Yale University

NIH Research Projects · FY 2024 · 2023-08

A foundational, unanswered question of neural metabolism is whether the dramatic reduction in glucose uptake observed during aging and Alzheimer's disease progression is a cause or consequence of the functional decline of neurons. Current methods for visualizing the dynamics and effects of metabolism in vivo are too limited to determine if this plays a role in eventual neuronal dysfunction. Notably, neurons are thought to primarily use glucose for redox protection via the pentose phosphate pathway (PPP), rather than for production of ATP by glycolysis, and when glycolysis is upregulated in neurons in vitro it leads to elevated redox damage and apoptosis. As neuronal stimulation can also temporarily increase glycolytic flux, this suggests there may be an uncharacterized competitive regulation of glucose use towards balancing either ATP production or redox protection. Therefore, the goal of this proposal is to develop and utilize genetically-encoded biosensors for key metabolites in C. elegans neurons to determine the relationship between elevated calcium activity, dynamic states of glucose metabolism, and redox balance at single cell resolution. Thus, aim 1 of this proposal will utilize the novel fluorescent sensor HYlight to dynamically measure changes in cellular glycolysis in vivo during conditions of energy stress, such as with neuronal stimulation. The main goals of this aim are to (1) determine the relationship between states of high ATP demand and induction of upregulated glycolysis, and (2) elucidate the molecular mechanisms that enhance glycolysis under these conditions. Aim 2 will determine whether states of high neuronal glycolysis also lead to increased redox sensitivity in single neurons. Genetically-encoded biosensors for ROS and NADPH will be used to read out cellular redox state and enable development of an assay for quantifying degradation in stimulation-induced calcium activity that results from cell-autonomous ROS accumulation. Finally, the effects of reducing glucose within a single neuron over the lifespan of aging worms will be compared in the context of calcium activity and behavior to determine whether these effects coincide with those induced by elevated redox damage. This system described herein would provide a new avenue for assessing the source and impact of ROS-induced decline and give significant insight into the importance of balancing glucose metabolism in maintaining neuronal function and behavior during aging. Additionally, it will provide important career development for the submitting candidate, who will train during the K99 phase under the mentorship of Dr. Daniel Colón-Ramos at Yale University. This lab has significant expertise in behavioral neuroscience, which will be critical for advancing the career goals of the candidate in linking single neuron biology to behavioral changes during aging. This award will also support the candidate’s career goals by providing an opportunity to learn critical research skills, mentorship expertise, and personalized guidance for navigating the faculty job search. With this training the candidate will be able to successfully complete the proposed research and transition to an independent faculty career.

NIH Research Projects · FY 2026 · 2023-08

Hybrid dysfunction offers an unparalleled window into the evolutionary drivers and genetic basis of divergence in key developmental processes. Hybrid dysfunction can also confer reproductive isolation and contribute to speciation. Intra-genomic conflicts may play a central role in this divergence, and underlie hybrid dysfunction, but empirical tests are few. A common source of conflict in viviparous organisms stems from conflict between dams and sires for resource allocation to developing offspring (e.g. parental conflict). Parental conflict may drive the evolution of genomic imprinting; an epigenetic phenomenon whereby alleles have parent-of-origin-specific expression. Under parental conflict, misregulation of imprinted genes causes abnormal placenta or endosperm development, and subsequently hybrid embryo death. This early onset inviability is common in both mammalian and plant hybrids, but the underlying genes are unknown. I will address this knowledge gap by pairing population and quantitative genomics, gene expression, and functional genetics to identify the genetic basis of hybrid seed inviability and test the role of parental conflict in generating this barrier. I recently discovered a species in one of the most widely studied models for genetics and evolution- the Mimulus guttatus species complex. Despite a recent split (~230KYA), M. decorus and M. guttatus are reproductively isolated via hybrid seed inviability caused by atypical endosperm development. Hybrid seed inviability has rapidly and repeatedly evolved in this group, with at least two independent incidences in ~230KYA. This exceptional variation in reproductive isolation, paired with the tremendous genetic and genomic resources of M. guttatus makes it an ideal system to study the genetic basis of hybrid seed inviability. By quantifying expression differences among species, I will characterize the epigenomic landscape of imprinting and assess if divergence in imprinting is driven by natural selection. In tandem, I will map the genetic basis of multiple incidences of hybrid seed inviability to assess the extent of overlap in inviability loci and estimate how repeatable conflict-driven evolution is. I will then identify candidate genes and functionally test them by constructing transgenic lines that alter imprinted gene expression and quantify if abnormal imprinted expression causes inviability; a central prediction of parental conflict. Lastly, using replicated contact zones, I will assess the dynamics of introgression between these species to estimate the efficacy of hybrid seed inviability as a barrier to gene flow. By using patterns of ancestry disequilibrium, I can also identify other putative incompatibility alleles. As I have generated a set of immortal inbred lines from these contact zones, I can then resurrect specific genotypes, perform manipulative crosses, and identify putative incompatibility phenotypes, thus bridging long term patterns of selection with specific hybrid phenotypes. This project will result in a holistic genetic dissection of a common and important reproductive barrier; from its molecular genetic basis to quantifying the role of these alleles in limiting introgression in nature. Given that many human diseases result from misexpression of imprinted genes, understanding the evolutionary forces that shape imprinted gene expression variation also has significant implications for human health.

NIH Research Projects · FY 2024 · 2023-08

Project Summary Stroke is the leading cause of disability in the United States. Patients with stroke are at increased risk of severe pneumonia and post-stroke pneumonia is associated with poor outcomes. This multi-PI project will leverage the complementary expertise of a Stroke Neurologist/Immunologist and a Pulmonary Critical Care physician/Immunologist to jointly tackle the mechanisms of increased risk of severe pneumonia in stroke patients and the mechanisms of dysregulated neuroinflammation and neurological recovery after stroke. Preliminary work in our experimental model of ischemic stroke and Klebsiella pneumonia demonstrates that bacterial inoculation into the lungs leads to reduced neutrophil recruitment and increased bacterial dissemination after stroke. Furthermore, pneumonia leads to enhanced neuroinflammation and increased interferon responses in the microglia and endothelial cells after stroke. The primary hypothesis is that stroke leads to an impairment in neutrophil function allowing for bacterial escape and dissemination through the body, which in turn leads to a delay in the resolution of neuroinflammation in the brain and further brain injury. The overall goal of the proposal is to identify new treatment targets to reduce post-stroke pneumonia and improve outcomes after stroke.

NIH Research Projects · FY 2024 · 2023-08

PROPOSAL SUMMARY The goal of the proposed K99/R00 award is to support Dr. Courtney Choy’s development into an independent community-engaged implementation scientist, with a focus on epidemiology and leveraging quantitative and qualitative data to address noncommunicable, cardiometabolic disease (CMD) risk in Pacific Islander children. Although they are among the most at risk of CMD, few efficacious interventions or innovations to address CMDs have reached Pacific Islander communities. The objective of this proposal is to develop and implement a context- specific, multi-component intervention to address CMD risk in Samoan children. Samoa, a middle-income Polynesian country with a large global diaspora, experiences among the highest CMD prevalence in the world. Existing data from the Ola Tuputupua’e (‘Growing Up’) cohort study, which this proposal will leverage, suggests that risk emerges early in life. Half of the enrolled children were affected by either obesity, elevated blood pressure, and/or high glycated hemoglobin (HbA1c) by the age of 9. During the K99 training phase, Dr. Choy will (1) extend her epidemiological training and use growth mixture modeling to inform intervention targets and timing and (2) gain new skills in qualitative methods, and implementation science to select and contextually adapt an intervention for use in Samoa. The Active Implementation Frameworks and evidence-based best practices in Pacific Islander communities will guide intervention development and implementation, which will take place in the R00 phase. The result will be a multi-component intervention for Samoan children that targets the age period in which markers of CMD risk (body mass index, blood pressure, and HbA1c) are most sensitive to modifiable behavioral and environmental risk factors. Further, the multi-component intervention will incorporate at least two evidence-based lifestyle/behavioral change strategies to address CMD risk factors, utilize existing infrastructure and local capacity (with an implementation team) and align with community-identified priorities. Preliminary evidence of the feasibility, acceptability, and effectiveness of the developed intervention among children in Samoa will produce a scalable program with the potential for replication in other Pacific Islander populations and comparable low-resource settings. To achieve career development goals and ensure a successful transition to independence, Dr. Choy has established an exceptional training environment, developed experiential learning activities to apply newly gained skills from didactic coursework, and assembled a multidisciplinary mentorship team with expertise in public health, implementation science, clinical trials, human biology, epidemiology, and Pacific Islander health. The mentorship, research training, leadership and career development activities proposed will ensure that at the conclusion of this award, Dr. Choy will be well-positioned to extend the methods developed to Pacific Islanders in the US, who are among the fastest-growing racial and ethnic minority groups and –because of their high CMD risk– are the ideal beneficiaries of early intervention to reduce health disparities.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT Alcohol Use Disorder (AUD) is a chronic relapsing illness associated with high rates of relapse, and thus, there is great need to develop and evaluate novel treatments to decrease relapse and improve alcohol use outcomes in AUD. We previously conducted a novel dose finding human laboratory, safety and pilot efficacy study to assess whether the neuroactive steroid (NAS) precursor pregnenolone (PREG) that influences GABAergic functioning may normalize alcohol-related stress disruption and improve alcohol use outcomes in AUD. Pilot data showed that PREG at 300mg/day reduced stress- and cue- induced alcohol craving, anxiety and normalized chronic alcohol-related disruption in stress biology and also reduced alcohol drinks/day (AvgD), percent drinking days and heavy drinking days (%DD and %HDD) compared to placebo (PBO) in an 8- week clinical study. On the basis of these findings, this project proposes a 12-week double blind, randomized Phase II clinical trial to evaluate the safety and efficacy of PREG treatment (300 mg/day) versus PBO in 150 AUD men and women. The following specific aims will be addressed: Aim #1: To establish the safety and tolerability of PREG (300mg/day) vs. PBO in men and women with AUD over the 12-week treatment period and at the 1-month follow up. Aim #2: To test the efficacy of PREG vs. PBO on the primary alcohol use outcome of PSNHDD and secondary drinking outcomes of HDD%, DD% and AvgD during the trial. Aim #3: To assess the effects of PREG vs. PBO on other secondary stress-related outcomes of alcohol craving, anxiety, depression and patient-related functioning during the trial. Aim #4: To assess the effects of PREG vs. PBO on PREG and other NAS levels and examine their relationship to primary and secondary alcohol use and related outcomes. Exploratory Aim 1: To assess enduring short-term treatment effect of PREG vs. PBO on primary and other secondary outcomes at a 1-month post-treatment follow-up. Exploratory Aim 2: To explore whether pre-treatment patient characteristics (sex, trauma history, AUD severity and co-occurring psychiatric disorders) influence PREG effects on primary and secondary outcomes. It is well known that chronic alcohol use downregulates GABA which plays a significant role in the stress pathophysiology of AUD and also in loss of control drinking. The proposed study is based on our novel preliminary findings and will test our innovative approach of boosting endogenous neuroactive steroid levels to increase their function and thereby improve AUD outcomes. If successful, the proposed research will establish PREG and neuroactive steroids as key targets in the treatment of AUD, and also provide data on neuroactive steroid levels and whether they may serve as biomarkers in AUD treatment.

NIH Research Projects · FY 2025 · 2023-08

Abstract Recent advances in human connectome research have led to the development of models that reveal the brain circuits associated with behaviors or symptoms. The networks that define these circuits yield functional phenotypes that can be measured in individuals and are unique to each individual. Such work holds tremendous promise for providing a biological basis for understanding brain function and brain disorders, it allows us to characterize trajectories of growth, development, and aging, to categorize patients according to their functional phenotype, ultimately aiding treatment decisions, and predicting outcomes. Building such connectome based predictive models, involves 3 distinct steps: 1) construction of the connectivity matrix summarizing the connections across the defined nodes/parcellation; 2) a subsequent association step linking edge strength to the behavior or clinical symptom of interest; 3) and finally a predictive model step for validation and to ensure the models generalize and the associations are not spurious. While many atlases are available, there has been no consensus on which atlas to use to define the nodes in building the connectome, making the sharing of models and validation across sites difficult. A second, often overlooked problem, is that the node configuration supporting one behavior may not be the same for a different behavior due to the functional flexibility in brain organization. Thus, while the parcellation and brain modeling steps have historically been treated separately, they are not independent and should not be treated as such. In this work we will develop a joint parcellation/brain-phenotype modeling approach that provides statistically powerful, analytically robust, and biologically interpretable Bayesian models that are not dependent upon the choice of the initial atlas. We will validate the models through measures of predictive power, reliability, and generalizability, and compare to existing state-of-the-art methods. Data for validation will include the healthy adult data from the human connectome project and a transdiagnostic sample of 450 individuals (after adding 150 subjects in this study) collected at Yale, spanning a range from healthy control subjects to those with psychiatric illnesses. Normative models for 16 behavioral measures and 6 clinical scores will be developed and shared with the neuroscience community. A key aspect of validation and reproducibility in research is the sharing of data and models. The use of approximately a dozen or so arbitrary atlases in the field inhibits the sharing of models. This work will move the field forward by improving the methodology of brain-phenotype predictive modeling, identifying the circuits supporting behavior, without a priori imposition of an arbitrary atlas. The results could advance our understanding of the brain networks supporting behavior and impact a wide range of psychiatric illnesses. Facilitating the release of generalized models to the research community will aid in understanding how to use these methods for assigning treatments and monitoring the response to treatment.

NIH Research Projects · FY 2024 · 2023-08

Project Abstract Each year in the U.S., millions of Spanish-speaking Hispanics experience negative consequences from harmful alcohol use. Hispanics suffer from alcohol use disorder (AUD) and alcohol-related liver disease at rates similar to non-Hispanic Whites. However, Hispanics with AUD are less likely to receive treatment than non- Hispanic Whites. Hispanics also face greater socioeconomic, cultural, and linguistic barriers to treatment for AUD than non-Hispanic Whites. These barriers include limited access to Spanish-speaking providers and culturally adapted evidence-based treatments. Computerized treatments and mobile technology have significant potential to increase access to evidence-based treatments in a cost-effective manner for Spanish speakers. A culturally adapted computerized treatment for Spanish-speaking Hispanics with substance use disorders has demonstrated efficacy at reducing rates of alcohol and drug use in a randomized clinical trial (Computer-Based Training for Cognitive Behavioral Therapy; Spanish CBT4CBT). Several other clinical trials have supported the efficacy and cost-effectiveness of English versions of CBT4CBT. Rapid, mobile delivery of CBT skills would facilitate treatment feasibility during risky periods for drinking in daily life (e.g. weekend evenings). But, CBT4CBT modules each require 35-40 minutes of sustained attention to complete and cannot be used quickly during high- risk drinking periods. This project proposes to develop a concentrated mobile version of Spanish CBT4CBT to rapidly deliver treatment content on high-risk drinking days: Aim 1) Identify Spanish CBT4CBT module topics associated with greater reductions in daily alcohol use and psychiatric symptoms. Aim 2) Develop a concentrated version of Spanish CBT4CBT consisting of a subset of content from the current modules. Aim 3) Test the feasibility and acceptability of using smartphones to deliver a concentrated version of Spanish CBT4CBT on high-risk drinking days. To complete these aims, my career development plan includes mentored training in the following domains: 1) conducting clinical trials of treatments for AUD; 2) designing culturally adapted treatments for Hispanics; 3) delivering treatment components through smartphones; 4) using mixed methods to explore stakeholders’ perspectives on treatments for AUD; 5) fostering professional development through grant writing, publishing, leadership, and networking. This award will provide me with hands-on experience in ongoing clinical trials of computerized treatments for Spanish speakers with AUD. I will participate in NIH training programs on digital technologies, minority health, and professional development, including the MOSAIC UE5 leadership forums. I will complete Yale University courses on clinical trial methods, qualitative research, and grant writing. These activities will build on my prior expertise with using mobile technology and advanced statistics to study the temporal dynamics of motivation for drinking and social processes. This award will support my transition to an independent research career developing mobile treatments for Hispanics to target drinking in real-time.

NIH Research Projects · FY 2024 · 2023-08

Project Summary/Abstract Axon degeneration is a conserved and tightly regulated process and is a driving force of later pathologies in many neurodegenerative diseases. There is a lack of treatments for neurodegeneration that results from disease, and axon degeneration is a target for early intervention and prevention. Elucidating the specific mechanisms that initiate axon degeneration could assist in identifying ways to protect axons in disease and ultimately treat neurodegeneration. C. elegans Mitochondrial Trafficking Mutants (MTMs) have no axonal mitochondria, resulting in axon degeneration. In preliminary experiments the applicant made a surprising discovery: inhibiting glycolysis in MTMs suppresses axon degeneration. Following this finding the applicant showed that degrading the enzyme responsible for lactate production rescues degeneration of axons, suggesting that lactate accumulation may drive axon degeneration. However, the molecular mechanism by which this occurs is unknown. This observation prompted the hypothesis that the product of glycolysis, lactate, accumulates in the absence of mitochondria and by creating a more acidic axonal environment instigates degeneration. Completion of this proposed work will elucidate the role of lactate and related metabolites on axon degeneration (Aim 1). Further, it will shed light on the consequences of loss of axonal mitochondria for neurons —a key feature of neurodegenerative diseases. Finally, these experiments may identify novel approaches for preventing morphological and functional axon degeneration (Aim 2). Work proposed here will establish the role of lactate in axon degeneration and further our understanding of the coupling between neuronal activity and lactate in health and disease. Upon completion of this fellowship the trainee will have received extensive training in an environment well equipped to support collaborative and cutting-edge research. The trainee will gain technical and analytical skills, and professional independence in preparation for an academic career in cellular and molecular neuroscience.

NIH Research Projects · FY 2024 · 2023-08

Prior to clinical evidence of fibrosis, microvascular injury occurs, presenting as an altered functional state of the endothelium, increased permeability, enhanced vasoreactivity, increased expression of adhesion molecules, excessive inflammation, and altered vascular wall growth. Microvascular rarefaction, or capillary dropout, is coincident with chronic fibrosis, and considered an accelerator of the disease. However little is known about the microvascular contribution to fibrotic diseases in which microvasculature are key to tissue health and homeostasis. Given the central role of the vasculature in barrier function, inflammatory regulation and interstitial tissue necrosis, it is likely that the microvasculature, specifically, mural perivascular cells (pericytes), are key contributors to fibrotic development and progression. Recent evidence suggests that microvascular dysfunction may be more directly influential to tissue remodeling than epithelial cells. Limited availability of human pericytes from a readily available human source has led to an incomplete understanding of the mechanisms underlying pericyte to myofibroblast transition that facilitate both microvascular and interstitial matrix remodeling. Our work, supported by that of others in this area, has led us to the hypothesis that pericytes cease homeostatic maintenance of the microvasculature by transition into a myofibroblast through the process of dedifferentiation and re-differentiation known as cellular reprogramming. As myofibroblasts, cells of pericyte lineage contribute to interstitial tissue fibrosis. Through three distinct aims we will show that, in response to growth factor, pericytes deposit extracellular matrix proteins to alternatively support vascular stability and fibrosis as they undergo phenotypic transition from microvascular pericytes to interstitial myofibroblasts. We also determine points in pericyte transition that may be key for therapeutic intervention. We utilize traditional molecular biology methods and biomaterials technology to determine the profibrotic mediators promote functional and phenotypic shifts in pericytes. Using 2- and 3-D bioengineered mechanically and biochemically tunable polymer based extracellular matrices, we decouple the role of biochemical and mechanical signals in regulation of PC to myofibroblast transition through the process of reprogramming. Results acquired through use of human cells in bioengineered structures will be validated with animal models of pulmonary fibrosis.

NIH Research Projects · FY 2025 · 2023-08

Abstract Hematopoietic stem cells and progenitors (HSPCs) are a rare population of self-renewing bone marrow (BM) cells that can generate all mature lineage blood cells for the lifespan of an organism. In adults, quiescent HSPCs reside within a hypoxic bone marrow niche (1-4% O2 physiologic hypoxia) and are capable of rapidly entering the cell cycle and differentiate to produce leukocytes in response to an invading pathogen. The mentor’s lab uncovered that resolution of inflammation is governed by spatial and temporal production of novel mediators and elucidated the specialized pro-resolving mediators (SPMs), a superfamily of autacoids that includes lipoxins (LX), resolvins (Rv), protectins (PD), and maresins (MaR). SPM biosynthesis structures and functions were established in the mentor’s lab and confirmed by others. SPMs are sub-nanomolar potent, stereoselective agonists that promote microbial clearance and containment, while enhancing host survival by accelerating host resolution mechanisms. This proposal is based on new findings from work in progress; we identified, using state- of-the-art metabololipidomics profiling, a specific SPM cluster in HSPC under physiologic hypoxia, which includes Resolvin D1 (RvD1), Resolvin D4 (RvD4), Resolvin E1 (RvE1), Maresin 1 (MaR1) and Lipoxin B4 (LXB4). Also, we recently identified a new SPM, Resolvin E4 (RvE4), in healthy human bone marrow. Tissues that experience physiologic hypoxic niches, such as BM, have high amounts of SPMs through undefined mechanisms. Thus, we propose to rigorously test the following hypothesis: In physiologic hypoxia, specialized pro-resolving mediators (SPMs) produced in BM are essential for regulating HSPC responses de novo and/or during infection at distal sites of invasion, as well as maintaining BM homeostasis. The following aims are proposed by using state-of-the-art omics platforms e.g., metabolipidomics (LC-MS/MS), mass cytometry (CyTOF) and single cell RNA sequencing (sc-RNA-seq) in murine and human HSPCs to: 1) determine the impact of physiologic hypoxia on endogenous novel SPM production in BM niche (K99), and 2) establish the regulation of HSPC differentiation and responses to infection by the SPM cluster, specifically RvD1, RvD4, MaR1 and the novel RvE4 (K99/R00). The K99 mentored phase will take place at Brigham and Women’s Hospital and Harvard Medical School under the mentorship of two internationally recognized scientists, Professor Charles N. Serhan, the leader in the structural elucidation of SPMs and their functions and Professor Leonardo Zon, the leader on hematopoiesis. Dr. Libreros will be further advised by a scientific committee/collaborator covering different aspects of the research proposal: Dr. Matthew Spite, Dr. Mark Perrella, and Professor David Scadden. The goal of this proposal is to provide a comprehensive scientific and career development plan for the applicant with the required skills to transition to an independent faculty position (R00). Results from these studies will yield fundamentally new insights into the functions of SPMs and novel pro-resolving pathways in HSPC biology. These discoveries can help develop novel treatments for hematological disorders and provide a basic understanding of how bone marrow supports resolution programs during infections.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Background. Sexual and gender minorities (SGM) experience among the largest mental health disparities of any population, at substantial cost to society. One driver has been the lack of evidence-based practice (EBP) addressing the distinct mechanisms underlying SGM's risk. For 10+ years, our team has developed LGBTQ- affirmative cognitive-behavioral therapy (CBT) as the first and only EBP specifically for SGM mental health, now in high demand. This proposal seeks to study optimal means of implementing this effective treatment at LGBTQ community centers nationwide to advance implementation science and health equity. Pilot Studies. Our pilot data across 50 LGBTQ community centers shows that (1) training can improve providers' LGBTQ- affirmative CBT skills, (2) directors of LGBTQ centers unanimously support this training, (3) providers perceive that ongoing supervision can help maintain implementation but that receiving learning materials is also invaluable. Still, knowledge gaps remain for implementation science, including: (1) whether train-the-trainer strategies can further sustain implementation, (2) whether features of low-resource settings (e.g., high staff turnover) predict optimal implementation strategies, and (3) the target mechanisms through which EBP training impacts implementation and client outcomes. The proposed research will answer these questions. Method. Drawing on our long-standing partnership with the US's coordinating hub of LGBTQ community centers, in this type 3 hybrid trial we will randomize 90 centers to receive one of three strategies for implementing LGBTQ- affirmative CBT to compare their effectiveness (Aim 1), identify center-level moderators of implementation success (Aim 2), and examine the impact of the three strategies on client mental health through theory- informed organizational and provider mechanisms (Aim 3). Drawing on social learning theory, mental health research in low-resource settings, and our pilot data, we will compare three implementation strategies: (1) a suite of digital learning materials (materials only condition); (2) these materials plus weekly webinar training for 12 weeks (direct training condition); or (3) the above plus 1-year of supervision from a local supervisor who will receive expert consultation in a train-the-trainer format (local supervision condition). Implementation outcomes will include objectively coded provider fidelity in LGBTQ-affirmative CBT and mixed methods assessments of its reach, adoption, and maintenance across centers. Effectiveness outcomes will include client depression symptoms. Hypotheses. We hypothesize that the three additive training strategies will predict respective graduated increases in implementation success up to 2 years post-training. We hypothesize center-level determinants of implementation success from the Consolidated Framework for Implementation Research. We hypothesize organizational and provider factors that will mediate the stronger impact of the more intensive training strategies on implementation success and client mental health. Dissemination. We will distribute scenario-based recommendations and budget impact to encourage maintenance across US LGBTQ centers.

NIH Research Projects · FY 2024 · 2023-08

Project Summary My lab uses a community-based participatory research approach to reduce health disparities in substance use among Black and Hispanic youth in urban communities. We primarily work in New Jersey (NJ) due to our close ties with Paterson and East Orange, NJ which both have the highest number of substance use disorders in the State and the largest group of racial-ethnic minorities (e.g. Black and Hispanic) in the state. My lab intentionally works on the community level as we have found that one-size-fits all approaches to ending the youth substance use epidemic will not work, particularly in communities that have been historically marginalized. Our recent work has discovered that targeting individual level behaviors to promote behavior change may not be enough to end the youth substance use epidemic and in fact, understanding the role of neighborhood characteristics may be a more plausible strategy. In our work, we have shown that predominantly urban communities such as Paterson, NJ and East Orange, NJ have some of the lowest neighborhood resources associated with healthy youth development and therefore can contribute to likelihood of using substances and becoming addicted. In addition, the use of complex statistical methods and study designs, may contribute to lack of mistrust of researchers, participation in studies and of the data by community members. We hypothesize that within predominantly urban communities, there is variability in structural risk and asset-based neighborhood characteristics associated with youth substance use. In line with using a social determinants of health approach, environmental and place-based factors have long been equated with health outcomes such as respiratory conditions (e.g. asthma) among youth. However, determining the exact resources within the community that contributes to substance use disorders have not been discovered. The field of addiction does not know the exact characteristics within a neighborhood that can serve as either protective or risk factors to substance use disorders within an urban community. In this Pioneer proposal which is responding to the RFA-DA-23-026, “NIDA Racial Equity Visionary Award DP1 mechanism”, we will combine innovative approaches and multiple forms of data to investigate neighborhood level factors by using participatory methods to co-create machine learning systems to predict and prevent substance use disorders with community members. We intend for this project to promote co- learning between community members and researchers that can lead to sustainable solutions for the community. The proposed work will shed light on the importance of place in addiction and also work towards eliminating racial bias in data sets and predictive algorithms by incorporating community members in all stages of the model development process. Findings from this study have the potential to change the way we as researchers conduct substance use and misuse prevention research and the way in which we formally engage with community members. This work can contribute significantly to achieving health equity for Black and Hispanic youth in urban communities.

NIH Research Projects · FY 2025 · 2023-08

In sub-Saharan Africa, heart failure (HF), a clinical syndrome that causes multiple debilitating symptoms and results in poor health-related quality of life, has a 34% 1-year mortality rate, the highest of any region globally. Major care gaps and sociodemographic disparities exist throughout the continuum of HF care. Self-care is an evidence-based intervention that can improve clinical and patient-reported outcomes in HF and is widely endorsed by international bodies. Self-care holds particular promise in low- and middle-income countries (LMIC) because it shifts fundamental care tasks from providers and facilities to patients while empowering patients to better care for themselves. However, self-care is underutilized in LMIC where providers lack knowledge, tools, and time to support patients in self-care and where differences in self-care implementation among patients and providers are not well known. The overarching goal of our work is to improve HF care in LMIC. In this Type 2 hybrid study, we will implement a digital-first, multi-component strategy for HF (Medly Uganda) to improve selfcare in HF at 6 Ugandan Regional Referral Hospital outpatient departments and evaluate both implementation and clinical outcomes. All study aims are guided by the RE-AIM framework and PRISM extension, involve mixed methods, and are designed to prioritize geographic and socioeconomic diversity. First, we will assess the implementation and clinical effectiveness of Medly Uganda to improve HF self-care in Uganda (Aim 1). To do this, we will conduct a stepped-wedge, cluster randomized trial that includes a control period followed Medly Uganda. The co-primary outcomes will be the Self-Care of HF Index (implementation) and the composite of mortality and HF hospitalization (clinical effectiveness). In parallel to this, we will conduct a mixed methods process evaluation to inform iterative adjustments to the implementation processes (Sub-Aim 1.1) using a community-engaged approach to systematically collect qualitative and quantitative process data at predetermined time points based on the Learn-As-You-Go design and make contextually appropriate implementation adaptations. Next, we will evaluate the implementation fidelity and context of Medly Uganda with a focus on reach, adoption, and maintenance (Aim 2) using a deviance approach based on Medly Uganda meta data from patients and providers, focusing on how social determinants might impact implementation. We will also explore patient and provider impacts beyond HF, such as mental health and healthcare autonomy. Then, we will explore cost, cost effectiveness, and sustainability factors for Medly Uganda (Aim 3), including an examination of cost and cost-effectiveness of Medly Uganda from a financial and societal perspective (Sub-Aim 3.1) and an exploration of patient and provider perspectives on the sustainability of Medly Uganda (Sub-Aim 3.2). This study will generate important insights to support implementation of innovative, locally relevant strategies to improve HF care in LMIC and aligns with NHLBI’s Center for Translation Research and Implementation Science priorities.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY The cutaneous vasculature is a crucial yet understudied component of the skin, responsible for essential functions such as tissue oxygenation, exchange of nutrients and soluble factors, and temperature control. While we have a significant understanding of vascular abnormalities present in various skin pathologies, we lack an understanding of both physiological remodeling and homeostatic mechanisms sustaining lifelong function. Specifically, we lack the resolution of the coordinated cellular behaviors that drive developmental vascular remodeling, as well as those that underlie vascular regeneration in the face of injury, particularly in the context of hemodynamic status. I hypothesize that network-wide coordination of EC behaviors in relation to hemodynamic changes regulates developmental and regenerative remodeling programs of the skin vasculature. To test this hypothesis, I have established an intravital imaging technique that allows for the longitudinal tracking and manipulation of the endothelial cells (ECs) that constitute the lining of all blood vessels in the skin of a live mouse. In Aim 1, I will investigate the neonatal vessel remodeling program and underlying EC behaviors that orchestrate the establishment of skin vascular network architecture and blood flow efficiency. Following establishment of adult vascular homeostasis, I will probe the cellular mechanisms that regulate the maintenance of adult vessel integrity via a targeted laser ablation approach, modeling the discrete membrane damage inflicted upon the endothelium due to shear and contractile forces. My preliminary data shows that EC migration within existing vessel structures is a critical EC behavior that underlies network-wide vessel regression during neonatal remodeling, as well as the reparative response of adult ECs to local damage. In Aim 2, I will transition my studies towards the understanding of the skin vasculature in the context of pathological states. Firstly, I will investigate the wound vascularization mechanisms of neonatal versus adult skin, and delineate the differential remodeling properties that enable the enhanced wound revascularization in neonatal skin that we have observed in preliminary experiments. Second, I will determine how coordination of flow-dependent EC rearrangement impacts the ability of adult wounds to revascularize via a genetic mutant model that uncouples the ability of ECs to polarize with respect to blood flow direction. To achieve these aims, I will use an integrated approach of cutting-edge imaging technology, transcriptomics, and genetic mouse models. This research is significant because we expect to uncover global cellular and molecular mechanisms that coordinate vascular development, homeostasis, and injury repair. My findings will likely drive innovation in related fields, given the ubiquity and crucial roles of the vasculature in all organs.

NIH Research Projects · FY 2026 · 2023-08

ABSTRACT Genome instability is generally deleterious to cell growth. Paradoxically, in cancer, it is associated with increased drug resistance, metastasis and immune evasion; all of which contribute to poor patient prognosis. The broader landscape of our research is to fully understand how the DNA damage response (DDR) pathway and mitotic machinery work in concert to promote faithful genome stability. A major component of the DDR pathway is Ataxia telangiectasia and Rad3 related (ATR) kinase. I previously discovered an unexpected, and novel DNA damage- independent, role for ATR kinase after mitotic entry. This newly revealed role for ATR is essential for genome stability. This unexpected role for ATR opened up many opportunities to understand cross-talk between the pathways that regulate genomic stability and is foundational to my lab. Our long-term goal is to make unique contributions by elucidating how the non-canonical roles of the proteins that make up the DDR pathway ensure genome stability. Our five-year goal is to further our understanding of the mechanisms by which ATR promotes genome stability outside of the DNA damage response pathway in both mitosis (program 1) and interphase (program 2). Our current understanding of the mitotic function of ATR is limited to our previous observations that ATR activates promotes proper Aurora B activity. This leaves a large gap of knowledge as to how ATR promotes proper chromosome segregation. We will explore the mitotic pathways that ATR regulates, focusing on putative direct substrates that we have identified (program 1). Additionally, our previous work uncovered two novel, DDR independent functions of ATR, which we aim to fully explore in this proposal (program 2). (1) ATR directly phosphorylates lamin A/C in interphase. This has prompted us to define the function of ATR on nuclear plasticity and nuclear envelope breakdown. (2) Basal ATR activity is necessary for proper centromere identity throughout interphase. We will focus on how basal ATR activity promotes proper centromere identity and function through its regulation of promyelocytic nuclear bodies. We expect that these projects will yield critical information on the role of ATR in mitosis and how it and the DNA damage response pathway promote faithful chromosome segregation independent of DNA damage and further our understanding of the mechanism that promote genome stability.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT This proposal describes a rigorous training program for the career development of Dr. Sang Kim as an independent physician-scientist. The principal investigator is a physician-scientist who completed his PhD in immunobiology and his clinical rheumatology fellowship at Yale University. His career goal is to become an independent investigator studying rheumatic complications induced by immune-based cancer therapeutics. He proposes to expand his training in T cell and cancer immunology through an intensive training research experience under the mentorship of Dr. Roza Nurieva, a world leader with unparalleled intellectual and technical insight into the role of T cells in cancers and autoimmune diseases. In addition, because interactions of tumors and the host immune system are critical in development of immune-related adverse events (irAEs) induced by cancer immunotherapy, and because cutting-edge genomic technologies are a powerful tool in understanding the complex biology of the immune system, Dr. Kim will also have an opportunity to study cancer/functional genomics under the mentorship of Dr. Andrew Futreal (co-primary mentor), a world-renowned scientist in cancer genomics. The research objective of this proposal is to investigate mechanisms of arthritis associated with immune checkpoint inhibitor therapy (arthritis-irAE). Preliminary data for this proposal revealed the predominance of Th1 cell signatures in the patients with arthritis-irAE. In addition, Th17 cells were expanded in arthritis associated with combined PD-1 and CTLA-4 inhibitor therapy with steroid resistance. Furthermore, Dr. Kim observed that more CD4+ T cells were polarized into Th17 cells in skewing conditions in the presence of combined PD-1 and CTLA-4 inhibitors than in the presence of PD-1 inhibitor alone. From these results, Dr. Kim hypothesizes that Th1 cells play a critical role in the pathogenesis of arthritis-irAE and that Th17 cells are pivotal in steroid-resistant arthritis-irAE induced by combined PD-1 and CTLA-4 inhibitors. To address the hypothesis, Dr. Kim will investigate mechanisms leading to development of arthritis-irAE, with special focus on Th1/Tc1, Th17/Tc17, and regulatory T cells, and determine mechanism-driven biomarkers to predict development of arthritis-irAE, reflect arthritis disease activity, and predict steroid resistance. This proposal serves as a training vehicle for Dr. Sang Kim to become an expert in rheumatic complications induced by immune-based cancer therapy, an explosively emerging and challenging clinical entity in rheumatology.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT. O6-Methylguanine methyltransferase (MGMT) reverts O6-alkylguanosine residues to guanosine via dealkylation by SN2 displacement. MGMT is ubiquitously expressed in healthy tissue but is silenced (referred to as “MGMT– “) in ~50% of glioblastomas (GBMs), most gliomas, and in up to 40% of colon cancers, 35% of small cell lung cancers, and 25% of non-small cell lung cancers. MGMT– tumors are sensitized to DNA alkylation agents, such as temozolomide (TMZ). This sensitization creates a therapeutic index (TI). TMZ prolongs survival of patients with MGMT– GBM by ~8 mo. The cytotoxicity of TMZ relies on an intact DNA mismatch repair (MMR) pathway. MMR silencing (referred to as “MMR–”) is the primary mechanism of acquired TMZ resistance, and second-line therapies are ineffective. Despite >20 y of research, efforts to overcome MMR silencing-based resistance have not been successful. Herein, we present 2-fluoroethylating agents (FEtAs) as novel, orally bioavailable com- pounds that selectively eradicate MGMT–/MMR– GBM in vivo, without systemic toxicity. Our data indicate FEtAs induce DNA interstrand cross-links (ICLs) only in MGMT– tumors by formation of O6-(2-fluoroethyl)guanosine (O6FEtG), slow cyclization to an N1,O6-ethanoguanine (EG) intermediate, and ring-opening by the adjacent cytosine. The slow rates of EG formation provide time for MGMT to reverse the initial alkylation in healthy (e.g., MGMT+) cells, leading to a high TI. In contrast, chloroethylation agents, such as mitozolomide, generate O6-(2- chloroethyl)guanosine (O6ClEtG), which cyclizes to EG competitively with MGMT reversal. This leads to the formation of ICLs or toxic DNA–MGMT cross-links, via opening of EG by MGMT, in healthy cells. Additionally, the chloroethylsulfide formed on MGMT reversal of O6ClEtG converts to a reactive episulfonium ion, which also cross-links MGMT to DNA, while MGMT reversal of O6FEtG creates a stable fluoroethylsulfide. Together, these differences lend a higher MGMT TI to FEtAs suggesting they are likely to display improved tolerability in humans. Since ICL toxicity is MMR-independent, FEtAs retain activity in TMZ-resistant, MMR– tumors. Here we will study the amount of O6FEtG formed from FEtAs and the rate of its reversal by MGMT. We will characterize the structure and reaction kinetics of the ICLs using oligonucleotides containing a single O6FEtG. We will conduct studies to improve FEtA CNS penetration. We will probe for synergy between FEtAs and DDR inhibitors, other DNA repair deficiencies, and radiotherapy. Completion of this research will lead to the identification of novel chemotherapies with high CNS penetration that operate by a novel, MMR-independent mechanism, thereby addressing acquired TMZ resistance. As FEtAs are structurally-related to TMZ, we expect rapid translation to the clinic. MGMT is silenced in a range of tumor types; this work will set the stage to evaluate FEtAs as treatments for other MGMT– tumors, alone or in combination regimes. Finally, to the best of our knowledge, the relative rates of DNA chemical modification and repair have not previously been exploited to obtain tumor specificity; we believe this “kinetic lethal” strategy may constitute the first iteration of a new approach to targeted chemotherapeutic design.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY/ABSTRACT As the prospect for the elimination of COVID-19 in the near future remains uncertain, non-pharmaceutical interventions (NPIs) such as limiting social gatherings, quarantine after exposure to the virus, and school closure, will continue to play important roles in mitigating the morbidity and mortality associated with the pandemic. Since these interventions impose immense economic, social, and health-related costs, their use should be recommended only when epidemic control benefits outweigh their adverse consequences. Our overall objective in this proposal is to develop an analytical decision tool to optimize the use of NPIs based on latest information related to the local epidemiology of COVID-19, the effectiveness of different NPIs, and the population’s stated disutility associated with these interventions. This decision tool is structured to provide a transparent mechanism to communicate the rationale for the current policy regarding the use of NPIs and the conditions under which the policy would change. To develop our decision tools, this proposal has three specific aims: 1) to develop state-level decision models that identify the optimal combination of NPIs, in real-time, and based on the projected loss in the quality-adjusted life-years (QALYs) and the disutility borne by the population under various combinations of NPIs under various combinations of NPIs; 2) to design, conduct, and analyze discrete-choice experiments to estimate the disutility weights of different NPIs as borne by population members due to social, economic, and health consequences of these programs; and 3) to estimate the societal tolerance for loss in QALYs due to existing infectious diseases without triggering NPIs. This tolerance threshold can be estimated using historical data related to past pandemic and seasonal influenza and will serve as a benchmark to decide when the burden of COVID-19 is low enough to lift all NPIs, at least for a short term. The research proposed in this project is innovative as it develops a novel, principled approach to consolidate real-time data from three different sources to optimize the use of NPIs: 1) COVID-19 cases, hospitalizations, and deaths as projected by existing and new predictive models of COVID-19 pandemic, 2) effectiveness of various NPIs in breaking the transmission of SARS-CoV-2, and 3) disutility weights of NPIs directly elicited from target populations. The proposed research is significant because it meets the critical needs of policymakers to identify evidence-based and real-time recommendations regarding the efficient use of NPIs to contain the burden of COVID-19. The methods and decision tools developed as part of this project could also be used in responding to other existing and future infectious threats where NPIs are employed.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY An evidence-based treatment that simultaneously addresses intensive care unit (ICU) sleep and circadian disruption (SCD) is desperately needed. Such treatment is needed because patients admitted to the ICU are at high risk for adverse outcomes resulting directly from acute SCD. It is well established among healthy controls that acute SCD is associated with immediate negative consequences such as metabolic, cognitive, cardiovascular, respiratory, skeletal muscle, and immune dysfunction. Normalization of sleep and circadian processes improves these dysfunctions. In the ICU, sleep and circadian processes cannot be segregated, and there are likely several overlapping domains of SCD (e.g., sleep duration, timing, architecture, and continuity, and circadian alignment and amplitude). Thus, a bundled approach to sleep and circadian promotion holds the most promise for reversing SCD, normalizing broader physiologic disruptions, and improving ICU outcomes. To date, ICU sleep promotion bundles have had limited success in documenting improved sleep, and sleep bundles have commonly ignored circadian disruption and circadian-based sleep promotion strategies. This is a critical gap. Translation of circadian principles to ICU sleep promotion is essential because alignment between biologic and clock time allows for subsequent strategic scheduling of behaviors, for example, scheduling sleep promotion during the biologic night to improve sleep duration and quality. In addition, circadian alignment has broader physiologic implications and related potential to improve function across a wide variety of organ systems, for example, scheduling eating during the biologic day to improve glucose tolerance. Investigations to date have not tested the effect of a multifaceted intervention that includes promotion of both circadian alignment via photic and nonphotic zeitgebers and overnight sleep via non-pharmacologic strategies (sleep chronobundle). The overall objective of this project is to test whether a sleep chronobundle, including daytime bright light, time-restricted daytime feeding, increased daytime mobility, and overnight sleep promotion mitigates ICU SCD. A mechanistic randomized controlled trial will be used to test our central hypotheses that a sleep chronobundle will (1) align biologic and clock day-night; (2) overlap behaviors (e.g., sleeping and eating) correctly with biologic time periods; and therefore (3) improve sleep and metabolic processes in the ICU. We are focusing on sleep and glucose metabolism metrics because of their high relevance to critical illness. Project aims include (1) Establishing the effect of the sleep chronobundle on circadian alignment via sampling of urine 6-sulfatoxymelatonin; (2) Determining the impact of the sleep chronobundle on overnight sleep using portable polysomnography; and (3) Defining the impact of the sleep chronobundle on glucose tolerance via continuous glucose monitoring. Ultimately, this work will provide mechanistic insights into ICU SCD and form the basis of studies that can test the impact of the sleep chronobundle on ICU outcomes.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract The overarching goal of this proposal is to develop innovative statistical methods for designing more effective HIV treatment and prevention interventions, along with more effective implementation strategies to deliver them. Due to HIV secondary transmission and social influence of preventive behaviors, the intervention received by one individual can have an effect (or spill over) on the risk for HIV infection, risk behaviors, retention in care and treatment adherence of other individuals. This mechanism, called ‘interference’ in causal inference, remains a major challenge for the evaluation of HIV interventions. Statistical methods accounting for interference are necessary for valid estimation of the individual effect of an intervention and of the overall population effect, as well as for understanding the extent to which social context plays a role though spillover effects and how it can be leveraged. This proposal will develop innovative methods to 1) disentangle individual and spillover effects of time-varying package intervention components in cluster random- ized trials with interference and non-compliance to the assigned components; 2) in network-based and cluster randomized studies, correct for bias due to misspecification of the interference sets, that is, the sets of individuals whose treatment affects the outcome of others. 3) identify individuals who are more likely to influence their peers to adopt behavioral changes and evaluate the improved effectiveness of strategies that target these individuals. This project will define novel causal estimands for the causal questions of interest, and extend marginal structural modeling methodology to adjust for confounding and spillover and to evaluate hypothesized strategies leveraging component-specific effects and influence heterogeneity. Bias correction for mismeasured transmission and social influence networks will be based on a main study/validation study approach comparing the use of phylogenetic- based clusters, social and sexual networks, and spatially-based networks and clusters. User-friendly software implementing the proposed methods will be developed and made publicly available to facilitate their uptake. To further facilitate dissemination, short courses about the new methods and software will be offered. The development of statistical methods will be motivated and applied to two large cluster randomized trials in Botswana (BCPP) and South Africa (TasP) and three network-based peer education studies (HPTN 037, CHAT, STEP), providing new insights into effective combinations of HIV interventions at the individual and community level and into novel strategies to leverage spillover and strengthen the impact of these interventions. The methods will be broadly applicable to many public health interventions for HIV and other infectious diseases. Our proposal fits within the mission of the National Institute of Mental Health - Division of AIDS Research, as well as that of a recent NOSI (NOT-AI-21-054), to advance the development and testing of behavioral and biomed- ical interventions by incorporating social context and behavioral science to understand and leverage transmission networks and social influence, as well as their social determinants.

NIH Research Projects · FY 2025 · 2023-07

Multiple system atrophy (MSA) is a fatal, progressive neurodegenerative disease that is characterized by demyelination in the corpus callosum and putamen due to the accumulation of alpha synuclein (α-syn) in glial cytoplasmic inclusions (GCI) within the oligodendrocytes. Previous data has shown that in post-mortem MSA brain, α-syn pathology is accompanied by MHCII expression and increased infiltration of peripheral T cells (CD4+). IFNγ released from CD4+ T cells enhances inflammation by binding to its receptor (IFNγR1) and, through the JAK/STAT pathway, activates MHCII antigen presentation. Other studies have shown, the neuroinflammation found in post-mortem tissue from MSA patients can be modeled in rodents through a modified AAV, Olig001-SYN which has a high tropism (>95%) for oligodendrocytes. Moreover, the Oligo001-SYN rodent model of MSA shows a similar robust CD4+ T cell response due to the oligodendrocyte α-syn expression. My preliminary results showed there was significant neuroinflammation via increase in IFNɣ and MHCII expression in the Olig001 mouse model. Additionally, when IFNɣ was knocked down there was a significant reduction in overall MHCII expression and general neuroinflammation and demyelination. IFNɣ is required for neuroinflammation and demyelination, however the cell specificity of IFNɣ remains unclear. Therefore, findings from this proposal can address if Th1 cells (CD4+ T cells that produce IFNɣ) are required for MSA pathology.

NIH Research Projects · FY 2025 · 2023-07

Abstract GSK3E functions as essential negative regulator of E-catenin, while E-catenin accumulation represents a central oncogenic driver in cancer. In solid tumors, E-catenin forms complexes with TCF7-factors for transcriptional activation of MYC1-5. In striking contrast to solid tumors, we found that B-cell leukemias and lymphomas are not only exempt from activating E-catenin lesions but highly sensitive to E-catenin accumulation and critically depend on its negative regulation by GSK3E-mediated degradation: Unlike other cancer types, inducible activation of E- catenin in B-cell malignancies suppressed MYC-expression and rapidly induced cell death. Instead of the transcriptional activator TCF7, our interactome studies in B-cell malignancies revealed that E-catenin formed a repressive complex with lymphoid-specific Ikaros zinc finger (IKZF)6-8 factors. Instead of MYC-activation, E- catenin assembled lymphoid-specific Ikaros factors and multiple members of the repressive nucleosome remodeling and deacetylation (NuRD)9-10 complex for transcriptional repression of MYC. GSK3E phosphorylates S/T-residues in E-catenin exon 3, to initiate E-catenin- degradation. To leverage lymphoid-specific repressive E-catenin-complexes as previously unrecognized therapeutic vulnerability, we examined GSK3E small molecule inhibitors. Strikingly, established GSK3E-inhibitors that are currently in clinical trials for the neurological disorders and solid tumors, were effective at low nanomolar concentrations in B-cell malignancies, induced massive accumulation of E-catenin, repression of MYC and acute cell death. Preclinical experiments based on patient-derived xenografts validated small molecule GSK3E-inhibitors for targeted engagement of lymphoid-specific repressive E-catenin-complexes to overcome drug-resistance in refractory B- cell malignancies in vivo. Based on this discovery, the central goal of this proposal is to repurpose GSK3E-inhibitors for targeted engagement of repressive E-catenin-complexes in refractory B-cell malignancies. Given that four GSK3E- inhibitors have already undergone full clinical development and demonstrated favorable safety profiles in eighteen clinical trials, we anticipate that this powerful new approach can rapidly be developed to benefit patients with refractory B-cell malignancies and be further extended to T-cell malignancies in the future. We propose two Aims to (1) elucidate the mechanistic basis of E-catenin-mediated tumor suppression and to (2) develop concepts to leverage targeted activation of E-catenin-Ikaros complexes for therapeutic intervention in refractory B-cell malignancies.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract: The pathologic hallmarks of systemic lupus erythematosus (SLE or lupus) are altered immune responses to nuclear autoantigens with autoantibody production and subsequent tissue injury. Studies have suggested a pathogenic role for innate immunity in lupus. Plasmacytoid dendritic cells and monocytes (MO) of innate immunity recognize ssRNA of the self antigen U1-small nuclear ribonucleoprotein (U1-snRNP) and dsDNA via TLRs 7/8 and 9, respectively, leading to the production of IFN-α, IL-1β and IL-18 which are linked to lupus pathogenesis and clinical manifestations. Regulated cell death can occur in distinct forms including pyroptosis and necroptosis. Pyroptosis and necroptosis cause inflammation by releasing inflammatory molecules such as cytokines, chemokines, and damage-associated molecular patterns (DAMPs). Pyroptosis is induced by the activation of the caspase-1-containing inflammasomes that cleave pro-IL-1β and - IL18 into IL-1β and IL-18, as well as gasdermin D (GASDMD) into the active form gasdermin D amino-terminal fragment (GASDMDN), the essential molecule for membrane pore formation in pyroptosis. Necroptosis that occurs independently of caspases is regulated by TNF-α, receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3), and downstream substrate pseudokinase mixed-lineage kinase domain-like (MLKL), the critical molecule for membrane disruption with the release of inflammatory molecules in necroptosis. RIPK3 and MLKL can also activate the NLRP3 inflammasome with IL-1β release, raising the possible interface of pyroptosis and necroptosis. Studies, including our own, support the implication of the inflammasome activation, pyroptosis, and necroptosis in lupus. We showed IL-1β and IL-18 production from human MO exposed to lupus U1-snRNP/anti- U1-snRNP antibody (Ab) or dsDNA/anti-dsDNA Ab immune complex (snRNP IC or dsDNA IC, also lupus IC refers to both) through the NLRP3 inflammasome activation which is enhanced by the cytokine macrophage migration inhibitory factor (MIF). However, there is a substantial scientific gap in our understanding of how pyroptic and necroptic pathways become activated, interface, and synergize to promote inflammation in lupus at molecular levels with contributing to unique clinical manifestations. Thus, we will test the hypotheses that lupus IC activate pyroptic and necroptic pathways in MO with synergistic interface in promoting inflammation and tissue injury and that the activation of such pathways alters in lupus contributing to clinicopathologic manifestations. The goal of the proposal is to address this hypothesis with: 1) Aim 1. Elucidate the mechanism and significance of the activation and interface of pyroptic and necroptic molecules in lupus; 2) Aim 2. Elucidate the “functional” alteration in monocytes that enhances pyroptosis- and necroptosis-mediated inflammation in lupus patients; and 3) Aim 3. Elucidate the role of macrophage migration inhibitory factor in promoting inflammation in lupus via regulating pyroptic and necroptic molecules. Our study would be highly informative in understanding lupus pathogenesis as well as in developing new approaches in evaluating and treating lupus patients.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Hookworm infection is a leading cause of anemia, malnutrition and growth delay in poor countries, especially in sub-Saharan Africa, where millions of people are infected. Current strategies to control hookworm rely on Mass Drug Administration of anthelminthic drugs, although evidence calls into question the long-term effectiveness of this approach to sustainably control or eliminate hookworm in populations at risk. Since 2007, Yale University and the Noguchi Memorial Institute for Medical Research at the University of Ghana have collaborated to characterize the epidemiology of hookworm in endemic communities. These studies have identified factors associated with hookworm infection status and response to deworming treatment. Preliminary data using human samples from Kintampo North, Ghana, suggests that host antibody (IgG) levels directed at hookworm adult worm excretory/secretory (ES) proteins are closely correlated with active infection. However, to date little is known about the targets of these antibody responses to hookworm antigens, which is a major gap in understanding of host-parasite interactions and pathogenesis. We hypothesize that host antibodies to specific hookworm proteins are predictive of infection status, and that a particular class of proteins, namely allergens, are the drivers of this response. Studies in Specific Aim 1 will validate the correlation between IgG antibody levels and infection status using 1,002 human serum samples and demographic data collected between 2007-2020 in Ghana field studies. Antibody levels will be measured by ELISA and analyzed for associations with hookworm infection status as determined by fecal microscopy. Data from human studies will be compared to results from controlled infection and treatment studies using the hamster model of Necator americanus. Studies in Aim 2 will focus on identifying the specific protein targets of host antibody responses using recombinant protein expression and Label Free Quantitative proteomics. Candidate hookworm allergen proteins will be expressed and purified, followed by screening for immunoreactivity using the human and hamster serum samples described in Aim 1. In addition, N. americanus adult worm ES proteins will be immunoprecipitated using highly reactive serum pools, followed by LC-MS/MS isolation and peptide mapping against hookworm/helminth genome sequence databases. These experiments will test the hypothesis that hookworm allergens are antigenic drivers of the host immune response that is associated with active infection. The overarching goals of the proposed research are to (1) correlate antibody responses with infection status and (2) characterize the role of hookworm allergens and novel antigenic proteins in host immune responses. These studies leverage a longstanding, productive collaboration between the University of Ghana and Yale that led to generation of novel preliminary data and adaptation of the first African hookworm strain in a laboratory model. These studies will enhance our understanding of hookworm pathogenesis and inform development of innovative tools to monitor deworming programs in endemic communities.

NIH Research Projects · FY 2025 · 2023-07

People with serious mental illness (SMI; psychotic and major affective disorders) experience significantly higher rates of morbidity compared with individuals of similar ages without these conditions. Among this broad population, the most vulnerable are Latinos living with SMI who are not engaged in treatment. Further, trauma among people with SMI, in general, is four to five times higher than for the general public, with the highest rates among Latinos in comparison to other racial and ethnic groups. Despite the availability of effective mental health treatment interventions, approximately 47% of Latino adults with SMI are not engaged in treatment. Those who are engaged have significantly lower rates of treatment adherence, retention, and satisfaction than White people. Barriers to treatment engagement for Latinos include lack of bilingual and bicultural clinical providers, the absence of culturally responsive interventions, lack of access to specialty services in Latino neighborhood, self-reliant attitudes, and cultural differences that impact the recognition of mental health concerns. Some evidence suggests that culturally tailoring interventions to Latinos may improve treatment engagement. The proficient use of Motivational Interviewing (MI), an evidence-based and person-centered treatment approach, has been shown to predict behavior change, such as treatment engagement, greater medication adherence and reductions in alcohol consumption, and additional outcomes among Latinos. However, no research to date has examined the cultural adaptation of the MI spirit or the full approach. To fully integrate Latino cultural values and MI, we need to pay attention not just to the intervention, but also to the method of service delivery. Latino peers may be in the best position to deliver MI to Latinos with SMI, given their lived experience with both the culture and the illness. Of the available research on Latino peers in recovery from SMI, evidence suggests that Latino peer navigators appear to enhance service engagement, recovery, and quality of life for Latinos in recovery from a SMI. Therefore, the overall objective of this career development proposal is to culturally adapt MI to integrate Latino cultural relational values and trauma informed care for delivery by peers in recovery from SMI to engage Latinos with SMI in mental health treatment. Specifically, the proposed research will 1) identify social and cultural factors that contribute to mental health treatment hesitancy and engagement among Latinos with SMI; 2) culturally adapt an MI treatment engagement intervention that is trauma informed and peer delivered for Latinos with SMI; and 3) provide evidence of the feasibility, acceptability, and engagement of target mechanisms associated with the intervention. My mentors, in combination with a world-class training environment at Yale University, will guide me towards my goal of becoming an independent bicultural and bilingual investigator of multi-level influences on mental health disparities, with expertise in designing and conducting rigorous, pragmatic research aimed at improving engagement, care, and clinical outcomes for Latinos and other racial and ethnic communities.