Yale University

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY A gap in knowledge remains regarding how structural organization of synapses, robustly established during development, is also flexibly modified during learning to sustain behaviors. Knowledge on how the cell biology of synapses is established and altered in the actuation of memories is of critical importance in our aspiration to understand how the building blocks of the nervous system come together to produce its functional output, behaviors. The key scientific premise of my research program is that our understanding of how complex behaviors emanate from the molecular building blocks of synapses could be meaningfully expanded by simultaneously interrogating structure-function relationships across scales, bridging knowledge on the molecular composition of individual synapses within single cells, the organization of single cells within circuits, and the coordinated activity of circuits within the brain of behaving animals. Achieving this integrated view is a challenge of critical importance best summarized in the BRAIN Working Group Report to the NIH Director, and requires new approaches that allow the integration of knowledge across 1) spatial scales, from the subcellular architecture of the synapse to the cellular architecture of the circuits governing behaviors, and 2) temporal scales, from the events leading to synaptic assembly during development, to the events leading to synaptic plasticity during learned behaviors. To address this challenge, my lab established strategic collaborations with network scientists, microscopists and computational biologists and pioneered integrated approaches that enable unprecedented access to the cell biology of the synapse in the thermotaxis circuit of C. elegans. Using these approaches we discovered concepts that reframed how we understand the cell biology of the synapse in vivo, and now position us to address three fundamental questions in neuroscience: 1) How is synaptic specificity achieved during the development of the nerve ring neuropil? 2) How are changing metabolic needs met at synapses to sustain function? 3) How are synapses modified to form memories and underpin behaviors? We propose to answer these questions for the thermotaxis circuit to define fundamental mechanisms that govern how synapses are precisely assembled, maintained, and modified to sustain behavior. Importantly, the resulting integrated understanding across scales will yield new concepts regarding the interplay between the programs that robustly establish synaptic architecture during development, and the plasticity programs that govern synaptic change to facilitate behavior. We anticipate, because of the molecular conservation of the examined pathways, that advancements in our understanding based on these innovations will result in transposable lessons of broad biological significance.

NIH Research Projects · FY 2026 · 2023-04

Melanoma is the solid tumor that metastasizes most frequently to the central nervous system (CNS). While major progress has been made in treating metastatic melanoma, advances in treating melanoma brain metastases (MBM) lag behind. This is due to near-universal exclusion of patients with untreated brain metastases from clinical trials, and only once a regimen is well established in extra-cerebral disease, is it studied in MBM patients. Moreover, pre-clinical studies of MBM have been hampered by a paucity of pre-clinical models and limited access to CNS specimens for analysis, as these patients are typically treated with radiation rather than surgery. We therefore developed infrastructure and resources which we propose to employ to better understand molecular, vascular and cellular underpinnings of brain metastasis. We started by studying activity of the PD-1 inhibitor pembrolizumab (pembro) in patients with untreated MBM on a phase II trial. Although the response rate was modest (26%), the two-year survival was 48%, and the incidence of radiation necrosis and/or neurologic symptoms related to edema was high. We therefore initiated a sequel trial of pembro plus bevacizumab in MBM; the response rate in the initial cohort was 55%, exceeding our expectations and warranting further evaluation. This response rate is similar to that seen with nivolumab and ipilimumab in MBM, but the toxicity profile is far superior with only minimal added toxicity from the bevacizumab. We propose to further study co-targeting the PD-1 and VEGF/VEGF-R pathways in MBM, and to determine the mechanism by which these pathways crosstalk to promote tumor growth within the brain. We will build on our success with anti-VEGF in a new trial involving pembro and a VEGF-R inhibitor (VEGFRi), lenvatinib. In parallel to our clinical research endeavors we developed novel immune-competent models of MBM that are modestly responsive to anti-PD-1, and developed in vitro models of the blood-brain-barrier to study effects of these drugs on vascular leak. These pre-clinical models will be used to test the hypothesis that the addition of VEGF pathway blockade to anti-PD-1 enhances T cell migration and function and directly decreases tight junction leakiness. We also hypothesize that anti-VEGF has different effects to VEGFRis. We will dissect the mechanism by which this occurs using our novel immune competent murine models and single cell transcriptomic and in situ proteomic strategies (Aim 1) and determine whether VEGF inhibiting drugs should be replaced with VEGF-Ris to treat MBM. We are expanding our ongoing clinical trial of pembro and bevacizumab by enrolling a second cohort of melanoma patients to verify our initial result. We have initiated a new trial of pembro and lenvatinib. We will analyze tumors and blood samples from patients on these trials in an attempt to validate mechanistic results from the murine experiments and identify predictors of response or resistance to this regimen (Aim 2). This proposal is responsive to PAR 21-033, which involves early phase clinical trials and biomarker and mechanistic studies. These studies can have important implications for other disease as well, such as lung cancer, which similarly metastasizes frequently to the CNS.

NIH Research Projects · FY 2025 · 2023-04

PROJECT TITLE Subcortical targets involved in the action of psilocybin in learned and innate escape behaviors PROJECT SUMMARY Mood disorders such as depression now represent a leading cause of disability throughout the world, with conventional first-line treatments failing to provide relief for most patients. In recent years, novel treatments with fast-acting properties have garnered significant interest as innovative therapies. The psychedelic psilocybin, in particular has recently gained attention for its ability to produce substantial and rapid antidepressant effects in early-phase clinical trials. This combination of increasing need and exciting preliminary clinical results has led to the recent ‘breakthrough therapy’ status designation for psilocybin to be studied as a treatment for major depressive disorder. Yet despite these exciting results in pilot clinical trials, the neurobiology underlying the effect of psilocybin remains less understood. Thus, a critical opportunity to further our understanding of the action of psilocybin will be addressed here. Among early studies examining the mechanisms of action of psilocybin, most have focused on the neocortex and hippocampus. However, previous cellular and circuit-level research has clearly implicated subcortical structures as key in the action of psilocybin. Our own preliminary work using whole-brain cFos mapping has identified several candidate subcortical regions that are modulated by psilocybin. Aim 1 will determine the activity of two of these key subcortical brain regions via in vivo recording with high-density silicon probes to determine how neural activity changes following systemic psilocybin administration. Aim 2 will use causal manipulation during psilocybin treatment to determine the relative role of subcortical regions during innate and learned escape behaviors in mice. Specifically, chemogenetics will be used to bidirectionally control brain region activity during a looming stimulus and learned escape behavioral paradigms. Cumulatively, this work will determine the relative roles of key subcortical brain regions in the action of psilocybin during escape behaviors and expand the framework for psilocybin’s use in clinical treatment.

NIH Research Projects · FY 2025 · 2023-04

The major β-hemoglobinopathies, sickle cell disease (SCD) and β-thalassemia, are two of the most common monogenetic diseases. β-thalassemia describes a heterogenous set of mutations, insertions, deletions, or substitutions of the β-globin gene, HBB, that result in no or decreased β-globin synthesis. SCD on the other hand is an autosomal recessive disorder that has a well-defined missense point mutation in the HBB gene, that changes a glutamic acid to valine residue at the sixth position in the polypeptide sequence. This mutation causes adult erythroid cells to express βs-globin and synthesize an abnormal form of hemoglobin, HbS, that is prone to polymerizing. Patients with β-hemoglobinopathies cannot synthesize normal β-globin, and thus cannot form normally functioning adult hemoglobin, HbA, the predominant form in mature erythroid cells. Elevating levels of fetal hemoglobin, HbF, in patients with β-hemoglobinopathies can alleviate clinical symptoms. Compared to HbA, which is composed of globin subunits α2β2, HbF is composed of α2γ2 subunits. After birth, hematopoiesis shifts from fetal hemoglobin expression in fetal liver to the bone marrow where adult erythrocytes are produced and express adult hemoglobin. The transition from fetal to adult hemoglobin, from γ-globin to β-globin expression, requires several critical transcription factors (TFs), namely BCL11A and ZBTB7A, that recruit the Nucleosome Remodeling and Deacetylase, NuRD, complex, and its catalytic component, CHD4, to the globin locus and repress γ-globin expression. A third TF, ZNF410, was recently found to be a unique activator of CHD4 expression. CRISPR/Cas9 knockout or shRNA knockdown of each of these three TFs induces HbF expression, and targeted therapies against these factors might offer a new therapeutic avenue for β-hemoglobinopathies. Immunomodulatory drugs (IMiDs) have been shown to recruit Cys2-His2 zinc-finger (C2H2-ZF) TFs to cereblon, the substrate recognition component for the Cullin-4 E3 ubiquitin ligase (CRL4) complex, for targeted protein degradation. The three TFs of interest all contain various numbers of tandem C2H2-ZF domains, and therefore, I hypothesize that IMiD-induced targeted protein degradation of one or a combination of BCL11A, ZBTB7A, or ZNF410, will lead to reactivation of the γ-globin gene and increase levels of HbF. Using a 1122 compound IMiD library built by the Crews lab, Aim 1 will determine which compounds from the library can serve as lead hit compounds that will be optimized by iterative rounds of structure-activity relationship studies and characterized in cellulo for target TF degradation. Aim 2 will examine the ability of these optimized compounds to reactivate γ-globin expression and induce HbF synthesis in HUDEP-2 and primary erythroid progenitor cells. Completion of this proposal will provide the preclinical framework to assess the therapeutic potential of targeted protein degradation against TFs critical in the γ-globin to β-globin switch as a novel treatment avenue for the major β-hemoglobinopathies.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY New mechanistic insights into placentation and the signaling pathways that maintain proper trophoblast differentiation and invasion of the endometrium will provide the foundation for new therapeutics against pre- eclampsia. Senescent cells are characteristic of the developing and mature placenta, and changes in the senescence-associated secretory phenotype (SASP) have been observed in placental dysfunction. In patients with pre-eclampsia, increased SASP factors are associated with their disease status. Senolytics and senomorphics have recently been proposed as therapeutic interventions for patients with pre-eclampsia, reducing the intensity of SASP. Histone variants organize the senescent genome, and a newly characterized histone variant, H2AJ, is enriched in the placenta and is known to control SASP. This proposal aims to perturb H2AJ in human embryonic stem cells and establish foundational knowledge about H2AJ and its role in trophoblast development and genome architecture. Our human trophoblast cells derived from human embryonic stem cells recapitulate many aspects of fetal placental development and upregulate H2AJ (Figure 5,6,7). This study will serve the long-term goal of identifying new regulators of trophoblast development by identifying protein partners of H2AJ that may serve as therapeutic drug targets. Differentiating human embryonic stem cells with and without H2AJ, we will examine transcriptional, chromatin, and secretory changes in the generated trophoblast lineages. Using protein immunoprecipitation, we will identify unknown interactors of H2AJ, providing new insight into the role of H2AJ loading in the genome. It has been shown that during the transition to senescence, cells undergo dramatic chromatin changes, segregating their heterochromatin into large aggregates in the nuclear interior while maintaining SASP genes in highly expressed euchromatin (Figure 8). The mobilization of heterochromatin away from the nuclear periphery and the maintenance of euchromatic boundaries are not well understood in trophoblast cells. While it is known that cells become senescent in the placenta, there are few studies describing their chromatin architecture. As H2AJ appears critical for the upregulation of transcription at specific SASP loci, loss of H2AJ may be associated with demarcation errors between the strict domains of euchromatin and heterochromatin. In this proposal, we will identify H2AJ's deposition in the genome and its role in transcription, characterizing the three-dimensional chromatin architecture of H2AJ depleted and control trophoblast cells. Furthermore, we will perform this system's first Hi-C and chromatin tracing studies. To date, this will be the first study to characterize H2AJ in human development and its role in trophoblast cells. This knowledge will be crucial for discovering new treatment modalities for patients experiencing pre-eclampsia.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY/ABSTRACT ABSTRACT Intracranial atherosclerotic stenosis (ICAS) is the most common cause of ischemic stroke in the world and continues to have the highest rate of stroke recurrence (>15% of patients in the first year). In comparison, carotid stenosis has a first-year stroke recurrence rate of <5% of patients, but the medical and surgical treatments that work for carotid stenosis are not as successful for ICAS. We propose that the failure to prevent ICAS stroke recurrence reflects that percentage stenosis alone is not the optimal approach to risk stratification, and that focusing on functional blood flow and the ICAS plaque itself will add critical information. Using MRI, we have shown that hemodynamic impairment and disruption caused by ICAS, measured with phase-contrast quantitative MRA and computational fluid dynamic wall shear stress, is correlated with the risk of recurrent stroke. We have also found an association between ICAS plaque enhancement on vessel wall MRI and recurrent ischemic stroke. To further explore our paradigm shifting approach of advanced MRI measurement of ICAS hemodynamic and plaque biomarkers, we will conduct an ancillary imaging study to CAPTIVA, a 3-arm trial of patients with symptomatic ICAS causing 70-99% stenosis randomly assigned to aspirin plus ticagrelor, rivaroxaban, or clopidogrel. Our ancillary study, CAPTIVA-MRI, will establish if there are MRI biomarkers that can identify ICAS patients who fail best medical management and could identify precision medicine treatment approaches for ICAS patients. We will perform multimodal MRI imaging in a subgroup of 300 CAPTIVA patients at the baseline and the 1-year follow-up visit. After decades of focusing on stenosis, the proposed approach could change ICAS research, based on mechanistic understanding of how ICAS causes stroke. We have assembled a team with the required expertise for a prospective multicenter MRI study, including primary investigators of the VERiTAS and MyRIAD prospective multicenter MRI studies of ICAS. The parent trial, CAPTIVA, does not include standardized brain or vascular imaging, presenting a clear opportunity. The proposed study will provide vital and actionable data for future research and will answer critical questions about ICAS progression and provide valuable MRI data for a wide variety of important analyses in the CAPTIVA trial dataset.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY Arrhythmogenic Cardiomyopathy (ACM) is a heritable disease that bridges the gap between the cardiomyopathies and the inherited arrhythmia syndromes. In its early “concealed” phase, ACM promotes the incidence of ventricular arrhythmias in the absence of overt structural or mechanical remodeling. As the disease progresses, myocyte loss, inflammation, and fibrofatty infiltration emerge, culminating in biventricular failure and further risk of sudden cardiac death (SCD). The pathophysiological significance of the disease is underscored by the fact that ACM is a leading cause of SCD in young individuals < 35 years of age. Mutations in desmosomal proteins account for the majority (approx. 60%) of ACM cases, and in this project we focus on a form of ACM known as Desmoplakin (DSP) cardiomyopathy (DSP-CM). DSP-CM has recently emerged as a unique clinical entity that engenders a severe left-dominant form of the disease. DSP-CM is now well recognized to be a heritable disease that is transmitted in an autosomal dominant pattern, albeit with incomplete and highly variable penetrance. Indeed, a major challenge in the field has been the lack of ability to distinguish whom amongst carriers of pathogenic DSP variants are truly at risk of SCD and whom will go on to live healthy and symptom- free lives. This issue takes on added urgency given that the prevention strategy for SCD in DSP-CM is exercise restriction, a rather draconian measure for young healthy individuals, often athletes. The highly variable penetrance associated with DSP-CM as well as the typical mode of SCD that these patients exhibit highlight the importance of gene-environment interactions in unmasking disease pathogenicity. Our own recent work has identified calpain-mediated desmoplakin degradation as a key factor linking DSP mutations with the development of ACM and its exacerbation by exercise. Our central hypothesis is that: 1) calpain-mediated loss of myocyte DSP protein is a key molecular event that is unmasked by exercise and β-adrenergic stimulation, and 2) the pathogenic effects of DSP degradation at the intercalated disc (ID) are exacerbated by abnormal stretch-related mechanotransduction leading to arrhythmias and heart failure. We will address this dual hypothesis using a multi- scale approach encompassing complementary studies in human engineered heart tissues (hEHT) and innovative genetic and surgical mouse models that are designed to address the complex interactions between external stressors (increased preload) and genetic predisposition (DSP mutations) in the manifestation of DSP- CM. Our studies will enable us to tease out contributions of separate aspects of endurance exercise to myocyte dysfunction and expose pathophysiological mechanisms by which calpain vulnerability is unmasked by external stressors to promote early onset arrhythmias and heart failure progression. Finally, we will test novel gene and small molecule-based approaches to inhibit exercise-related calpain vulnerability while avoiding toxicity.

NIH Research Projects · FY 2026 · 2023-04

Summary Alzheimer's disease (AD) is a common neurodegenerative disease characterized by a progressive loss of memory and cognitive decline. Over the last decade, the prevalence of AD and AD-related dementia (ADRD) has been rapidly growing. It is predicted that there will be 150 million AD patients by the year 2050, tripling the number in 2018. This will cause severe economic and social burdens. It has been estimated that speeding up the onset of dementia by even one year would increase the worldwide prevalence of dementia by 10%. However, currently, little is known about causes or mechanisms for this rapid increase in AD population. Many factors, particularly environmental factors, have been proposed as potential contributors to this rapid increase. Noise is a common high-risk environmental factor for human health and also a common deafness factor. Noise can induce hearing loss; hearing loss can induce and accelerate cognitive decline. In particular, recent studies demonstrate that noise can induce hidden hearing loss (HHL), which is caused by noise-induced inner hair cell synapse degeneration leading to difficulty of speech understanding in communication and therefore eventually social isolation. Currently, our world is becoming more and more noisy due to traffic, TV, and wide use of personal audio and video devices. We hypothesize that noise is a high-risk factor for AD development and may play an important role in AD population growing. To test this novel hypothesis, we will investigate whether noise can accelerate AD development and progression in AD mice (Aim 1). We will also investigate whether AD can impair the cochlear efferent system, which plays a critical role in the protection of hearing from noise, to increase susceptibility to noise and in turn to accelerate AD development and progression (Aim 2). In Aim 3, we will further investigate whether deficiency of ATP-purinergic function can accelerate AD development and progression, since our previous study found that deficiency of ATP-purinergic signaling function could induce hearing loss and increase susceptibility to noise. ATP-purinergic signaling also plays an important role in neuroinflammation, which is a consequence of noise exposure and plays a critical role in AD development and progression. Therefore, ATP-purinergic receptors have been considered an excellent potential target as well for AD prevention and treatment after anti-amyloid clinical trials have failed. These proposed studies will help to identify high-risk factors or contributors to the rapidly growth of the AD population and elucidate underlying mechanisms, thereby laying the foundation for development of new preventive and therapeutic interventions for AD and ADRD. Particularly, recent studies reported that visual and auditory stimulations with gamma oscillation cycles could reduce Aȕ expression in the brain and improve memory in AD mice, further indicating that the auditory system has a critical role in AD development and progression.

NIH Research Projects · FY 2026 · 2023-04

Project summary Protein secretion plays a central role in developing and maintaining multicellular organisms. Specialized cell types in tissues secrete proteins by regulated or constitutive secretion. Regulated secretion occurs in response to an extracellular stimulus that elicits the release of signaling molecules, while constitutive secretion facilitates the deposition of extracellular matrix components that provide tissue integrity. Even though these processes are highly significant for human health, features that determine whether a protein is secreted by regulated or constrictive secretion remain unknown. A central regulator of intracellular protein distribution is the trans-Golgi Network (TGN), which sorts and packages secretory proteins into specific vesicular carriers targeting them to intracellular storage granules (regulated secretion) or the cell surface (constitutive secretion). The identification of the mannose-6-phosphate receptor (M6P-R) that recognizes M6P tags of lysosomal led to the idea that specific sorting receptors also sort secretory proteins. However, conserved recognition signals or cargo receptors remain unknown. How are these molecules recognized and sorted for targeting the correct destination? The concept of concentrating macromolecules into biomolecular condensates by liquid-liquid phase separation (LLPS) has revolutionized modern cell biology. Human cells use this principle to organize biochemical processes spatially without a membrane. Our recent research raises the novel possibility that the segregation of secretory proteins in the TGN lumen follows this concept. Our work has shown that purified chromogranins (CGs) or Cab45 undergo liquid-liquid phase separation (LLPS) in the milieu of TGN. Both proteins have been suggested to co-aggregate with secreted proteins (clients) to facilitate their sorting and packaging. We show that CG or Cab45 liquids, not solid aggregates, are essential for client sorting and packaging. Nonetheless, the underlying mechanisms of LLPS-dependent client packaging remain elusive. Therefore, our long-term goal is to understand the molecular basis of LLPS-dependent cargo sorting for regulated (by CGs) and constitutive (by Cab45) secretion. Our proposal aims at identifying the mechanisms of LLPS-dependent sorting in reconstituted systems that recreate the milieu of the TGN lumen. We will include model membranes to examine if and how these condensates associate with the luminal leaflet of the TGN. We will use cell culture models of regulated (P12 cells) or constitutive (skin fibroblasts) secretion to validate our in-vitro results in living cells. Our concept will establish the molecular requirements for condensate formation, the mechanisms of client recognition and vesicular formation in regulated and constitutive secretion. These results will provide a fundamental understanding of an exciting new paradigm in cell biology and impact the research of pathologies caused by defective protein secretion, such as psychiatric disorders or cancer.

Fonds de recherche du Québec – Nature et technologies · FY 2023-2024 · 2023-04

Volet: Bourses de doctorat en recherche; Domaine: Environnement; Objet: Biodiversité et biocomplexité; Objet: Paysage et restauration; Application: Sciences et technologies; Application: Environnement; Mots-clés: CLIMATE CHANGE MITIGATION, FOREST SUCCESSION, FACILITATION, COMPLEMENTARITY, DIVERSITY THEORY, COMPETITION

NIH Research Projects · FY 2025 · 2023-04

Project Summary An estimated 40.3 million adults and adolescents needed treatment for substance use disorders in 2020. Yet only 6.5% of those needing treatment received it. Access to high-quality care remains a significant challenge. In the past decade, private equity (PE) firms have acquired numerous substance use disorder (SUD) treatment facilities, although evidence on the extent of these acquisitions is scarce. SUD treatment centers are attractive targets for private equity acquisition due to increased need for SUD treatment, increased health insurance coverage, and expansions in coverage of SUD treatments, all of which increase demand for treatment. Opportunities for efficiencies due to the fragmented nature of the market and changes to regulations regarding buprenorphine prescribing have the potential to increase profits. Commentators have raised concerns that a focus on short term profits may lead to quality declines in private equity-acquired facilities. Similar acquisitions in other areas of health care have resulted in declines in quality and increases in mortality. In this proposal, we will, for the first time, comprehensively catalog and describe SUD facility acquisitions by private equity investors from 2010-2022. We will we use a quasi-experimental difference in differences research design that leverages differences in the timing of ownership changes to study changes in the delivery of care and patient outcomes. Our interdisciplinary team consisting of faculty from the Yale Schools of Medicine, Management, and Public Health is uniquely suited to study this issue with the methodological and substantive expertise to assess the complex treatment and policy implications. Our specific aims are: (1) To catalog and describe changes in private equity ownership of SUD treatment facilities (2010-2022) and the predictors of such changes; (2) To determine whether private equity acquisition is associated with changes at the organization level including changes in treatments offered (more facilities offering medication for opioid use disorder (MOUD) treatment; fewer facilities offering primary care services or services attractive to complex patients) and changes in the types of insurance accepted; (3) To determine whether private equity acquisition is associated with changes in MOUD use, quality and patient insurance type in acquired facilities. Pressure to increase the volume of commercially insured patients given the significantly higher reimbursement rates may lead to reductions in the number of Medicaid patients treated; (4) To determine whether private equity acquisition is associated with changes in OUD treatment quality and opioid-related adverse events in communities with acquisitions. Ultimately, a goal of an SUD treatment facility is to improve health outcomes. Using commercial insurance data, we will examine changes in treatment quality and health outcomes (OUD-related emergency department visits). To our knowledge no information about the extent and impacts of this change have been documented in the peer-reviewed literature.

NIH Research Projects · FY 2026 · 2023-04

Nicotine addiction among individuals with chronic pain is a serious public health concern with significant health- care expenses and lost productivity. Chronic pain-associated negative affective state, such as anxiety, is a risk factor for tobacco use. Greater chronic pain intensity increases sensitivity to anxiety, which is, in turn, associated with increased smoking and vaping. Therefore, I hypothesize there is a neurobiological mechanism in which chronic pain decreases mesolimbic dopaminergic signaling to causally induce a negative affective state, thereby increasing the risk of nicotine use. To investigate this causal mechanism in the comorbidity of chronic pain and nicotine use, I propose modeling this comorbidity in rats and the following aims: Aim 1: Evaluate ventral tegmental area – nucleus accumbens (VTA-NAc) dopamine (DA) signaling in a model of chronic pain to determine whether decreases in DA signaling underlie the chronic pain-induced negative affective state. Aim 2: Determine whether nicotine has increased reinforcing efficacy in chronic pain states compared to pain-naïve states. This proposal combines behavioral, pharmacological, neurochemical, and optogenetic approaches to reveal whether chronic pain-associated decreases in VTA-NAc DA signaling underlie chronic pain-induced negative affective states, and whether nicotine is more rewarding in chronic pain states compared to pain-naïve states due to decreased baseline DA signaling, thereby exacerbating nicotine’s effects on DA signaling. My overall career goal is to become an independent academic scientist, identifying neurobiological mechanisms underlying these phenomena to develop new interventions for patients with co-morbid pain and nicotine addiction. The proposed K01 trainings will increase my knowledge of neurobiology and circuitry of the mesolimbic reward pathway, allowing me to investigate its mediation in chronic pain-induced negative affect and nicotine addiction. My primary mentor Dr. Addy, an expert in neurobiology and neurochemistry of substance abuse, will provide training on mechanisms of drug reinforcement and in vivo voltammetry. My co-mentor Dr. DiLeone, an expert in neuronal circuits controlling reward-related behaviors, will provide training on neuromodulation and in vivo optogenetics. My co-mentor Dr. Porreca, an expert in pain-induced affective and motivational behaviors and reward circuits, will provide training and oversight on modeling and understanding brain reward circuitry in chronic pain and negative affect. My co-mentor Dr. Picciotto, the director of the Junior Faculty Mentoring Program for the Department of Psychiatry at Yale, will mentor my career enhancement trainings together with Dr. Addy. My consultant Dr. Ditre, an expert in human comorbidity of chronic pain and nicotine addiction, will provide insights in the discussions of human data. I will also receive extensive training by attending courses, seminars, conferences, and workshops. Through the proposed K01, I will effectively gain new technical skills and perspectives, ultimately facilitating a successful transition to become a principal investigator.

NIH Research Projects · FY 2026 · 2023-04

Physician-scientists play a critical role in the biomedical workforce, yet there is a well-recognized shortage of those with dual expertise in medicine and scientific research. Growing the physician-scientist workforce is a national priority; the United States Congress has formally asked the NIH to recognize the vital importance of training physician-scientists. NIH has cited inadequate professional development during medical school as a barrier to physician-scientist development. There is growing attention in medical education to the importance of professional identity formation, defined as the development of values, beliefs, aspirations, and sense of affiliation aligned with the norms of the profession. There is evidence that experiences during medical school play a critical role in shaping physician-scientist professional identity formation, yet little is known about training practices and organizational characteristics of medical schools with the greatest success in physician-scientist professional identity formation among medical students. We propose a multi-stage mixed methods study to identify the training practices and organizational characteristics of MD programs with greatest success in physician-scientist professional identity formation among single-degree medical students. This objective will be achieved through the following specific aims: 1) use data from the Association of American Medical Colleges [AAMC] Graduation Questionnaire [GQ] to identify MD programs which consistently demonstrate greatest success in physician-scientist professional identity formation among medical students; 2) develop hypotheses about best practices and organizational characteristics for promoting physician-scientist professional identity formation by conducting in-depth qualitative interviews at MD programs demonstrating the greatest success in physician-scientist professional identity formation, and; 3) evaluate associations between practices and organizational characteristics identified in Aim 2 and success in physician-scientist professional identity formation through a national survey to identify best practices and characteristics that are broadly applicable to U.S. MD programs. This study is significant because it will address the lack of evidence-based approaches to research training and inform efforts to grow the physician-scientist workforce. Dissemination will take the form of scholarly publications and real-world communications (toolkit, website, podcasts, etc.) in partnership with high-profile national organizations, e.g., AAMC, for maximal impact.

NIH Research Projects · FY 2025 · 2023-04

PROJECT SUMMARY Despite continuing advances in medical genetics, medical imaging, and surgical interventions, thoracic aortic aneurysms (TAAs) are increasingly responsible for significant morbidity and mortality. Large clinical studies reveal the complexity of the disease, which typically presents sporadically in older individuals, with uncontrolled hypertension amongst the key risk factors, while also presenting in younger individuals having genetic or congenital predispositions. Standard methods (including multivariate regressions) have failed to improve prediction of life-threatening acute aortic syndromes (dissection and rupture) and current AHA/ACC guidelines based on maximum aortic diameter fail to predict risk. Further complicating the situation, recent data show that, although life-saving, surgical repair of the proximal aorta with a prosthetic graft increases incidence of distal aortic disease and acute events, thus emphasizing the need to time surgery appropriately – that is, either unnecessary delays due to adherence to current guidelines or pre-mature intervention may increase risk to patients. There is a dire need for a better approach for predicting thoracic aortic growth and potential outcomes. This proposal is significant for it is designed to resolve this unmet clinical need; it is innovative for we propose a novel mechanobiological and biomechanical data-driven approach to develop a next-generation (neural operator based) machine learning tool that can better predict TAA growth and certain outcomes, including drug efficacy. We will combine a novel repurposing of extant murine and human data, generation of ~25000 new synthetic data sets, and collection of unique new murine data (12 models of TAAs) to identify the best machine learning approach, then combine extant and prospective clinical imaging data (~300 patients) to train and test the final neural network (a deep operator neural network, or DeepONet). Our proposed unique meta-learning framework is simply not possible with standard neural networks. We will exploit multi-fidelity training so that both low resolution data and relatively inaccurate models can be used in training when combined with high-fidelity real or synthetic data and uncertainty quantification via functional priors (the most informative Bayesian priors) that are learned by combining historical data, biophysical models, and GANs (generative adversarial networks). This unique combination allows us to learn posteriors with few samples (e.g., 2 or 3 new medical images), hence predictions can be made for new cases with minimal (clinical) information. This project is possible given our highly collaborative team of physician-scientists, bioengineers, and applied mathematicians having a strong track record of successful research (grants, papers) and training of diverse students, post-docs, and residents.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY The increased use of both prescription and illicit opioids among women of child-bearing age over the past two decades has resulted in a five-fold increase in neonatal opioid withdrawal syndrome (NOWS). The requisite monitoring and management of infants with NOWS prolong postnatal hospitalization, resulting nationally in hospital costs that currently exceed $560 million annually. Multiple approaches have been investigated to reduce the substantial care and cost burden of treating infants with NOWS postnatally, including through dyadic approaches such as the Eat, Sleep, Console protocol. Far less attention has been paid to understanding the clinical needs and subsequent trajectory of care for infants with NOWS during the remainder of their infancy— including their risk of hospital readmission and the associated costs. A clearer understanding of these issues is integral to developing protocols to assist mothers with opioid use disorder (OUD) in caring for their infants during a time when the health care needs of both the mother and the child are great. Mothers with OUD are at high risk of relapse and of overdose during the first postpartum year. In turn, the risk of abuse or neglect is highest for all children in the first year of life. Thus, the objective of this career development proposal is to understand the challenges of caring for infants with NOWS in the year following discharge from the birth hospitalization. The first study aims to quantify the risks and costs of hospital readmissions during the first year of life for infants with NOWS. The second study will focus on the challenges of keeping infants with NOWS healthy and safe in the year following discharge from the birth hospitalization, a vulnerable time for both mother and child. Long-term, my goal is to use these findings to inform the development of an evidence-based model of care within the home-visiting framework that addresses the unique needs of families affected by NOWS following discharge from the birth hospitalization. In applying for this award, I am seeking to develop the skills I need to successfully transition into the field of NOWS research. As an epidemiologist with a background in pediatric nursing, I have spent the past 12 years investigating the impact of the opioid crisis on adults (and to a lesser extent, children and adolescents) through secondary analyses of large datasets. I need further training related to 1) the clinical care of infants with NOWS and skillfully working with mothers with OUD, 2) economic evaluations, 3) qualitative research methods, and 4) implementation science. I have assembled a mentorship team led by experts in parental substance use and child abuse that addresses each area of training and leverages the resources available at Yale. The training and research outlined in this proposal promise to have a high impact on my career, as they will provide me with the skills and knowledge that I need to meet my long- term career goal of developing expertise in designing substance use interventions that are evidence-based, cost- effective, and informed by the lived experience of families.

NIH Research Projects · FY 2025 · 2023-04

Abstract Psychiatric disorders are a set of chronic, non-communicable diseases (NCDs), a global public health problem of enormous economic, social, and medical cost to the international community. Stress, alcohol use and other factors (social and environmental; physical disorders) often exacerbate psychiatric disorders. Low and middle income and developing countries are disproportionately affected due to both economic disadvantages and the less well-established institutional infrastructures dedicated to the understanding, treatment and prevention of psychopathology. We propose a collaborative research training program in the multidisciplinary, translational research of stress, alcohol use and psychopathology between Faculty of Medicine, Chulalongkorn University in Bangkok, Thailand (the lead Thai site) Phramongkutklao (PMK) Hospital, also in Bangkok, and Prince of Songkla University (PSU) in Hat Yai, Thailand; and the Yale University School of Medicine in the U.S. The project is directed by leaders in Thailand and the US, Dr. Kalayasiri at Chulalongkorn, and Drs. Potenza and Gelernter at Yale, mPIs for this application. Three major training mechanisms are proposed for Thai trainees, including long- term (1-2-yr) predoctoral and postdoctoral fellowships, and short-term (1-6 month) fellowships. During the five- year program, training responsibility increases at Chulalongkorn, PMK, and PSU (as experience builds) and decreases at Yale. This program builds on an extensive research program in Psychiatry at Yale University School of Medicine, and on existing training and research collaborations between investigators at Yale and investigators at Chulalongkorn in the study of the genetics and clinical correlates of psychopathology (especially substance use disorders) in Thai populations. The program will contribute to the capacity of investigators at Chulalongkorn, PMK and PSU and more generally in Thailand to conduct state-of-the-art research in interdisciplinary and translational research in psychiatry through a range of training experiences (genetics, molecular biology, clinical, developmental, psychopharmacology, clinical trials, neuroimaging, implementation, community, and policy research); and has strong ongoing potential for clinical translation. The explicit long-term goal of the program will be to build a critical mass of scientists, health professionals, and academics with expertise and a sustainable research environment at the collaborating Thai institutions to better understand, treat, and prevent the NCDs of psychiatric illnesses. This training program is also specifically designed to serve a major public health purpose by enhancing Thailand’s capacity to confront major health concerns including alcohol and other stress-related disorders that are unprecedented in Thailand’s history. The context of this program is a long-term research and training collaboration between Yale and Chulalongkorn that focused on drug dependence which was co-led by at Yale by Drs. Gelernter and Malison. This new program with a new focus is expanded in scope to address the needs of Thailand better and include a Thai mPI, and is now named “The Robert T. Malison Yale-Chulalongkorn Stress, Alcohol Use and Psychopathology Training Program” for Dr. Malison, who passed in July, 2020.

NIH Research Projects · FY 2026 · 2023-04

Abstract- The dorsolateral prefrontal cortex (dlPFC) mediates working memory and top-down control, but is impaired by acute or chronic stress, and is dysfunctional in most mental disorders. Stress exposure increases norepinephrine (NE) release, which strengthens amygdala emotional responses via (β-AR and α1-AR, but weakens the dlPFC via α1-AR, while the role(s) of β-AR are unknown. Based on this work, the general α1-AR antagonist, prazosin, and β-AR antagonist, propranolol, are used to treat Post-Traumatic Stress Disorder (PTSD). However, prazosin and propranolol are not always effective, and findings suggest that some subtypes of α1-AR and β-AR may benefit PFC, and thus antagonists that block all subtypes may be counterproductive. The proposed research will perform the first study of α1-AR (α1A-AR, α1B-AR, α1D-AR) and β-AR (β1-AR, β2- AR, β3-AR) subtype actions in rhesus monkey dlPFC, using multiple label immunofluorescence to localize α1- AR and β-AR subtypes on pyramidal cells, GABAergic interneurons, astrocytes and microglia, and immunoEM to reveal ultrastructural locations, e.g. at pre-synaptic release sites or on dendritic spines in layer III dlPFC. We will use iontophoresis coupled with single unit recordings of dlPFC neurons in monkeys performing a working memory task to determine how stimulation of α1-AR and β-AR subtypes alters task-related neuronal firing, and their second messenger actions. We will also use systemic administration of α1-AR and β-AR subtype selective antagonists to block stress-induced working memory deficits, and test whether selective agent(s) are more potent and efficacious than the currently used, nonselective agents, prazosin or propranolol, and whether low doses of more selective antagonists can restore cognition with fewer side effects. Aim 1 will characterize the roles of α1-AR subtypes, examining their localization (Aim 1A), physiological actions in dlPFC (Aim 1B), and effects on working memory performance during a mild, acute stressor (Aim 1C). Preliminary data indicate that the α1A-AR subtype markedly reduces working memory-related dlPFC neuronal firing, and that a selective α1A-AR antagonist potently blocks stress-induced cognitive deficits, suggesting a superior strategy for therapeutics. Aim 2 will characterize β-AR subtypes, examining their localization (Aim 2A), physiological actions in dlPFC (Aim 2B), and effects on working memory performance during a mild, acute stressor (Aim 2C). Preliminary data indicate that the β1-AR subtype markedly reduces working memory-related dlPFC neuronal firing, and that a selective β1-AR antagonist blocks stress-induced cognitive deficits. In heart muscle, the “fight or flight” stress response is mediated by β1-AR opening of voltage-gated Cav1.2 Ca2+ channels (encoded by CACNA1C), and our preliminary data indicate that detrimental β1-AR actions in primate dlPFC involve similar actions, helping to explain why gain-of-function mutations in CACNA1C increase risk of mental disorders with impaired dlPFC function, including PTSD. Identifying the subtypes of α1-AR and β-AR that impair dlPFC function will help the design of more effective therapies for stress-related mental disorders.

NIH Research Projects · FY 2026 · 2023-04

Molecular mechanisms that regulate insulin resistance and its progression to type2 diabetes (T2D) are not understood and might reveal a therapeutic opportunity to reduce the burden of T2D. We recently identified causative loss-of-function mutations in the CELA2A gene (Esteghamat & Mani, Nat. Genet. 2019), which encodes the pancreatic elastase Chymotrypsin-like ELAstase 2A in kindreds with extreme phenotypes of metabolic syndrome, type 2 diabetes, and early-onset atherosclerosis. Interestingly, the genome-wide association studies have also shown a strong association between common variants in the CELA2A gene and blood pressure, LDL cholesterol and BMI, underscoring its role in the disease of the general population. Although only known for its function as a pancreatic exocrine enzyme, we found it to be a circulating protein that is expressed in the extrapancreatic tissues, including gut. Its plasma levels rise after each meal in parallel to plasma insulin levels in healthy humans. Our characterization of CELA2A functions revealed its ability to trigger insulin secretion and sensitivity. In contrast, the catalytically inactive mutant CELA2A proteins found in the cohort increased platelet aggregation and reduced insulin sensitivity. Mechanistically, CELA2A was found to proteolytically cleave GPCRs such as PAR2 and induce PAR2-dependent activation of AMPK, while mutant CELA2As trigger different PAR2-dependent pathways, resulting in increased ERK and reduced AMPK activation. Thus, CELA2A appears to be an endogenous ligand of PAR2, a GPCR that has been implicated in the regulation of glucose homeostasis. These functions underscore CELA2A’s role as a novel risk factor and an attractive therapeutic target for the treatment of T2D. We will investigate the physiological functions of CELA2A and explore its molecular mechanisms of action in glucose homeostasis in global, acinar cell- and gut-specific Cela2a knockout mice, with a focus PAR2-dependent pathways. Cela2a KO mice are ideal for this goal as they show impaired insulin sensitivity and increased hepatic and plasma triglycerides that match the phenotypes of humans.

NIH Research Projects · FY 2025 · 2023-04

Project Summary N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels composed of transmembrane GluN1, GluN2 (A-D), and GluN3 (A-B) subunits that mediate Ca2+ influx into the dendritic spine. Importantly, the unique intracellular tail of the GluN2B subunit is essential for learning and memory. Furthermore, autism spectrum disorder (ASD) and schizophrenia (SCZ) associated variants are found within the GluN2B intracellular tail. My overall goal is to elucidate a novel mechanism by which GluN2B tails contribute to the function of GluN2B- containing NMDARs (GluN2B-NMDARs). Previous experiments showed that actin-targeting drugs impact NMDAR activity, but the underlying mechanisms are unknown. My preliminary data strongly support the primary hypothesis of this project: a direct interaction between actin filaments and the GluN2B intracellular tail regulates NMDAR activity. The objective of my proposed project is to elucidate the biochemical basis for an interaction between the GluN2B tail and actin filaments and determining how this interaction regulates NMDAR function. I will achieve this objective by pursuing three highly complementary aims: Aim 1. Define the minimal fragment of GluN2B intracellular tail that mediates high affinity binding to actin filaments and the interfaces on GluN2B and actin that mediate binding. My preliminary data show a direct interaction between GluN2B tails and actin, but the key interfaces that mediate this interaction between the proteins is unknown. I will use recombinant proteins to perform quantitative binding assays and cross-linking assays to identify the minimal fragment of the GluN2B tail needed to mediate a high affinity interaction with actin. Aim 2. Characterize how Ca2+ and genetic variants impact GluN2B:actin binding. Ca2+ decreases NMDAR activity in cultured hippocampal neurons, but whether Ca2+ selectively impacts GluN2B-NMDAR activity is unknown. My preliminary data suggest Ca2+ strengthens the GluN2B tail:actin interaction. Genetic variants that lie within GluN2B tail regions that I found bind actin have been identified in patients with ASD and SCZ. Clusters of conserved and positively charged residues can contribute to actin binding of proteins. I will perform quantitative binding assays to identify how Ca2+ levels, disease-associated variants, and clusters of conserved and positive residues impact the GluN2B tail:actin interaction. Aim 3: Determine how manipulations of the actin cytoskeleton and GluN2B tail impact GluN2B-NMDAR activity. Changes in actin polymer state impact NMDAR activity in neurons. However, whether actin selectively impacts GluN2B-NMDARs or is mediated by GluN2B tail:actin interactions is unclear. I will monitor changes in GluN2B-NMDAR mediated currents in HEK293 cells to determine the impact of 1) actin-targeting drugs and 2) disrupting the GluN2B tail:actin interaction. After mastering these techniques, I will monitor how actin dynamics and GluN2B tail:actin interactions impact NMDAR activity in cultured hippocampal neurons.

NIH Research Projects · FY 2025 · 2023-04

Project Summary Mammography screening for breast cancer has clear, substantial benefits, including significantly reduced breast cancer mortality and improved treatment options for early detected cancers. However, regular mammography screenings subject women to several potential harms, including high false positive rates, with over 60% of women experiencing a false positive finding after 10 years of annual screening; high false negative rates, with more can- cers missed in dense breasts which obscure tumor appearance; and high recall rates, causing undue anxiety and unnecessary, potentially invasive workup for women with a false positive screen. New 3D mammography technology called digital breast tomosynthesis (DBT) has shown increased cancer detection and decreased re- call rates, but radiologists require longer interpretation time and may lack experience. The clinical workflow could potentially be enhanced with computer aided detection systems. However, current methods only focus on a single mammogram exam, ignoring crucial decision-making information that a radiologist would consider, such as prior mammograms, patient demographics, and personal history. Conversely, established breast cancer risk models rely only on patient demographics and personal/family history, excluding mammographic history. Toward the overarching goal of reducing the harms and increasing the benefits of mammography screening, we propose to increase accuracy of breast cancer detection and predict future cancer development from serial 3D mammogram screenings using a novel deep learning model that jointly incorporates spatial, temporal, and non-imaging clinical information. Our method adopts attention-based neural networks, i.e., Transformers, which learn complex depen- dencies between different elements in a sequence and automatically attend to the most relevant information. In addition to the potential for improved performance, the attention mechanism provides built-in model interpretation to better understand the inputs that are important for the model’s predictions, instilling user confidence in the model and facilitating extraction of mammographic biomarkers for breast cancer detection and development. Our specific aims are to: 1) develop a powerful deep learning model for simultaneously leveraging spatial, temporal, and non-imaging clinical information from DBT exams; 2) create a new tool to detect breast cancer from lon- gitudinal DBT screenings; and 3) develop a new model for predicting development of breast cancer based on longitudinal DBT studies and extract 3D mammographic biomarkers associated with cancer development. Be- yond the direct benefit of improved breast cancer detection and risk estimation, this work could reduce radiologist reading time and workload, inform new individualized screening protocols, further our understanding of the role of breast architecture in cancer risk, and guide development and monitoring of preventive treatments. Finally, the developed deep learning methodology will have wide applicability to spatiotemporal analysis in other medical conditions and imaging domains.

NIH Research Projects · FY 2025 · 2023-04

Despite many available evidence-based interventions (EBIs) to prevent HIV, new infections and mortality in Georgia have increased by 30% and 335%, respectively. Georgia is a LMIC with HIV prevalence of 0.4% and concentrated in key at-risk populations (KAPs) like MSM and PWID, where the epidemic is growing fastest. Though all the elements of the HIV prevention toolkit are present in Georgia (voluntary counseling/testing [VCT], pre-exposure prophylaxis [PrEP], opioid agonist therapies [OAT], syringe services programs [SSPs] and treatment as prevention [TasP]), they are markedly under-scaled in terms of coverage and, importantly, are not adequately implemented in KAPs. In addition to suboptimal implementation of HIV prevention, Georgia’s ability to control its epidemic has been hampered by its lack of expertise in biostatistics. This expertise is need not only to analyze a rich array of databases that could guide more effective implementation in real-time, but in its ability to rigorously design, implement and analyze data from implementation trials, which are urgently needed in the Georgian context where the epidemic is concentrated in KAPs and where stigma and discrimination abounds. To meet this need, we propose to develop and institutionalize the Georgian Implementation Science Fogarty Training (GIFT) program at Ilia State University (ISU), the leading research institution in Georgia, in collaboration with Yale University. GIFT will leverage the combined resources of Yale University (Medicine and Public Health) with ISU, through provision of long-, mid-, and short-term training. GIFT will focus on HIV prevention through advanced training in biostatistics and implementation research methods. Specifically, GIFT adheres to three core aims, including to: 1) train 4 GIFT faculty in Year 1, followed by 6 doctoral students recruited in Years 2 and 3, who will focus their training on HIV prevention and who will receive advanced training in biostatistics and implementation research methods through a hybrid learning program; 2) transfer knowledge and build capacity within ISU, the Caucasus and Eastern Europe and Central Asia through short-term training at a Summer Bootcamp; and 3) develop the HIV Implementation Science and Biostatistical Methods Resource Hub to consolidate resources and build partnerships for better population outcomes. All doctoral students will be paired with ISU and Yale mentors and gain real-world experience analyzing data from HIV implementation trials now underway or completed at Yale. Training resources will be contextualized for the HIV epidemic in Georgia. Building on prior experience with Fogarty training programs and existing successful collaborations and partnerships between Yale and ISU, we are poised to train the next generation of public health researchers more effectively, recognizing the need for better implementation with HIV prevention in KAPs. GIFT will provide advanced training to produce a phased transfer of knowledge, skills and expertise from Yale to ISU over the course of five years, resulting in a model curriculum for training at Georgia’s top research university, with strong support by regional and local faculty and commitment by community, government and international partnerships.

NIH Research Projects · FY 2026 · 2023-04

Aberrant metabolism is increasingly recognized as a hallmark of cancer. The Warburg effect is a well-known example of such abnormal cancer metabolism, which entails a shift away from oxidative to glycolytic glucose metabolism (despite the presence of oxygen) and usually also increased glucose uptake. The detection of this increased glucose uptake, via a radioactive analogue (2-18F-fluoro-2-deoxy-D-glucose, FDG) with positron emission tomography (PET), is often used for diagnosis, staging, and evaluating disease progression of tumors outside the brain. However, in patients with brain tumors FDG-PET is frequently inconclusive because the normal high glucose uptake in healthy brain is comparable to that in tumors, thereby obscuring the tumor-to-brain image contrast. As a result, FDG-PET is not frequently used in these patients. That leaves brain tumor patients without the benefits of metabolic imaging, which has a significant negative impact on the management of their disease. The recently developed MRI-based method, deuterium metabolic imaging (DMI) can be an alternative strategy to detect abnormal glucose metabolism. DMI is based on 3D deuterium (2H) magnetic resonance spectroscopic imaging (MRSI). After administration of the nonradioactive deuterated glucose, DMI can detect both glucose and its downstream metabolites lactate and glutamate. In cancer cells that show the Warburg effect the 2H-labeling in lactate and glutamate reflects the typical shift from oxidative to glycolytic metabolism. DMI can detect this 2H-labeling and reveal the cancer-specific glucose metabolism with high tumor-to-brain image contrast. Because of these features and the ease of use of the method, DMI can become a robust metabolic imaging technique for brain tumors that so far has been missing. The goal of this proposal is to validate DMI of glucose metabolism as a potential imaging tool for neurooncology, particularly for glioblastoma, the most common and lethal primary brain tumor. We envision that, for patients with brain tumors, DMI can provide a similar benefit as FDG-PET has for many patients with tumors outside of the brain. In Aim 1 we therefore seek to validate the 2H-labeling pattern in lactate and glutamate detected with DMI as surrogates of the Warburg effect, by comparing them with absolute measurements of the Warburg effect in rodent models of GBM. Aim 2 is focused on the potential of DMI to provide an early biomarker of response to standard of care chemotherapy. To confirm the improved performance of DMI relative to current clinically available methods, in Aim 3 metabolic maps generated by 1H MRSI, FDG-PET and DMI, are compared for tumor-to-brain image contrast in patients with GBM. The proposed aims will provide better understanding of the fundamental processes underlying the DMI-based image contrast, provide the first insight in its value for monitoring therapy and disease progression, and benchmark its performance as a new metabolic imaging method. These achievements will strengthen the foundation for further development of DMI as a clinically viable technology for metabolic imaging.

NIH Research Projects · FY 2026 · 2023-04

Project Summary Acute kidney injury (AKI) significantly increases the risk of developing progressive kidney fibrosis and chronic kidney disease (CKD). T cells and polymorphonuclear neutrophils (PMNs) have been shown involved in pathogenesis of AKI; however, their role(s) during AKI-to-CKD transition remain uncertain. Our recent publication and preliminary data revealed that failure of resolving tubular injury from unilateral ischemia/reperfusion injury (U-IRI, a rapid AKI-to-CKD transition mouse model) led to not only macrophage persistence but also a second wave of T cells and PMNs infiltrating into tubulointerstitium, which closely associated with a proinflammatory milieu. Concomitantly, the tubular cells from U-IRI kidney expressed higher level of injury marker, vascular cell adhesion molecule 1 (Vcam1), and exhibited a dedifferentiated expression profile, correlating with kidney atrophy. Clinically, we found that increasing numbers of T cells and PMNs in the renal interstitium at the time of renal biopsy in patients with AKI negatively correlated with 6-month recovery of GFR. In the U-IRI mouse model, we found that depletion of T cells and PMNs attenuated the second wave of tubular injury and partially restore tubule mass, suggesting that T cells and PMNs promote secondary tubular injury and kidney atrophy, and that blocking T cells and PMNs recruitment can attenuate CKD from AKI. Our chemokine/receptor pair analyses from the scRNA-seq dataset on the IRI kidneys identified that CXCL16 and MCP-2 (Ccl8) are the top homing signals to recruit T cells (CXCR6+) and PMNs (CCR1+) and that persisted macrophages are the primary source of CXCL16 and MCP-2 during AKI-to-CKD transition. Together, these findings have led us to hypothesize that in the setting of failed tubular repair, macrophage-expression of CXCL16 and MCP-2 promotes a second wave of T cells and PMNs infiltrating into the injured kidneys and that tubular VCAM-1 enhances T cells adhesion and retention, which together lead to secondary tubular injury. Thus, targeting the CXCL16/CXCR6 and MCP-2/CCR1 as well as VCAM-1/T cell signaling after kidney injury holds great potential for the treatment of CKD progression. To test this hypothesis, we propose to define the importance of CXCL16/CXCR6 and VCAM-1 signaling in T cell homing and adhesion (SA1) and the importance of MCP-2/CCR1 signaling in PMN homing (SA2) during AKI-to-CKD transition and then to translate our understanding of these homing signals into developing polyamidoamine dendrimer nanoparticles that can selectively deliver siRNAs to knockdown these homing signals to slow or even prevent CKD progression (SA3). This work will provide preclinical data defining how to prevent the second wave of immune activation and transition from AKI to CKD.

NIH Research Projects · FY 2026 · 2023-03

Project Summary Obesity, which is defined as the excess accumulation of white adipose tissue (WAT) is associated with the development of numerous metabolic, inflammatory, and cardiovascular derangements. However, it is the accumulation of visceral adipose (VWAT) that is associated with metabolic diseases, while the accumulation of subcutaneous adipose (SWAT) is thought to protect against the development of obesity-associated disease. Sex hormones are known to influence both the accumulation of adipose, adipose distribution between VWAT and subcutaneous adipose (SWAT), and the development of metabolic disease, but the precise mechanisms by which sex hormones influence adipose function and distribution remain unclear. We have previously shown that there is an obesity-specific mechanism of adipogenesis that drives adipocyte hyperplasia in a sex-specific pattern. Estrogen plays a role in this process, where exogenous estrogen drives a female-like patterning (both VWAT and SWAT) of adipocyte hyperplasia in male mice, while the absence of systemic estrogen in female mice results in male-like patterning (VWAT only response). Our preliminary data indicates that in the absence of systemic estrogen, estrogen signaling still plays a direct role in VWAT adipocyte hyperplasia at the onset of obesity. We show here that it is VWAT endothelial cells that express aromatase and thereby produce the estrogen required for adipocyte hyperplasia in males and ovariectomized females. We propose that aromatase expression in VWAT endothelium is controlled by glucocorticoids, linking the VWAT-specific actions of estrogen to the hypothalamic-pituitary-adrenal (HPA) axis. Based on our preliminary data, we hypothesize that estrogen and glucocorticoids participate in an endothelial cell-adipocyte precursor axis that regulates obesogenic adipogenesis, thereby influencing distribution of WAT and impacting metabolic disease. Here we will establish the role of adipose- produced estrogen in obesity and metabolic disease, determine the mechanisms that regulate aromatase in VWAT endothelial cells and define the molecular mechanisms of that regulate estrogen action in adipocyte precursors.

NIH Research Projects · FY 2026 · 2023-03

Project Summary/Abstract: Inflammatory bowel disease (IBD) is largely characterized by dysregulated cytokines and antimicrobial responses. Innate mechanisms are the initiating drives of host responses to microbes and the resulting cytokine and antimicrobial responses need to be carefully balanced. Mitochondrial pathways play a key role in mediating these innate responses and a dysregulation in mitochondrial mechanisms has been increasingly recognized to play a role in IBD. The focus on the mitochondrial dysregulation in IBD has been predominantly in epithelial cells. However, mitochondria contribute to innate immune outcomes through a variety of mechanisms, including metabolic pathways, reactive oxygen species, communication with the endoplasmic reticulum (ER), and mitochondrial DNA (mtDNA) release. Of the >240 IBD-associated loci a number of genes within these loci modulate host innate responses and mitochondrial function through both direct and indirect mechanisms. As such, upon encounter of human macrophages with microbial products, we have found IBD-associated genes that regulate glycolysis and in turn macrophage polarization, the mitochondrial respiratory chain and mtROS, and ER stress. We further find that upon human macrophage stimulation by a range of pattern recognition receptor (PRR) ligands, release of mtDNA is dramatically increased along with activation of the cGAS- STING pathway. The cGAS-STING pathway then serves to promote responses across the many PRRs. We have preliminary data that at least one IBD- associated gene which partially localizes to the mitochondria, LACC1, modulates PRR-induced activation of the cGAS-STING pathway, and in turn, downstream PRR-initiated downstream outcomes. We hypothesize that the cGAS-STING pathway amplifies responses across a broad range of PRRs through a variety of intracellular mechanisms, that the threshold of this regulation is important in susceptibility to intestinal inflammation and might be therapeutically targeted under conditions of intestinal inflammation, and that IBD- associated geneticvariants regulate these outcomes, thereby influencing key innate immune outcomes. Relevance: These combined human cell and mouse studies will provide insight into mitochondrial mechanisms regulating key outcomes in macrophages, the manner in which these mechanisms are altered in IBD patients and in the context of IBD risk variants, and how these mechanisms might be modulated during intestinal inflammation in order to improve outcomes in vivo. These comprehensive and mechanistic studies will establish a foundation for additional studies to therapeutically target mitochondrialpathways shared across innate immune responses so as to restore innate immune dysregulation.