Johns Hopkins University

NIH Research Projects · FY 2025 · 2024-04

Project Summary/Abstract Alternative splicing (AS) is the fundamental mechanism of generating isoform diversity in eukaryotic cells. AS occurs at an especially high frequency in the retina and other nervous system tissues, contributing to many important cellular and physiological functions within the cells, including tightly regulated and complex processes like neuronal development. Moreover, dysregulation of AS can have a substantial impact on retinal survival and function. Retinitis pigmentosa (RP) is a group of inherited retinal diseases that cause dysfunction and degeneration of the light-sensitive photoreceptor layer of the eye, resulting in irreversible vision loss and blindness in over 1.5 million people worldwide. Yet, therapeutic options for these patients remain limited. Many mutations that cause mis-splicing of a gene cause RP. Furthermore, defects in the regulation of AS, including mutations in spliceosome components and other associated splicing factors, also cause disease. It is unknown why retinal cells are so particularly, and often exclusively, susceptible to aberrant splicing, despite these defects often occurring in either ubiquitously expressed genes or from mutated splicing factors found in all tissues of the body. By combining novel long-read RNA-sequencing technology with CRISPR engineering and 3D human stem cell organoid models, we can acquire a detailed understanding of AS and learn how dysregulated AS contributes to retina-specific disease. To further investigate the AS landscape in human retinal development and disease, this proposal aims to 1) comprehensively examine the AS events in rod and cone photoreceptors that occur during differentiation of human stem cell-derived retinal organoids, 2) investigate how splicing factor mutations alter normal AS in the retina compared to other neurons, and 3) understand how dysregulated gene expression that results from aberrant splicing causes retina-specific degeneration. During the mentored phase of this proposal, I will take advantage of the many strengths of my multidisciplinary team of mentor/co-mentors, advisors, and collaborators to perform and analyze the single cell long-read transcriptomic experiments with the developing retinal organoids and acquire the training needed regarding brain organoid differentiation and CRISPR methodologies to successfully transition myself to the independent research phase. In the independent phase, I will use the splicing factor mutant cell lines generated in the Zack lab to further understand the mechanism(s) of retinal-specific degeneration caused by dysregulated AS events. The experiments proposed will not only provide the retinal field with a more complete understanding of AS in the retina, knowledge which can possibly be harnessed to design new treatment options for RP, but the training plan we have developed will also provide a robust pathway to establishing my successful and productive independent research career that extends well beyond the aims of this grant.

NIH Research Projects · FY 2025 · 2024-04

Racial disparities in health arise in complex ways. Multicomponent interventions to address them (often implemented by healthcare systems, employers, and community organizations) are often designed to address multiple mechanisms (e.g., medication adherence, lifestyle behaviors). However, not all such interventions are effective in reducing disparities in health. Designing effective interventions requires knowing which barriers to health, when acted upon, may greatly reduce disparity. Standard approaches (e.g., mediation analysis, Oaxaca-Blinder decomposition) are often used to generate this evidence base but do so under severe limitations, including their inattention to concepts of appropriateness (e.g., they often over-adjust measures of racial disparity for factors implicated in disparity such as socioeconomic status), and their inattention to matters of causal inference (e.g., confounding). Causal decomposition methods, in contrast, answer the question: how much disparity in an outcome (e.g., hypertension control) would change if we could remove the racial disparity in a barrier to health (e.g., clinical inertia in treatment decision-making). They consider what variables should separately be accounted for when measuring disparity in an outcome, when removing disparity in a barrier, and while addressing confounding. This new approach is being taken up by applied researchers, but often they are interested in more general settings than current methods allow. The goals of the proposed methodological project are to extend causal decomposition methods to settings relevant for cardiovascular health research and provide guidance and tools to facilitate their adoption and use. This methodological project will extend and tailor causal decomposition methods to: 1) accommodate longitudinal and time-to-event data structures; 2) accommodate aspects of patient-specific clinical needs; 3) accommodate generalization beyond the study sample to specific populations of interest. A key focus of the work will be to disseminate existing and newly developed causal decomposition methods to applied researchers in cardiovascular health through the development of tutorials, publicly available software tools, a website, and courses and workshops at research conferences. The methods development and dissemination will be informed by three illustrative data sets: the Coronary Artery Risk Development in Young Adults study, the RICH LIFE Project, and the All of Us Research Program. If this proposal is successful, the methods developed herein could support the design of effective, targeted, and multicomponent interventions that reduce disparity.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Hyperinflammatory gene expression upon tissue injury is a major health risk and the cause of significant morbidity and mortality, particularly if it occurs in the lung. The polarization and injury-responsiveness of macrophages (MF) is tightly regulated to ensure proper control of homeostatic, inflammatory, anti-inflammatory and regenerative processes. The transcriptional and epigenomic determinants of (hyper)responsiveness are poorly understood. We have recently determined that the nuclear hormone receptor, Peroxisome Proliferator Activated Receptor gamma (PPAR) is part of a transcription factor cascade in Interleukin (IL)4 polarized MFs, downstream of the Signal Transducer and Activator of Transcription (STAT)6 induced Early Growth Regulator (EGR)2. Remarkably, PPAR proved to have ligand-dependent, but also apparently ligand-independent, epigenomic activities controlling alternative polarization. In addition, we can show that IL-4 polarized MFs establish a unique epigenome and inflammatory gene expression program upon Toll-Like Receptor (TLR)-ligand exposure. A newly identified part of this interaction, we termed extended synergy, is dependent on the expansion of Nuclear Factor (NF)κB-p65 cistrome and increased enhancer activity. The previously alternatively polarized MFs produce immune-modulatory factors, including Chemokine (C-C) Ligand (CCL) 2, IL6 at an extremely high level in vitro and in vivo in a murine Th2-type airway inflammation and lung injury model upon lipopolysaccharide (LPS) exposure leading to exacerbation of the response. The extended synergy and the LPS response of several genes, including Ccl2 and Il6, depend on the presence of PPAR and some can be modulated by its synthetic and selective ligand, Rosiglitazone. We hypothesize that PPAR acts as signal-dependent epigenomic bookmark, selecting and safeguarding enhancers during MF-extended synergistic inflammatory gene expression by modulating the amplitude of the response. The receptor acts via two distinct mechanisms (1) actively selecting and inducing enhancers genome-wide and (2) also as epigenomic bookmarker and architectural factor altering gene expression only upon non-cognate inflammatory signals. We are examining these hypotheses by systematically mapping the transcriptional and epigenomic changes requiring PPAR, the receptor’s role in the dynamically changing 3D chromatin architecture and in mouse models of lung injury, Th2 inflammation and the combination of the two leading to a hyperinflammatory response leading to exacerbation of these disease processes. Using cutting-edge epigenomics and transcriptomics technologies, with innovative genetic and chimeric mouse models analyzed by high dimensional flow cytometry and single cell technologies will provide mechanistic insights into this novel mode of action of the nuclear receptor PPAR and may lead to the identification of novel pathways to alleviate lung injury and disease progression.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY/ABSTRACT Chronic Chagas cardiomyopathy (CCC) is a devastating disease that develops in 30-40% of individuals decades after initial infection with Trypanosoma cruzi, resulting in heart failure, cardiac arrhythmia, stroke, pulmonary embolism, and sudden cardiac death. Currently, there is no effective treatment for CCC, there is no way to predict which patients will go on to develop CCC, and the mechanisms that underly the disease progression are very poorly understood. A better understanding of CCC pathogenesis is therefore urgently needed to develop novel treatment strategies and allow for risk stratification of patients in need of personalized interventions. A compelling hypothesis to explain the pathogenesis of CCC centers around a hyperinflammatory immune response that results in excessive cardiac damage over time. This study focuses on investigating how B cells and their interactions with other cells can potentiate proinflammatory and autoimmune responses to mediate the cardiac pathology seen in CCC. In the first aim, I will use a combination of high-throughput single- cell RNA sequencing (scRNAseq) and spatial transcriptomics of human CCC samples to characterize the immune pathways mediating CCC. Using scRNAseq on peripheral blood mononuclear cells from a longitudinal cohort of patients that are known to develop CCC, I will characterize immune cell alterations that precede CCC at a high resolution, potentially revealing early processes that mediate disease progression. Spatial transcriptomics will be employed on end-stage human CCC heart tissue to determine the spatial association of proinflammatory gene expression with regions of cardiac damage. This will help characterize the immune cell subtypes and pathways that are most likely mediating direct tissue damage in human Chagas disease. Together, these data will highlight potential therapeutic targets for both early and advanced CCC. The analyses of these high-throughput sequencing experiments will have a strong focus on evaluating pro-inflammatory immune cells, including B cells, and their signaling pathways. The second aim of this project seeks to determine whether B cell depletion can provide a therapeutic benefit in a murine model of CCC. B cells will be depleted in two different groups: 1) before cardiac manifestations develop, to evaluate the feasibility of immunotherapy for halting disease progression; and 2) after cardiac manifestations have occurred, to evaluate whether targeted B cell depletion can provide a therapeutic benefit in the advanced form of the disease. Overall, the proposed study will transform our understanding of the immunologic mechanisms underlying CCC pathogenesis. Long term, this work can serve to change the way in which we manage CCC, allowing us to ultimately treat Chagas disease as an immunologic disorder rather than an infectious one. Long term, this work will serve to inform strategies for diagnosis, prevention, and management of this important cardiac disease.

NIH Research Projects · FY 2026 · 2024-04

Poor dietary quality and high rates of diet-related chronic diseases such as type-2 diabetes are urgent national public health priorities. Strategies to effectively shift dietary patterns in the United States (US) toward healthy diets are urgently needed to improve dietary quality and prevent diet-related chronic diseases. Fast-food restaurants are a key environment to promote healthier food choices given the frequency of fast-food consumption, the poor dietary quality of fast foods, and their adverse health effects. Numerous restaurants and food companies use non-nutrition labels that communicate to consumers about qualities of menu items (e.g. grown in the USA, local, gluten-free, pesticide-free). Such menu labels are also garnering interest among policymakers. Existing research shows that nutrition labels can change behavior, but we lack evidence on whether other labels can promote healthier food choices as well. Non-nutrition labels could improve dietary quality via lower red and processed meat intake, but could also promote undeserved perceptions that unhealthy food items are healthy (i.e., a ‘health-halo’ effect). Given the rapid development and growing food industry and policy interest in non-nutrition labels, there is a critical need for timely, rigorous evaluation of the real-world effects of such labels and the optimal design to maximize behavior change. The primary objective of this application is to conduct two sequential randomized controlled trials (RCTs) to determine the degree to which menu non-nutrition labels influence the healthfulness of purchases at fast-food restaurants and overall dietary intake. The first study aim is to conduct an RCT to compare the effects of different menu label designs on the healthfulness of hypothetical fast-food meal orders in a nationally representative sample of 6,000 US adults. We will randomly assign 6,000 adults to view menus from two fast-food restaurants (one burger, one sandwich) that display one of five types of label designs: 1) control (QR code); 2) green, positively framed label; 3) red, negatively framed ‘warning’ label; 4) grade label and 5) traffic-light label. The second aim is to conduct an RCT to evaluate the effect of repeated exposure to menu labels on the healthfulness of real fast- food meal orders, delivered to the participant, over an eight-week period. We will randomly assign 450 adults to one of two label conditions: control label vs. the label from Aim 1 that led to the largest improvements in the healthfulness of menu items ordered. In the third aim, we will evaluate the longitudinal effect of menu labels on overall dietary quality (Healthy Eating Index scores) assessed via multiple 24-hour dietary recalls. Results will provide policy relevant evidence to identify how menu labels, a proven public health policy approach to change consumer behavior, can be used to improve diet quality and diet-related health of Americans.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Acute Respiratory Distress Syndrome (ARDS) is a devastating illness with an annual incidence of 200,000 and a mortality of 40%. Endothelial barrier dysfunction is critical for the pathogenesis of vascular permeability and the resulting tissue edema and profound hypoxia seen in ARDS. Endothelial barrier dysfunction in ARDS is a complex phenomenon involving cytoskeletal changes in response to pro-inflammatory mediators, which can also precipitate endothelial cell apoptosis, a programmed form of cell death. While cytoskeletal changes are reversible and are eventually followed by recovery of the endothelial barrier, endothelial cell apoptosis represents a final cellular fate and determinant of endothelial barrier disruption. In fact, after apoptotic-endothelial injury, restoration of barrier function requires endothelial cell proliferation, migration and/or endothelial progenitor cell seeding. Therefore, a transition point likely exists between pro-apoptotic signaling (injury phase) and pro-proliferative signaling (recovery phase) leading to endothelial barrier restoration; further insight into which would identify therapeutic targets that not only reduce severity of ARDS but also facilitate recovery. Our laboratory has identified non-canonical functions for caspase 3, a terminal enzyme of the apoptotic cascade. Traditionally, activation of caspase 3 had been considered as the point of no return in the execution of apoptosis. We identified a disconnect between activation of caspase 3 and the execution of apoptosis. In initial work, we found loss of the signaling molecule mitogen activated protein kinase activated protein kinase 2 (MK2), resulted in prevention of both apoptosis and endothelial barrier dysfunction despite activation of caspase 3. Interestingly, in these experiments active caspase 3 was sequestered in the cytoplasm. We have also shown caspase 3 enhances barrier integrity is via cytoskeletal changes that increase centrifugal forces, reminiscent of changes required for cellular migration. We have exciting preliminary data showing caspase 3 promotes migration and proliferation. Further, our preliminary data suggests caspase 3 functions via yes activated protein (YAP), a known regulator of migration and proliferation implicated in angiogenesis and lung development. However, there is little known about the role of YAP in endothelial barrier recovery following lung injury and the molecular regulators that initiate YAP signaling in this process. Based on our previous findings and preliminary data, we hypothesize the association between caspase 3 and MK2 is necessary for nuclear translocation of caspase 3 and the apoptosis-promoting function of caspase 3. We further hypothesize that inhibiting the association between active caspase 3 and MK2 converts caspase 3 to a pro-proliferative factor, via YAP signaling, thereby accelerating endothelial barrier recovery. Thus, the Aims of this study are: 1) determine the mechanisms by which MK2 facilitates transport of caspase 3 into the nucleus in vitro. 2) determine the mechanism(s) of active cytoplasmic caspase 3 in promoting endothelial cell proliferation in vitro. 3) elucidate the role of cytoplasmic endothelial caspase 3 in accelerating recovery following lung injury.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Parkinson's disease (PD) is one of the most prevalent neurodegenerative disorders, affecting nearly one million people in the United States as of 2020. The core etiopathology comprises intracellular accumulation of Lewy body-like α-synuclein (α-syn), followed by progressive loss of motor-controlling midbrain dopaminergic (mDA) neurons in the substantia nigra pars compacta. There is growing evidence that the dysfunction of the cerebral vasculature called the blood-brain barrier (BBB) is involved in PD progression and, more importantly, implicated in altered drug efficacy. PD patients suffer from a wide spectrum of clinical syndromes such as movement disorders/parkinsonism, dementia, and autonomic nervous system dysfunction. Current standards-of-care are not curative and focus on symptom management. Thus, a treatment option that can modify the disease pathology is sorely needed. One of the major challenges in developing such a therapy is the absence of an experimental model recapitulating human PD pathology, which is needed for quick and reliable screening of candidate therapies prior to clinical evaluation. Even though recent advances in human induced pluripotent stem cell (hiPSC) technology makes it possible to derive mDA neurons of PD patients, current 2D culture models do not recapitulate the native microenvironment around the neurovascular unit and thus cannot model the pathophysiology of late-onset human PD, which is induced by accumulated aberrant α-syn aggregation. To this end, we will develop and validate an advanced high-throughput in vitro human PD model that reflects pathological vascular and tissue changes observed in the brains of PD patients. Specifically, we will engineer and optimize a multi-well microfluidic tissue on-a-chip platform integrated with vascular perfusion and a blue light module to reconstruct the complex microenvironment at the interface of the human neurovascular unit and parenchyma tissue in human brains (Aim 1). We will then induce the core etiopathology of brain-borne α-syn aggregation in the PD patient-derived mDA neurons using our novel light-inducible pathogenic protein aggregation system (i.e., optogenetics-assisted alpha-synuclein aggregation induction system, OASIS) and α-syn preformed fibrils (Aim 2.1). In parallel, we will investigate the effect of blood-borne pathogenic α-syn aggregates, observed in patients, on PD progression in our model (Aim 2.2). We will validate our newly engineered human PD model by its comparison to clinical observations, including α-syn accumulation, neuroinflammation, progressive neuronal death, and pathological changes of the BBB. Finally, we will demonstrate that our PD model can serve as a testbed to evaluate the delivery efficiency and therapeutic efficacy of highly versatile drug-loaded delivery platforms (i.e., human ferritin nanocages) (Aim 3). If successful, the developed platform will ultimately allow us to build patient-specific disease models instrumental to the development of personalized medicines to treat PD patients.

NIH Research Projects · FY 2025 · 2024-04

Mitochondria play an essential role in cellular function and impact human health through varied mechanisms, including energy metabolism, cell signaling, and apoptosis. Many aging-related diseases have a mitochondrial (MT) component, with studies highlighting associations with cardiovascular disease, frailty, and overall mortality. In addition to inherited variation, mitochondria experience somatic mutations at much higher rates than the nuclear genome, introducing a state of heteroplasmy, defined as having more than 1 mtDNA allele within a cell. Using whole-genome sequence data from ~200,000 samples in the UK Biobank (UKB), we found that increased levels of MT heteroplasmy are associated with all-cause mortality, with individuals harboring a heteroplasmic nonsense mutation having a 1.7-fold increased risk of death, even after adjusting for the total number of heteroplasmies. These data indicate that the functional nature of the heteroplasmic mutation is a key driver of increased mortality risk. We propose to directly test the impact of heteroplasmic nonsense mutations across a range of variant allele fractions (VAF) on MT function through base editing of the MT genome followed by a comprehensive suite of MT functional assays. Given the strong link between primary MT disorders and cardiac function, we further propose to test the impact of these variants on cardiomyocyte electrical activity using human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs). In the ~200,000 UKB participants, we have identified 47 ultra-rare heteroplasmic nonsense single base-pair changes. Nineteen of the variants are targetable by existing MT base editing technologies. We propose to optimize constructs to target all 19 bases in HEK293T cell lines, which have served as the model system for MT genome editing, and then select up to 2 constructs per gene for downstream functional analyses in additional cell lines. To functionally characterize heteroplasmic nonsense mutations, we will first expand the range of VAF through a combination of single-cell expansion and the use of mitoTALENs and test the hypothesis of a dose-response relationship between VAF and MT function. We will then use a series of high-throughput assays to comprehensively characterize the mutant cell lines by assessing MT function (e.g., cellular respiration, glycolytic flux) and quantity (mtDNA-CN, nucleoid density, mass). We will then use hiPSC-CMs to test whether deleterious heteroplasmic variants across a range of VAFs modify electrical excitability, repolarization, or conduction, or compromise the development and maturation of cardiomyocytes. The increasing recognition of the role of MT heteroplasmic mutation in aging-related disease has created an urgent need to functionally characterize these mutations. Indeed, while this proposal focuses on heteroplasmic nonsense mutations, we have already identified ~1000 ultra-rare heteroplasmic missense and non-coding mutations at highly constrained sites that likewise increase risk for all-cause mortality. Thus, the proposed experiments will provide the foundational framework for future studies to distinguish benign from deleterious MT heteroplasmic mutations at these 1000s of potentially deleterious heteroplasmic sites.

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY Endemic Burkitt lymphoma (eBL) outcomes in children with advanced stage disease are markedly inferior to those in children with early-stage disease. eBL is one of the most rapidly growing tumors. As a result, diagnostic delays of days-to-weeks contribute to late presentations, and directly affect stage and curability of disease. BL is the most common childhood cancer in Uganda. A child with a large jaw mass will be readily recognized as having eBL, and will typically be treated before histologic confirmation. However, a child with a lesser jaw swelling, or an abdominal mass, may be treated with empiric antimicrobials or observed for weeks. Even after procedures are performed for suspected BL, final histopathology is typically not available for another two weeks. EBV is consistently associated with eBL, and is detectable in blood and saliva of eBL patients. Although EBV DNA holds promise as an early detection marker for eBL, diagnostic utility is limited by poor specificity. EBV DNA not related to tumor is present in blood in a small fraction of latently infected normal lymphocytes and EBV virions may be present in plasma. EBV virions are also frequently detectable in saliva. We hypothesize that the specificity for malignancy would be markedly enhanced by enriching specimens for circulating tumor-derived EBV DNA. This may be accomplished by modifying preanalytical variables in specimen preparation, including upfront cell stabilization to limit viral DNA leakage from latently infected cells, elimination of normal lymphocytes (and epithelial cells in saliva) that may harbor latent EBV DNA, and enrichment for CpG methylated DNA, which effectively reduces virion DNA since that is never CpG methylated. Enrichment for methylated EBV DNA is a particularly innovative approach to improve assay specificity. We will test these hypotheses in two aims. In Aim 1 (blood), PBMC-derived viral DNA will be reduced by analyzing plasma, as opposed to whole blood or buffy coat cells, collected in cell-stabilizing tubes to minimize ex vivo cell lysis. Virion EBV DNA will be excluded via CpG methylated DNA separation. The effect of specimen transport temperature, time, and other preanalytical variables on cell-free DNA (cfDNA) recovery and EBV DNA measurements will be evaluated. In Aim 2 (saliva), collection techniques that minimize oral epithelial cells and lymphocytes and minimize ex vivo cell lysis along with exclusion of virion EBV DNA will be assessed. The effect of specimen transport temperature, time, and additive on cell-free DNA (cfDNA) recovery and EBV DNA measurements will be also be evaluated. These studies aim to define the optimal specimen preparation methods that will be feasible in African settings, and elsewhere, and can serve as the basis for future standards for the biospecimen science community at large. Improved diagnostic specificity of EBV DNA measurements achieved by optimizing preanalytical procedures should translate directly into earlier diagnoses and higher cure rates for eBL. Improved specimen handling also has the potential to improve eBL treatment monitoring, and could extrapolate to the diagnosis/monitoring of other EBV-associated malignancies.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY In the U.S., chronic respiratory diseases are a leading cause of death and disability. Occupational exposures substantially contribute to the burden of chronic lung diseases, resulting in >$10 billion in annual medical expenditures. To address this significant public health burden, there is a pressing need for early identification and mitigation of workplace risk factors, particularly in at-risk groups experiencing elevated exposures to respiratory toxicants. Hairdressers are an understudied, at-risk population for whom respiratory health is a major concern. Over 700,000 U.S. hairdressers are exposed to chemicals which may impact lung health. Our pilot data show that hairdressers of color (Black and Latina) have twice the national asthma prevalence and that, compared to U.S. women, they have elevated exposures to mixtures of chemicals of concern, including phthalates and volatile organic compounds (VOCs). Limited animal and in vivo studies report that the few phthalates and VOCs studied may affect epithelial cells leading to airway remodeling, have immunomodulatory and adjuvant properties, and may be linked to oxidative stress and inflammatory responses, mechanisms underlying respiratory diseases. Still, no studies to date have conducted robust exposure characterization of these chemicals and assessed their role on the respiratory health of U.S. hairdressers, despite prevalent respiratory health concerns. The overarching goal of the Measurement of Exposures, Lung hEalth, and functioN in hAirdressers: The MELENA Study is to comprehensively assess personal and workplace behaviors and workplace exposures to phthalates and VOCs and elucidate their role on lung health. We will use robust exposure characterization approaches to identify opportunities for actionable interventions to mitigate occupational exposures and associated respiratory health risks. To this end, we will leverage our strong community partnerships and multidisciplinary team of experts to execute the following aims in a demographically diverse cohort of hairdressers: (1) assess exposures to phthalates and VOCs and identify exposure risk drivers; (2) determine if exposure to phthalates and VOCs is associated with increased upper airway and systemic oxidative stress and inflammatory markers; and (3) determine if phthalates and VOCs are associated with worse lung function and respiratory symptoms. This will be the first study to comprehensively characterize exposures to phthalates and VOCs (individually and as mixtures using novel mixtures methods) and associated lung health risks among U.S. hairdressers. While work is a social determinant of health that can drive health disparities, it is often overlooked as an avenue for public health interventions. Thus, identifying modifiable workplace contributors in this population will provide key foundational evidence for disease prevention and control. Findings will have direct public health impact by informing effective interventions and federal policies to address exposures and protect vulnerable populations.

NIH Research Projects · FY 2025 · 2024-03

1 Project Summary/Abstract (30 lines) 2 Extracellular vesicles (EVs) are nanoscale balloon-like particles found in the body that play crucial roles in cell communication 3 and can be used as diagnostic markers for complex diseases. However, the lack of reliable methods to purify, analyze, and 4 manipulate EVs has hindered their widespread use in research and clinical settings. To overcome these limitations, we propose 5 the development of a groundbreaking system called STEADI (System for Translational Exosome Analysis and Diagnostics 6 through Integrating electroporation and single-molecule detection) to enable comprehensive analysis of EVs. 7 STEADI integrates a microfluidic electroporator with a high-throughput size-based particle sorting method and a highly sensitive 8 optical molecular detection technique. By utilizing microbeads of different sizes associated with specific EV surface proteins, 9 STEADI allows precise sorting of EVs based on their surface profiles after efficient loading of molecules into the EVs using 10 electroporation. The loaded and sorted EVs are then released from the carrier microbeads and subjected to detailed analysis 11 using a highly sensitive single-molecule detection technique, enabling accurate quantification and characterization of the diverse 12 contents inside the EVs. The primary objective of STEADI is to provide a user-friendly platform for comprehensive exosome 13 analysis and effective preparation of EVs for research and clinical applications. By combining microfluidic electroporation with 14 a multi-laser cylindrical illumination confocal spectroscopy (CICS) system, STEADI allows for the rapid production of precisely 15 loaded EVs and quantitative profiling of their internal contents. 16 STEADI offers several advantages over existing methods. Firstly, it eliminates laborious pre- and post-purification steps of EVs 17 for molecular loading, molecular detection, and downstream assays, thereby streamlining the process. Secondly, the system 18 enables clean and efficient loading of molecules into EVs, preserving their molecular concentrations and membrane integrity 19 and facilitating the detection of lowly expressed molecules that would otherwise be undetectable. Thirdly, the integration of 20 CICS with microfluidic devices provides enhanced single-molecule detection capabilities, allowing for detailed characterization 21 of exosomal contents with single-EV and single-molecular resolution. 22 The STEADI system has broad applications in biological and clinical research. It facilitates the study of exosome biology and 23 their roles in various physiological and pathological processes. Additionally, it opens doors to exploring EVs' diagnostic and 24 therapeutic potential. Accurately quantifying the molecular properties of rare biomolecules within EVs is crucial for advancing 25 our understanding of disease mechanisms and developing targeted therapies. 26 In conclusion, the STEADI system stands as a remarkable leap forward in EV research, elevating its potential for seamless 27 translation into clinical applications. By providing a comprehensive and user-friendly platform that combines microfluidic 28 electroporation with CICS, it offers new avenues for studying the biology of EVs and their applications in research and medicine. 29 This innovative system has the potential to enhance our understanding of exosome functions and contribute to the development 30 of novel diagnostic and therapeutic strategies across various fields.

NIH Research Projects · FY 2026 · 2024-03

Congenital heart disease (CHD) is the most common type of birth defect, affecting ~1% of all newborns, with 1 in 4 children requiring cardiac surgery. Children who require surgery for CHD are at an increased risk for long- term hypertension, chronic kidney disease (CKD), end-stage renal disease, and mortality. An excess burden of hypertension, CKD and death has also been reported in adults with CHD suggesting that pre-disease pathways and incident kidney disease may begin in childhood and progress into adulthood. These elevated risks, along with the dramatically improved survival of children after cardiac surgery, require urgent focus on long-term health in the rapidly growing population of pediatric and adult patients with CHD. Improved phenotyping of the relationship between CHD with hypertension and CKD is needed to better understand the epidemiology and risk factors of these outcomes in CHD. The objective in this application is to recruit and retain children with CHD in a cohort entitled the Congenital Heart disease In Children: Kidney-AssociateD Conditions with Epidemiologic Endpoints (CHICKADEE) study. We propose enrolling 350 children 4-8 years after their first cardiac surgery for CHD at 4 U.S. children’s hospitals. This cohort will be target enriched enrollment for hypoplastic left heart syndrome (HLHS) and other single-ventricle cardiac lesions, as well as those with a history of severe cardiac defects associated with the highest risk of kidney disease. In-person visits with ambulatory blood pressure monitoring, kidney function assessment, and cardiac and kidney biomarker measurement will be conducted at enrollment and then annually for at least 3 total visits. The specific aims are to: 1) Characterize ambulatory hypertension, albuminuria, and CKD in children with CHD and compare to non-CHD forms of CKD using a well- phenotyped and established external cohort of pediatric CKD. This aim will include the entire cohort, but particularly focus on children with HLHS and their blood pressure and glomerular filtration rate trajectories. We will also extract clinical genetic data and samples for future genetic analyses; 2) Identify perioperative factors and clinical characteristics associated with increased risk of hypertension, albuminuria, and CKD. Retrospective and prospective data collection will be combined to assess associations between the number of lifetime acute kidney injury events, medication exposures, and specific echocardiographic features with increased risk of hypertension and CKD; and, 3) Develop panels of urine and plasma glomerular, tubular, and inflammatory biomarkers to quantify the impact of CHD on kidney health. This aim will yield a clinical panel for early detection of kidney injury. A biorepository will be established as a resource for current and future investigations. These studies are critical for identifying children at the highest risk for hypertension, albuminuria, and CKD. In the future, the developed biomarker panel will be used for prompt diagnostic and therapeutic actions. The findings will also be applied to create consensus guidelines for kidney follow-up after cardiac surgery and data to directly support the development of novel interventions and improve long-term outcomes and quality of life for children with CHD.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY Globally, new female HIV infections are concentrated in women outside of known key populations. Identifying women at high risk for HIV acquisition and successfully engaging them in HIV prevention services, particularly pre-exposure prophylaxis (PrEP) programs, is an urgent priority for sustaining epidemic control, protecting U.S. investments and reducing the risk of future cross-border transmission. However, these tools have low to moderate sensitivity, missing many women at high risk for HIV. This is partly due to underreporting of risk factors, but also because many women are at heightened HIV risk solely through relationships with high-risk male partners. Moreover, many women enrolled into PrEP programs stop using PrEP within months of initiation. Decades of research shows that curable sexually transmitted infections (cSTI) are objective markers of future HIV risk, but cSTI testing largely has been omitted from HIV programs in high burden settings. With the advent of lower cost multiplex cSTI testing and point of care diagnostics, there is new opportunity to determine whether integrating female cSTI testing services into HIV programs can improve HIV epidemic control. Here, we propose an individually randomized effectiveness implementation trial of SRST plus cSTI diagnostic testing for chlamydia, gonorrhea, trichomonas, and syphilis compared to SRST alone to increase PrEP use among women aged 15-39 years. We hypothesize that cSTI testing will increase PrEP use primarily through (i) improved identification of women at high risk for HIV and (ii) enhancement of self-perceived HIV risk. The proposed research will be nested within the Rakai Community Cohort Study, a population-based HIV surveillance cohort. To support Aim 1, ~4,500 HIV-negative women will be individually randomized 1:1 to PrEP screening based on SRST plus cSTI diagnostic testing (intervention) versus PrEP screening based on SRST alone (control arm). Both arms will be offered syndromic case management for cSTIs and syphilis testing for pregnant women (standard of care). The primary outcomes will be PrEP uptake, adherence, and persistence, assessed through clinical records and drug level testing. To support Aim 2, we will perform a mixed-methods, implementation science evaluation using qualitative and quantitative methods to assess the mechanisms, barriers, and facilitators to improving PrEP outcomes through cSTI testing and how this varies by cSTI pathogen, SRST outcomes, and demographic profiles. To support Aim 3, we will use mathematical models to evaluate different cSTI testing approaches to reduce HIV incidence at a population level. We will also model broader health benefits of cSTI testing. Results will provide actionable, RCT-level, and population-level empirical evidence to inform the strategic delivery of high-impact HIV interventions, including U.S. developed innovations such as lenacapavir, not to mention strengthening U.S. leadership and investments in the prevention and treatment of HIV and other STIs.

NIH Research Projects · FY 2026 · 2024-03

ABSTRACT Early life exposure to air pollution (AP), which disproportionately affects underserved communities, is thought to disrupt cognitive, emotional, and behavioral development. Human studies show that exposure during the prenatal and early postnatal periods are associated with developmental delays, autism, more severe attention- deficit hyperactivity disorder, depression, and anxiety. Increasing evidence also suggests that exposure to AP is associated with variation in the human gut microbiome, which other evidence suggests can alter brain physiology and cognitive development. Despite these prior findings, no studies have considered the role of the gut microbiome as a potential mediator of the effects of AP exposure on brain development and neurodevelopment outcomes in early life. Further, human studies have focused on the associations of AP exposure with cognitive outcomes at a single timepoint, and very few have examined AP-induced changes in brain structure and function. Our overarching hypothesis is that AP exposure adversely impacts brain and neurodevelopmental outcomes, and that these effects are partially explained by alterations in the gut microbiome. Our preliminary data show that AP exposure is associated with a) poorer Bayley’s motor scores at 24-months, b) adverse gut bacterial and fecal metabolic profiles at 6-months, and c) brain tissue microstructure and blood flow; and d) the newborn gut microbiome is associated with brain measures. Our multidisciplinary team of investigators proposes to test our hypothesis in a cohort of 200 Latino mother-infant pairs, with detailed assessments of maternal health and nutrition, infant growth, and early feeding practices at 1, 6, 12, 18, and 24-months of age. Child cognitive, language, and motor capacities were previously assessed using Bayley Scales at 24 months. With separate NIH funding, we are currently collecting follow-up measures at 6yr, including anthropometric measures, nutritional information, and current and cumulative environmental exposures to ambient and near-roadway AP. We will use archived (1, 6, 12, 18, 24-months) and newly collected stool samples (6yr) to examine gut bacterial species and fecal metabolic pathways (SCFA, lipid, amino acid, bile acid) that can alter brain development. Our specific aims are to determine the extent to which early life exposure to AP is associated with neurodevelopmental outcomes and brain measures (Aim 1), and with the gut microbiome and fecal metabolome (Aim 2) in early- and mid-childhood. We further aim to determine whether air pollution-associated gut microbial profiles and fecal metabolic pathways mediate the associations of AP exposure with neurodevelopmental outcomes and brain measures (Aim 3). This study offers a unique opportunity to advance our understanding of the harmful effects of early life exposure to AP. Results may also suggest interventions that could prevent or attenuate neurodevelopmental disorders, such as limiting prenatal and early life AP exposure or other novel interventions to promote or quell growth of specific gut bacteria.

NIH Research Projects · FY 2026 · 2024-03

Burns affect >11 million people worldwide annually. A fourth of all burn injuries occur in children under age 16 and burns are a major source of morbidity and mortality in children. Treatment of large full thickness burns is a major challenge due to limitations of autogenous skin, wound infection, severe metabolic stress and other associated injuries. The long time to burn wound closure results in susceptibility to infection, prolonged pain and long hospitalization. Human deceased donor skin allografts are a temporizing option for skin cover following severe burn injury, but the grafts are soon rejected. While modern treatment provides survival from severe burns, the healing leaves an imperfect result with scarring, disfiguration, loss of critical skin functions, and increased vulnerability to late, cosmetic, psychological, and physical defects, especially in children. A breakthrough to achieve regeneration of perfect skin has thus become a major aim in wound healing. In response to these challenges, we propose to induce in situ skin regeneration through repopulation of skin allografts using our recently discovered novel stem cell mobilizing therapy. In search for better therapy for organ transplantation patients, a new two-drug combination (AMD3100=A, low-dose-FK506=F) or MRG was discovered serendipitously that enabled long-term liver and kidney allograft survival with short-term treatment and freedom from immunosuppression. This tolerance was associated with allograft chimerism (Host repopulation) and local down regulation of the immune response. AF treatment also accelerated skin wound healing and promoted hair follicle neogenesis. We hypothesize that in situ skin regeneration can be realized by host repopulation of skin allografts through pharmacological mobilization of endogenous bone marrow stem cells with a mechanism similar to protection from liver/kidney allograft rejection. Rejecting skin allografts may create a local environment which facilitates recruitment of mobilized host stem cells, while recruited stem cells may differentiate into components of skin and repopulate the allografts. Thus transplanted skin eventually become “self”. This “allograft chimerism” approach represents a paradigm shift in which skin allografts are treated as “biological scaffolds” which the host repopulates with circulating bone marrow derived stem cells, during which time the allografts are protected from rejection. The main goals of this proposal are to understand the mechanisms of skin regeneration through repopulation of skin allografts in small animal models and to demonstrate the efficacy and feasibility of our novel stem cell mobilizing therapy in repopulation of skin allografts in a preclinical large animal (swine) model of full thickness burns. This work, if successful, will cause a paradigm shift using skin allografts and stem cell mobilizing therapy to heal severe wounds/burns through scar free skin regeneration.

NIH Research Projects · FY 2026 · 2024-03

Project Summary Chromatin context defines all DNA transactions, from gene regulation to genome maintenance. Changes in chromatin composition are hallmarks of cancer cells that contribute to malignant transformation and have been studied extensively using a wide range of Next Generation Sequencing (NGS) approaches. However, conventional genome-wide mapping efforts are limited to the detection of individual targets of interest, impeding the study of their often diverse biological roles. Multi-subunit chromatin-regulatory complexes as well as protein interactions with histones or noncanonical nucleic acid structures are largely defined through correlative analyses of separate mapping efforts, which are unable to determine physical interaction, proximity on the same DNA fragment or even presence in the same cell. These limitations underline an urgent need for improved, context- dependent chromatin mapping and characterization efforts. In this application, we will establish a versatile and broadly applicable technique that allows for the genome-scale analysis of two-component interactions on chromatin. The proposal builds on our extensive experience in the study of genome-wide chromatin responses to DNA damage, as well as the expertise of co-Investigator Dr. Michael Seidman in the analysis and visualization of close-proximity molecular interactions in the context of DNA replication stress. Aim 1 will combine this complementary expertise to develop Proximity-based Chromatin Immunoprecipitation (ProxiChIP), a tool to characterize functional subsets of a given chromatin feature based on its interacting partners, shifting the current ChIP paradigm towards combinatorial feature mapping. Using well-characterized, interacting chromatin binding proteins as a proof of principle, we will convert a method currently restricted to the imaging-based detection of protein interactions (proximity ligation assay, PLA) to a broadly applicable biochemistry tool suitable for immunoprecipitation and a diverse set of downstream applications including NGS. In Aim 2, we will apply this methodology to advance the epigenomic characterization of pathological RNA:DNA hybrids, a complex and poorly understood feature of many cancer genomes thought to contribute to DNA replication stress, cancer genome instability and therapy response. RNA:DNA hybrids have been mapped genome-wide using DNA:RNA immunoprecipitation (DRIP) and related methods. However, existing approaches fail to distinguish between physiological and pathological RNA:DNA hybrid subsets. ProxiChIP-based mapping of RNA:DNA hybrids in the context of replication stress is expected to define the genomic features that underly pathological R loop formation, which presents an essential step towards understanding their impact on genome integrity and malignant transformation.

NIH Research Projects · FY 2026 · 2024-03

Treatment of parasitic nematode infections in humans and livestock relies on a small arsenal of anthelmintic drugs that have historically reduced parasite burdens. However, anthelmintic resistance is increasing, and little is known about the molecular and genetic causes of resistance to most drugs. The free-living roundworm Caenorhabditis elegans has proven to be a powerful model to identify and characterize the molecular targets of the three major anthelmintic drug classes (i.e., benzimidazoles, macrocyclic lactones, and nicotinic acetylcholine receptor agonists), demonstrating its value as a model to understand mechanisms of resistance (MoR). Despite this knowledge, resistance against all anthelmintic drug classes is widespread. Thus, a new approach to combat nematode infections is needed. Emodepside is a “resistance-breaking” anthelmintic with a distinct mode of action (MoA) not found among commonly deployed anthelmintics. Studies in the C. elegans laboratory-adapted strain, N2, have shown that SLO-1 is essential for emodepside sensitivity and that SLO-1 loss-of-function mutations are resistant to emodepside. Further, biochemistry showed that the drug opens SLO-1 channels. However, variation in slo-1 has not been linked with resistance in natural populations, where the MoR remains unknown, highlighting this critical moment to focus on emodepside resistance before it becomes a burden. Preliminary data using C. elegans genome-wide association studies (GWAS) have shown that additional genes, beyond slo-1, are involved in emodepside resistance across natural populations. First, to identify genetic variation in candidate genes, I will introduce variants using CRISPR-Cas9 genome editing, use established high-throughput assays (HTA) and competition assays to test the effects of genetic variants in each strain and identify emodepside resistance genes. Second, gene expression patterns, drug metabolites, and conjugates involved in the biotransformation of emodepside will be measured in the emodepside-resistant C. elegans slo-1 deletion strain, to understand how slo-1 contributes to emodepside’s MoR. We must uncover the xenobiotic metabolizing enzymes (XMEs) involved in modulating the biological activity and behavior of emodepside. The proposed research provides a unique opportunity to use an integrative approach to obtain novel data about emodepside’s MoR and encourage appropriate use of emodepside before resistance is widespread. The proposed training plan will bridge my graduate experience in metabolomics with my interest in host-parasite biology and genetics. Completing this research and training will aid my development as an independent scientist and allow me to create a neglected tropical disease (NTD) research program using integrative omics approaches. My focus will be on making discoveries to improve our understanding of parasite biology, host-parasite interactions, and our ability to treat parasite infections.

NIH Research Projects · FY 2025 · 2024-03

PROJECT SUMMARY Major depressive disorder (MDD) is a debilitating psychiatric disorder with a high lifetime prevalence, imposing a severe economic burden on society. Despite a number of clinically effective treatments for MDD, many patients exhibit resistance to current antidepressants. Thus, novel interventions based on pathological mechanisms of MDD are needed. We recently discovered that glutaminase (GLS1), the enzyme which catalyzes the hydrolysis of glutamine to glutamate, is highly upregulated in activated microglia in the brain of mice subject to Chronic Social Defeat Stress (CSDS), a well-established rodent model used to study stress-induced mood disorders, including depression. We then reported that inhibiting the elevated glutaminase activity with JHU083, a glutamine analog prodrug, dramatically inhibited the stress-induced microglial glutaminase upregulation, inflammatory cytokine induction, and normalized the CSDS-induced social avoidance and anhedonia. Although JHU083 showed therapeutic efficacy, its translational potential is hampered by its propensity to cause gastrointestinal toxicity likely associated with its reactive diazo group and its non-selective inhibition of all glutamine-utilizing enzymes, not just GLS1. Given the significant clinical potential of this mechanism, we propose to overcome this limitation by utilizing our recently developed selective GLS1 inhibitor JHU29 and directly targeting microglial GLS1 by attaching JHU29 to hydroxyl-dendrimer nanoparticles delivery system. Our team discovered that hydroxyl-terminated poly(amidoamine) (PAMAM) dendrimers (~4-10 nm) selectively target activated microglia in the injured brain while showing minimal uptake in healthy brains. This is a highly translational approach, as targeted dendrimer delivery has been demonstrated to be well-tolerated and efficacious in multiple large and small animal models and was recently shown efficacy in a Ph2 clinical trial (NCT04458298). We have assembled a highly experienced team with extensive expertise in neuroinflammatory mechanisms and preclinical models of chronic stress (Zhu), dendrimer nanoparticles (Rangaramanujam), pharmacokinetics (Rais), glutaminase drug discovery and clinical translation (Slusher). Together we will develop a dendrimer-glutaminase inhibitor conjugate (Dendrimer-JHU29) for chronic stress-associated psychosocial behavior deficits by implementing the following three aims: AIM 1- Synthesize and characterize generation-4 (G4) and generation-6 (G6) Dendrimer- JHU29 conjugates. AIM 2- Assess their oral pharmacokinetics, microglial target engagement, tolerability, and biodistribution. AIM 3- Using the optimal conjugate and dosing paradigm from Aim 2, evaluate its therapeutic and tolerability profile in CSDS and vicarious defeat stress (VDS) mice models. Successful execution of these aims will result in a Dendrimer-JHU29 conjugate ready for Investigational New Drug (IND)-enabling studies to support future clinical studies to combat chronic stress-associated depression.

NIH Research Projects · FY 2026 · 2024-03

Modified Project Summary/Abstract Rapid globalization and urbanization continue to escalate the burden of non-communicable diseases (NCD) across the world. Control of the major three NCD risk factors – high blood pressure (BP), high blood glucose (BG) and smoking—are dismally low, particularly in urban areas, due to multiple levels of adverse health determinants. One reason for poor control is a dearth of physicians and nurses. Community health workers (CHWs) can fill this void. To date, trials have documented that task-sharing with CHWs reduces systolic BP and fasting BG and achieves smoking cessation. However, most trials have been done in rural areas, and most trials of CHWs tested management of a single condition, e.g. just hypertension. Furthermore, despite demonstrated impact in systematic reviews, task-sharing with CHWs is far from being fully implemented in health care delivery systems. In this context, we propose to conduct the first implementation research study on task-sharing with CHWs for concurrent management of hypertension, diabetes, and smoking in urban Nepal, where there is a well-established CHW programs with robust governance and operational system, making it an ideal place to test. Our overarching goal is to scale up our demonstrated evidence-based task-sharing interventions to address three NCD risk factors in which we engage key stakeholders and partners, and use contemporary mobile health (m-health) tools. This study, a type 2 hybrid effectiveness-implementation research study, will be conducted in 33 study sites in Pokhara, the second largest city in Nepal. Building upon our teams’ three CHW-led, home-based interventions from the study area that significantly reduced BP and fasting BG, and improved smoking cessation rate in semi-urban areas as well as an m-health intervention that lowered systolic BP in an urban area in Nepal, we will develop the intervention package named SCALE-NCD. Our specific aims are 1) Establish a partnership with stakeholders in order to institutionalize and sustain the intervention, 2) Understand reach, determinants and structured barriers for scale-up of the intervention, and 3) Determine the effectiveness of the SCALE-NCD intervention, and its impact on reach, adoption, fidelity , sustainability, and cost. This study will be led by exceptionally strong team of implementation scientists, epidemiologists, clinicians, and anthropologists in the field of NCDs who have built trusted relationship with stakeholders over time, including policy makers, implementors, advocates, health professionals, CHWs, and patients with NCDs. Our study in Nepal provides a valuable model for the U.S. by demonstrating how task-sharing with CHWs can effectively manage hypertension, diabetes, and tobacco use in resource-constrained settings. Leveraging Nepal’s long-standing and well-functioning CHW systems, the study offers practical implementation insights that are relevant to underserved communities in the U.S., where access to care remains uneven. Our team has already initiated pilot work in the Washington, DC–Maryland–Virginia (DMV) area to assess current care delivery and identify opportunities and models for CHW integration, enabling concrete next steps toward adopting integrated comorbidity management in the U.S. The findings also contribute to advancing implementation science methodologies and developing scalable frameworks for managing multiple chronic conditions through team-based care; an area of growing priority in U.S. Results will be widely disseminated through domestic conferences, further contributing to implementation scientists. Finally, given the disproportionate cardiovascular disease burden among South Asian populations in the U.S., this research offers culturally and epidemiologically relevant strategies that could be adapted to improve outcomes in these high-risk population.

NIH Research Projects · FY 2026 · 2024-03

Prostate cancer (PCa) remains an incurable disease once progression to the metastatic castration-resistant (mCRPC) state occurs; killing >30,000 U.S. men/yr. Unfortunately, each of the FDA-approved agents for mCRPC produces only modest increases in overall survival followed by the emergence of a resistant and more aggressive phenotype. Thus, there urgent unmet need for innovative therapies with novel mechanisms of action that allow discrimination between normal and PCa cells. Herein, we put forward an innovative strategy to selectively exploit selective vulnerabilities in neuroendocrine prostate cancer (NEPC), a highly aggressive variant of mCRPC with poor prognosis, using an orally-dosed and highly specific lysine-specific demethylase 1 (LSD1) inhibitor, bomedemstat, in clinical development for unrelated indications to maximize near-term patient benefit. The prevalence of NEPC is increasing in mCRPC patients following prolonged treatment with potent androgen receptor (AR) axis-targeted therapies (e.g., abiraterone, enzalutamide) as an adaptive mechanism of resistance. Comprehensive genome-scale analyses integrated with pharmacogenomics pipelines will provide a rich mechanistic understanding of the genetic and epigenetic features underlying the sensitivity differential between NEPC and AR+ prostate cancer (ARPC) subtypes to LSD1 inhibition (LSD1i). It should be noted that although NEPC is more sensitive, LSD1i also has significant activity against ARPC tumors, which has important clinical implications for the growing number of advanced mCRPC patients with heterogeneous lesions encompassing mixed molecular and pathological subtypes. LSD1-depedent vulnerabilities are identified that will guide patient selection for a precision medicine approach. Additionally, combination therapy targeting LSD1-induced synthetic lethal susceptibilities are rationally designed to provide more potent and durable responses to extend the clinical utility of these well-tolerated clinically-tested agents with favorable safety and pharmacokinetic profiles. Our proposed work has both conceptual and technical innovations. We will 1) validate LSD1 as a target in NE+ and AR+ tumors via comprehensive mechanistic interrogation using unique patient-derived xenograft and organoid (PDX/O) models; 2) compare responders vs. non-responders across subtypes to inform patient selection in future biomarker-driven clinical trials using state-of-the-art genome-wide profiling technologies; 3) dissect the functional effects of LSD1i in advanced preclinical model systems using these same advanced profiling strategies; 4) identify rational combination therapies based on LSD1i-induced synthetic lethal vulnerabilities to produce durable responses using a high-throughput PDO screening platform; and 5) use clinical-grade drugs to maximize near-term therapeutic potential against this highly aggressive lethal mCRPC variant emerging as a resistance mechanism to standard-of-care therapies with increasing frequency.

NIH Research Projects · FY 2025 · 2024-03

Project Summary/Abstract Mitragyna speciosa (kratom) is a plant indigenous to Southeast Asia with over 40 bioactive alkaloids, two of which, mitragynine (MG) and 7-hydroxymitragynine (HMG), act at mu opioid receptors (MORs). Use of kratom and kratom products increased in the US contemporaneous a critical change-point in the opioid crisis (when painkiller prescribing became more conservative), with many US adults initiating kratom use for putatively therapeutic indications, such as mitigation of withdrawal from prescribed or nonprescribed opioids, and as a self-treatment for symptoms of pain, fatigue, or psychiatric or substance use disorders (SUDs). The number of US users in the past few years is likely >10 million—and increasing. Yet we have little understanding of the balance of, or determinants of, beneficial or adverse effects of kratom either proximal to use (e.g., sedation, nausea, analgesia) or over time (e.g., symptoms of SUD). Preclinical work shows that MG and HMG act as partial biased at MORs but also have non-opioid mechanisms of action and that these and other alkaloids may have therapeutic potential for the treatment of pain and SUDs with less risk than traditional opioids. This converges with self-report data, where there is remarkably consistent therapeutic benefit attributed to kratom with minor-moderate side effects and fewer indicators of abuse potential than might be expected from a substance that, for some, reportedly substitutes for opioids. Yet this work is limited to cross-sectional surveys, case reports, and social-media analyses, which may suffer from self-selection and recall bias and sheer lack of information. Many large surveys of regular users are outdated, given rapid expansion of kratom products, and diverge from findings in smaller samples, particularly with respect to prevalence of kratom addiction and withdrawal. Given kratom’s complex pharmacology and novelty (in the US), and the accompanying near- vacuum in policy and in clinical recommendations, more work is needed to understand its risks and potential benefits. So, too, is scientific consilience in developing a systematic line of kratom research. The aims of this K99/R00 work towards both. For the K99, we aim to, using a sample of kratom-using US adults: (1) conduct momentary assessment of individual instances of kratom product use in daily life; (2) determine associations of momentary responses with directly assayed content of samples of participants’ kratom products; (3) evaluate relationships among kratom effects (including withdrawal-like effects), the kratom products used, and alkaloid concentrations in biospecimens; (4) explore narrative accounts of kratom use. For the R00, I will (5) systematically evaluate effects of kratom discontinuation. Collectively, these studies will be a bridge to randomized interventional studies to be proposed in an R01. These studies also begin the interdisciplinary and interinstitutional collaboration I believe is needed in kratom research, reflected by the diversity of knowledge and expertise of my K99 mentors and collaborators who will ensure that my enhanced training places me on the strongest foundation for developing into one of the top leaders in the field of human kratom research.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY The guidelines for the cardiovascular care of spinal cord injury (SCI) have remained essentially unchanged since mean arterial pressure (MAP) management at 85-90 mmHg was recommended 20 years ago. Due to the heterogeneity in SCI, there is a critical need for a precision medicine treatment. A promising target is impaired autoregulation, or the ability of blood vessels to regulate organ blood flow across a wide range of MAP. This results in altered blood flow through the spatial domain, with hypoperfusion at the permanently damaged injury epicenter (umbra) and gradually increasing blood flow in the surrounding vulnerable tissue (penumbra). To capitalize, our team is developing an ultrasound implant to monitor spinal cord blood flow in humans without contrast. Ultrasound is enabled by removal of the posterior aspect of the vertebra via laminectomy, a common procedure after SCI to relieve pressure. Autoregulation can then be measured through metrics such as transfer function analysis (TFA), presenting a diagnostic tool for the acute care physician after SCI, with quantification of injury extent and severity. Therapeutically, the spatial distribution of blood flow can provide feedback for targeted MAP management. We hypothesize that after SCI, autoregulation as measured by ultrasound will be impaired around the injury, extending into the penumbra. Concurrently, systemic MAP management will differentially affect blood flow over distance from injury, with outcomes depending on penumbra flow. We have made progress towards this goal and will address this hypothesis through the following Specific Aims: Preliminary Data: We have developed an algorithm, FlowMorph, which can extract single-vessel flow parameters from ultrasound data and track them over time. We have also implemented a rigorous measure of autoregulation, TFA, and detected the limits of autoregulation capacity in healthy spinal cord. Finally, we have characterized the spatial distribution of blood flow in spinal cord without MAP management. Aim 1: Establish autoregulation as a clinical marker of vascular injury following SCI: We will use the novel techniques listed above to quantify autoregulation through distance from SCI and link it to functional outcomes. Autoregulation as a prognostic marker will reveal the penumbra as a clinically important area vulnerable to MAP changes. Moreover, it will provide mechanistic insight into using MAP management to optimize blood flow. Aim 2: Use MAP management to protect the penumbra: Following SCI, we will treat rats with MAP management at various levels, both pharmacologically- and volume-induced. We will measure the spatial distribution of flow using FlowMorph and relate it to functional outcomes. Different MAP levels will result in variable levels of flow through the umbra, penumbra, and healthy tissue due to altered autoregulation. We expect to find that penumbra flow will be the strongest predictor of outcomes, providing a therapeutic target in the clinic. This study is significant because it will be the first to use metrics of autoregulation as a clinically useful diagnostic tool in SCI. Therapeutically, it will provide a blood flow-based personalized target for MAP management.

NIH Research Projects · FY 2026 · 2024-03

Diabetic retinopathy is a major cause of blindness worldwide. Current treatments are available only advanced stages of DR. There is a great need for identifying regulators and therapeutic targets. Research advances have expanded our understanding of DR. These include the role of protective factors in DR, the pathophysiologic effect of oxidative stress, and the multi-faceted nature of DR as a disease of the neurovascular unit that involves multiple cell types. This includes neuroretinal dysfunction and neurodegeneration that are early abnormities in DR which can contribute to disease progression. Soluble guanylate cyclase (sGC) is a key enzyme for nitric oxide (NO) signaling. Oxidative stress is known to inhibit sGC in systemic settings, including diabetes, via oxidation of the sGC prosthetic heme, leading to its inactivation and eventual loss. This is notable since oxidative stress is known to be an important driver of DR progression. Drugs promoting sGC are in use for some systemic conditions and are continuing to be actively developed, highlighting the translational potential of targeting this molecule. Our lab has found that an sGC activator drug exerts neuroprotective effects in rodent models of diabetic retinopathy. We have further found evidence for a functional role for sGC in the retina. In this proposal, we seek to expand our studies of sGC with respect to its role in diabetic retinopathy. We will use a combination of in vitro and in vivo approaches to explore the potential inactivation of sGC in retina by diabetes and its stressors. In addition, we will explore the cell-specific roles of sGC in the retina using conditional knockout mice. The proposal could advance sGC as an important regulator and target in DR and also increase understanding of the interplay of retinal cell types in this condition, shedding further light into DR as a disease of the neurovascular unit.

NIH Research Projects · FY 2025 · 2024-03

Project Summary: Photoreceptor (PR) subtype patterning in the retina generates primate-specific trichromatic color and high-acuity spatial vision. Networks of factors drive cells along poorly understood developmental trajectories to reach terminal PR fates. Misspecification of PRs results in diseases causing blindness. My central goal is to determine the mechanisms that specify PRs and how these change to generate PR diversity among primates. PRs express light-sensitive proteins called opsins, which confer distinct functions. Short-wavelength sensitive cones (S cones) express S-opsin, long/medium-wavelength sensitive cones (L/M cones) express L/M-opsin, and dim light sensitive rods express Rhodopsin (Rho). In humans, opsin expression follows a temporal order: S opsin, then L/M opsin, and lastly Rho. The Johnston Lab found that retinoic acid (RA) and thyroid hormone (TH) signaling specify cone subtypes in human retinal organoids. Early, low TH and high RA signaling specify S cones. Later, high TH and low RA yields L/M cones. The mechanism by which TH and RA signaling regulate PR development is unclear. Comparative developmental biology is a powerful approach for revealing general mechanisms of development and how tweaking these mechanisms generates diversity between species. In contrast to humans, the initiation of opsin expression in the marmoset is inverted: Rho, then L/M opsin, followed by S opsin, suggesting that marmoset PR subtypes are born in a different order and/or mature at different rates compared to human PRs. I developed primate retinal organoid technology and determined that opsin expression in organoids recapitulates species-specific developmental patterns. I will genetically and pharmacologically manipulate retinal organoids to compare mechanisms of PR specification between primates and identify changes that diversify retinal patterning between species. During the K99 phase, I will determine how TH and RA regulate PR subtype specification and maturation in human and marmoset organoids. During the R00 phase, I will elucidate how TH and RA signaling regulates the cell fate trajectories of human and marmoset PRs during development. I will identify and validate candidate cis-regulatory changes that drive differences in PR specification, by knocking out associated genes, reciprocally swapping putative divergent regulatory elements in stem cells, and assessing effects in human and marmoset organoids. These comparative approaches will untangle the regulatory networks underlying the divergence of PR development between primate species. My goal is to become a leader in the field of neuronal fate specification, with a focus on understanding mechanisms of cell fate determination in primate retinal development. My mentoring team and the Johns Hopkins University community will provide the ideal environment for achieving in this goal.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY Glaucoma is a major cause of blindness, affecting over 80 million people worldwide. Glaucoma is a neurodegenerative optic neuropathy caused by the loss of retinal ganglion cells (RGC), leading to loss of vision. Current therapies are all directed at lowering intraocular pressure (IOP), and yet RGC loss still continues in many patients despite IOP lowering. The identification of an agent that complements IOP lowering by promoting RGC survival would be a significant advance toward improving the visual outcomes of patients with glaucoma. Using cultures of primary RGC, we screened more than 10,000 compounds and identified candidates with potent neuroprotective properties, including a drug that is FDA-approved for an unrelated indication. We further characterized the novel pathway through which these compounds act to protect RGC, thus identifying a novel drug/drug target combination for neuroprotection. We have also developed a novel thermosensitive gel-forming eye drop drug delivery system that provides efficacious drug delivery to the posterior segment, even in large animals (rabbits, pigs). Importantly, we observed that the combination of more effective intraocular drug penetration provided by the gel-forming eye drop with a drug that binds to melanin in the eye, led to RGC protection in vivo with once weekly topical dosing. In this application, we are screening additional neuroprotective drugs for melanin binding, cell uptake, and intraocular penetration to compare head- to-head for pharmacokinetics and efficacy in an optic nerve crush rat model and a bead injection mouse model of IOP elevation. The goal is to develop an efficacious eye drop for neuroprotection that requires once weekly, or ideally once monthly maintenance dosing. In Aim 1, we will make further formulation changes in the eye drop to increase intraocular drug absorption, and formulate additional melanin-binding neuroprotective drugs. In Aim 2, we will characterize the pharmacokinetics and efficacy of a variety of dosing regimens and formulations to identify the most effective dosing approach with the lowest dosing frequency. In Aim 3, we will perform dose-ranging studies in rabbits to achieve similar drug concentrations in target tissues that were shown to be effective in Aim 2. We will also perform safety evaluations with longitudinal dosing, including fundus exams, IOP, and retinal morphology analyses. The demonstration of efficacy in rodent models of neurodegeneration along with similar pharmacokinetics and no overt toxicity in the rabbit eye, would provide evidence of the therapeutic potential of our neuroprotective drug delivery strategy for the treatment of glaucoma.