Rutgers Biomedical And Health Sciences

NIH Research Projects · FY 2025 · 2021-06

Project Summary Tuberculosis (TB), caused by pathogenic bacteria Mycobacterium tuberculosis (Mtb), is causing significant morbidity and mortality to humans across the world. Live, attenuated M. bovis Bacillus Calmette-Guérin (BCG), is the only TB vaccine currently licensed by the World Health Organization for use in humans. Although BCG prevents severe disease in children with variable efficacy, it fails to protect against pulmonary TB in adults, who are the primary source of transmission of Mtb in the community. Moreover, BCG may cause disease in immune- compromised individuals, such as those co-infected with HIV. To control the development of active disease and to break the chain of Mtb transmission, a new, safer and more effective vaccination approach is urgently required. The development of “paradigm-shifting” protective measures against TB will significantly be aided by the optimization of safe and effective combinatorial platforms, such as integrating novel vaccines with adjunct host-directed therapy (HDT) and/or antimycobacterial drugs. This strategy is aimed at inducing appropriate innate immunity along with potent and durable T cell responses, both of which are necessary for effective control of TB. Such an integrated approach is urgently needed to control the pathology of active, cavitary TB cases and transmission of Mtb, as well as to prevent reactivation of latently infected individuals, estimated to be about a quarter of the world population, who are Mtb-infected and mostly asymptomatic but can reactivate the disease upon immune suppression. Selection and usage of a relevant animal model that recapitulates the pathophysiology of cavitary TB, as seen in humans is vital to screen novel and better intervention strategies to combat the disease, including potent vaccine and drug candidates. We have established a rabbit model of aerosol Mtb infection that mimics the range of human manifestations of pulmonary TB, from cavitary (transmissible) disease to latent infection. Dr. Subbian has established a rabbit model of cavitary TB and the sub-award PI, Dr. Kupz has developed a tractable and reproducible mouse model to study the reactivation dynamics of latent Mtb infection following the loss of CD4+ T cells as it occurs in HIV co-infected individuals. Using these two models, we propose to determine the ability of a novel recombinant BCG strain (BCG::ESAT- 6-PE25SS developed in Dr. Kupz lab), in combination with mTOR inhibitor (everolimus) and/or two first-line antibiotics, isoniazid and rifampicin, to protect against progression to cavitary TB (rabbit) and/or induce sterilizing immunity in latency (mice). To compare our approach, we will test individual components in these model animals. We will also define mucosal (lung) and systemic (blood) immune parameters that predict protection against Mtb challenge in our model system. The results of these studies can contribute towards the development of new generation vaccine platforms for targeting other intracellular pathogens, in addition to Mtb.

NIH Research Projects · FY 2025 · 2021-06

PROJECT ABSTRACT Anemia and inflammation often co-occur in chronic diseases including inflammatory bowel disease (IBD), infection, and cancer. Anemia is often refractory to treatment in these diseases, and the impact of anemia and anemia therapies on disease outcome is poorly defined. Accordingly, there is a significant medical need to better understand the causal links between anemia and inflammatory diseases. One of the best defined links between anemia and inflammation is a peptide hormone called hepcidin, which critically inhibits iron release from intracellular stores. Hepcidin levels typically increase dramatically during inflammation, and can cause one form of anemia termed anemia of inflammation (AI). Conversely, inflammatory diseases associated with heavy bleeding cause a distinct form of anemia known as iron deficiency anemia (IDA), in which hepcidin levels are suppressed. The fundamental focus of this research proposal is to: i.) investigate the impact of hepcidin on inflammatory disease, ii.) identify cellular populations expressing hepcidin and its partner ferroportin during inflammation, and iii.) determine the impact of iron modulation by distinct cellular population on the resolution of inflammatory diseases. I will employ innovative technical approaches and develop new tools to define the role of hepcidin and ferroportin during chemically-induced intestinal damage, intestinal infection, and in inflammation-induced cancer. Collectively, results from these studies will define the regulation and functional significance of novel cellular mediators of iron within the intestine. These findings will critically inform ongoing efforts to develop therapies targeting tissue repair and anemia in the context of inflammatory diseases.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY The purpose of this Mentored Research Scientist Development Award (K01) is to provide support for me to become an independent investigator with a multidisciplinary research program in HIV at the intersection of prescription drug use, comorbidities, and aging. In this project, I propose to leverage existing administrative claims data from Medicare beneficiaries ≥65 years of age from 2007-2018 to determine how high-risk prescription opioid use, e.g. high dose use (≥120 mg/day) or prolonged use (≥90 days), affects health outcomes and health care utilization in older people living with HIV (PLWH). Building on my strong foundation in HIV epidemiology and HIV comorbidities across the lifespan, the training from this K01 will allow me to (1) obtain proficiency in the analysis of large-scale, longitudinal administrative claims data; (2) attain content expertise in prescription drug utilization research and pharmacoepidemiology; and (3) develop expertise in the application of machine learning methods. My career development plan includes specific coursework, seminars, conferences, directed readings, and tailored mentoring from a multidisciplinary team comprised of experts in administrative claims data, substance use epidemiology, pharmacoepidemiology, and machine learning methods. Rutgers University provides an exceptional environment for completion of this training, with research support and infrastructure for analyzing large, multiyear datasets (including Medicare claims) and conducting high-impact HIV research. The proposed research is significant given that a substantial gap exists in understanding how prescription opioid use affects a growing population of aging PLWH who commonly report chronic pain, and have multiple comorbidities, increasing polypharmacy, and increased risk for untoward drug-drug interactions. This research is critical to target appropriate prevention and treatment programs to optimize health outcomes for this population. To fill this gap, specific aims are to: (1) Assess the associations between HIV infection and a) adverse health outcomes (e.g. falls/fractures, dementia, mortality) and b) health care utilization (e.g. emergency department use, inpatient hospitalizations, and outpatient visits) among older adults, and estimate the interaction between HIV infection and high-risk prescription opioid use on adverse outcomes and utilization; (2) Among older PLWH, estimate drug-drug interactions between high-risk prescription opioid use and specific antiretroviral drug classes or sedatives (e.g. benzodiazepines) on risk of adverse health outcomes and health care utilization; and (3) Examine the feasibility of applying existing machine learning approaches to predict adverse health outcomes and high health care utilization among older PLWH based on patient profiles and opioid prescription patterns in a large administrative claims database. Findings from this project will generate valuable new information and tools to support clinical decisions and more precisely target prevention and treatment interventions to improve health for older PLWH who are prescribed opioids, and directly inform an R01 application to study drug-drug and drug- disease interactions between widely used prescription drugs and common comorbidities among older PLWH.

NIH Research Projects · FY 2025 · 2021-06

ABSTRACT The endoplasmic reticulum (ER) of eukaryotic cells is an essential organelle with many critical functions including, protein secretion. Its function is closely tied to its morphology. Work in higher eukaryotes has shown that mutations in key proteins required to generate the ER tubular network cause specific growth and developmental defects. In contrast to higher eukaryotes, little is known of how the ER is shaped in early eukaryotes such as protozoa. ER structure in the protozoan parasite, Plasmodium is dynamic and stage-specific but its molecular determinants are unknown. To understand how the ER acquires its shape in different stages of Plasmodium, we identified homologs of key ER-shaping proteins including ones that contain a reticulon homology domain. One of these protein induces membrane curvature in vitro. P. berghei parasites lacking the protein have dysmorphic ER, an enlarged digestive vacuole, are severely attenuated in the asexual cycle but infect hepatocytes normally. We hypothesize that the putative Plasmodium ER-shaping proteins we identified have stage-specific roles in maintaining proper ER structure/function. This proposal will determine the contributions of these proteins in shaping the ER of erythrocytic and hepatic stages of Plasmodium, using morphological and ultrastructural studies of P. berghei gene-knockouts. It will determine the effect of their loss on a key ER function, protein trafficking in the parasite. Our study will provide the first causal link between ER architecture, protein trafficking and the ability of the malaria parasite to reside in different host environments.

NIH Research Projects · FY 2025 · 2021-05

Abstract There is ample evidence that homelessness is associated with inadequate access to essential health services and that African American, Hispanic/Latinx and rural populations are at high risk for homelessness and/or its consequences. However, there is insufficient knowledge about gaps in use of specific types of healthcare among homeless adults, whether such gaps are greater among minority and rural populations, and the potential of permanent supportive housing (PSH) programs to mitigate the gaps. This study aims to: 1) quantify the contribution of homelessness to gaps in essential health services use among Medicaid beneficiaries by race/ethnicity and rural residential status; 2) evaluate the extent to which the gaps are mitigated by placement in PSH programs; and 3) identify Medicaid and PSH policy and programmatic strategies for improving access to essential health services and reducing associated racial/ethnic and rural disparities. This study will overcome shortcomings in prior research by using novel large scale, population- based, long-term data, combined with integrated quantitative-qualitative research methods. The study will address the first two aims using 10 years (2011-2020) of linked homeless services and Medicaid administrative data for New Jersey and Pennsylvania. The contribution of homelessness to healthcare gaps and disparities will be measured by comparing utilization and spending for a broad spectrum of community-based and hospital health services among adults experiencing homelessness to matched Medicaid beneficiaries who have not been homeless. PSH will be evaluated by comparing trends in healthcare use and disparities by race/ethnic and rural residential status among those receiving housing placements to adults in similar circumstances who did not receive such placements. Specific actionable strategies for improving policy and practice will be identified in focus groups with front-line PSH professionals during which quantitative findings will be shared and discussed.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY/ABSTRACT Alzheimer's disease (AD) is the most common form of dementia in the elderly, and currently there exists no disease modifying therapy. Familial forms of AD (FAD) are caused by mutations in Amyloid Precursor Protein (APP), whose processing can result in the formation of amyloid beta (Aβ), or by mutations in Presenilin 1/2 (PSEN1/2), which comprise in part the γ-secretase complex that cleaves Aβ from fragments of APP. My long- term career goal is to study the mechanism of neurodegeneration in Alzheimer disease. The more proximate goal, as put forward in this proposal, is to characterize the neurodegeneration in a new rat knock-in model of Presenilin1 dysfunction. Psen1-knockout (Psen1-KO) mice and knock-in (KI) mice with homozygous FAD- associated L435F mutations (Psen1LF/LF) are embryonic and perinatally lethal, precluding a more rigorous examination of the effect of AD-causing Psen1 mutations on neurodegeneration. Given the better suitability of rats as a model organism, with regards to surgical interventions and behavior testing, we generated a rat KI model of the Psen1LF mutation. We find that, unexpectedly and in contrast to Psen1LF/LF, Psen1LF/LF rats survive into adulthood despite a loss of γ-secretase activity. The survival of these rats affords the opportunity to examine the effect of homozygous Psen1 AD mutations on neurodegeneration.

NIH Research Projects · FY 2026 · 2021-05

ABSTRACT. Influenza A virus (IAV) causes annual epidemics and dangerous pandemics involving millions of cases of illness and deaths worldwide. The main cause of pathology from IAV is excessive inflammation, therefore, the overarching goal of this proposal is to learn how mechanisms of inflammation can be manipulated to promote disease tolerance to virus infection. Cytokine production, a chief contributor of inflammation, is regulated at the post-translational level to balance between efficient antiviral responses and damaging inflammation. A major molecular regulatory mechanism involves ubiquitination of signaling components. The specific goal of this proposal is to identify mechanisms of regulation of inflammation by the ubiquitin (Ub) system during IAV infection in vivo. We recently reported that the E3-Ub ligase, TRIM6, catalyzes the synthesis of unanchored poly-Ub chains, which promote antiviral IFN-I responses. However, the role of TRIM6 in regulating other inflammatory cytokines is not known. We generated TRIM6 knockout mice (Trim6-/-), which provides a unique tool to identify novel immune pathways regulated by TRIM6 and unanchored Ub in vivo. Our preliminary data show that Trim6-/- mice have fewer signs of pathology even though there are increased IAV titers at early time points post-infection. We also found reduced expression levels of CXCL1, a well-known neutrophil chemo-attractant, which correlated with reduced neutrophil infiltration to the lungs of IAV-infected Trim6-/- mice. We found that TRIM6 and unanchored Ub form a complex with PI3K/AKT signaling components, and their phosphorylation is impaired in Trim6-/- cells. Our data also suggest that TNFα produced by infected cells induces pathogenic CXCL1 in bystander cells to recruit neutrophils. Neutrophils are known to be recruited to the lung during IAV infection and can play both protective and detrimental roles. However, what factors drive neutrophils to cause tissue damage during infection are not well-understood. Therefore, there is a gap in knowledge on the mechanisms of regulation of neutrophil recruitment and their roles in the balance between protective responses and pathogenic inflammation. Our hypothesis is that TRIM6 is activated by TNFα signaling and promotes early CXCL1-mediated pathogenic inflammation, thereby inhibiting disease tolerance. In Aim 1, we will determine the cellular source of TRIM6-induced CXCL1, and its role in neutrophil recruitment to the lungs, during IAV infection. We will demonstrate the role of early CXCL1 production in pathology and whether TNFα is involved in inducing TRIM6-mediated CXCL1. In Aim 2, we will determine the mechanism by which TRIM6 and Ub modulate the activation of PI3K-AKT for downstream signaling and how TRIM6 is activated during infection. The outcomes include the identification of the cellular source of pathogenic CXCL1, and the mechanism by which TRIM6 is activated for signaling. This information will guide the development of therapeutic approaches by targeting TRIM6 and CXCL1-producing cells to reduce inflammatory diseases.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY Macrophages underlie the progression of inflammatory diseases, including atherosclerosis, autoimmune diseases, chronic wounds and cancer. Metabolic rewiring of activated macrophages to alter their function has become an attractive therapeutic strategy, however in vivo evidence is lacking to support its therapeutic potential. The research proposed here will address the metabolic regulation of macrophage effector function in physiologically relevant settings by fluorescence lifetime imaging of metabolic coenzymes NAD(P)H and FAD, using zebrafish as an in vivo model of inflammation and wound healing. Importantly, this non-invasive imaging modality measures intracellular metabolic state while maintaining macrophages in their native microenvironment, unlike traditional approaches. This study will monitor changes in the metabolic profile of macrophages at various wound models over time, and test the functional requirement of metabolic regulators, Irg1, Stat3 and mitochondrial ROS, in macrophage-dependent wound healing. Metabolomics analysis will also be performed using wounded tissue to gain further mechanistic insight into the metabolic reprogramming of macrophages in vivo. Collectively, this study will develop imaging-based tools to probe in real time the temporal and spatial metabolic regulation of immune cell functions in live animals that can inform development of new therapies to mitigate macrophage-mediated inflammation. The proposed study in this career development award application will be conducted under the primary mentorship of Dr. Anna Huttenlocher and the co-mentorship of Dr. Melissa Skala during the K99 phase of the award. The Huttenlocher lab at UW-Madison is an ideal environment for these studies due to the leading expertise in leukocyte biology in inflammation and wound healing, as well as live imaging strategies in zebrafish embryos. Dr. Skala is a leader in developing autofluorescence lifetime imaging of metabolic coenzymes and its applications. I will benefit immensely from the mentorship of Drs. Huttenlocher and Skala, and will bridge the expertise of both mentors toward elucidating the mechanisms of metabolic reprogramming of macrophages in complex in vivo environments. I am committed to a career as an independent investigator at an academic institution studying leukocyte biology; in particular, studying the metabolic regulation of leukocyte effector functions in context of inflammatory disorders. In addition to the already excellent resources I am afforded by my mentors, UW-Madison provides a wealth of other scientific and career development opportunities to support my academic growth. Moreover, I am actively involved with the metabolic community at the Morgridge Institute for Research, located on UW-Madison campus, to further enhance my training in immunometabolism. Taken together, my mentors and UW-Madison provide an ideal environment to fully support my scientific pursuits and ensure that I achieve my long term career goals.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY This goal of this project is to create a unique and comprehensive research repository of aging trajectory da- tasets, related resources, and analytic methods that can be used to answer new and important questions in aging and related sciences. Specifically, by harmonizing and merging multiple data sets this project will gener- ate the data infrastructure needed to understand change over time in care settings, geriatric syndromes, physi- cal functioning, and shared risk factors at multiple levels (patient, provider, community, healthcare system, and society) and across multiple domains (biological, behavioral, sociocultural, and physical/built environments) including chronic conditions and history of acute illness such as COVID-19, exposure to air pollution, neighbor- hood socioeconomic, and health care system factors (Aim 1). Analytic strategies will be developed for user- defined cohorts and their propensity score-matched controls, e.g., older adults who were living with chronic conditions including Alzheimer's disease and related dementias (ADRD), diabetes, heart failure, end-stage re- nal disease, metastatic cancer, and HIV. State-of-the-art analytic methods are used to identify patterns of ag- ing trajectories (care setting, geriatric syndromes, physical functioning) experienced by older adults during the final years of life and their association with shared risk factors and distal outcomes (Aim 2). From the assem- bled trajectory file in Aim 1, cohorts are derived by aligning an originating index time such as age cutoff point and time at diagnosis (e.g., ADRD, stroke, chronic kidney disease). Both a model-based approach and ma- chine learning algorithms are then used to discover multilevel and potentially interactive predictors of trajecto- ries (e.g., rapid functional decline in independent living beneficiaries) and specific outcomes (e.g., respiratory ventilator usage among Medicare beneficiaries diagnosed with COVID-19) (Aim 3). The unique resources are then shared to disseminate resources including datasets, documentation, source code, and methodology (Aim 4). At the end of this project, the research infrastructure to investigate the relationship between shared risk factors and aging trajectories will be ready to use and replicate, giving investigators unprecedented ability to solve new challenges in aging science. This will allow researchers to understand the underlying processes and systems associated with reversible periods of disability across care settings, and interventions that may be used to support recovery of function and reduction of geriatric syndromes including cognitive decline, for the purpose of reducing burdensome care transitions, and maintenance of functional independence. This project will also create the resources and methods needed to evaluate the impact of innovations and interventions im- plemented at the patient, provider, community, healthcare system, and society/policy levels to improve care quality and outcomes for older adults.

NIH Research Projects · FY 2025 · 2021-05

Mechanisms of the BRCA-network in Tumorigenesis and Therapeutic Response (OVERALL) Abstract: The faithful repair of DNA damage and efficient resolution of stalled replication forks are fundamental mechanisms by which mammalian cells maintain DNA sequence fidelity and chromosomal integrity during DNA replication and in response to exogenous DNA damage. Defects in DNA repair mechanisms not only contribute to genomic instability and subsequent tumorigenesis, but also can alter the epigenetic landscape of cells and impart therapeutic vulnerabilities that can be exploited clinically. The investigators participating in this P01 project share common interests in understanding the mechanisms by which cells maintain genomic integrity to suppress tumorigenesis, and in exposing tumor vulnerability to therapy based on mechanistic understandings of the genomic consequences of impaired DNA repair. One particularly strong area of research offered by this team is a multi-disciplinary approach to understanding the basic mechanisms by which the BRCA1-PALB2-BRCA2 complex and associated regulators participate in regulating DNA replication and repair choice. Regulators of the BRCA-network include, 53BP1, RNF4, BARD1, TOPBP1, EHMT2 (G9a), MCM10, SLFN11 (mouse Slfn9), and BCCIP. Some of these factors have been a long-standing research focus for investigators in the project team. The research collaboration is formed around the central themes of how members of this large network of proteins interface with each other to maintain genome integrity, suppress tumor development and modulate tumor response to cancer therapy. Four projects, two Shared Resource Cores, and an Administrative Core are proposed to achieve three scientific goals: 1) to reveal novel mechanisms by which the recruitment and function of the BRCA1-PALB2-BRCA2 network is regulated by chromatin context mediated by methylation, sumoylation, and ubiquitination; 2) to refine the roles of the BRCA network in DNA replication, tumor suppression and define the genomic consequences of BRCA dysfunction; and 3) to explore new opportunities to target defects in the BRCA network for therapeutics in medulloblastoma and breast cancer.

NIH Research Projects · FY 2025 · 2021-04

Project Summary Cardiovascular disease (CVD) is a major health concern facing the U.S. population, with the impact of risk factors accumulating over the lifespan and beginning well before older age. Increasingly, the study of cardiovascular health for people living with HIV is vital given the growing number of middle- and older-aged adults living with HIV as a chronic condition and its complex effects on cardiovascular health directly, and indirectly through HIV's impact on inflammation. In the general population, poor or inadequate sleep has been linked with increased risk for CVD, in part through its impact on inflammation and other physiological mechanisms. In our own work, among sexual minority men living with HIV, poor sleep has also been linked with lower rates of adherence to antiretroviral (ART) medications. Therefore, the study of cardiovascular health among people living with HIV requires special consideration of the pathways from poor sleep through worsened HIV health to increased CVD risk both directly, and indirectly through inflammation (principally, IL-6, TNF-alpha, and CRP). Further, we hypothesize that, contributing to this larger picture of physiological pathways from sleep to CVD risk, is a day-level psychosocial-behavioral dynamic whereby experiences of minority stress affect subsequent ART adherence through the impact of minority stress on sleep. Accordingly, in the proposed observational, longitudinal study of 240 racially-diverse sexual minority men living with HIV, aged 45-64, we aim to use longitudinal data to test the hypotheses that poor sleep longitudinally predicts greater CVD risk among SMM-LWH aged 45-64, in part through the impact of poor sleep on HIV health, and in part through the impact of poor sleep on inflammation. Additionally, we aim to test the day-level hypothesis that the impact of multiple, intersecting sources of minority stress (sexual minority stress, racial/ethnic minority stress, and/or HIV-related stress) on next-day ART adherence in SMM-LWH operates, in part, through the day-level impact of minority stress on poor sleep. This proposed study aligns with the growing recognition of the importance of sleep in numerous mental, behavioral, and physical health outcomes, and also contributes to our understanding of how minority stress “gets under the skin” (here, through its impact on sleep) to affect physical health outcomes in individuals with marginalized identities.

NIH Research Projects · FY 2025 · 2021-04

Project Abstract Long noncoding RNAs (lncRNAs) regulate gene expression. Peripheral nerve injury dysregulated their expression in the pain-related regions including dorsal root ganglion (DRG). However, the role of most identified lncRNAs in neuropathic pain is still uncertain. Identifying novel lncRNAs and exploring their contribution to neuropathic pain may provide novel strategies for management of this disorder. We recently used a next generation RNA sequencing approach and identified a large, native, full-length non-coding RNA in mice and human. Because it is expressed highly in the DRG, we named it as DRG specific long noncoding RNA (DS-lncRNA). Our preliminary data revealed that peripheral nerve injury downregulated DS-lncRNA likely due to a decrease in the expression of a transcriptional activator Pou4f3 in the injured DRG. Rescuing this downregulation attenuated the nerve injury-induced pain hypersensitivity, likely through blockade of the increased interaction between RALY (a transcription co-factor) and the RNA polymerase II (RNA II) and consequent silence of the RALY/RNA II-triggered expression of Ehmt2 mRNA and its coding protein G9a (a key player in neuropathic pain) in the injured DRG. Given that DS-lncRNA can directly bind to RALY, our preliminary results indicate that DRG DS-lncRNA downregulation is required for neuropathic pain likely through negative regulation of DRG RALY/RNA II-triggered G9a expression. This proposal will further examine whether and how DS-lncRNA contributes to neuropathic pain. In Specific Aim 1, we will first investigate whether rescuing downregulation of DS-lncRNA in the injured DRG attenuates neuropathic pain development and maintenance. We will then examine whether mimicking nerve injury-induced downregulation of DRG DS-lncRNA leads to neuropathic pain symptoms in the absence of nerve injury. In Specific Aim 2, we will examine whether peripheral nerve injury results in time-dependent downregulation of DS-lncRNA and its transcription factor Pou4f3 in the DRG. We will also examine whether DS-lncRNA downregulation is attributed to a decrease of Pou4f3 expression in the injured DRG after peripheral nerve injury. In Specific Aim 3, we will test the effect of DS-lncRNA on the expression of Ehmt2 mRNA, G9a and their downstream pain-related genes in the injured DRG after peripheral nerve injury. We will also determine whether DS-lncRNA downregulation enhances the binding of RALY to RNA II leading to RALY/RNA II-triggered Ehmt2/G9a increase and G9a-controlled pain-related gene decrease in the injured DRG after peripheral nerve injury. Our study will likely identify a previously unknown regulatory mechanism for neuropathic pain. Given that virus- mediated gene therapy has been used in clinical trial, the present study will have a potential clinical application in neuropathic pain management.

NIH Research Projects · FY 2025 · 2021-04

Rutgers Cancer Institute of New Jersey (RCINJ) provides an outstanding environment for training in basic, clinical, behavioral or population-based cancer research and brings together an exceptional group of investigators organized in multi-disciplinary research teams focusing on a broad spectrum of cancer research. The Cancer Metabolism and Growth (CMG) Program, at RCINJ has robust research strengths bolstered by strong collaboration between researchers at Rutgers and our formal consortium partner Princeton University. The proposed program leverages these unique research strengths and resources of RCINJ to provide training in translational research in Cancer Metabolism and Growth and Tumor Host Interactions. The primary goal of this T32 training program is to provide postdoctoral candidates with the highest quality training and research experience so that they are competitive in developing research careers in academia, government, and the private sector. To achieve this goal, trainees will engage in mentored cancer research, co-curricular and professional career development activities for two years. Program candidates are Ph.D. recipients who wish to develop careers in cancer research. The program will recruit a cohort of 2 new trainees each year from year 1-5. Thus, the program will have 2 trainees in year 1 and 4 trainees in year 2 through 5 of the training grant. High achieving post-doctoral fellows (evidenced by research productivity in the form of number of publications, fellowship awards, presentations etc.), may be supported for an additional third year of training through institutional funds. Faculty members with active, well-funded research programs and extensive mentoring experience will support program trainees. All of the faculty mentors chosen for this program have significant R01-level peer reviewed funding in tumor metabolism and host interactions focused research. Experienced leadership team with track-record of research productivity and mentoring will ensure successful implementation of the program. Both formative and summative evaluation will be integral parts of the proposed program. We will annually track the educational and professional activities of trainees for at least 10 years after completing research training. We will disseminate results from the comprehensive evaluation on the program website. Additional dissemination will occur through articles published in peer-reviewed journals and through presentations at regional and national conferences, by program staff as well as participants. Our program benefits from the multi-disciplinary research environment and robust educational resources available at RCINJ (the state's only NCI designated Comprehensive Cancer Center) as well as the consortium relationship with Princeton University. 19

NIH Research Projects · FY 2025 · 2021-04

Tumor suppressor p53 plays a central role in tumor prevention. p53 is frequently mutated in human cancer, including colorectal cancer (CRC). Many mutant p53 (mutp53) proteins not only lose tumor suppressive function of wild-type p53, but also gain new oncogenic activities to promote tumorigenesis, which is defined as mutp53 gain-of-function (GOF). Maintaining metabolic homeostasis is a novel and critical mechanism of p53 in tumor suppression. Cancer cells often display lipid metabolic reprogramming, which contributes greatly to cancer progression. Currently, the role and mechanism of mutp53 in cancer metabolic reprogramming are poorly defined. Our preliminary studies suggest that mutp53 drives lipid metabolic reprogramming as a critical GOF in CRC cells, and targeting lipid metabolic reprogramming compromises mutp53 GOF in colorectal tumorigenesis. Based on our preliminary results, we hypothesize that GOF mutp53 drives lipid metabolic reprogramming as a critical mechanism to promote colorectal tumorigenesis, which can be targeted for therapy in CRC carrying mutp53. In this proposed study, we will determine the role (Aim 1) and mechanism (Aim 2) of GOF mutp53 in driving lipid metabolic reprogramming in CRC. We will further assess targeting mutp53-driven lipid metabolic reprogramming as a potential therapeutic strategy for CRC carrying mutp53 (Aim 3). The goal of this study is to determine the mechanism of GOF mutp53 in CRC to provide effective targets and strategies for CRC therapy. Metabolic reprogramming and p53 mutations are common events in cancer, and have become extremely attractive targets for cancer therapy. We expect that the results from this proposed study will deepen our understanding of the role and mechanism of mutp53 in metabolic reprogramming and tumorigenesis, and provide the rationale and base for the development of new therapeutic targets and strategies for cancers carrying mutp53.

NIH Research Projects · FY 2025 · 2021-04

Project Summary: Ovarian cancer (OC) is associated with the highest mortality rate of all gynecologic malignancies in the United States. The low rate of survival is mainly due to two factors: 1) the advanced stage of the disease at diagnosis, and 2) the inadequate efficacy of available therapeutic options, especially for recurrent metastatic disease. The standard-of-care for patients with primary OC includes debulking surgery (removal of ovaries and visible intraperitoneal tumors) followed by chemotherapy with platinum-based drugs (e.g., cisplatin) and paclitaxel (PTX). However, approximately 90% of patients after suboptimal resection and 70% of patients after optimal cytoreduction will experience relapse within 18-24 months. Unfortunately, there is no effective standard-of-care for recurrent patients who return to the clinic with drug-resistant metastatic disease. As a result, their survival rate is very low. The objective of this research is “to develop a non-surgical, targeted, and clinically translatable stem cell-based platform that can overcome drug resistance in recurrent and metastatic ovarian cancer”. The success of the developed stem cell-based platform will be measured by not only demonstrating the eradication of metastasis and inhibition of relapse, but also providing long-term survival benefits. To achieve this objective, we genetically engineered and isolated a unique adipose-derived stem cell (ASC) clone that overexpresses secretory human carboxylesterase 2 for targeted enzyme/prodrug therapy of cancer, and nanoluciferase for quantification of response to therapy and evaluation of cancer relapse. Using bioluminescent imaging (BLI) complemented with magnetic resonance imaging (MRI) and immunohistochemistry, we demonstrated that the engineered ASCs migrate and localize at both ovarian tumor stroma and necrotic regions. Our published data also show that the engineered ASCs are able to target and kill the drug-resistant OC cells that are rich in cancer stem-like cells (CSCs), overexpress MDR-1/ABCG2 drug efflux pumps, and have high ALDH enzyme activity. Statistical analyses of tumor burden and survival rates showed that administration of the engineered ASCs in combination with the prodrug irinotecan provided complete tumor response and survival benefits in 80% of treated mice. To transform this ASC-based technology into a platform with a broad application in targeted therapy of recurrent OC, we will use epithelial OC cells that are obtained from patients who have received various treatment modalities but have returned to the clinic with drug-resistant disease. The biodistribution and tumor tropism of the engineered ASCs will be determined by BLI, MRI, and immunohistochemistry. The tumor response to therapy, inhibition of cancer relapse, and long-term survival benefits will be determined in immunocompromised mice. Tumor tissues from non-responsive groups will be collected and characterized at molecular, cellular and genomic levels to understand the mechanisms underlying their escape and to help develop corrective measures. Adverse effects during treatment and toxicity to healthy tissues will be studied by histopathology & hematology.

NIH Research Projects · FY 2025 · 2021-03

PROJECT SUMMARY/ABSTRACT. This Mentored Research Scientist Development Award (K01) proposal will develop Dr. Greta Bushnell’s career as an independent investigator, and enable her to advance into a new line of research focused on evaluating misuse and abuse related morbidity of medicines prescribed to young people and the policies and practices that can mitigate these harmful outcomes. This new direction of research requires distinct training in substance abuse policy, misuse and abuse liability of prescription drugs, adolescent development of substance use, analysis of state-level policies, and advanced methods to address confounding control. With a training program supported by an experienced team of investigators, Dr. Bushnell will complete the proposed research, filling key gaps surrounding benzodiazepine (BZD) misuse and abuse related harms in young people and modifiable factors to reduce these harms. This research focus comes at a pertinent time of rising BZD-related morbidity, continued prescribing, lack of safety data in youth at developmental risk for substance use problems, and current opportunities to leverage opioid-related policies to reduce BZD-related harms. Overdose deaths involving BZDs significantly increased in recent years with »11,500 deaths in 2017, partially attributed to the opioid crisis, and in adolescents and young adults there were »39,000 emergency department visits for BZD-related poisonings in 2016. Despite BZDs being frequently prescribed to adolescents (3%) and young adults (6%), there are no estimates on overdose risk following BZD treatment and it is unknown whether and to what extent BZD prescribing to youth inadvertently increases risk of BZD or other substance misuse later in life. Prescription drug monitoring programs (PDMPs) represent a potentially important, but understudied, means to mitigate BZD- related morbidity. PDMPs are state-run surveillance systems collecting patient details on prescriptions for controlled substances with capabilities to detect risky prescribing. Dr. Bushnell will use large, national administrative claims data (2000-2018) covering privately and publicly insured young people (10-29 years) to evaluate whether selected state PDMP features decrease BZD-related harms and risky BZD prescribing in young people (Aim 1). To evaluate individual-level risks, she will quantify overdose risk following BZD treatment in young people compared to alternative treatments and by prescription details (Aim 2) using the national claims datasets with measures from Area Health Resources Files. She will then evaluate whether adolescent BZD treatment increases downstream risks of BZD misuse or harmful substance use in early adulthood (Aim 3) using the nationwide Monitoring the Future sample of high school seniors with panel surveys into early adulthood (»2,450/year, 1976-2016). The proposal will inform an R01 submission on identifying youth-specific risk factors in the progression from treatment with BZDs, or other controlled substances, to abuse and leveraging this knowledge to inform interventions. Addressing these questions and developing this new line of research are only feasible with intensive training, support, and protected time, which the K01 can provide.

NIH Research Projects · FY 2025 · 2021-03

Project Summary/Abstract The current opioid epidemic is a major health crisis that has contributed to decreased life expectancy in the U.S. A main cause of morbidly and mortality is opioid reuse and relapse in chronic cases. Understanding the neurocognitive mechanisms and factors underlying reuse vulnerability is thus a pressing need. Leveraging a novel combination of neurocognitive tools and a multi-session longitudinal design, our recent work in opioid use disorder (OUD) has begun to delineate a precise decision making mechanism for opioid reuse by showing that treatment-engaged patients are at higher risk for reuse when they exhibit increased tolerance of unknown probabilistic outcomes (ambiguity tolerance) in a financial choice task (Konova et al., 2019 JAMA Psychiatry). But why patients become more tolerant of ambiguous uncertainty in periods preceding reuse remains unknown. One potential explanation consistent with decision theory is that, in these periods, they become overoptimistic about ambiguous outcomes, which leads them to overestimate the probability of good outcomes (or underestimate bad outcomes) when faced with a decision to reuse, and therefore more likely to do so. Here, we propose a multi-level, convergent test of this framework by using well-defined, quantitative measures of this presumed “optimism bias”, alongside quantitative measures of uncertainty tolerance, which we propose to collect with concurrent high-resolution fMRI recordings, and yoked to longitudinal clinical assessments. In Aim 1, we aim to establish the relationship between uncertainty tolerance and optimism bias in patients with OUD and matched controls by studying these behaviors across a set of choice and estimation tasks (the latter designed to capture optimism about simple financial and more complex outcomes, tapping into drug-choice-relevant domains such as health outcomes). We also examine for potential moderation by various psychopathological dimensions in a large, unselected population of online (MTurk) subjects. In Aim 2, we collect fMRI data during the same choice and estimation tasks to delineate the mechanism by which optimistic neural representations of uncertainty might drive behavioral tolerance of this uncertainty, and reuse, in OUD. In Aim 3, we use a multi- session longitudinal design to understand the interaction between optimism bias and uncertainty tolerance as they relate to opioid reuse, session-to-session, allowing us to elucidate the specific timescale and nature of this interaction. With this project we aim to provide an answer to why patients become more uncertainty tolerant in periods preceding reuse and, in doing so, hope to uncover an upstream mechanism (centered on optimism bias) of this vulnerability, including its neural implementation. In addition to this conceptual advance, this work will provide a novel set of cognitive tools to precisely and objectively measure these processes with potential to predict poor outcomes such as reuse, in a way that can be easily implemented in clinical settings. Finally, the findings from this work will inform novel therapeutic interventions by providing precise neurocognitive targets, as well as their ideal timing, with the goal of mitigating reuse risk and improving long-term patient outcome.

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY/ABSTRACT Congestive heart failure is one of the leading cause of morbidity and mortality in the USA and the World. However, despite the major advancements in research and therapeutic developments, there has been no improvement in death rates over the years. This necessitates reinvestigating the basic mechanisms that govern the progression heart failure. Adaptation of gene expression is the earliest fundamental response during overload. We have shown the widespread regulatory influence of promoter-proximal RNA polymerase II (pol II) pausing on gene transcription in heart. However, the underlying mechanisms that control and synchronize the release of paused pol II for active transcription are unclear and conflicting, and its contribution to development of cardiac hypertrophy and failure still unknown. Negative elongation factors (Nelf), comprising of five subunits (NelfA to NelfE) has been implicated in pol II pausing, with NelfA subunit identified as an essential component for pausing. Our preliminary data shows increase in NelfA expression with cardiac hypertrophy, which is required for compensatory increase in gene expression in these hearts. Conversely, NelfA levels decline in failing hearts, suggesting downregulation of NelfA could be contributing to decompensation and progression of failure. Our genome-wide sequencing data shows widespread NelfA occupancy on active promoters including inducible and constitutively expressed essential genes. Interestingly, immunoprecipitation of chromatin bound NelfA shows association with chromatin remodelers and pre-mRNA processing proteins. In this study, we investigate the role of NelfA in pol II dynamics, chromatin remodeling and gene expression, and its impact on progression of heart failure. We have hypothesized that adaptation of gene expression during cardiac hypertrophy is achieved by phosphorylation dependent increase in the rate of clearance of paused pol II from essential gene promoters, and de novo recruitment of NelfA and assembly of paused complex at inducible promoters. Loss of NelfA results in disrupted paused complex, altered chromatin remodeling, inefficient transcript processing and inhibited gene expression that precipitates heart failure. We have proposed two robust specific aims to test our hypothesis – 1. To examine the mechanisms regulating NelfA -dependent pol II pausing in gene transcription during cardiac hypertrophy. 2. To investigate the effects of loss of NelfA on gene expression and progression of heart failure in conditional NelfA-KO model subjected to pressure overload.

NIH Research Projects · FY 2025 · 2021-02

Regulation of Ku70 Methylation and Functions by SETD4 (Abstract) The objective of this study is to elucidate a novel regulatory mechanism of Ku70 functions that are controlled by lysine methylation. Ku70 is a critical protein in DNA damage repair, especially during the initiation of non- homologous end-joining after irradiation. This function is carried out by its dimerization with Ku80 and encircling of DNA at the break sites. The free form of Ku70 is known for its anti-apoptosis activity in the cytoplasm, due to its binding with the pro-apoptosis protein BAX. Our preliminary studies suggest that SETD4, a putative non- histone methyl-transferase, methylated Ku70 to cause Ku70 relocation to the cytoplasm. Over-expression of SETD4 suppressed apoptosis, while SETD4 depletion sensitized it. SETD4’s chromatin-binding was dependent on Ku70, but not vice versa. SETD4 can be recruited to DNA damage sites, but only at a relatively mid-late time point after DNA damage. Based on these novel findings, we hypothesize that Ku70 methylation by SETD4 plays a critical role for the functional translocation of Ku70 from DNA double strand breaks (DSB) to the cytoplasm. We have generated highly specific antibodies against methylated Ku70 and SETD4, and several Ku70 and SETD4 knock-in mouse lines. We strive to use a combined approach that integrates biochemistry, cell and molecular biology, and mouse genetics to test our hypothesis. In Aim 1, we will focus on Ku70-methylation and its anti-apoptotic and DNA repair activities. First, the consequence of Ku70 methylation on Ku70/Ku80 dimer stability and its binding to DNA will be determined. Second, the cytoplasmic activity of methylated Ku70 in apoptosis will be verified with non-DNA damaging agents. Third, the direct effect of Ku70 methylation on Ku70 recruitment and retention at DNA damage sites, DSB repair efficiency, and cellular sensitivity to ionizing radiation will be measured. Lastly, we will use in-house developed Ku70 knock-in mice to characterize the functions of the lysine-containing SAP domain of Ku70 and its methylation in vivo. In Aim 2, we will focus on how SETD4 regulates apoptosis and DNA damage response through Ku70. First, we will identify the structural elements that are critical for SETD4 to methylate Ku70. Second, the consequence of SETD4 modulation on apoptosis will be measured in cells incapable of Ku70 methylation. Third, we predict that, while Ku70 is required for SETD4 recruitment to DNA damage sites, the SETD4’s enzymatic activity may be required for Ku70 disassociation from DNA damage sites. Thus, the mutual roles of SETD4 and Ku70 on their recruitment and/or retention at DNA damage sites will be determined, and their effects on DNA repair will be assessed. Lastly, we have tagged the floxed mouse Setd4 allele with V5 and Flag (V5F) epitopes. We will use this mouse line to systematically analyze SETD4’s role in Ku70 methylation, and its subsequent contributions in development and tumorigenesis. These studies are expected to elucidate a previous unknown mechanism that coordinates Ku70 functions in the nucleus and cytoplasm. The success of this project is ensured by our unique reagents and animal models as part of a rigorous approach.

NIH Research Projects · FY 2025 · 2021-02

Abstract/Summary This T32 IMSD proposal aims to increase the pool of PhD scientists from underrepresented (UR) backgrounds who contribute and have a significant impact on the health-related research needs of the nation. The proposal builds on an R25 award, managed (1996-2013) by the former University of Medicine & Dentistry of New Jersey, and since 2016 by Rutgers Biomedical and Health Sciences. From 1996-2013, the program enrolled 89 students, of whom at least 86% are pursuing a research, science and/or medicine career. Since 2016, IMSD appoints and trains UR students from 9 graduate programs (GPs) representing all biological/biomedical disciplines across the Rutgers-New Brunswick campuses, with 21 students currently in training. The IMSD partner programs have a robust pool of eligible applicants, committed and productive faculty members, and resources to provide outstanding cutting-edge training in health-related disciplines relevant to the NIH mission. The GPs provide their trainees broad knowledge and research experiences needed to advance in their chosen fields, coupled with rigorous research design, data analysis and interpretation. Students are trained to conduct research responsibly, ethically and with integrity, and to think critically and independently. IMSD will play a central role in providing support, complementary to that of the individual GPs, to promote the success of UR students, whether IMSD-funded or not. We are requesting 5 NIH-funded predoctoral positions per year for 5 years, with 2 years of support for each trainee. The IMSD trainees are integral to the community of UR predoctoral recruits who are funded using university and GP resources. Aim 1 is to recruit, train and graduate with PhD UR trainees, with important metrics, such as retention, time-to-degree and publication rate, comparable to or better than non-UR American trainees, used to measure success. To this end, IMSD will provide: (1) close mentoring/advising; (2) a 0- credit, required course in years 1-4 that will focus on annual student research presentations and related professional and lay communication skills development; (3) career and skills development workshops; and (4) an annual research symposium for trainees and mentors. IMSD activities will reinforce student training in responsible conduct of research and standards/methods related to rigor and reproducibility. In Aim 2 we will implement evidence-based and/or pilot strategies that improve the training and persistence of UR graduate students, build scientific and professional identity, and increase awareness and readiness for transition to productive and successful careers in biomedical research and research-related careers. Programs will focus on: (1) student wellness; (2) development of writing and communication skills, including submission of a fellowship application; (3) participation in career-oriented programs; and (4) community engagement. In addition to the direct impact on our trainees, the IMSD, through its inclusion of all UR students from GPs across the campus, will contribute strongly to a sense of community and belonging.

NIH Research Projects · FY 2025 · 2021-01

ABSTRACT The uterine circulation and placenta are specifically designed to regulate the flow of blood and transport of es- sential nutrients to the fetus. Disruption of maternal hemodynamic regulation during pregnancy can adversely impact fetal health, resulting in miscarriage and intrauterine growth restriction (IUGR). Current treatment op- tions for IUGR patients are extremely limited, focusing primarily on early delivery; thus, putting the mother and child at risk for complications associated with preterm birth. Epidemiological studies indicate that pregnant women exposed to fine particulate matter (PM) have a heightened risk of fetal loss and development of IUGR. We have reproduced this phenomenon in laboratory rodent models, wherein animals exposed to nanosized titanium dioxide (nano-TiO2) aerosols develop IUGR and suffer a greater number of ‘miscarriages’ (fetal reabsorptions). We have demonstrated that acute and chronic exposures significantly impair uterine vascular endothelium-dependent dilation, severely limiting maternal-to-fetal blood flow and impacting fetal growth. An understanding of the mechanisms underlying dysregulation in uterine and placental blood flow is critical for developing treatments and reducing IUGR. Based on previous findings, we hypothesize that maternal inhalation of nano-TiO2 aerosols during pregnancy promotes the development of IUGR by disrupting endothelium-dependent NO and AA signaling cascades, resulting in reduced uterine vasodilation and blood flow. Moreover, folic acid (FA) supplementation will rescue this utero-placental hemodynamic imbalance and prevent IUGR through its action in NO signaling. Using novel approaches and methodolo- gies, these studies will: (1) evaluate uterine nitric oxide-driven vasodilation, (2) determine whether alterations in arachidonic acid metabolism impair uterine vascular reactivity and impact placental perfusion, and (3) assess the therapeutic benefit of dietary folic acid supplementation to improve utero-placental blood flow and attenuate the development of IUGR after maternal exposure to nano-TiO2 aerosols. These studies are conceptually innovative as we will utilize our unique resources to identify mechanistic targets within the utero-placental mi- crocirculation and test directed nutritional interventions for IUGR. This work is technically innovative as we will use novel methodologies developed for the evaluation of environmental toxicity in maternal-fetal medicine. Overall, the successful completion of these studies will: (1) create the conceptual framework to identify environmental exposure as a risk factor for the development of IUGR; (2) reveal new mechanistic insight into the vascular pathogenesis resulting from nanomaterial exposure; (3) provide a molecular basis to identify how nanomaterial exposure manifests as vascular disruptions; and (4) identify mechanistic targets for therapeutic strategies to ameliorate microvascular dysfunction and improve utero-placental blood flow. These intervention- al strategies are not only limited to PM, but are widely applicable to understanding the role of a spectrum of environmental toxicants in the pathophysiological development of IUGR.

NIH Research Projects · FY 2025 · 2020-09

Project Summary – Restoring Sight to the Blind: Neural Imaging with Retinal Prostheses Retinal prostheses restore sight to the blind by electrically stimulating still viable cells in the retina. These devices consist of a microstimulator array attached to the retina that is driven by video input from a glasses-mounted camera. Retinal prostheses have been shown to restore basic visual functions such as the recognition of shapes and rudimentary navigation. However, patients show significant variability in visual skills, and currently fall short of expected visual capabilities. The postdoctoral research proposed in this study focuses on explaining these patient limitations by investigating the sensory reorganization that occurs during blindness. Neural reorganization during blindness enables auditory and tactile tasks to be processed in visual brain regions. This type of reorganization in brain sensory regions was shown to be a key limiting factor in the use of the cochlear implant for artificial audition. In Aims 1 and 2 of this proposal, Dr. Noelle Stiles will evaluate blindness- induced cortical reorganization and adaptation with neural imaging in retinal prosthesis patients, which in turn could allow for better selection of patients for visual restoration. Dr. Stiles’ postdoctoral work is also focused on investigating the interaction of artificial vision with the natural senses, such as audition. Her research has already shown that artificial vision influences auditory localization in ways similar to natural vision’s influence on audition. In Aim 3, she will expand this research to determine whether enhanced auditory and tactile perception developed during blindness are retained or reduced following the restoration of vision. This project will provide a more complete understanding of blind brain reorganization and the effects of artificial vision. Aim 1 will be completed during the K99 phase (pilot data collection is complete). Aims 2 and 3 will continue through the K99 and R00 phases, allowing for fellow training and data piloting. The proposed research is designed to prepare Dr. Stiles for successful transition to a tenure- track faculty position. She will receive training in structural neural imaging data collection, processing, and visualization from Prof. Yonggang Shi. She will also be trained in ophthalmological retinal imaging by her principal mentor Prof. Vivek R. Patel at USC, and in biomedical engineering by her co-mentor Prof. James D. Weiland at the University of Michigan. All of these fields are critical to the study of visual prostheses. She will be mentored by an advisory committee including Profs. Mark S. Humayun, Arthur W. Toga, and Yonggang Shi. Both USC and Michigan have retinal prosthesis implantation and behavioral testing programs involving clinicians, engineers, and visual neuroscientists, in addition to state of the art neuroimaging facilities, making them ideal locations for this research.

NIH Research Projects · FY 2024 · 2020-09

Project Summary/Abstract Acute respiratory distress syndrome (ARDS) develops in some individuals as a sequela to indirect stress on the lung from systemic infection (sepsis/endotoxemia). However, it is unclear why only some patients with sepsis develop ARDS. One possible risk factor leading to ARDS in patients with sepsis is exposure to air pollutants such as ozone. Recently, FDA acceptable environmental levels of ozone exposure have been directly linked to the development of ARDS. Our overall goal is to elucidate the mechanisms underlying the increased risk of developing ARDS following exposure to oxidants such as ozone. ARDS develops, in part, due to an accumulation of dead and dying neutrophils and neutrophil-derived proinflammatory apoptotic bodies in the lung. Under homeostatic conditions, these are removed by macrophages via a process known as efferocytosis. We hypothesize that the increased risk of ARDS following ozone exposure is due impaired efferocytosis. Moreover, this is exacerbated in individuals with genetic deficits in the pulmonary collectin, surfactant protein D (SPD), which controls macrophage efferocytosis. To test this, we developed a novel experimental model in which mice are exposed to inhaled ozone followed by intravenous (i.v.) lipopolysaccharide (LPS), a bacterial-derived toxin released into the blood during sepsis (endotoxemia). Our aims are to (1) Determine if ozone exposure and decreased SPD activity exacerbate inflammation and acute lung injury (ALI) by impairing macrophage efferocytosis and (2) Determine if decreased SPD activity exacerbates ozone-induced impairment of macrophage efferocytosis in humans. Wild type and lung-specific conditional SPD knock out mice will be treated with ozone followed by LPS. Macrophage efferocytosis will be measured by flow cytometry. The mechanistic pathways associated with oxidative stress, which is important in ozone toxicity, will be identified using RNA sequencing (RNAseq). We will analyze lung inflammation and macrophage efferocytosis in human subjects, stratified according to single nucleotide polymorphisms within the SPD gene, following controlled ozone exposure. The results of these experiments will provide novel mechanistic insights into the relationship between ozone exposure, macrophage function, SPD variation, and susceptibility to ARDS. These studies are significant, as oxidants such as ozone have been implicated as a risk factor the development of ARDS. The experiments, coursework, and structured mentorship proposed in this application will provide the basis for an NIH R01 grant and initiate the PI's career in independent translational research.

NIH Research Projects · FY 2024 · 2020-09

Enterovirus D68 (EV-D68), a viral pathogen associated with moderate to severe respiratory illness in children, also infects the nervous system in rare cases and causes neurological complications such as polio-like acute flaccid myelitis (AFM). Despite a significant disease burden, no approved antiviral drugs or vaccines are available for EV-D68. Due to its medical importance, EV-D68 is currently listed as a priority pathogen by the National Institute of Allergy and Infectious Diseases. Because the occurrence of the next EV-D68 outbreak cannot be predicted, the need for a potent, broadly acting, and safe EV-D68 inhibitor is urgent. The objectives of this proposal are to explore novel viral proteins as antiviral drug targets and to develop corresponding chemical probes as tools for target validation. This proposal is built upon our recent discovery that the EV-D68 2A protein exerts cysteine protease activity that is specific for the viral polyprotein VP1-2A junction and its protease activity can be inhibited by an FDA-approved oral drug, telaprevir. We further showed that telaprevir had submicromolar to low micromolar potency against several contemporary human EV-D68 strains from clades A and B in different human cell lines, including human neuronal cells. The antiviral potency of telaprevir (IC50) against EV-D68 was similar to that of HCV, indicating clinical relevance of repurposing telaprevir as an EV-D68 antiviral. The mechanism underlying the inhibition of the EV-D68 virus by telaprevir was independently elucidated using a serial viral passage experiment, leading to the selection of an N84T mutation near the active site of EV-D68 2Apro protein that reduced the potency of telaprevir in enzymatic and cellular antiviral assays. To validate the hypothesis that EV-D68 2Apro is essential for viral replication both in vitro and in vivo, our goals are to develop 2Apro inhibitors with favorable pharmacokinetic properties and to use them as chemical probes to validate EV-D68 2Apro as a viable antiviral drug target in cell culture as well as in a neonatal mouse model of EV-D68-induced paralytic disease. Specifically, we propose to design more potent and selective EV- D68 2Apro inhibitors through structure-activity relationship studies of telaprevir. All designed compounds will be systematically tested in in vitro enzymatic and cellular assays and profiled for pharmacokinetic properties. Prioritized lead compounds will be selected to test in EV-D68 mouse models. The expected outcomes of the proposed research are to validate EV-D68 2Apro as a viable antiviral drug target and demonstrate the in vivo antiviral efficacy of EV-D68 2Apro inhibitors.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Helminth parasites, including hookworms, infect approximately 2 billion people worldwide and represent a significant public health concern. To combat these parasites, the mammalian immune system has evolved mechanisms to maintain a delicate balance between promoting beneficial inflammation needed to reduce parasitic burdens, but also subsequently restricting that inflammation once the infectious threat is eliminated. When properly regulated, this allows for protective immunity to be achieved without the development of unwanted immunopathology. It is well established that type 2 inflammation, characteristic of helminth-induced immune responses in humans and mice, is initiated via the production of type 2 cytokines by group 2 innate lymphoid cells (ILC2s) and type 2 T helper (TH2) cells. The activation of both innate and adaptive lymphocytes results in the induction of smooth muscle contraction, eosinophilia, mucus production and the population expansion of basophils. Despite our knowledge of the factors that promote type 2 inflammation, the mechanisms that restrict its ability to promote immunopathology remain poorly defined. Our preliminary studies revealed that helminth-induced ILC2 responses, type 2 cytokine production, lung eosinophilia and mucus production are significantly elevated following the depletion of basophils. Moreover, depletion of basophils resulted in dramatic lung pathology and decreased lung function. Strikingly, our new studies also revealed that ILC2s activated in the absence of basophils failed to upregulate expression of the receptor for the neuropeptide neuromedin b (Nmb). Further, delivery of Nmb to helminth-infected mice resulted in reduced ILC2 responses, eosinophilia and mucus production. These data suggest that Nmb is a potent inhibitor of type 2 inflammation. Nmb belongs to the bombesin-like family of neuropeptides consisting of neuromedin B, N, S and U. Importantly, neuromedin U was recently shown to be an important positive regulator of helminth- induced ILC2 responses. Collectively, our studies suggesting that Nmu and Nmb operate as neuropeptide ‘rheostat’ that properly balances helminth-induced inflammation. Based on our strong preliminary studies and generation of novel Nmbr-floxed and Nmur-Cre mouse models, three specific aims will address the following questions: (i) Do helminth-induced basophils regulate Neuromedin b receptor expression on immune cells, (ii) Does Nmb restrict the activation of multiple immune cells in a manner that properly regulates helminth-induced inflammation, and (iii) Do Nmu and Nmb directly counterbalance each other and operate as a neuropeptide rheostat? Collectively, these studies will interrogate novel mechanisms through which type 2 cytokine- mediated immunity and inflammation are negatively regulated. Defining the mechanisms through which basophils initiate a Nmu/Nmb-mediated rheostat may inform new therapeutic strategies to treat helminth- induced immunopathology.