compounds that successfully target CA assembly and stability have recently been developed as part of long-acting drug regimens and show great promise clinically. Following their release into the cytoplasm of target cells, HIV-1 cores undergo an uncoating process in which CA subunits are shed from the core. It is now evident that proper uncoating is crucial for subsequent steps in virus replication, including reverse transcription, nuclear entry, and integration. We have recently demonstrated that destabilization of the CA lattice through mutations and CA-targeting compounds increases the propensity to form aberrant virus particles. In these particles, the gRNA and enzymes are localized between the CA lattice and viral envelope. Interestingly, this phenotype has striking similarities with the eccentric virions that are generated by inhibition of integrase (IN)-gRNA interactions. We have shown that the lack of protection by the CA lattice in both circumstances results in premature loss of gRNA and IN in a proteasome-independent manner. However, the mechanism by which IN and gRNA are degraded upon loss of CA protection remains unclear. Furthermore, recent studies have implicated that CA may shield viral nucleic acids from the host sensor proteins that initiate antiviral responses. I hypothesize that tampering with the stability of the HIV-1 CA lattice will result in premature exposure and sensing of viral nucleic acids in infected cells. Here, I propose to determine how prematurely exposed viral ribonucleoprotein complexes (vRNPs) are degraded in target cells (Aim 1). I plan to determine if altered CA stability elicits a more robust innate immune response against HIV-1 and the mechanism by which viral nucleic acids are sensed (Aim 2). Together, the results of these experiments will contribute to a better understanding of the proposed role of CA in virus replication and evasion of innate immune sensing.

NIH Research Projects · FY 2025 · 2022-09

Our laboratories recent studies (Stegh and colleagues, Cell Report, 2017; Wahl and colleagues, Cancer Research, 2017) indicated that IDH1 wild-type (IDH1-wt) is overexpressed in 2/3 of HGG (referred to here as `IDH1-wthigh GBM') that lack IDH1R132H mutation. Both alone while genetic and pharmacological inhibition of wt-IDH1, and in combination with radiation therapy (RT) slows the growth of patient-derived HGG xenografts 5,6 overexpression of wt-IDH1 promotes intracranial HGG growth molecular levels, wt-IDH1 high , . On GBM produce excess NADPH, which serves as a rate-limiting reductant that drives the biosynthesis of mono- unsaturated fatty acids (MUFAs). In addition, enhanced NADPH production increases glutathione (GSH) level, reduces reactive oxygen species (ROS), activates phospholipidperoxidase glutathione peroxidase 4 (GPX4)-drivenlipid repair, and dampensthe accumulation of polyunsaturated fatty acid (PUFA)-containing lipid peroxides, known executioners of ferroptosis. Based on these findings, we hypothesize that wt-IDH1 through enhanced lipid repair, heightened MUFA biosynthesis and displacement of oxidizable PUFAs from plasma membrane phospholipids antagonizes ferroptosis, a recently discovered form of cell death has rapidly gained recognition as a paradigm shifting strategy to specifically target cancer cells. We further hypothesize that wt-IDH1 inhibition cooperates with known inducers of ferroptosis, including RT and immune-mediated checkpoint inhibition, to antagonize HGG progression. For the pharmacological inhibition of wt-IDH1, we have used and characterized 13i, a first-in- class competitive ???-unsaturated enone, developed by AbbVie. 13i potently inhibits wt-IDH1 enzymatic activity, by covalently binding to the NADP+ binding pocket. Our data indicate that 13i promotes ferroptosis, is brain-penetrant, and like genetic ablation, reduces progression and extends the survival of IDH1-wthigh HGG bearing mice, alone and in combination with RT. We will test these hypotheses in three Specific Aims: Aim 1: Determine how wt-IDH1 impacts de novo fatty acid biosynthesis and membrane phospholipid composition to inhibit ferroptosis. Aim 2. Determine how wt-IDH1 promotes GPX4-dependent lipid repair and antagonizes ferroptosis. Aim 3: Determine if genetic and pharmacological inactivation of wt-IDH1 amplifies ferroptosis in response to RT and immune checkpoint blockade and antagonizes HGG progression. Objectives and long-term goals. We will credential wt-IDH1 as regulator of ferroptosis in HGG and will validate the pharmacological inhibition of wt-IDH1 using a novel NADP+ competitive inhibitor as a therapeutic strategy. Results from these studies are expected to inform the design of IND-enabling studies evaluating the potential of 13i as adjuvant for anti-HGG therapy.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY Just as gene and protein expression are common characteristics to identify a cell, lineage is an important aspect of cell identity. In the past, lineage tracing has been used to determine what cells arise from a specific cell type, as defined by the expression of a cell-type-specific gene; however, the establishment of single-cell genomics techniques has ushered in lineage tracing at single-cell resolution. New technologies for lineage tracing can track the progeny of single cells, regardless of their initial gene expression. The Morris lab has developed a single-cell lineage tracing (scLT) method, called CellTagging. CellTagging demonstrated the ability of scLT approaches to identify similarities in cells based on lineage and offer mechanistic insights to cell reprogramming. CellTagging and other virus-based scLT technologies still present limitations, though, in that they require multiple transductions to increase lineage resolution, and may fail to capture biologically relevant bifurcation events due to cell labeling at discrete time points. These technologies are also subject to transgene silencing in certain cell models, such as iPSC-derived organoids, rendering them ineffective for use in many models of development and disease. To overcome these limitations, it is necessary to develop new scLT tools that can be applied without repeated manipulation of cells and be used in iPSC differentiation and reprogramming systems without silencing hindering the readout of lineage information. Here, I propose to utilize a CRISPR-Cas12a-guided cytidine deaminase as a method to continuously record heritable lineage data through targeted cytidine to thymine editing. I have developed and validated the ability of a novel CRISPR-Cas12a-guided cytidine deaminase to accrue base edits on a targeted synthetic DNA region over time in vitro and recovered these synthetic sequences via single-cell RNA-sequencing (scRNA-seq). These two outcomes are a promising proof- of-concept that scLT can be performed with these accrued single base edits. Here, I propose to (1) increase the resolution of CellTagging to capture bifurcation events, using this novel DNA editor to constantly edit single bases in a targeted editing region (TER) and dispense with the need for multiple transductions, and (2) integrate this base editor system into a safe harbor locus within an iPSC line to shield the transgenic components of the technology from silencing, validating this approach in kidney organoid differentiation. My proposed developments of the CellTagging technology increase the potential for discovery because they can be broadly applied to model systems that are either not amenable to multiple manipulations or are prone to transgene silencing. By making all plasmids, cell lines, protocols, and analysis tools for these systems publicly available, I aim to provide a valuable resource across several areas of cell biology. These resources will provide an experimental toolkit for anyone working with in vitro developmental, reprogramming, and disease models to interrogate single-cell lineage at high resolution.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY/ABSTRACT In stark contrast to the widespread prevalence and devastating outcomes associated with alcohol use disorder, currently available treatment options are only moderately effective. The large heterogeneity in AUD presentations may obfuscate etiology and individualized treatment options. In this 5-year K01 mentored research scientist award application, I propose to characterize the behavioral, molecular, and genetic correlates of AUD biotypes (subtypes determined by biology) across the lifespan. To this end, I will apply semi-supervised machine learning algorithms to structural brain imaging data from the largest available AUD and alcohol use datasets from childhood to old age (total n=61,428). I will examine the stability of these biotypes across the lifespan, including among substance-naïve children and adults with heavy alcohol use, as well as their correlates with neurobehavioral stage-based constructs of addiction (i.e., impulsivity, negative affect, cognition) and with alcohol involvement trajectories. I will then conduct a genome-wide association study of AUD biotypes to disarticulate their genetic architecture, genetic correlates, and potential molecular pathways that may be leveraged for drug repositioning. This grant develops my skillsets in semi-supervised machine learning, AUD heterogeneous presentations, multivariate genome-wide methods, and multi-omic analytic approaches. These skill sets will serve as a backdrop for a planned R01 submission that will leverage my background in large-scale data analysis to translate across biological modalities in substance use research.

NIH Research Projects · FY 2025 · 2022-08

HIV is more common among adolescents and young adults (AYA, 13–24 years old) in the United States. Low uptake of HIV prevention services suggests a missed opportunity for implementing evidence-based interventions such as pre-exposure prophylaxis (PrEP) and sexually transmitted infection testing among this important population. Most research institutions in the United States have limited opportunities for AYA training, mentorship, and capacity building activities. Over the past three years, our multi-disciplinary team organized AYA implementation HIV research as part of ITEST, Innovative Tools for Expanding HIV Self-Testing. This study uses crowdsourcing methods and implementation science strategies to develop innovative HIV self-testing services for AYA. Crowdsourcing has a group of people solve all or part of a problem, then implement exceptional ideas. Our study discovered that crowdsourcing methods could also be used to help identify highly qualified trainees through open calls, build capacity for youth-led research using hackathons, and sustain these benefits through participatory learning communities. These approaches break new ground in HIV training using participatory methods that will help AYA to become junior leaders while building institutional capacity for AYA HIV research. Extending from this strong foundation, we propose the “Stimulating Training and Access to HIV Research Experiences” (STAR) Institute. The project brings together a highly qualified group of research mentors at Washington University in Louis, UNC Chapel Hill, Georgia State University, Northeastern, and Wake Forest University. Specific aims of this project include: 1) identify and recruit AYA interested in HIV research for STAR Institute; 2) develop crowdsourcing and implementation research capacity at participating US institutions; 3) initiate and sustain enduring AYA research capacity through a digital participatory learning community. Each year we will identify 10 trainees who will join our intensive six-week summer training program and a year-long virtual learning community. The proposed R25 training program complements existing training opportunities and will provide unique resources to build capacity for AYA studies, implementation research, and crowdsourcing.

NIH Research Projects · FY 2026 · 2022-08

PROJECT SUMMARY Many age-related neurodegenerative diseases—including Alzheimer's disease (AD)—are associated with misfolded protein deposition that promote inflammatory responses, neuronal dysfunction, and cognitive deficits. We have recently identified that aging and AD both display impaired meningeal lymphatic function, which ultimately results in impaired CSF drainage to deep cervical lymph nodes, as well as CSF perfusion into the brain parenchyma, collectively promoting waste build up and A-induced pathologies in AD mice. Our preliminary data demonstrate that aging is also associated with the accumulation of IFNγ-producing CD4 and CD8 T cells in the dural meninges, closely associated with the meningeal lymphatics. IFNγ signaling represents a transcriptional hallmark of aged meningeal lymphatics and augmentation of this axis in young mice attenuates their functional drainage of CSF. We therefore hypothesize that during aging and in AD, elevated expression of IFNγ from meningeal CD4 and CD8 T cells impairs meningeal lymphatic function function via direct signaling on their IFNγ receptors, leading to meningeal lymphatic deterioration. Such deterioration later results in impaired brain perfusion by cerebrospinal fluid (CSF), subsequently leading to the accumulation of debris and worsening progression of AD. We further hypothesize that using cytokine neutralizing antibodies, we can preserve meningeal lymphatics in aged mice and prevent or reduce the age-associated brain dysfunctions and augment existing immunotherapy strategies in AD patients. Addressing our hypotheses of this proposal will illuminate mechanistic pathways underlying age-related meningeal lymphatic dysfunction, and identify new promising avenues for therapeutic interventions intended to reduce AD-related pathology.

NIH Research Projects · FY 2025 · 2022-08

Project Summary Diabetes affects more than 34 million people in the US alone. It is the leading cause of non-traumatic lower limb amputation, largely due to the development of chronic diabetic wounds. While various therapies have been explored to treat diabetic wounds, effective treatment remains a challenge as current therapies cannot efficiently address the key intrinsic causes of slow diabetic wound healing, i.e., chronic inflammation, abnormal skin cell functions (particularly migration), and delayed angiogenesis. To address these causes, it is crucial to control TGFβ signaling. TGFβ1/p38 pathway is directly associated with prolonged inflammation, and impaired cell migration in wounds. Meanwhile, TGFβ1/Smad2/3 pathway is required to regulate a critical cell type for wound healing, myofibroblasts. As such, inhibiting TGFβ1/p38 pathway without affecting TGFβ1/Smad2/3 pathway will simultaneously address the 3 key intrinsic causes, leading to accelerated diabetic wound healing. However, this cannot be achieved by any existing approaches. In this project, we propose to create a new wound dressing to achieve the goal. It will consist of a peptide-based TGFβ receptor II (TGFβRII) inhibitor ECG, and a reactive oxygen species (ROS)-scavenging hydrogel. The ECG will be gradually released from the hydrogel to continuously inhibit TGFβ1/p38 pathway so as to improve cell migration and decrease tissue inflammation. The enhanced endothelial cell migration will lead to accelerated angiogenesis. The hydrogel will scavenge upregulated ROS in the diabetic wounds to further decrease inflammation. Notably, ECG will not affect TGFβ1/Smad2/3 pathway under high glucose condition. To the best of our knowledge, none of the existing TGFβ receptor inhibitors have shown capability of inhibiting TGFβ1/p38 pathway without downregulating TGFβ1/Smad2/3 pathway. In our preliminary study, application of a single dose of wound dressing into excisional wounds in young diabetic mice significantly accelerated wound closure. The wounds completely closed at day 14. In contrast, the wound size remained >53% for the hydrogel-treated, and untreated wounds. The wound dressing also decreased ROS content, M1 macrophage density and p-p38 expression, and increased vessel density in the wounds. These preliminary results demonstrate that ECG-releasing wound dressing is promising for diabetic wound healing. It is yet to test whether the wound dressing can promote diabetic wound healing under aged condition, as aging itself impairs cell migration and angiogenesis. We hypothesize that the wound dressing based on ECG and ROS-scavenging hydrogel will significantly enhance skin cell migration, stimulate tissue angiogenesis, and decrease tissue inflammation, leading to accelerated healing of diabetic wounds under young and aged conditions. Aim #1 will test the hypothesis that optimal wound dressings will simultaneously scavenge ROS, increase skin cell migration, promote endothelial lumen formation, and attenuate inflammatory cytokine secretion under TGFβ and high glucose conditions. Aim #2 will test the hypothesis that the developed wound dressings will accelerate diabetic wound healing under young and aged conditions. This project is innovative because the proposed wound dressings will simultaneously address the 3 key intrinsic hurdles for diabetic wounds to heal, by differentially regulating TGFβ signaling, i.e., inhibiting TGFβ1/p38 pathway, while not affecting TGFβ1/Smad2/3 pathway that is essential for diabetic wound healing.

NIH Research Projects · FY 2025 · 2022-08

Project Summary Epigenetic mechanisms have been linked to many human disorders and diseases, most notably cancer. However, in recent years more and more human diseases have been found with possible epigenetic insults. In this regard, our collaborators at the Children’s Hospital of Philadelphia have through exome sequencing discovered germline-line mutations to the H3FA and H3FB genes in pediatric patients that suffer from similar neurological disorders and craniofacial abnormalities. These two genes encode for the histone variant H3.3, and thus represent the first germ-line mutations found on histone H3 in humans (neurohistone H3.3 mutations). Histones are small basic proteins that bind DNA to give rise to our chromatin structure. Along with DNA methylation and long non-coding RNA, post-translational modifications to histones regulate gene expression patterns (epigenetic mechanisms) and chromatin organization. Histone H3.3 is a specialized histone variant linked to active genes, and somatic mutations to this histone have been found in different brain cancers exclusively on the N-terminal tail. Our preliminary data have found that these neurohistone H3.3 mutations are spread across the entire protein from the N- to C-terminus. Therefore, we hypothesize that these mutations while resulting in similar phenotypes, do this by disrupting different epigenetic mechanisms involving histone H3.3. In this proposal, we aim to understand how these neurohistone mutations lead to neurodevelopmental problems. First, we aim to determine if these neurohistone mutations affect global or local histone modification patterns on wild-type or mutant histone variants using novel mass spectrometry (MS) approaches in cellular models. Next, we look to determine if these neurohistone mutants affect chromatin structure or conformation, or cause misincorporation of the H3.3 variant in the genome. Lastly, we will characterize proteome expression in a mouse model of one of the mutations using a novel in utero stable isotope labeling approach. It is our goal to determine how these neurohistone H3.3 mutations affect epigenetic and cellular signaling mechanisms to disrupt neurodevelopment and maintenance leading to neurological disorders in these pediatric patients.

NIH Research Projects · FY 2025 · 2022-08

SUMMARY Depression is a leading cause of morbidity and mortality worldwide and it is projected to become the second leading cause of disability by 2030. Despite these results indicate the urgent need to address depression as a public-health priority to reduce disease burden and disability, current pharmacotherapies for depression require prolonged administration (weeks if not months) for clinical improvement and they are often associated with high non-response rate. In contrast, recent clinical evidence has shown that a single sub-anesthetic dose of ketamine induces a robust and rapid (within matter of hours) antidepressant effect in 70% of treatment-resistant patients. Notably, ketamine is the first rapid-acting antidepressant with efficacy for treatment-resistant symptoms of major depression disorder such as anhedonia. Anhedonia, defined as diminished pleasure from, or interest in, previously rewarding activities is commonly precipitated by exposure to chronic stress and it is well suited to study in laboratory animals. Whereas ketamine’s primary molecular target is under debate, there is broad consensus in the literature that activation of the AMPAR as well as induction of synaptogenesis driven by de novo protein synthesis-dependent mechanisms are required for ketamine’s ability to ameliorate stress-induced anhedonia. Nevertheless, major technical barriers have hindered a circuit and synaptic-level dissection of such mechanisms. Therefore, understanding the detailed circuit and synaptic mechanisms and establishing a causal link between ketamine- evoked AMPAR-mediated synaptic plasticity and specific behavioral outcomes is crucial for designing novel and safer therapeutic targets. Here, we will tackle this question by using electrophysiology, optogenetics and recently developed technologies that offer the unprecedented opportunity to block AMPAR as well as de novo protein synthesis within genetically specified cells. Accordingly, the following hypotheses will be tested: (i) increased AMPAR-mediated synaptic transmission on D1-MSNs mediates the anti-anhedonic effects of ketamine, (ii) mPFC->NAc input is necessary for ketamine-mediated amelioration of stress-induced anhedonia, and (iii) ketamine-induced de novo protein synthesis-dependent plasticity in D1-MSNs drives ketamine-mediated amelioration of stress-induced anhedonia. Altogether, results from these studies will increase our understanding of the mechanisms of action of ketamine and might lead to new potential targets to treat stress-induced anhedonia.

NIH Research Projects · FY 2025 · 2022-08

Abstract Alzheimer disease (AD) is the most common form of dementia and neurodegeneration affecting more than 5 million Americans with no current effective treatment. Several Mendelian mutations and risk variants have been identified. We and others have shown that AD is associated with changes in brain cell proportion and transcriptomic changes, some of them are also cell specific. Additionally, the latest genetic studies implicate cell-specific pathogenic events that lead to disease. Pathogenic variants in APP, PSEN1 and PSEN2 affects APP processing leading to Aβ aggregates and neuronal death. Genetic variants in TREM2 and MS4A modify AD risk by affecting microglia activity. To fully understand and characterize the role of transposable elements (TE) in AD pathogenesis there is a need to novel and multidisciplinary approaches. Here we will combine novel genomic approaches in human brain tissues, direct converted neurons and iPSC-derived microglia (iMGL) to identify cell-specific TE and downstream (chromatin accessibility, transcription) changes implicated in AD. We will leverage a large and unique resources of human brain samples and fibroblast from individuals with mutations in APP, PSEN1, PSEN2, as well as risk variants in TREM2, MS4A or APOE. We will also use the direct converted neurons and iMGL, together with new genomic editing approaches to target and characterize the mechanism by which TE contribute to disease.

NIH Research Projects · FY 2024 · 2022-08

PROJECT SUMMARY This proposal defines a 5-year plan to prepare Aaron Ver Heul, MD, PhD, to reach his long-term goal of independence as a physician-scientist studying the neuroimmunology of allergic diseases. He is currently an Instructor in the Division of Allergy and Immunology at Washington University doing post-doctoral studies in the laboratory of Dr. Brian Kim. There, Dr. Ver Heul has studied basic mechanisms of itch to complement his clinical interest in chronic spontaneous urticaria (CSU), an itchy skin condition characterized by exaggerated mast cell (MC) responses. He has demonstrated that the cytokine interleukin (IL)-33 enhances itch in a MC-dependent manner and proposes to extend these studies to CSU. Thus, the primary scientific goal of this proposal is to understand the role of IL-33 in CSU and itch. Washington University is an ideal place to perform the proposed training and research, with outstanding resources and expertise readily available. Dr. Brian Kim, the primary mentor, is an expert in neuroimmunology, atopic dermatitis, and itch. Dr. Steve Brody, the co-mentor, is an expert in translational models of epithelial biology. Both have strong records of continuous funding and training successful scientists. Dr. Ver Heul has also assembled an advisory committee with expertise in immunology, neuroscience, functional genomics, and in vivo microscopy, who will provide crucial scientific training and career guidance. He will take didactics in genomics, microscopy, and scientific writing. He will have multiple opportunities to present his work locally and to the larger scientific community. Dr. Ver Heul’s immediate objectives during the award period are to acquire requisite skills to complete the proposed research, publish the results, and successfully obtain independent funding. IL-33 is increased in a variety of allergic diseases including CSU, which affects 1% of the population. MCs are highly responsive to IL-33, and exaggerated MC activation is a major contributor to CSU pathogenesis. Recently, two distinct pathways by which MCs can be activated to cause itch were identified. One responds to allergens, while the other responds to a variety of small molecules including neuropeptides and many common drugs (known as basic secretagogues). Taken together with the demonstration that IL-33 enhances histaminergic itch, Dr. Ver Heul now proposes to test the hypothesis that IL-33 broadly amplifies different forms of MC-mediated itch responses. By completing the proposed aims, Dr. Ver Heul will define 1) how IL-33 enhances allergen- mediated itch and 2) how IL-33 enhances basic secretagogue-mediated itch. The proposed studies determine mechanisms of IL-33-enhanced MC activation in vitro and in mouse models of itch and extend these findings to analyses of CSU patient samples. Fulfilling the aims will provide new insight into mechanisms of CSU and itch and establish Dr. Ver Heul as an independent investigator in allergy and immunology.

NIH Research Projects · FY 2025 · 2022-08

PROJECT ABSTRACT: This proposal seeks to understand the impact of genetic and social risk factors on Alzheimer's disease (AD) age at onset, disease progression and biomarkers trajectories in Hispanic populations. Under the guidance of primary mentor Dr. Randall J. Bateman, the training and research plan will build upon Dr. Jorge Libre's expertise in behavioral neurology and epidemiology of AD in Hispanic populations to prepare him for an independent career that integrates genetics, social science, and life course epidemiology into the study of AD. Dr.Llibre will pursue a mentored research scientist program of training at Washington University, that will advance his knowledge and skills in (1) enhanced knowledge of the genetic architecture of Alzheimer's disease, genetics epidemiology and advances in the analysis of molecular genetic data, (2) statistical methods for longitudinal analysis, (3) clinical and biomarkers assessments of AD and (4) use large data for the study of AD. These are the areas needed to further develop his expertise and become a successful, independent investigator. The research proposed in this application was designed to significantly contribute to the field of AD in Hispanics populations. The research plan will capitalize on deeply phenotype cohorts and large populations studies including Caribbean-Hispanics (10/66 study, Caribbean American Dementia and Aging Study [CADAS], Dominantly Inherited Alzheimer's Network [DIAN]), Central and South American (10/66 study, DIAN) and Hispanics living in the US (US-Health and Retirement study [HRS], DIAN), which may provide a unique understanding of disease onset, progression, and biomarker rate of chance in different populations subgroups. Furthermore, we will explore AD trajectories in Hispanics with Dominantly Inherited AD (DIAD) and sporadic AD (sAD), which may provide a full spectrum of AD in Hispanic populations. The proposed datasets include detailed information on education, employment histories, cardiovascular risk factors, rich neurocognitive assessments and biomarkers. DIAN recently received an Alzheimer Association award to study Familial AD in Hispanic populations by enrolling research participants from Latin America (Mexico, Colombia, Brazil Argentina), which Dr. Llibre will use on his K award and as the starting point for his eventual R01 application. The proposal will explore the influence of genetics and social disparities of health on age at onset, cognitive profiles, and biomarker trajectories in Hispanic populations. This research will begin to fill a critical gap in AD knowledge by uncovering novel genetic and gene by environment interactions pathways leading to dementia in Hispanic populations, of which Dr. Llibre will use to determine future areas of independent research in Hispanics. This evidence can help identify strategies most likely to reduce the population burden of AD in Hispanics populations, as well as the disparities therein.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT Glioblastoma (GB), the most common malignant primary brain tumor in adults, is invariably fatal. Thus, there is an urgent need to discover new and meaningful therapeutic strategies for this lethal disease. A major reason for our current lack of effective therapies is the extensive genetic and epigenetic heterogeneity of GB tumors. Within the epigenetic diversity of GB cells, accumulating evidence has now firmly established the existence of a clinically important subpopulation of GB cancer cells, called GB stem cells (GSCs), which represents a key cellular substrate for treatment resistance and disease recurrence. Thus, targeting the GSC pool in tumors is an important conceptual strategy, which has the potential to generate novel, durable treatments. We and others have previously shown that the pluripotency-related transcription factor SOX2 plays a critical role in the expression of malignant GSC phenotypes, including self-renewal capacity, invasiveness, and in vivo tumor growth. Remarkably, despite the significant genetic heterogeneity between tumors, SOX2 is expressed in almost all GB tumor cells, including GSCs, implicating this transcription factor as a common epigenetic driver in GB. Thus, the identification of the mechanisms that regulate SOX2 function in GSCs will not only advance our knowledge about the fundamental biology of SOX2 in the clinically relevant GSC subpopulation but also lead to the discovery of SOX2-dependent therapeutic targets that may be effective across different GB tumors and cell types. Based on published work and preliminary data, we will examine two key mechanisms controlling the SOX2 program in GSCs: post-translational control of SOX2 stability (Aim 1) and specification of SOX2 target gene expression through local and long-range chromatin interactions (Aim 2). To study these mechanisms, we will take advantage of two complementary human model systems: a well-characterized library of patient tumor- derived GSCs and a novel, isogenic, human neural stem cell-based GSC model with defined genetic mutations. The long-term goal of this project is to develop novel SOX2-directed therapeutic strategies to disrupt malignant tumor growth and amplify the efficacy of current treatments.

NIH Research Projects · FY 2025 · 2022-08

Abstract – Inhibition of T-Cell Receptor Signaling for Treatment of Adult T-Cell Leukemia Lymphoma Human T-cell leukemia virus type 1 (HTLV-1)-associated adult T-cell leukemia-lymphoma (ATLL) is an aggressive lymphoproliferative malignancy. Despite aggressive chemotherapy, this disorder is fatal in almost all individuals. Our preliminary results, and those of others, uncovered a high rate of mutations in the T-cell receptor (TCR) signaling pathway in ATLL, with frequent mutations in phospholipase Cγ, protein kinase Cβ (PKCβ), and caspase recruitment domain containing protein 11 (CARD11) leading to activation of transcription factors nuclear factor κB (NFκB) and interferon-regulatory factor 4 (IRF4). We hypothesize that this pathway drives ATLL development and/or progression and targeted therapy against this pathway will synergize with combination chemotherapy. We also suggest that lenalidomide represses IRF4 expression in ATLL. We propose to assess: Aim 1: Role of IRF4 activation in ATLL through RNAseq and functional analysis of IRF4-dependent gene targets. We will also determine if repression of IRF4 mediates the cytotoxic effects of lenalidomide in ATLL. Aim 2: Phase 1 study of lenalidomide in combination with EPOCH chemotherapy for HTLV-ATLL is conducted through the ETCTN, and correlative studies performed in the current project to assess whether efficacy of lenalidomide is mediated through effects on IRF4, and depend on mutations in TCR pathway components. HTLV load, expression, and clonality assays will be used to monitor the efficacy of therapy. Aim 3: Role of PKCβ activation in ATLL will be examined as an alternative therapeutic target, based on sensitivity of ATLL cells to inhibitors, enzastaurin and midostaurin. We will assess whether sensitivity to these inhibitors are affected by mutations in PKCβ or CARD11, which often co-occur in ATLL. This study will provide in-depth knowledge of the role of TCR signaling in ATLL, and new therapeutic targets.

NIH Research Projects · FY 2024 · 2022-08

ABSTRACT Sustained retention in HIV care is critical for viral suppression, but people living with HIV (PLWH) frequently transition in and out of care over time, putting them at high risk for ongoing viremia and drug resistance. Even when PLWH return to care after loss to follow-up (LTFU), rates of becoming LTFU again in the future are very high. Our preliminary data from Zambia suggests that 30% become LTFU again within 6 months, and that 50% of those who are currently LTFU have previously cycled in and out of care. The time of reengagement in care is thus a critical period to intervene in this high-risk population, and strategies to improve post-return care delivery are urgently needed to break ongoing cycles of disengagement. After returning to care, PLWH often report being scolded, health care workers (HCW) delay ART re-initiation due to adherence concerns and do not consistently monitor virologic response, and PLWH likely still experience the life situations (e.g., competing obligations, travel away from home, or psychosocial factors) that led to their initial LTFU. Strategies to 1) target HCW behaviors (i.e., welcoming returning patients, prompt ART re-initiation) using implementation strategies such as practice facilitation and audit and feedback and 2) offer PLWH support services tailored to their initial reasons for LTFU (e.g., extended refills, transfer coordination, peer-navigation) may be promising. To be successful, however, we need to understand how to effectively implement these approaches into existing care, including 1) how to engage HCWs in attitude- and behavior-change strategies, 2) what support services to prioritize for different barriers, and 3) how to integrate activities into existing workflows. This R34 proposal will build the necessary foundation for implementing a multicomponent reengagement strategy to support sustained reengagement after returning to HIV care. In Aim 1a, we assess patient and HCW needs and preferences for reengagement strategies with patient semi-structured interviews (n=20) and focus group discussions with HCWs and clinic leadership (4 FGDs, n=8-10 each). In Aim 1b, we conduct discrete choice experiments (200 PLWH, 100 HCWs) to quantify relative preferences for the contents and attributes for our reengagement strategy. In Aim 2, we undertake a human- centered design process that engages key stakeholders (PLWH, HCWs, clinic leadership, Ministry of Health) in co-developing the content and features of a multicomponent reengagement strategy focused on 1) improving the patient reengagement experience, 2) optimizing ART re-initiation and viral load monitoring post-return, and 3) providing tailored services that target individuals’ specific reasons for LTFU. In Aim 3, we will pilot the developed strategy at one urban and one rural clinic in Lusaka, Zambia. We will use a mixed-methods approach to assess the implementation (e.g., acceptability, appropriateness), service delivery (e.g., time to ART re- initiation, viral load monitoring), and clinical outcomes (e.g., retention and viral suppression at 6 months post- return). Results from this proposal will directly inform a future R01 to formally test this health-system intervention for patients reengaging in HIV care in a Type II hybrid implementation-effectiveness cluster randomized trial.

NIH Research Projects · FY 2026 · 2022-08

Individualized brain biomarkers of late life depression: contributions to heterogeneity and resilience Project Summary/ Abstract Approximately 10% of people aged 60+ suffer from depression. Such late-life depression (LLD) is linked to broader adverse health outcomes such as stroke and dementia. Many brain correlates of LLD have been reported, but each explains only a small amount of interindividual variance in LLD symptoms, likely due to a many-to-one mechanistic mapping in which multiple neural mechanisms contribute to similar symptoms. Heterogeneity in clinical presentation arises from between-patient differences in acute severity, symptoms, chronicity, and age of onset. Few patients are matched in all clinical domains and therefore heterogeneity in conventional research samples is often unavoidable. Over and above clinical heterogeneity, additional risk/resilience factors may alter the experience of LLD at the individual level. Population data from the UK Biobank offers an unprecedented opportunity to fully disentangle clinical heterogeneity by curating clinically homogeneous subject groups. We will distinguish brain profiles, longitudinal changes, and resilience/vulnerability factors that are uniquely linked to LLD clinical domains of: acute severity, mood symptoms, somatic symptoms, chronicity, and late onset LLD. Sixty brain measures associated with LLD, including cortical thickness, gray matter volume, fractional anisotropy, white matter hyperintensities, and resting state connectivity will be used for all aims. In Aim 1, we will curate five homogeneous groups of UKB subjects with shared clinical presentation, focusing on: late onset LLD, acute severity, lifetime chronicity, mood symptoms, and somatic symptoms. Using normative models trained on never-depressed UKB subjects, we will distinguish normative brain deviation profiles associated with these different domains of clinical heterogeneity. In Aim 2, we will curate new groups of UKB subjects with shared longitudinal changes in acute severity, chronicity, mood symptoms, or somatic symptoms to test whether changes over time in brain measures are linked to longitudinal changes in clinical presentation. This aim therefore offers an independent validation of aim 1 and differentiates between state and trait markers of LLD. In Aim 3, we will test the hypothesis that cumulative environmental and psychological stressors alter the experience of LLD at the individual level. We will obtain individual-specific statistical estimates of resilience/vulnerability based on the difference between predicted and actual depression scores (‘brain depression gap’). The brain depression gap will be linked to stressors separately in each homogeneous subject group (same as aim 1) to determine factors that promote LLD resilience or vulnerability. Public health significance: this proposal is expected to move the field closer to a full understanding of LLD heterogeneity by combining theory-driven subject groups with data-driven population prediction models. Gaining a better understanding of LLD heterogeneity may inform improved strategies for treatment and prevention of LLD, which could positively influence broader health outcomes in older age.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract: CLN1 disease or Infantile Neuronal Ceroid Lipofuscinosis (INCL or Infantile Batten disease) is one of the earliest onset and most rapidly progressing forms of neuronal ceroid lipofuscinosis (NCL or Batten disease). CLN1 disease is caused by deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1). This deficiency has a devastating and rapidly progressing effect upon affected children that starts within the first year of life, and because there is no effective therapy available CLN1 disease is always fatal. We have been able to dramatically improve therapeutic outcomes in PPT1-deficient mice by targeting adeno-associated viral (AAV)-mediated gene therapy to the central nervous system (CNS) regions that are most affected, including the spinal cord. However, unlike other forms of NCL, such therapeutic effects are limited to the CNS regions that are transduced. As such, successfully translating gene therapy for CLN1 disease into the clinic will be a significant challenge in the much larger and more complex brain of a child. To overcome this obstacle, we shall use a novel CRISPR/Cas9 generated CLN1 R151X sheep model to refine our therapeutic strategy, assessing the delivery, dosing, safety and efficacy of gene therapy in a larger species that is ideally suited for translating these advances. We recently published that these CLN1 R151X sheep display pronounced CLN1 disease-relevant phenotypes. Our preliminary data extend these observations, revealing an earlier onset of neurologic disease, and widespread histologically and radiologically detectable pathology that is more pronounced than in PPT1-deficient mice. We have also shown that an intracranial injection of an AAV9 vector expressing PPT1 raises expression of this enzyme in the brain of sheep to supraphysiological levels. We believe CLN1 R151X sheep not only more accurately model human CLN1 disease, but also provide an ideal testing ground for optimizing the dosing and delivery of gene therapy in a fashion that is not possible in mice. We now propose to characterize the progression of these disease phenotypes in CLN1 R151X sheep, in order to provide detailed landmarks of disease progression using cognitive and neurologic testing, and MRI imaging, correlating these data with histological findings (Aim 1). We will also define the parameters of vector dosing and delivery routes to achieve widespread transduction of the sheep brain and spinal cord, and elevate PPT1 activity to levels predicted to be capable of producing therapeutic benefit (Aim 2). Finally, we shall determine the therapeutic efficacy, minimum effective dose, safety and clinical response to this optimized delivery of scAAV9-CCAG-PPT1 to the brain and spinal cord of CLN1 R151X sheep (Aim 3). These data will allow us to refine our gene therapy approach, and position us for entry into the CREATE-Bio development program towards clinical translation of the first effective treatment of this devastating disease.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY In Alzheimer’s disease (AD), Aβ accumulation and plaque formation precedes dementia by decades, suggesting that other downstream pathophysiological processes are responsible for precipitating symptomatic disease. Prior studies in humans reveal that brain metabolism is impaired in early AD, including an initial regional energy deficit with a superimposed, marked metabolic shift away from whole-brain and regional glycolysis. However, it is not yet clear how amyloid-induced metabolic dysfunction manifests at the cellular level and affects different cell types, how cellular metabolic dysfunction relates to tissue energy deficit and disruption of functional brain organization, and if and when this might be reversible. These questions have been difficult to answer due to technical challenges in spatiotemporally assessing cell type-specific mitochondrial function and energy metabolism, along with plaque deposition, at the microscopic and mesoscopic levels in vivo. Our central hypothesis is that plaque deposition induces metabolic dysfunction localized to specific cell types and/or cellular components. We further hypothesize that specific cellular changes in metabolic dysfunction differentially affect metabolism at the tissue level and functional brain organization at the regional and global levels. To test these hypotheses, our team has developed several technologies in mice including two-photon fluorescence lifetime imaging microscopy (TP-FLIM), multi-parametric photoacoustic microscopy (PAM), and wide-field optical imaging (WFOI). We will use these methods to measure concentrations of nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), cerebral metabolic rate of oxygen (CMRO2), and neural and hemodynamic activity. In addition to indicating overall mitochondrial activity, the ratio of NADH to FAD (N/F ratio) provides an optically-accessible index of metabolic shifts towards or away from glycolysis in vivo, a key early aspect of AD-related metabolic dysfunction. Since brain amyloid clearance is now readily achievable in both mice and humans, our approach will further allow us to determine whether the metabolic dysfunctions discovered from the efforts above are reduced following amyloid clearance. In the project, we aim to (Aim 1) determine the in vivo relationship between amyloid plaque deposition and cellular N/F ratio in AD mice at the microscopic level using TP-FLIM; (Aim 2) determine how amyloid plaque deposition and cellular metabolic dysfunction affect regional and global measures of tissue metabolism and functional brain organization using PAM and WFOI; and (Aim 3) determine whether amyloid plaque clearance reverses the metabolic abnormalities identified in Aims 1 and 2. Understanding the spatiotemporal relationship between Aβ accumulation, metabolic dysfunction, and functional brain organization from the cellular to systems level will be critical to revealing the mechanisms by which amyloid deposition affects downstream processes, and ultimately lead to neurodegeneration and symptomatic AD. Moreover, our study will reveal whether the metabolic dysfunction in AD is reversible or not.

NIH Research Projects · FY 2025 · 2022-08

Cellular mechanisms of bioenergetic plasticity The long-term goal of our research program is to understand how cells fine-tune their metabolic programs to meet their ever-changing energetic needs. Many cell types in the body, from muscle fibers to neurons, have evolved unique metabolic programs that are essential for survival and proper function. Even within a single cell, specific processes are energetically coupled to mitochondria or the glycolytic machinery for specialized metabolic support. However, the underlying molecular basis of metabolic plasticity and its relationship to cellular function are poorly understood. Understanding the mechanisms of metabolic regulation is highly relevant to many disease states, including diabetes, myopathies, and Leigh syndrome, where metabolic dysfunction is heavily implicated. In eukaryotic cells, energy, in the form of ATP molecules is primarily produced by glycolysis and mitochondrial oxidative phosphorylation. My laboratory combines optical imaging of biosensors in live cells with genomics and transcriptomic analysis to investigate metabolic regulation in cellular compartments. With these tools, we have been able to discover novel pathways for stimulation of mitochondrial and glycolytic ATP production in active neurons during electrical activity. We now seek to understand how energy metabolism is locally regulated in subcellular compartments, and uncover metabolic specialization of functionally distinct neuronal types. To carry out this work, we plan to utilize our strength in cellular imaging of metabolic function along with new technological advances to: (1) determine how subcellular organization of the glycolytic machinery regulates synaptic vesicle endocytosis, and (2) elucidate molecular mechanisms of metabolic specialization using the available transcriptional profiles of neuronal subtypes. Our study will shed light on both local and global mechanisms of metabolic plasticity at the subcellular level and across cell types. As such, our findings will be broadly relevant to the scientific community studying cellular metabolism and its implications in disease states.

NIH Research Projects · FY 2025 · 2022-08

SUMMARY Chronic inflammation affects millions of Americans each year and can manifest in a variety of chronic pain conditions where normally innocuous stimuli produce pain symptoms. Long-term use of current pain therapeutics, including NSAIDS, corticosteroids, and opioids, can cause unwanted side effects, and addiction potential can limit their utilization. Recent studies have demonstrated a clear link between chronic low-grade inflammation and the increase in Chronic Inflammatory Pain (CIP) conditions. Alternative therapeutic targets are needed for the treatment of these painful conditions. Nuclear receptors, ligand-activated transcription factors that regulate a variety of physiological processes including metabolism, inflammation, reproduction, and development, represent key drug discovery targets (second to GPCRs). The REV-ERB proteins are nuclear receptors which function as transcriptional repressors and direct regulators of NLRP3 inflammasome components and proinflammatory cytokines (IL-1, IL-18), and regulate the activity of macrophages at sites of cellular damage. To date, the role of REV-ERB in relation to the manifestation of chronic pain symptoms has not been elucidated. Due to its role in NLRP3 inflammasome and proinflammatory cytokine regulation, we hypothesize that REV-ERB is a viable drug target for the treatment of inflammatory pain. Our strategy will leverage the known physiological functions of REV-ERB in chronic inflammation and use a chemical biology approach to identify novel REV-ERB ligands, with superior pharmacological profiles, to advance this potential therapy toward clinical trials. Our new preliminary data shows that total loss of REV-ERB in mice increases mechanical hypersensitivity. Our previous studies demonstrated that pharmacological activation of REV-ERB had no negative effects in preclinical mouse reward models, suggesting that targeting of REV-ERB may benefit many chronic pain conditions.

NIH Research Projects · FY 2025 · 2022-08

The overall goal of this project is to enable new methods in neurocritical care through the development of a quantitative framework for modeling human brain dynamics from electrophysiological data. This goal is timely because of an increasing emphasis in neurocritical care on tracking and predicting secondary injury and optimizing functional outcomes, needs that are challenging, unmet and consequential. Indeed, secondary neurological injuries and neurological worsening, including seizures, contribute to cognitive deficits and life-long impairments. The high prevalence of traumatic brain injuries means that the societal burden of these sequalae is significant, and their economic burden is measured in the tens of billions of dollars. Thus, technology that can help clinicians anticipate secondary injuries would provide tremendous leverage for improving the care of critically ill patients. Electrophysiological monitoring, such as the electroencephalogram (EEG), can provide a means to noninvasively examine brain electrical activity in high-risk populations, thus providing a pathway to such technology. However, many paradigms in EEG signal processing and informatics struggle with patient individuality and variation, compounded by the idiosyncrasy of neurological disease. As a result, many current approaches are limited to population-level characterizations and are susceptible to bias in datasets. In the proposed work, we will pursue a new approach to modeling human brain dynamics that is interpretable and robust to individual variability. We will develop and use dynamical systems models that generate simulated EEG activity, then fit these models to patient data in a minute-to-minute timescale. These models capture the internal biophysics of populations of neurons and are thus explanatory from a physiological perspective, setting the stage for innovative clinical applications. Specifically, we will: (i) develop the modeling framework and use it to quantify dynamical effects of brain injury in individual patients, (ii) track changes in internal brain dynamics and states through the course of injury, to predict neurological worsening, and (iii) use the model as an in silico surrogate to predict the response of patients to clinical interventions. We will validate our technical approach through retrospective and prospective studies in a heterogenous dataset spanning injuries and age. If successful, this work would represent a first-of-its-kind demonstration of a data-intensive approach to provide actionable clinical information about individual neurocritical care patients.

NIH Research Projects · FY 2024 · 2022-08

Project Summary Use of vaping products (e.g., electronic nicotine delivery systems, e-cigarettes) has been increasing rapidly, particularly among teens and young adults. With limited information on the long-term effects of vaping products, health information about vaping has been somewhat unclear in regards to associated health risks. Teens and young adults may be reluctant to disclose their use of vaping products to parents or health providers and instead turn to social media to share and seek out information regarding vaping risks and cessation supports. Given the ubiquitous use of social media platforms among this population and the ability for advanced artificial intelligence (AI) and natural language processing (NLP) technologies to analyze content shared on social media platforms, there is strong potential for this information to be leveraged and used to detect and reach out to those most at risk for negative health outcomes caused by vaping. Thus, our current proposal outlines the use of detection models to identify teens and young adults socially networking about vaping, the use of a chatbot to screen for the needs of eligible users, and the use of a digital intervention system (i.e., quitSTART with an embodied chatbot) aimed to support vaping cessation efforts by increasing risk awareness and decreasing pro-vaping attitudes. In Aim #1a, we will develop an intelligent detection system by leveraging state-of-the-art machine learning, deep learning, and NLP techniques for mining massive social media data on vaping with clinical inputs. This detection system will implement multiple functionalities on both Twitter and Reddit social media platforms to identify posts regarding the use of vaping products, negative health outcomes experienced, and interest in vaping cessation. To evaluate the validity and specificity of the detection model developed on both platforms, we will also conduct surveys among a subsample of those identified (N=100) to rule out false positives and to gather data on vaping behaviors, social media content generation about vaping, motivations for vaping product use, and interest in vaping cessation to refine the developed models in Aim #1b. In Aim #2, we will develop both a chatbot to screen individuals identified on social media as well as an in-app chatbot to guide users to tailored content, conduct daily assessments and check-ins, motivate and encourage their cessation efforts, and promote sustained user engagement within a widely-used evidence-based mobile application (app) intervention for combustible smoking, quitSTART. We will conduct usability and acceptability testing on both levels of the chatbot among a sample of participants (N=30) recruited in Aim #1b. In Aim #3, we aim to integrate the developed detection model, chatbot screener, and adapted mobile app into a streamlined outreach and intervention system, and conduct a randomized controlled trial (N= 189) to evaluate user engagement with and preliminary efficacy of the digital intervention on vaping behaviors among teens and young adults. This integrated system has the potential to improve public health outcomes related to vaping and to inform the feasibility of such chatbot tools to sustain mHealth intervention engagement.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT Heart failure with reduced ejection fraction (HFrEF) affects ~3 million people in the U.S. and is a mortal and morbid disease. HFrEF impairs a patient’s ability to exercise and perform activities of daily living. Although weak cardiac pumping ability clearly contributes to chronic disability associated with HFrEF, abnormal skeletal muscle function in patients with HFrEF is also a key debilitating factor. Skeletal muscle is therefore a vital target for treatment of HFrEF. Dietary inorganic nitrate enhances aerobic exercise capacity and muscle power in patients with HFrEF, as demonstrated in our preliminary studies. The overarching aim of the proposed clinical trial is to determine whether inorganic nitrate in a once-a-day oral gelcap format, offers a new, safe, and effective treatment for ameliorating the impaired exercise performance due to HFrEF. The Aim of the R61 component of this grant is to set up the infrastructure necessary for the phase II clinical trial and to begin the trial. This includes compounding the inorganic nitrate (KNO3)/placebo capsules and all safety testing, obtaining Institutional Review Board approval, ClinicalTrials.gov registration, REDCap database development, Recruitment Enhancement Core (REC) engagement, Clinical Translational Research Unit (CTRU) project approval, enrollment and assessment and dose administration of the first subjects. The Aims of the R33 component are to determine the effectiveness of acute (2 hours after a single dose) and chronic (after 6 weeks of once-a-day dosing) KNO3 treatment (10mmol) vs. placebo on quadriceps muscle power and on aerobic exercise performance (V̇O2peak) in patients with HFrEF (left ventricular ejection fraction <45%). We hypothesize that both acute and chronic dosing of 10mmol of KNO3 will improve exercise performance in HFrEF. To test this hypothesis, we will perform a randomized, double-blind, placebo-controlled, parallel-arm design study. Patients with stable New York Heart Association class I-III HFrEF will undergo screening and phenotyping before completing baseline peak muscle power and aerobic performance measurements. On visit 2, subjects will receive a single dose (1 capsule) of their assigned treatment (10mmol KNO3 or placebo) and will repeat the measurements of exercise performance after 2h. Subjects will then continue with their assigned treatment (KNO3 or placebo, 1 capsule daily) for 6 weeks. On visit 3, subjects will repeat the tests of exercise performance. The primary endpoints are maximum quadriceps muscle power, measured using an isokinetic dynamometer, and aerobic exercise performance, based on V̇O2peak. Max exercise time and max muscle velocity, tolerability and safety of KNO3 vs. placebo treatment and their effects on hemodynamics, plasma nitrate, plasma nitrite, breath nitric oxide (NO), and heart failure symptoms (using well-validated questionnaires) will also be quantified in this phase II, single-center clinical trial. The potential impact of finding a new, safe and effective treatment that helps millions of patients beat the physical disability of HFrEF is enormous.

NIH Research Projects · FY 2026 · 2022-08

PROJECT SUMMARY / ABSTRACT UTIs occur in 8% of pregnant women, affecting ~500,000 women annually in the U.S. Serious maternal and infant consequences include pyelonephritis, sepsis, preterm labor, and low birth weight. Guidelines recommend screening and empirical treatment of UTIs (including asymptomatic bacteriuria) in early pregnancy. This approach has become standard of care, yet there is a lack of rigorous evidence to inform antibiotic selection or duration in pregnant women. Most evidence about the benefits and harms of antibiotic regimens has been generated by randomized clinical trials that excluded pregnant women, and which are further limited by small sample size, short follow-up, and heterogeneous treatments. Despite clear guidelines for non-pregnant women, treatment standards for pregnant women are vague. The American College of Obstetricians and Gynecologists (ACOG) recommends nitrofurantoins and sulfonamides as first-line during the second and third trimesters, but provides no guidance for first trimester management. Uncertainty remains about the teratogenicity of nitrofurantoins and sulfonamides due to limitations of previous observational studies. In addition, ACOG does not define appropriate antibiotic duration, instead recommending the “shortest effective duration.” Overall, the lack of guidance about antibiotic selection and treatment duration leads to substantial practice variation and the potential for harm. For example, clinicians commonly prescribe broad- spectrum agents despite antimicrobial stewardship initiatives that recommend narrow-spectrum agents. Providing optimal antibiotic agents and durations could prevent avoidable adverse events, microbiome disruption, and antibiotic-resistant infections. A comprehensive evaluation using real-world data is needed to quantify the benefits and risks of commonly used antibiotic regimens to treat pregnant women with UTI. We will conduct a non-experimental study to examine the utilization, effectiveness, and safety of several commonly used antibiotic regimens in a real-world setting of diverse pregnant women in the U.S. We will apply modern epidemiological study design and analytic approaches to study almost two million pregnant antibiotic recipients and a subset of their infants, from two national databases as well as EHR data from three regional healthcare systems. Our utilization analysis will use microbiology results to characterize variability in antibiotic prescribing within the context of local uropathogen susceptibility patterns. Our antibiotic effectiveness analyses will provide insight into whether the risks of antibiotic treatment failure outcomes vary by antibiotic regimen. Our safety analyses will compare the risk of birth defects and other perinatal complications by antibiotic regimen. Exploratory analyses will examine additional adverse drug events (e.g., Clostridioides difficile diarrhea). This large study will generate evidence to address critical gaps in knowledge about optimal treatment of UTIs in pregnant women. Our results will inform clinical decision-making and reduce suboptimal antibiotic prescribing, which will ultimately prevent adverse events, improve perinatal outcomes, and minimize antibiotic resistance.

NIH Research Projects · FY 2025 · 2022-08

Project Summary Diabetic retinopathy is an increasingly common cause of visual impairment and blindness among adults. Modern therapy has become increasingly effective, but remains insufficient to prevent vision loss in a sizable proportion of patients. Early-acting and efficacious new remedies are needed, especially since the prevalence of worldwide disease is increasing. A barrier to accomplishing this goal is a poor understanding of the earliest causes of retinal injury in diabetes. In this application, we will address this barrier by studying early changes in retinal metabolism during diabetes – changes that are likely to contribute to disease onset and that can be targeted for therapeutic purposes. Hyperglycemia is the hallmark of all forms of diabetes and is directly related to its complications, including diabetic retinopathy. Since glucose is the primary fuel of the retina, we investigated what pathological effects might occur due to its excess supply in diabetes. Specifically, we discovered that diabetes is associated with a fundamental shift in retinal metabolism away from tissue break down (catabolism) and towards tissue building (anabolism). Among the largest changes is that of lipid biosynthesis, a pathway responsible for generating a ubiquitous medium-chain fatty acid in mammalian cells, palmitate. In diabetes, retinal palmitate synthesis is elevated by 70% compared to non-diabetic controls. Using targeted genetic manipulation of the enzymes in the synthesis pathway, we determined that reduction of palmitate prevents vision loss in diabetes whereas elevating its production accelerates the onset of visual abnormalities. We now ask how such signals are related to disease development and what specific molecules are involved. Towards these goals, we recently found that excess palmitate in the diabetic retina impacts several retinal enzymes that are regulated by S-palmitoylation. The largest change was seen in retinal Ryanodine Receptor 2 (Ryr2) – an intracellular ion channel that regulates calcium homeostasis – as it is hyper-palmitoylated in diabetes compared to non-diabetic controls. In this application we will determine whether this molecular change is associated with pathology and whether it can be reversed for therapeutic effects. We will address three major aims: (1) define the effect of diabetes on retinal Ryr2 palmitoylation and its functional consequences; (2) delineate whether Ryr2-associated calcium flux in rods is dependent on retinal lipid biogenesis; and (3) determine whether improving retinal lipogenic signaling in diabetes reduces diabetic retinopathy severity. By accomplishing these aims, we could uncover essential root causes of diabetic retinopathy and we may introduce novel targets for therapy directed at a very early stage of the disease process.