Rutgers Biomedical And Health Sciences

NIH Research Projects · FY 2025 · 2024-08

Project Summary Accumulating evidence from individual clinical trials and conventional trial level meta-analysis suggests that restrictive transfusion threshold of 7 to 8 g/dL is as safe and effective as the 9 to10 g/dL threshold, based largely on an analysis of the primary outcome of mortality. However, it is much less clear whether the safety of restrictive hemoglobin thresholds applies to all patient groups. In this revised application, we focus the aims on patients with underlying cardiovascular disease given recent results that suggest patients with acute myocardial infarction could be harmed by restrictive transfusion strategy and that it is especially important to evaluate restrictive transfusion in patients with pre-existing cardiovascular disease. Individual clinical trials do not have adequate sample size and conventional trial level meta-analysis lack the specific detail to examine the effect of transfusion in specific subgroups. An individual patient data meta-analysis (IPDMA) will have the power and detail to fully explore the effects of transfusion thresholds across clinically important subgroups. For this IPDMA, randomized clinical trials that assigned patients red blood cell transfusions based on transfusion threshold (sometimes also referred to as trigger) have been identified from systematic searches of the literature. The search will be updated and supplemented by direct query of experts in the field prior finalizing the studies that are included. Data use agreements and letters of support document the commitments of investigators to provide individual patient data from 89% of the participants included in these trials. The IPDMA will be combined into a single comprehensive analysis database. The aims of the analysis are to 1) Primary Aim: To estimate the treatment effect of liberal versus restrictive transfusion thresholds in patients with cardiovascular disease including those with myocardial infarction, pre-existing cardiovascular disease and cardiac surgery. We will evaluate clinically important pre-specified risk factors including older age, sex, heart failure, type of MI, baseline hemoglobin concentration, and others on primary and secondary outcomes. 2) Secondary Aim: To estimate the treatment effect of liberal versus restrictive transfusion thresholds in other clinically important pre-specified subgroups with severe chronic illnesses including cancers and chronic renal diseases, GI bleeding and by age and sex. 3)Exploratory: Use a personalized medicine approach to create models designed to identify risk factors and combinations of risk factors that modify the effect of transfusion strategy on the primary and secondary outcomes. The results will advance our knowledge about the impact of transfusion threshold in cardiovascular and other clinically important patient groups, where there is concern about the safety of applying a universal `restrictive' hemoglobin threshold for transfusion. If liberal transfusion is shown to be superior to restrictive transfusion in patient groups, guidelines will be updated and clinical practice will need to change.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY We propose to develop an undergraduate research program that promotes the advancement of students from underrepresented (UR) groups in the field of addiction research: the Training in Research Undergraduate Experience through the Rutgers Addiction Research Center, the “TRUE RARC” Scholar Program. The lack of diversity among addiction scientists is associated with real opportunity costs, from hindered scientific innovation to persistent disparities in outcomes of substance use treatment for diverse populations. Undergraduate research programs are necessary to remedy underrepresentation, as they build a bridge to advanced STEM degrees by helping students envision a future career in research, learn foundational research skills, and build competencies and self-efficacy. Rutgers is an ideal environment in which to build an undergraduate addiction research training program. As The State University of New Jersey, Rutgers enrolls >45K undergraduates across three campuses, each of which ranks highly in diversity and social mobility for low-income students. Further, Rutgers is home to the largest comprehensive addiction research center in the United States, consisting of >150 researchers representing more than 40 departments and schools, that closely coordinates with Rutgers Health, which provides substance use services across the state of New Jersey. The specific aims of our proposal for the TRUE RARC Scholar Program are: (Aim 1) Develop an innovative three course sequence (Data Science & Addiction Research 1 & 2, followed by a Capstone course) to teach students about the foundations of addiction research and data science methods, culminating in a digital badge representing measured competencies. Our course sequence will make use of the Adolescent Brain Cognitive Development (ABCD) study data, introducing students to a hallmark NIH resource, while teaching them about interdisciplinary addiction research, including genetics, neuroscience, and social/ environmental influences on substance use outcomes. (Aim 2) Upon completion of their foundational coursework, engage 10 or more students from underrepresented backgrounds in six-month, mentored research experiences in RARC faculty labs, culminating in a research presentation at the annual RARC symposium. (Aim 3) Provide professional development opportunities for career readiness and graduate school matriculation by collaborating with our robust network of academic, industry, state, and health care partners. (4) Create a national resource for addiction research education by building a web repository to publicly disseminate core aspects from our proposed program, and that of other RARC-affiliated and NIDA/NIAAA- sponsored training programs, including coursework and professional development content. This will expand the impact of our undergraduate training program beyond the Rutgers campuses. In summary, our program will build an undergraduate pipeline into a range of graduate programs, and create associated resources for the field, to significantly remedy underrepresentation among addiction scientists.

NIH Research Projects · FY 2024 · 2024-08

Project summary Posttranslational arginylation on proteins installed by arginyltransferase ATE1 is a critical modification for cancer progression and metastasis. ATE1 is a beneficiary regulator, inhibition of which often results in elevated metastasis (e.g., prostate cancer) and poorer outcomes. Unfortunately, the physiological roles of ATE1 (and its arginylation activity) in cancers remain poorly understood, mostly due to the lack of analytical methods to discover the protein substrates of ATE1 from cancer samples. Arginylation regulates protein half-lives in cellular systems through the Arg/N-degron pathway and represents one of the three (arginine, proline, and acetylation) N-terminal degradation signals (N-degrons) for protein turnover. Here, we aim to discover the protein substrates of ATE1 in cancers for the development of targeted protein degradation (TPD) by hijacking ATE1 and its arginylation activity. We have developed an unbiased proteomic profiling method to discover the ATE1 substrates and their precise arginylation sites, the method is termed activity-based arginylation profiling (ABAP). Using ABAP, we have successfully profiled arginylation from 12 samples including 4 cancer cell lines. In this proposal, we would like to establish a library of cancerous substrates from commonly used cancer cell lines using the NCI60 pellets. To take advantage of arginylation for targeted protein degradation against cancers, we have engaged ATE1 with protein of interest (POI) through our model Halo-FKBP system. We discovered, for the first time, that engagement of ATE1 with cancer targets (e.g., SHOC2 and RAD52) successfully induced POI degradation in cellular models. We termed this technology arginylation targeting chimera (ArgTAC). Our preliminary data on degradation data from the first few POIs encouraged us to further develop this technology as a novel approach in the TPD field. We will apply a series of state-of-the-art approaches including proteomics and molecular biology to characterize the mechanism of action of induced POI degradation after engagement with ATE1. A series of molecular events will be characterized including cellular proximity between ATE1 and POI, the arginylation and polyubiquitination of POI, and the POI degradation dependency on UBR E3 ligases and proteasome. To achieve optimal degradation of broad cancer targets, we will expand our tool compounds designed for the Halo-FKBP system. We will also further develop new ATE1 binders/recruiters through peptide optimization based on LIAT1 (ligand of ATE1) binding domain to ATE1 using screening and structure/computer modeling-based drug discovery. More ArgTACs targeting other POIs will be synthesized and tested for degradation activities in cell models to expand the application of ArgTACs. The proposed ABAP and ArgTAC platform here will likely generate a catalog of oncoproteins regulated by arginylation, and offer a general and revolutionary TPD technology targeting oncoprotein degradation as a therapeutic strategy for cancers.

NIH Research Projects · FY 2024 · 2024-08

PROJECT SUMMARY This proposal aims to develop and pilot test a theoretically-grounded, behavioral intervention to promote the development of a therapeutic alliance between oncologists and Latino advanced cancer patients. This intervention will be grounded in the theoretical model of the TA and a conceptual model of the TA in Latino cancer patients that we will develop through the proposed formative research. Based on data from the literature and our preliminary studies, this intervention will seek to improve oncologist TA by promoting trust, empathy, shared decision-making, and patient-centered communication. The intervention framework will be based on Self Determination Theory (SDT), which posits that behaviour change (improvement in TA) is more likely when conditions of autonomy, competence, and relatedness are met. First, mixed methods will be used to create a conceptual model of the TA between oncologists and Latino cancer patients to determine targets for the intervention (Fig 1). Aim1a will leverage data from an ongoing funded multi-site, prospective observational R01 on medical/religious/cultural beliefs, TA, and EOL care outcomes (n>270). For Aim 1b, we will conduct in-depth interviews and focus groups with oncologists (n=9) and Latino cancer patients (n=35) in order to gain a more nuanced understanding of the TA and refine our conceptual model. Once the intervention has been created, we will then refine the intervention using stakeholder (Aim 1b participants) feedback regarding its feasibility and acceptability. For Aim 3, an independent set of 8 oncologists and 50 patients will be studied, half (n=4 oncologists, 25 patients) will be randomized to training in the TA intervention, and the other half (n=4, 25) will be randomized in to usual care. We will examine feasibility (rate of intervention completion), acceptability (oncologists’ and patients’ satisfaction), and efficacy of the TA intervention for improving the TA between oncologists and Latino cancer patients, as measured by patient THC scores as the primary outcome. Secondary outcomes will include engagement in ACP and EOL care. Dr. Tergas’s long-term career goal is to establish a leading, independent program of research focused on the reduction of disparities in EOL care by implementing and disseminating effective, empirically-supported interventions for use in clinical practice. As a foundation for her independent research program, her short-term career goal is to develop an implementable, evidence-based behavioral intervention for improving therapeutic alliance between oncologists and Latino cancer patients. This K08 award application represents the cornerstone of her strategy to achieve my short- term goal and consists of cohesive and complementary research aims and training objectives. Specifically, in order to achieve her career goals, she needs training in intervention development and implementation science, as well as continued training and mentorship in EOL care disparities. Conducting the research detailed in my research aims will help me achieve this training and experience, as well as provide preliminary data for a future R01-level grant application.

NIH Research Projects · FY 2025 · 2024-08

Project Summary/Abstract Overview: The methylation of DNA and histone tail residues tunes chromatin structure to control gene expression programs. Methionine supplies one-carbon (1C) units for this S-adenosyl-methionine (SAM)-mediated methylation. These 1C units can come 1) directly from dietary methionine or 2) indirectly from serine, glycine or another 1C donor to regenerate methionine. Both 1C metabolism and this epigenetic landscape are critical in the development of and for healthy maintenance of differentiated tissues. Importantly, a common hallmark of aging, cancer and other diseases is altered levels of these amino acids that provide 1C units and/or DNA and histone methylation. Goals: Critically, what is not understood is how homeostasis of SAM metabolism is achieved across mammalian tissue types and in proliferating cells through the dynamic interplay between production from available nutrients versus consumption by methylation reactions. Further, it is not known how much SAM consumption is required to support epigenetic regulation. Our objective is to apply modern quantitative methods to understand the nutrient sources of the 1C units used for SAM synthesis and the contribution of SAM consumption to global DNA and histone methylation. This will be accomplished by addressing the following questions: 1) What are SAM production and consumption rates? We will use liquid chromatography-mass spectrometry (LC-MS) paired with stable isotope tracing to measure SAM turnover fluxes to understand how SAM levels are sustained in each major tissue types in vivo and in proliferating cells in vitro. 2) What are the nutrient sources of the 1C units used for SAM synthesis upon altered methionine and 1C unit availability? Using tracing, we will quantify contribution of the 1C unit for SAM synthesis from exogenous methionine versus endogenous production from other nutrients. 3) How much SAM consumption supports DNA and histone methylation? We will adapt established approaches in quantitative metabolic analysis to measure rates of global SAM-consuming methylation of DNA and histones. 4) How does SAM availability alter DNA and histone methylation dynamics? We will measure the influence of altered SAM production on DNA and histone methylation fluxes. Vision: Through the studies proposed in this R35 MIRA grant, our team will advance quantitative tools to interrogate novel roles for 1C metabolism in controlling SAM homeostasis and its influence on the epigenetic landscape across tissue types and in proliferating cells. This comprehensive knowledge will provide fundamental insights into how metabolic homeostasis integrates supply of available nutrients to support epigenetic-driven gene expression. Such knowledge is critical for developing therapeutic strategies to manipulate SAM metabolism in diseases with aberrant changes in epigenetic methylation.

NIH Research Projects · FY 2023 · 2024-08

ABSTRACT Initiation of alcohol use typically occurs well before the legal drinking age, which is concerning as early use is associated with short and long-term adverse outcomes including acute and prolonged neurobiological effects. For youth, the media constitutes a primary source of learning about alcohol. Entertainment media frequently features images of and references to alcohol, associating alcohol use with social, sexual, and financial success, with little depiction of the hazards of drinking. Moreover, alcohol content is easily accessible via new (digital) media platforms such as social media and YouTube which are highly interactive, allowing for user engagement through exchange and manipulation of information. Adolescents are high consumers of media and with the proliferation of smartphones and tablets, youth have anytime, anywhere, on-demand access to media alcohol content. Yet, virtually no information is known about in vivo exposure to such content in terms of frequency/duration, medium/format, and context. Study Aim 1 will quantify and characterize in vivo exposure to alcohol content in entertainment media (film, TV, popular music) and new media (social media, YouTube, internet, video gaming). Aim 2 will examine bi-directional prospective associations between in vivo exposure to alcohol media content and alcohol use across short-term and longer-term timeframes and Aim 3 will identify mechanisms of this association; an exploratory aim will test moderation of these associations by key individual and contextual risk factors. Etiological research points to several cognitive and social mechanisms underlying the association between in vivo media alcohol exposure and drinking, including perceived norms, cognitions (expectancies, drinker prototypes), identity, and attitudes (favorability, evaluative conditioning). Using a 3-wave measurement burst design, 300 youth age 15-18 will complete a 3-week ecological momentary assessment (EMA) protocol using a Smartphone app. Participants will be asked to provide event-level information upon encountering alcohol content and if possible, to upload a screenshot, photo, or text description of the exposure. Event surveys are paired with random-prompted EMA surveys as well as weekly surveys assessing alcohol use and longer surveys prior to each burst to measure risk factors and media utilization. Existing media intervention programs are unequipped to handle new media, fail to target adolescents, a group arguably at greatest need for intervention, and do not target media as it is experienced in vivo on portable devices. Fine- grained ecological studies such as this are needed to shape the content of just-in-time (JiT) interventions and to inform the best timing of intervention delivery, with the goal of producing reductions in underage alcohol use. In Aim 4, focus groups with a subset (n=48) of participants will provide understanding of the perceived impact of alcohol-related media content on behavior and cognitions to inform intervention development. This project uses a timely ecological approach to answer the fundamental question of how media alcohol content elevates underage drinking risk to optimally inform next steps in preventive media literacy intervention research.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Coronavirus disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been a major challenge for public health since the first case was reported in December 2019. In May 2023, the WHO and CDC marked the end of the COVID-19 public health emergency. However, COVID-19 remains a threat due to continuously evolving new variants, and fully vaccinated people remain susceptible to infection by the newer variants of the virus. SARS-CoV-2 entry is primarily mediated by binding of the SARS-CoV-2 spike protein (receptor-binding domain, RBD) to the human angiotensin-converting enzyme 2 (ACE2) receptor. Although ACE2-expressing cells support robust SARS-CoV-2 viral replication, ACE2 expression profiles are not completely associated with clinical manifestations or immune responses. Furthermore, SARS-CoV-2 infects organs or cells that do not express ACE2, suggesting the involvement of alternative receptors for SARS-CoV-2. Our and other laboratories have identified ACE2-independent alternative receptors for SARS-CoV-2, and infection via alternative receptors (e.g. CD147) is resistant to monoclonal antibodies against spike RBD, which is the target for several SARS-CoV-2 vaccines to block ACE2 binding. Our preliminary data show that oral epithelial, salivary gland, and gingival epithelial cells are susceptible to the replication-competent SARS-CoV-2 and pseudotyped SARS-CoV-2 Omicron variant despite low or undetectable expression of ACE2. These oral epithelial cells do however express high levels of alternative receptors CD147 or AXL, suggesting the role of alternative receptors in SARS-CoV-2 infection of oral epithelial cells. We hypothesize that these alternative receptors play a critical role in SARS-CoV-2 infection and virus-mediated immune activation in region-specific oral epithelial cells. In Aim 1, we will determine repertories of receptors for SARS-CoV-2 and infection profiles in region-specific oral epithelial cells and tissues from healthy subjects and subjects with oral inflammation pre- and post-treatment. In Aim 2, we will determine the contribution of specific receptors to SARS-CoV-2 infection and virus-mediated immune activation in oral epithelial cells. Considering that the virus will continue to infect humans regularly, it is critical to understand the role of alternative receptors for SARS-CoV-2 in the oral mucosa, the potential portal of SARS-CoV-2 entry, to develop anti-viral therapeutics and strategies to dampen virus-mediated immune activation and disease outcomes, especially as regards emerging variants.

NIH Research Projects · FY 2026 · 2024-08

ABSTRACT Non-alcoholic fatty liver disease (NAFLD) is linked to insulin resistance, type 2 diabetes mellitus (T2DM) and several other major comorbidities. Hepatic carbohydrate and lipid metabolism is maintained by the balance between anabolic hormones, primarily insulin, and catabolic hormones including glucagon and catecholamines. Our novel findings show that norepinephrine (NE) resistance occurs within a week of high fat diet (HFD) feeding in mice. This proposal seeks to determine the molecular mechanisms leading to the suppression of NE signaling and the effect this has on hepatic metabolism and the pathogenesis of NALFD and T2DM. NE and related hormones act through G-protein coupled receptors linked to Gq/11 and phospholipase C to generate the second messengers inositol 1,4,5 trisphosphate (IP3) and diacylglycerol, which induce cytosolic Ca2+ oscillations in individual hepatocytes. These dynamic Ca2+ signals are integrated in the intact liver by intercellular propagation of Ca2+ waves across entire hepatic lobules, from the periportal (PP) to the pericentral (PC) zones. Glucagon synergizes with Ca2+ to promote PP glycogenolysis and gluconeogenesis and inhibit PC de novo lipogenesis. Our data show that short-term HFD attenuates NE-induced Ca2+ signaling in hepatocytes and impairs the propagation of intralobular Ca2+ waves in intact perfused liver. PP hepatocytes specialize in gluconeogenesis, whereas PC hepatocytes are the primary site of lipogenesis and the first to show steatosis in NAFLD. We propose that this early lesion in Ca2+ signaling is responsible for the loss of Ca2+-dependent inhibition of lipogenesis selectively in PC hepatocytes. The present studies build on the unique expertise of the investigators and several experimental tools developed by the team. The aims to be investigated are: 1. Identify the molecular mechanisms by which HFD suppresses Ca2+ signaling. We will investigate the role of novel Protein Kinase C (nPKC) isoforms in suppressing Ca2+ signaling by negative feedback on hormone- induced IP3 generation. In vivo knockdown and direct manipulation of nPKC isoforms will test the hypothesis that HFD impairs Ca2+ signaling through constitutive activation of nPKC as a consequence of lipid accumulation, giving rise to a self-sustaining cycle of progressive steatosis. 2. Investigate zonal differences in Ca2+ signaling and how they are affected by HFD. We will examine the effect of feeding HFD on the zonal organization of Ca2+ signaling in intact liver perfused with Ca2+-dependent hormones, and how this modifies the synergistic regulation of hepatic metabolism by glucagon. In addition, isolated hepatocytes will be zonally stratified to elucidate mechanisms of Ca2+ signaling dysfunction. 3. Test whether HFD affects zonal regulation of metabolism by Ca2+ to promote pericentral lipogenesis. We will determine the effect of HFD on the zonal regulation of key enzymes of lipid metabolism by Ca2+-linked hormones. We will also investigate the effects of HFD on mitochondrial Ca2+ signals and the lobular organization of mitochondrial metabolism.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY HPV-associated malignancies include cancers of the cervix, oropharynx, anus, vulva, vagina, and penis. They cause thousands of deaths in the United States each year. Treatments for metastatic disease rarely provide lasting palliation and have minimal curative potential. First-line therapy increasingly includes immune checkpoint blockade in combination with chemotherapy, limiting the options for subsequent treatment. Hence, there is a compelling need for more effective treatments for advanced-stage disease. HPV-associated cancers harbor the oncogenic HPV E6 and E7 proteins. These antigens are attractive targets for immunotherapy due to their uniform expression by tumors and absence of expression by healthy tissues. This expression pattern permits targeting the HPV oncoproteins without toxicity and with the intent to eliminate cancer cells and cure the patient. Specific immunotherapeutic targeting of HPV oncoproteins with cancer vaccines has not been effective, likely due in part to the reliance of vaccines on host T cells, which vary in precursor frequency and functional avidity. We have developed a new type of treatment for HPV-associated cancers that targets the E7 oncoprotein by administering high-avidity autologous T cells directed against E7 by a gene-engineered T-cell receptor (E7 TCR-T cells). E7 TCR-T cell therapy has demonstrated safety and clinical activity in treatment-refractory, metastatic HPV- associated metastatic cancers. In a phase I clinical trial, no dose-limiting toxicity was identified. Clinical activity included regression of widespread, bulky cancer and complete regression of most tumors in some patients – including immune checkpoint blockade-refractory tumors. Translational research identified immunomolecular mechanisms of resistance, which now permits for screening of patients to treat only those whose tumors demonstrate an absence of resistance markers. This project is for a two-center, phase II clinical trial of E7 TCR- T cell therapy for metastatic HPV-associated cancers. This trial will leverage the strengths of Rutgers Cancer Institute of New Jersey and the NIH Clinical Center to implement novel immunomolecular screening, next- generation (two-week) engineered T-cell manufacturing, and recruitment and treatment of patients from diverse geographic regions and backgrounds. The short-term goal of this research is to assess the response rate for E7 TCR-T cell therapy in the treatment of metastatic HPV-associated cancers. The long-term goal is to develop a new strategy to treat these malignancies with the aim of increasing survival and curing some patients. Aim 1 will determine the response rate for E7 TCR-T cell therapy by conducting the phase II trial. This research will also further characterize the safety profile of the treatment. Aim 2 will conduct transitional research to reveal the mechanisms by which E7 TCR-T cells mediate tumor regression and elucidate tumor evasion mechanisms that can guide next-generation therapeutic approaches and biomarker discovery.

NIH Research Projects · FY 2025 · 2024-07

Project Summary Anxiety and anxiety-related disorders (AARD) are notable risk factors for alcohol use problems (AUP) and their co-occurrence (AARD-AUP) results in tremendous health costs and societal burden. The self-medication model states that those with AARD use alcohol to numb or cope with arousing and distressing negative emotional states; however, longitudinal evidence for self-medication is mixed and psychological treatments integrate cognitive-behavioral therapy (CBT) skills for substance use disorders with CBT interventions for AARD have demonstrated modest success. The heterogeneity in these findings is likely influenced by dynamic and person-specific factors. We hypothesize that results from person-specific analyses can improve our understanding of this heterogeneity and can be harnessed to personalize interventions for AUP. Personalized interventions have the potential to increase symptom reduction and rate of change, as well as therapy retention and alliance. Modeling person-specific patterns of psychopathology has been challenging due to historical limitations with data collection and statistical analyses. However, increasing access to digital phenotyping – an innovative method for collecting single-subject intensive ecological momentary assessment (EMA) integrated with passive sensing smartphone data streams (e.g., GPS data, accelerometer, Bluetooth/call/SMS data) – offers a compelling solution for the development and evaluation of truly personalized psychological interventions. Such data can be used to generate statistical models of a single individual’s pattern of behavior over time. Previous data-driven (i.e., feedback-based) interventions have demonstrated success; however, it is unclear whether using person-specific models to adapt an active intervention reduces symptoms or improves therapeutic efficiency (i.e., rate of change), therapeutic retention, or therapeutic alliance. This K99R00 award will provide training in passive sensing data collection methods and conduct of clinical trials, as well as continued training in alcohol research. In the K99 phase, I will build on previous expertise constructing person-specific models and identifying person-specific factors testing self-medication models of AARD-AUP, developing a method for personalizing a CBT skills-based intervention. In the R00 phase, the feasibility of this intervention will be tested in a pilot three-group parallel randomized clinical trial (RCT), which will test the feasibility of a personalized CBT skills-based interventions compared to two control conditions testing the relative efficacy of personalization and personalization method, respectively. In the personalized condition, the therapist will review identified influential factors from the person-specific model, treat these factors using CBT skills, and track them using the mindLAMP digital phenotyping platforms – testing a data- driven method for personalizing and adapting the content of each session. Findings will inform future R01 applications testing personalized interventions for problematic alcohol use.

NIH Research Projects · FY 2026 · 2024-07

Suicide among Black youth has increased rapidly over the last two decades, reaching unprecedented levels. While there have been substantive gains in knowledge related to the etiology of suicide ideation and behaviors (SIB), most of this work has included predominantly White samples or have been cross-sectional or short-term longitudinal studies. Less is known about the course of SIB during the adolescent and emerging adult years, and malleable individual-specific and social ecological precursors and correlates associated with this course, among low-income, urban Black youth. Such work is vital to informing developmentally-sensitive preventions and interventions aimed at mitigating this major public health problem. The proposed R01 application aims to address the aforementioned limitations by leveraging data collected among Black youth (N = 630) drawn from Baltimore City over a 20-year period spanning the critical developmental windows of adolescence and emerging adulthood, during which SIB often emerges. Data on mental health, academic and social competence, and life events have been collected annually during early childhood (i.e., ages 6-8), adolescence (i.e., 12-18) and emerging adulthood (i.e., 19-26). In addition, SIB, substance use and social ecological constructs (i.e., life events, racial discrimination, neighborhood characteristics, and social support) have been measured during adolescence and emerging adulthood. Specific aims of the study include: (Aim 1) to identify trajectories of SIB across adolescence and young adulthood, and whether SIB trajectories during adolescence are associated with SIB trajectories in emerging adulthood; and (Aim 2) to determine risk and protective individual-specific and environmental factors during the early childhood, adolescent, and emerging adult years that are associated with SIB trajectories during adolescence and emerging adulthood. We also plan to explore whether the identified risk and protective longitudinal factors in Aim 2 influence the transition or changes in SIB trajectory membership from adolescence to emerging adulthood. Determining salient factors associated with SIB continuity during adolescence and emerging adulthood may inform the identification of pathways that could be promoted or interrupted, which may have long-term implications for SIB across the life course. The investigative team brings together expertise in the etiology and prevention of SIB among Black youth, prevention science, mental health and substance use of adolescent and adult populations, and longitudinal latent variable modeling; thus, we are extremely well- positioned to accomplish the study aims. This study directly responds to recommendations offered in the Congressional Black Caucus’ Ring the Alarm report as it seeks to: 1) identify subgroups of Black youth at increased risk for SIB across the lifespan; 2) determine risk and protective factors that are amenable to early intervention; and 3) examine non-psychiatric factors (e.g., social and structural determinants) associated with SIB course, which if found to be significantly related to SIB, might be the focus of targeted upstream suicide preventions among Black youth.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Smoking is the most dangerous way to use nicotine but fewer than 8% of cigarette smokers successfully quit each year. While quitting all tobacco and nicotine use is best, completely switching to a noncombustible nicotine product, like e-cigarettes, can reduce exposure to harmful chemicals and risks for those who cannot quit. However, many people inaccurately believe that e-cigarettes are as harmful as combustible cigarettes, which may suppress switching. Limited research exists on underlying reasons for these beliefs, and few studies have identified educational messages that may be used for adults who smoke to address these beliefs. More research is needed on comprehensive messages that can target beliefs and effectively communicate the relative risks of e-cigarettes versus cigarettes, and to assess their potential effects on beliefs, intentions, and e- cig use. Research is also needed on methodological tools that can be used to identify communication that can effectively increase message-consistent beliefs and behaviors. Extant research suggests that tobacco message testing may be enhanced by using neurocognitive measures of message effectiveness, which have been shown to explain additional variance in intention and behavioral outcomes compared to self-reported measures alone. Functional near-infrared spectroscopy (fNIRS) is one method for measuring neurocognitive response that provides unobtrusive, subconscious, objective measures of cognitive processing predictive of behavior change. The overall aims of the proposed research are to: 1. Identify latent classes of beliefs about cigarette and e-cigarette harms to inform message development; 2. Develop, pilot test, and refine e-cigarette educational messages that target prevalent belief classes using neurocognitive and interview responses, and 3. Test finalized messages to assess effects of belief class messages on neurocognitive response, belief and intention change, and actual e-cigarette use. This study will examine the effectiveness of comprehensive e- cigarette educational messages tailored to prevalent belief patterns, and whether neurocognitive message responses can predict message-consistent beliefs, intentions, and behaviors. A comprehensive training plan is proposed to ensure the implementation of these aims, with objectives focused on 1) refining skills in neuroimaging techniques, experimental design, and analysis, 2) obtaining experience in designing and testing relative risk communications, and 3) expansion of skills in advanced quantitative and qualitative methods. The research and training plans will help me to establish an independent line of research applying communication neuroscience methodology to tobacco regulatory science research, and to support my pathway to independence.

NIH Research Projects · FY 2026 · 2024-07

Summary/Abstract: Initial cocaine abstinence is associated with severe sleep disturbances. Poor sleep is one of the strongest predictors of return to use (relapse), suggesting potential overlap in brain arousal and reward networks. Separate literatures link orexin (hypocretin) neurons in lateral hypothalamus (LH) to the regulation of wake and drug motivation, yet how these neurons might commonly contribute to sleep disturbances and subsequent drug use in CUD is not well understood. In rats, the intermittent access (IntA) schedule of cocaine self-administration increases cocaine demand, a behavioral economics measure of drug motivation. These motivational changes are persistent and are further enhanced following protracted abstinence. We showed that cocaine IntA is associated with an increase in the number and activity of orexin producing neurons, and that these increases are causally linked to cocaine demand. Our preliminary studies indicate that cocaine IntA rats also exhibit sleep dysregulation during initial abstinence, characterized by excessive wakefulness and a corresponding reduction in NREM (non-rapid eye movement) sleep. In Aim 1, we test if these sleep disturbances persist throughout protracted (28d) abstinence following cocaine IntA, and how they are related to population activity of orexin neurons throughout the inactive period. Using a chemogenetic approach, we predict that normalizing orexin neuronal activity during the inactive period will improve sleep outcomes and reduce cocaine demand following protracted abstinence. In Aim 2, we test if orexin projections to ventral tegmental area (VTA) are involved in mediating excessive wakefulness during cocaine abstinence. This is based on our preliminary data showing that waking is associated with increased orexin binding in VTA, as determined by the novel OxLight1 sensor. We predict OxLight1 signal in VTA will be enhanced in IntA rats during early and protracted abstinence, and that chemogenetic inhibition of VTA-projecting orexin neurons will normalize both sleep and cocaine demand outcomes at both timepoints. We will also determine how IntA changes levels of orexin 1 (Ox1R) vs orexin 2 (Ox2R) receptors in VTA and their functional role in mediating arousal and cocaine demand during abstinence. Finally, in Aim 3, we test the potential utility of repurposing the FDA approved dual Ox1R/Ox2R antagonist suvorexant for use in CUD. We predict that daily dosing with suvorexant, administered immediately prior to the inactive period, will normalize sleep and reduce drug motivation in IntA rats. Based on data indicating that suvorexant can persistently reduce orexin levels, we also predict that chronic suvorexant will normalize orexin cell numbers and their input to VTA, and thus reduce drug demand beyond the treatment period. Together, these studies combine several cutting-edge approaches to determine how orexin neurons, and their inputs to VTA, might serve as a neurobiological link between sleep disturbances and drug motivation during abstinence. This project is therefore an important step forward in the development of an orexin-based treatment for CUD.

NIH Research Projects · FY 2025 · 2024-07

EXECUTIVE SUMMARY/ABSTRACT After decades of increasing production of and reliance upon plastic materials, six billion metric tons of plastic waste has been deposited in our environment. Through environmental, commercial, and municipal degradation processes all plastics are eventually fragmented into micro-nanometer scale plastic particles and fibers known as micro-nanoplastics (MNPs). Consequently, MNPs have become a nearly ubiquitous contaminant of our environment and food web, to the extent that concentrations of MNPs in meat, dairy, seafood, and grains range from 10 to over 3,000 µg/mL. Yet little is known about the hazards of MNP ingestion exposures. A major concern is the ability of ingested MNPs, demonstrated by a growing number of studies, to reach the circulation and breach biological barriers to enter virtually any tissue. They have been found in human blood (at up to 13 µg/mL), colectomy samples, and placenta, and a recent study in our lab showed that MNPs ingested by pregnant rats reach the livers, hearts, lungs, kidneys, and brains of fetal pups within 24 hours. Yet the impacts of MNP ingestion on human health, including absorption, biodistribution, toxicity, and inflammation remain unclear, constituting major knowledge gaps that impede any science-based risk assessment of this emerging contaminant. To address these knowledge gaps, the proposed project, comprising three interconnected aims, will assess toxicity, uptake, and biodistribution of environmentally relevant MNPs, and the roles of MNP properties and intestinal inflammation in each. In Aim 1 we will develop and fully characterize 5 environmentally relevant gold core – plastic shell MNPs of three major plastic polymers (polyethylene, polyethylene terephthalate, and polystyrene). The pristine core-shell MNPs will be photo-aged to simulate decades of environmental exposures. The Au cores will enable accurate quantification of MNPs in cells and tissues, and intracellular localization in Aims 2 and 3. In Aim 2 we will use a triculture small intestinal epithelium model and an “intestine- on-chip” (IOC) model with organoids from both healthy donors and donors with inflammatory bowel disease (IBD) coupled with simulated digestions to determine the role of iMNP physicochemical properties in iMNP toxicity, inflammation, uptake, and translocation, and the role of inflammation in iMNP uptake and toxicity. Specific molecular inhibitors and siRNA silencing will also be used to determine the cellular mechanisms involved in MNP translocation. In Aim 3 we will assess in vivo MNP uptake, toxicity, biodistribution, and the role of inflammation in each, in healthy and IBD-susceptible intestinal Hnf4a knockout mice. These studies will provide validation and translational assessment of in vitro approaches as well as detailed data on iMNP organ and tissue biodistribution. The data generated from these studies will allow health risk assessors and policymakers to assess the potential risks of iMNP exposures and provide the basis for future mechanistic and epidemiological studies.

NIH Research Projects · FY 2026 · 2024-07

SUMMARY Duchenne muscular dystrophy (DMD) is incurable. Lack of dystrophin is the culprit. While the ultimate solution to this devastating disease is to restore dystrophin, the scientific and medical communities actively seek to discover and fix key secondary factors. We study a corrective role of phosphorylated Connexin-43 (Cx43) as a potential secondary factor in DMD cardiomyopathy. Cx43 forms gap junction channels at the intercalated disc (ID) of the cardiomyocytes (CMs). The gap junction helps the heart to beat in unison. We found that dystrophic CMs exhibit pathological Cx43 remodeling – that is Cx43 lateralization away from the ID. Lateralized Cx43 proteins remain as hemichannels, allowing for an undesired passage of ions and metabolites to and from the extracellular space of the CM. Cx43 remodeling contributes to arrhythmic events, stress-induced lethality and long-term fibrotic cardiomyopathy in DMD. Our long-term goal is to improve cardiac function in DMD patients by identifying secondary targets for pharmaceutical agents to protect against heart failure. In this grant proposal, we postulate that the microtubule (MT) network is the connector between dystrophin loss and Cx43 remodeling and that phospho-Cx43 can fix the dystrophic MTs. Our central hypothesis is that Cx43 remodeling and pathology are linked to a dystrophic, disorganized microtubule that triggers X-ROS elevation and oxidation of CaMKII to affect the phospho-status of Cx43. Reciprocally, changes in the phospho-status of Cx43 affects the phospho-status of b-tubulin. As a result of this loop, Cx43/MT normalization corrects downstream effectors, with an ultimate impact on arrhythmias and long-term fibrosis. We also believe that, in addition of suppressing Cx43 pathological remodeling, phospho-Cx43 stabilizes critical members of the intercalated disc. We will first determine if Cx43 remodeling mediates pathology triggered by a dystrophic microtubule cytoskeleton (Aim1). We will investigate if a phospho-mimic form (serine 172) of b-tubulin phenocopies the beneficial effect of colchicine (an inhibitor of MT polymerization) mediated by phospho-Cx43 in dystrophic MTs. We will also test an alternative role for phospho-Cx43 as a scaffold stabilizer. We expect to demonstrate a regulatory interplay between MT and Cx43, in which one phospho-player rescues the other. We will then determine if and to what extent NOX2 and oxidized CaMKII promote Cx43 remodeling and pathology. We will also test if Cx43 is a direct target of CaMKII (Aim2). We expect that important aspects of the Cx43-mediated pathobiology of the hearts of dystrophic mice will be prevented when X-ROS and oxidized-CaMKII are targeted. Impactfully, the microtubules link the lack of dystrophin with Cx43 remodeling. Phospho-mimic mutations appear to correct the dystrophic microtubule hyperdynamics, suppress Cx43 remodeling, and to protect the intercalated disc. The study of phospho-Cx43 will lead to new treatments of DMD-cardiomyopathy.

NIH Research Projects · FY 2024 · 2024-07

PROJECT ABSTRACT Rutgers, The State University of New Jersey (RU)-New Jersey Medical School (NJMS) requests funds to purchase and outfit an automatic watering delivery system (AWS) to serve the needs of the current as well as future expanding numbers of rodent cages in the Cancer Center (CC) building vivarium in Newark. The current facility was built in 2004 with 2200 Individually ventilated cages (IVC) with city water provided through water bottles. NJMS has seen a big increase in biomedical research and with it the need for use of animals in the research. To accommodate the growing number of NIH funded investigators using animals, we have outfitted the facility with high-density IVC caging, increasing the cage capacity of the facility to 6000 cages within the existing footprint. Currently, the water source for the animals is city water that is bottled and autoclaved prior to providing them to the animals. This task is very labor intensive and time consuming. Also, the increased amount of water bottles increases the chances for repetitive motion injuries to our animal care staff due to the many tasks associated with providing the water bottles- removing caps, putting on the caps, carrying heavy baskets with the autoclaved water etc. An AWS that supplies chlorinated reverse osmosis (RO) water proposed in this project will significantly reduce the labor costs (estimated 2 FTEs), improve worker safety, provide constant and consistent high quality water and increase the rigor and reproducibility of research. The animal care technician time savings will be redirected to more animal care needs. The labor cost savings will result in less per diem costs for the investigators. The equipment requested fits existing facility dimensions and utilizes most of the existing physical infrastructure. To ensure equipment operation Rutgers University will pay for the annual maintenance contract for proper maintenance of the equipment.

NIH Research Projects · FY 2024 · 2024-07

PROJECT SUMMARY/ABSTRACT Perceived loneliness is an independent risk factor for both diabetes and dementia. Older adult populations are a high-risk group for the metabolic and cognitive consequences of social isolation due to frailty, loss of mobility and lack of affordable transportation. However, the neural mechanism underlying the relationship between social connection, glucose homeostasis, and cognitive health is poorly understood. To address this gap, my F99/K00 proposal builds on preclinical and clinical evidence demonstrating that social isolation negatively impacts glucose homeostasis. Glucose regulation is critical in the progression of both diabetes and dementia. Oxytocin is a neurohormone which modulates social behavior and energy balance. The oxytocin receptor (OTR) is highly expressed in the ventromedial hypothalamus (VMH), a brain area critical to glucose homeostasis. VMH glucose sensing neurons respond to fluctuations in extracellular glucose to regulate peripheral glucose homeostasis. However, the role of oxytocin in modulating VMH glucose sensing and the extent to which social isolation disrupts VMH glucose sensing has never been investigated. Our preliminary data demonstrate that:1) single-housed mice are hyperglycemic compared to co-housed mice; 2) VMH OTR neurons are inhibited by glucose; 3) VMH OTR neurons co-express neuronal nitric oxide synthase (nNOS), a marker for glucose-inhibited (GI) neurons. Together, this evidence supports our central hypothesis that oxytocin modulates VMH glucose sensing neurons, and that social isolation amplifies this modulation leading to a hyperglycemic phenotype. AIM 1 F99 Phase: I will investigate the mechanism by which social isolation induces hyperglycemia. I will perform whole-cell patch clamp electrophysiology in brain slices to determine whether oxytocin modulates GI neurons. I will use in vivo fiber photometry calcium recordings to determine if VMH-OTR activity increases during induced hypoglycemia. I will use CRISPR/Cas9 to selectively knock down the VMH OTR receptor in co-housed mice and determine glycemic differences compared to controls. These F99 experiments will contribute mechanistic insights regarding the role of oxytocin in social isolation-induced glucose dysregulation. My training plan will provide the conceptual and technical neuroscience foundation necessary for the K00 phase of my career development plan. AIM 2 K00 Phase: I will determine if social contexts affect cognitive aging via oxytocin control of glycemia AIM 2A will use an aging rodent model to determine how different patterns of endogenous oxytocin release affect glucose homeostasis and memory performance. These experiments will allow me to build on my F99 skillset and gain expertise in optogenetics, social behaviors, and cognitive testing assays. AIM 2B will use secondary analysis methods to model human longitudinal data and identify the specific social relationships that mediate the link between loneliness, diabetes, and cognitive function in diverse patient sub-populations

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Schizophrenia (SZ) is a chronic condition characterized by cognitive impairment, atypical beliefs, and reduced reward/goal pursuit, which contribute to ongoing functional impairment even when other active phase symptoms subside. Symptom severity has been associated with poor decision-making with respect to integrating information about potential rewards and associated uncertainty, often manifesting as maladaptive unwillingness to tolerate the risk of failure. This risk-aversion limits opportunities to gain new or additional rewards or resources (i.e., nothing ventured, nothing gained). How an individual attends to and accrues information critically impacts subsequent decisions. While research has identified neural circuits implicated in impaired decision-making during choice in SZ, less is known about the processes by which information is attended to and accrued from the environment and integrated before a choice is made. This project will identify how decision- making in SZ is impacted by attentional, behavioral, and cognitive processes preceding decision choice, and whether associated neural signals predict daily, real-world decisions. Isolating these antecedents of impaired decision-making allows for the implementation of ameliorative interventions, empowering those with SZ to make choices that could reduce the burden of illness. First, leveraging concurrent functional magnetic resonance imaging (fMRI) and eye-tracking, the project will characterize attentional processes underlying evidence integration prior to decision choice. It will determine if SZ is associated with visual attentional biases toward aversive, and away from potentially rewarding, stimuli, and whether these biases underlie differences in associated brain activation in regions such as the nucleus accumbens (NAcc), insula, and anterior cingulate. Second, during fMRI it will test whether implementing cognitive strategies prior to decision-making upregulate reward-related brain activation and ameliorate disadvantageous choice in SZ. During upregulation via cognitive strategies, those with SZ are hypothesized to improve decision-making while increasing connectivity between NAcc and prefrontal regions, and during decision-making they are predicted to exhibit increased NAcc activation underserving adaptive reward pursuit. Such a result would suggest the feasibility of teaching these strategies as a clinical intervention for excessive risk aversion. Third, real-world daily behavior will be assessed using experience sampling methods to ascertain how daily decisions are impacted by perceived risks and symptom expression. Those with negative symptoms are predicted to endorse greater perceived risk of failure before potential activity engagement and those with positive symptoms are predicted to have less perceived risk of failure, despite impaired engagement. Finally, combined data will establish whether brain activation and task behavior predict real-world decisions to pursue rewards despite potential risk of failure. The project will establish the foundation for a novel interventional program of research into improving decision- making in SZ by elucidating distinct targets of behavioral and neuromodulatory intervention.

NIH Research Projects · FY 2026 · 2024-07

Project Summary/Abstract Mu opioids, such morphine and fentanyl, remain in the mainstream for moderate and severe pain management. However, they also produce many side-effects such as tolerance and addiction, leading to the development of opioid use disorder, a main cause of the opioid epidemic and climbing opioid overdose death. Mechanisms underlying mu opioid actions, particularly their side-effects such as tolerance and reward, are extremely complex and involve multiple distinct systems or diverse signaling pathway. Fat mass and obesity-associated protein (FTO) was initially identified as a demethylase of N6-methyladenosine (m6A), a modified nucleotide in mRNA. Later, it was found that Fto exhibits significantly higher catalytic activity in demethylation of N6,2'-O- dimethyladenosine (m6Am) compared to m6A. Accumulating evidence suggests that FTO influences gene expression by modulating mRNA stability, alternative splicing, translation, and epigenetic modulation. Notably, FTO has emerged as an important molecular player in various neuronal functions, and neuropsychiatric disorders. Recently, we investigated the involvement of FTO in mu opioid actions, specifically tolerance and reward in mice. Our preliminary data demonstrated that Fto depletion or inhibition significantly reduced morphine and fentanyl tolerance and morphine reward measured by conditioned place preference (CPP). Furthermore, depleting Fto in the nucleus accumbens shell (NAcSh) by microinjecting AAV-Cre in Ftofl/fl mice or the dorsal root ganglion (DRG) by inducing Cre expression in the DRG by tamoxifen in Ftofl/fl:Advillin-CreER2 mice led to attenuation of morphine CPP and tolerance, respectively. Additionally, we performed a preliminary transcriptome study using the NAcSh of Fto-NAcSh-depleted mice, which revealed alterations in gene expression and alternative splicing. Collectively, these strongly support an overarching hypothesis that Fto contributes to mu opioid reward and tolerance in the NAcSh and DRG, respectively, through distinct molecular mechanisms. FTO inhibition or modulation of a key molecule or pathway downstream of FTO in a specific region may therefore represent a new approach for preserving mu opioid analgesia while mitigating specific unwanted side effects. To test this hypothesis, we propose the following three specific aims. Aim 1. Determine the role of Fto in the NAcSh and DRG on mu opioid tolerance and reward by CPP and intravenous self-administration. Aim 2. Determine if the role of Fto in the NAcSh and DRG on mu opioid reward and tolerance is mediated by increased m6Am or m6A by using double-floxed mouse models, Ftofl/flPcif1fl/fl and Ftofl/flMettl3fl/fl. Aim 3. Investigate molecular mechanisms underlying the role of Fto in the NAcSh and DRG on mu opioid reward and tolerance using several cutting-edge approaches, including m6Am/m6A mapping, miCLIP, SLAM-seq, GLORI, ribosome profiling and RNA-seq. The proposed studies promise to provide new insights into in vivo molecular mechanisms in mu opioid action regarding RNA methylation and identify new targets downstream of FTO, which may have therapeutic potentials for developing a novel strategy to reduce mu opioid tolerance and reward in pain management.

NIH Research Projects · FY 2026 · 2024-07

Project Summary/Abstract Cellular metabolic and biosynthetic demands change throughout the cell cycle, which critically impacts cell growth, proliferation, and human disease. Understanding how cellular metabolism is modulated in different cell cycle phases is fundamental to understanding the molecular mechanisms governing cell growth and proliferation. Yet, very little is known about how metabolism changes throughout the cell cycle and how these changes mechanistically link to the cell cycle machinery. The master metabolic regulator Mechanistic Target of Rapamycin Complex 1 (mTORC1) is an evolutionarily conserved protein kinase complex that integrates upstream growth factor and nutrient signals to stimulate anabolic cell growth. mTORC1 is activated in most, if not all, proliferating eukaryotic cells, but the role of mTORC1 in controlling cellular metabolism has not been studied in distinct cell cycle phases. Thus it is unknown whether the metabolic program induced downstream of mTORC1 is differentially regulated throughout the cell cycle, or whether mTORC1 can play unique roles in specific cell cycle phases. We tracked mTORC1 activity across the full cell cycle and found that mTORC1 is acutely and differentially regulated, with its activity peaking in S/G2 and lowest in mitosis and G1. We hypothesize that mTORC1 is a crucial effector through which the cell cycle orchestrates metabolic changes, dynamically modulating metabolic pathways in a phase-specific manner to meet changing biosynthetic requirements. In this planned research program, we will elucidate the cell cycle phase-specific functions of mTORC1 by combining metabolomics and metabolic flux analysis with detailed mechanistic studies. Based on our preliminary data, we anticipate that these studies will uncover new functions of mTORC1, along with new regulatory mechanisms, that are not evident in studies on asynchronous cell populations, thus providing critical new insights into the fundamental mechanisms that integrate cell cycle control with cellular metabolism.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Both healthy individuals and those with addictive disorders experience cravings, a strong urge for a particular substance. Yet despite this and the recognized impact craving has on some individuals, such as those with food addiction, leading to overconsumption of highly palatable, high-calorie/high-fat foods, little is known about the neural mechanisms underlying craving and its impact on decision making. Past fMRI studies have identified canonical value and emotion/interoceptive processing regions of the brain that are sensitive to craving. However, these studies rely on cue-reactivity paradigms that do not examine subjective value computations, an essential component of behavioral intent understood to dictate - and potentially bias - decision making under craving. Work in my lab has shown craving scales subjective value (i.e., how much an individual feels an item is worth) in a multiplicative manner along a similarity dimension. Meaning people disproportionately value a craved item and those similar to it more than dissimilar items during craving. However, the precise neural mechanisms supporting this biased increase in subjective value in food addiction remain unknown. To address this, I will induce craving via a multisensory food activity and have subjects complete willingness-to-pay probes during fMRI so I may track how individuals’ subjective value changes pre- and post-craving. Furthermore, I will use the Yale Food Addiction Scale to select for normative (control) and addictive food consumption (food addiction) subjects to compare how craving may differentially exert its effects in food addiction. In Aim 1, to test how craving differentially exerts its effect in food addiction, I will examine how subjective value changes for craved and non- craved foods pre- and post-craving induction with the hypothesis that the food addiction group will exhibit more disproportionate increases in subjective value for both the craved snack and non-craved snacks compared to controls who will display a more targeted effect. Furthermore, to determine what neural bases may be primed in food addiction to produce these disproportionate increases in subjective value, in Aim 2 I will assess whether patterns of neural activity (i.e., neural representations) for the value of the craved and non-craved snacks are less distinct in food addiction compared to controls, serving to drive more generalized overconsumption. The training and research outlined in this F31 application will enhance my predoctoral training by: (1) solidifying my understanding of motivational and decision neuroscience (neuroeconomics) methods and theory and (2) increasing my competency in craving and behavioral addictions. The activities planned are aimed at advancing my theoretical understanding of how motivational states interact with decision making, neuroimaging skills, and clinical knowledge, all of which will be essential to achieving Aims 1 and 2, results from which would provide novel insights into the precise neural mechanisms that underlie craving in food addiction.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Opioids derived from opium are amid the oldest medications dating back to the 1800s, originally used for gastrointestinal distress and now are among the most effective treatments for pain management. It wasn’t until the mid-1800s that the abuse potential for opioids was evident, and this problem has since multiplied on an international scale. Within the last decade, the number of opioid overdose deaths per year in the United States alone has more than tripled. It is therefore urgent to not only find solutions to combat opioid overdoses, but to also develop new treatments for pain management. Opioid-induced respiratory depression (OIRD), the main cause of opioid overdose deaths, is primarily mediated through mu opioid receptors, particularly in two small brainstem regions, the preBötzinger complex (preBötC) and the parabrachial complex (PBN) that are sufficient and necessary to drive OIRD. Recent reports have also highlighted important cell types and downstream molecular mechanisms within these brain regions that mediate OIRD. Moreover, several laboratories within the last few decades have been exploring the function of the mu opioid receptor gene, Oprm1, which undergoes extensive alternative splicing to generate two classes of splice variants: Exon 1- (E1) and Exon 11- (E11) associated variants. The functional relevance of these splice variants has been demonstrated in mediating the actions of various mu opioids, including analgesia, tolerance, physical dependence and reward. However, it remains unknown how the two sets of Oprm1 splice variants influence OIRD. Preliminary data and unpublished data from our laboratory demonstrate differential actions of E1- and E11-associated variants in OIRD from varying doses of fentanyl and morphine in our rat models. Therefore, the overarching hypothesis is that the two classes of Oprm1 alternatively spliced variants (i.e., E1- and E11-associated variants) in the preBötC and PBN differentially influence OIRD. Aim 1 examines the role of E1- and E11-associated variants within two key brain regions modulating respiration, preBötC and PBN, on OIRD in rats, and will be completed in Year 1 of this fellowship training. Aim 2 identifies the expression of E1- and E11-associated Oprm1 variants within various cell types of the preBötC & PBN in rats (Year 2). To test these aims, we generated two gene targeted rat models, in which Oprm1 E1- or E11-associated splice variants can be conditionally deleted. Comprehension of the distinct roles of E1- and E11-dependent variants on OIRD in these two brain regions and in key cell types within these regions is expected at the end of this proposed project. Investigation of how these effects are altered by different opioids, doses, and sexes will also be assessed. The proposed study will advance our understanding of Oprm1 alternative splicing mechanisms and their role in OIRD, allowing us to develop therapeutic strategies to combat OIRD.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY In mammals, majority of nitrogen waste is produced by gut microbiome in the form of ammonia. Ammonia is a neurotoxin and is cleared by two major pathways: 1) conversion via the urea cycle enzymes (UCEs) into nontoxic urea for excretion; and 2) assimilation into glutamate (Glu) by glutamate dehydrogenase (GDH) then into glutamine (Gln) by glutamate ammonium ligase (GLUL), also termed glutamine synthetase (GS). In recent years, UCEs and Glu/Gln synthesis have been found to be involved in tumor growth/development in various tissues. Partially elevated UCE activities are implicated in tumor promotion via mechanisms such as enhanced pyrimidine and polyamine production. Likewise, Glu/Gln biosynthesis is generally thought to be tumor- promoting in various cancers. Curiously, while the liver is the main organ that handles ammonia waste, the roles of the two ammonia handling pathways in liver cancer remain systematically unexplored. In hepatocellular carcinoma (HCC), the major histological subtype of liver cancer, UCE expression is often down- regulated and correlates with worse prognosis, and GS expression is frequently elevated yet correlates with better outcomes. Using mouse models of HCC driven by oncogenic -catenin, we recently reported that - catenin led to decreased expression of UCEs and increased expression of GS. Increased ammonia levels were observed in the plasma and tumor interstitial fluids, which were exacerbated by the ablation of hepatic GS. These data indicate that oncogenic -catenin leads to the disruption of nitrogen homeostasis, and that defective ammonia-clearance promotes HCC growth. The main goals of this application are to study the roles of the UCEs in HCC, and to test the hypothesis that lowering ammonia burden can be a therapeutic strategy for HCC treatment/prevention. We propose three Specific Aims. Aim1: Test the hypothesis that suppression of UCE expression contributes to the growth of HCC driven by -catenin. This will be done by 1) overexpressing or knocking down UCEs in various HCC mouse models; 2) studying the mechanism how down-regulation of UCEs may promote HCC. Aim 2: Study how oncogenic -catenin regulates UCE transcription. We will test how -catenin interacts with other transcription factors such as C/EBP and HNF4a, and test how they interact with the UCE promoter/enhancer regions to regulate expression. Aim 3: testing that lowering ammonia burden can be a therapeutic/preventive strategy for HCC. This will be done by feeding mice with low protein diet and test the effect on growth signaling and HCC progression in vivo. Successful accomplishment of the project will determine the role of UCEs in HCC, identify hyperammonemia as a risk factor, and help establish dietary intervention as a strategy for treating/preventing HCC.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Asthma progression is associated with reduced growth and an increased decline in lung function (LF) that is thought to arise from a complex interplay of genes and environment. While several asthma genetic risk loci have been identified to-date, an in-depth utilization of how environmental factors interact with these loci remains limited. The methylome and the metabolome are both heavily influenced by the environment and recent studies confirm a link between both omes. Integrating DNA methylation with the metabolome could be a powerful approach to obtain converging evidence of specific pathways influencing asthmatic lung function trajectories at the genome-wide level. While previous studies have investigated the role of the metabolome or methylome with regard to lung function, none have investigated both “omes” simultaneously for lung function outcomes in asthmatics. The central hypothesis of this proposal is that DNA methylation (CpG sites) plays a critical role in modulating downstream metabolites and thus metabolic pathways, some of which may be driven by an underlying genetic effect on lung function in children with asthma. We want to specifically investigate multi-omic data from Wnt, Hippo and sphingolipid pathways, known to affect asthmatic lung function generally and in our preliminary data. We capitalize on the genetic, methylomic and metabolomic data generated from three large prospective childhood cohorts including two from the Trans-omics for Precision Medicine (TOPMed) consortium: Childhood Asthma Management Program (CAMP) and The Genetic Epidemiology of Asthma in Costa Rica (GECRA) study and an independent cohort: The Vitamin D Antenatal Asthma Reduction Trial (VDAART). This study seeks to integrate multiple omics using innovative and state-of-the-art methodologies: quantitative trait loci (QTL) mapping to identify genome-methylome associations with LF (Aim 1), QTL mapping and causal inference testing to evaluate methylome-metabolome associations with LF (Aim 2), correlation-based network methods to identify a highly correlated set of omic-driven biomarkers in dysregulated pathways (Aim 3). Priyadarshini Kachroo, PhD, MS is a bioinformatician whose long-term career goal is to transition towards becoming an independent data scientist with expertise in utilizing multi-omic approaches to complex disease phenotypes. As Dr. Kachroo completes these aims, her career development plan will support her training goals: 1) deepen clinical understanding of asthmatic LF phenotypes; 2) expand on the statistical skills including causal inference testing 3) develop skill-set of network methods for integrating multi-omic datasets 4) enhance skills in study-design, mentorship and the ethics of scientific conduct and communication of research. Dr. Kachroo’s strong quantitative and methodological background well position her to accomplish these goals, complete the aims of this proposal and prepare her for an independent research career. The support of a diverse and world- class mentoring and advisory team in the Channing Division of Network Medicine at Brigham and Women’s Hospital and Harvard Medical School complement the research areas and ensure success of this proposal.

NIH Research Projects · FY 2024 · 2024-07

SUMMARY/ABSTRACT Macrophages (Mφs) are innate immune cells, phagocytes and professional antigen presenting cells, which are crucial for tissue homeostasis and adaptive immune response regulation. The high phenotypic and functional plasticity and the high penetration rate in several solid tumor types including pancreatic ductal adenocarcinoma (PDAC) make Mφ cell therapy ideal for solid tumor treatment. To overwrite their immune suppressive program once infiltrated in solid tumors, Mφs can be engineered with chimeric antigen receptors (CARs) to redirect their specificity and function. Phosphatidylserine (PS) is a negatively charged amino-phospholipid mostly confined to the inner leaflet of the plasma membrane bilayers, but flipped to the outer membrane under lethal stress. PS is a universal “eat-me” antigen of apoptotic cells and often abnormally externalized by cancer cells. We hypothesize that PS targeted CAR Mφs (α-PS CAR-Ms) may be a universal “third-party, off-the-shelf” adjuvant cell therapy of traditional therapies for solid tumor treatment, because cancer treatment modalities such as targeted therapies can induce transient but massive tumor cell apoptosis. To verify it, we employed KRAS-addicted PDAC models for pilot studies and have created an α-PS CAR that can provoke antigen specific phagocytotic and cytotoxic activities in Mφs against KRAS inhibitor (KRASi)-treated PDAC cells. Due to structure conserveness of PS in human and mouse, α-PS CAR-Ms reacted with both mouse and human PDAC cells post KRASi treatment. In the project, we propose to demonstrate the anti-tumor effect of α-PS CAR-Ms as an adjuvant therapy of KRASi in human PDAC xenograft models and mouse spontaneous PDAC models and dissect the working mechanisms of α-PS CAR-Ms. Aim 1. Demonstrate α-PS CAR activities in human PDAC models. Aim 1a will generate α- PS CAR-Ms using human primary Mφs and perform functional assays in vitro to determine antigen-specific phagocytosis and cell killing. In addition, we will explore whether combination with inhibitors targeting natural PS receptors on Mφs and blockage of the “don’t-eat-me” signal will further enhance tumoricidal activities of α-PS CAR-Ms. Aim 1b will determine the polarization status of α-PS CAR-Ms by flow cytometry analysis and dissect the molecular reprogramming by -omic analysis. Aim 1c will demonstrate the tumoricidal activity of α-PS CAR- Ms as an adjuvant therapy of KRASi in human PDAC xenograft models. Aim 2. Determine the tumoricidal activity of α-PS CAR-Ms in immunocompetent mouse models. We hypothesize that murine α-PS CAR-Ms can not only kill PDAC cells directly but may also rewire the tumor immune microenvironment to further provoke anti-tumor immune response. Aim 2a will produce murine α-PS CAR-Ms and determine their phagocytotic and killing activities against KRASi-treated PDAC cells. After validation, we will examine whether α-PS CAR-Ms are effective to impair tumor growth and prevent tumor recurrence as an adjuvant therapy of KRASi in spontaneous PDAC mouse models. Aim 2b will characterize the TME remodeling by α-PS CAR-Ms via immunophenotyping and delineate the potential intercellular crosstalk within immune cell types.