University Of Alabama At Birmingham

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY/ABSTRACT: High blood pressure is a leading cause of morbidity and mortality worldwide and greatly contributes to a multitude of cardiovascular disease. Although the renal sympathetic nerves have been the focus of many basic and clinical studies, the role of the renal afferent (sensory) nerves in mediating hypertension remains poorly understood. Activation of renal afferents can potentially either excite or inhibit global sympathetic nerve activity and thereby increase or decrease blood pressure, but the precise mechanisms controlling the balance of the excitatory vs. inhibitory reflex actions of the renal afferent nerves are currently unknown. We have recently determined that the endothelin-1 (ET-1) system significantly modulates renal afferent nerves. Our central hypothesis is that endothelin A (ETA) receptor activation on renal afferent nerves increases sympathetic nerve activity and blood pressure, and that endothelin B (ETB) receptor activation on renal afferent nerves decreases sympathetic nerve activity and blood pressure. We will test this hypothesis using rodent models in the settings of both normal physiology and hypertension. ETA or ETB receptors on renal nerves will be directly activated in rats instrumented with radiotelemetry devices, which allow for recording of blood pressure and markers of sympathetic nerve activity in unrestrained, conscious animals. These experiments, which will also include in vitro culturing, imaging, and electrophysiological examination of renal afferent nerves will provide important new information about the mechanisms of afferent nerve activation. High salt and high fat content are common features of the typical Western diet and are well-known to play a role in the development of many cardiovascular diseases such as hypertension. Our preliminary evidence indicates that these factors also increase renal afferent nerve expression of ET-1 and ETA receptors. Because of this connection to increases in abberant renal afferent nerve activity and blood pressure, we will also test the hypothesis that high salt or high fat diet increases afferent nerve ET-1 expression and a preponderance of ETA to ETB receptor signaling promoting increased afferent nerve activity, sympathetic tone, and blood pressure. This hypothesis will be tested through the use of rat models with specific pharmacological inhibition of ETA and/or ETB receptors on renal afferent nerves, and animals will be fed a high salt or high fat diet. Together, these studies will provide a mechanistic understanding of how excitation versus inhibition of renal afferent nerves contributes to hypertension and thus could lead to specific and efficient therapeutic interventions for the treatment of hypertension. This research study and the proposed mentored training plan will provide the applicant with the specific scientific training needed for successful transition into independence.

NIH Research Projects · FY 2024 · 2021-08

PROJECT SUMMARY Macrophages are a class of immune cells important in the prognosis and treatment of several diseases, including cancer. CD68 is the established clinical biomarker for pan-macrophage. Currently, there are no non- invasive probes specific for human or mouse CD68. We propose to develop novel probes for human and mouse CD68 for PET imaging of macrophages. This project involves phage display screening of a human Fab library (Aim 1a); binding of resulting “hits” to CD68 proteins (Aim 1b) validating in CD68-positive macrophages to select lead huFabs (Aim 1c); and evaluating the function of the resulting lead radiolabeled huFabs in appropriate mice models (Aim 2). From this work, we will produce a PET imaging toolkit for imaging human and mouse macrophages with high binding affinity, specificity, and pharmacokinetic properties suitable for same-day imaging. Our long-term goal is to apply our novel PET probes to quantify macrophage density in vivo in different diseases in humans and mice. Specifically, we envision that co-clinical trials will employ these probes for imaging human subjects and corresponding mice models of disease to monitor response to treatments that modulate macrophage activity.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Skeletal muscle atrophy and muscle wasting is associated with both acute and chronic pathological conditions such as traumatic spinal cord injury and inpatient bedrest. Decreases in muscle mass from the atrophy is associated with power outcomes to other comorbidities, and increased susceptibility to obesity and diabetes. Current pharmaceutical interventions to increase muscle mass have been limited in their effectiveness. This poor efficacy is in part due to the limited understanding of the different mechanisms that contribute to decrease muscle mass. Mitochondrial dysfunction has been proposed as one of the contributors to skeletal muscle atrophy. However, the precise mechanisms that contribute to impaired mitochondrial functionality and the development of skeletal muscle atrophy is unknown. Mitochondrial dynamics have emerged as key regulators of both physiology and pathology in skeletal muscle. We have recently reported that induced adult skeletal muscle deletion of both mitofusin 1 and 2 have a profound effect on exercise capacity. Furthermore, preliminary analysis of these animals exhibit signs of decrease muscle mass and the induction of the unfolded protein response (UPR) and atrophy genes. We also observed elevated levels of FGF21 in skeletal muscle and circulation. These data suggest that adult skeletal muscle mitochondrial dysfunction and elevated muscle-derived FGF21 contributes to the development of muscle atrophy. Furthermore, utilizing a spinal cord injury (SCI) model, which develops pathological skeletal muscle atrophy, we observe elevated levels of skeletal muscle Fgf21 mRNA. We hypothesize that the observed elevated skeletal muscle derived FGF21 in circulation further contributes to the observed atrophy. Therefore, the overall objective of this proposal is to understand the contribution of mitochondrial dysfunction in skeletal muscle to the development of skeletal muscle atrophy. Using genetic models and translatable therapeutic interventions we will attempt to address this very important question. Results from this proposal have broad implications for our understanding of the molecular changes that contribute to the development of skeletal muscle atrophy. The specific aims are to: 1.) Establish the requirement of FGF21 signaling for skeletal muscle atrophy in response to muscle mitochondrial dysfunction; 2.) Reveal the contribution of elevated FGF21 in the development of skeletal muscle atrophy in response to a contusion spinal cord injury (SCI); 3.) Determine whether pharmacologic inhibition of FGF21 signaling after spinal cord injury (SCI) prevents skeletal muscle atrophy. This proposal will to provide much needed insights into our understanding of molecular pathogenesis of skeletal muscle atrophy.

NIH Research Projects · FY 2025 · 2021-08

Project Summary/Abstract Most bacteria are found living within complex polymicrobial environments where they must compete for nutrients, space, oxygen, and defend themselves against exogenous, host, or bacterial derived antimicrobials. Bacterial community dynamics and behavior can be greatly influenced by reactive nitrogen species (RNS), which are important signaling molecules, immune mediators, and antimicrobials. Unfortunately, there is limited understanding on how RNS regulate the structure and function of polymicrobial communities and what impact this interplay has on host immunity, which has created enormous gaps in knowledge. Previous studies that explore the role of RNS on microbial physiology predominantly use single species models instead of polymicrobial models, which better represent the lifestyle of microbes. Our preliminary findings indicate that commensal or beneficial bacteria induce the production of RNS to outcompete pathogenic bacteria and maintain homeostasis within a host. Yet, there are no studies that have clearly defined the molecular mechanisms that govern synergy between commensal bacteria and RNS, and how they dictate the pathogen- host relationship. Over the next five years, the studies outlined in this MIRA application will use molecular genetics, systems biology tools (metabolomics, proteomics, and RNA sequencing), and a Drosophila melanogaster invertebrate model to determine how RNS regulate interactions between commensals and pathogens and also elucidate what impact this has on the host. Critically the field of RNS as an effector of bacterial population dynamics is vastly understudied yet is vital to understand because it has significant health consequences. The findings of this research proposal will advance the field of bacterial-bacterial interactions, as well as the role of RNS responses in controlling and modulating the host-pathogen interface. Our studies could potentially provide valuable mechanistic data that can be harnessed for the development of commensal- mediated anti-infection therapies.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY/ABSTRACT Viruses are able to infect every kind of organism on the planet. They play an integral part in maintaining normal ecosystems but sometimes cause perturbations that have adverse impacts on human health, agriculture, and the wider environment. Understanding the role that viruses play in ecosystems and developing approaches to mitigate their undesirable effects represents a major goal of virologists. Organisms can only be understood in the context of their relationships. This begins with the delineation of common and distinguishing properties and is formalized by the science of taxonomy which creates an ordered, hierarchical system of classification and nomenclature. Taxonomy is critical for building a holistic understanding of the biology of the organisms that inhabit this planet. The International Committee on Taxonomy of Viruses (ICTV) provides a coherent framework for understanding viruses by classifying them on the basis of their kinships as described by the virus taxonomy. The ICTV taxonomic database and programs form the bedrock used by virologists worldwide to understand the global virome, standardize the nomenclature of the huge influx of newly discovered viruses, and promote both research and education in a rapidly changing environment. This proposal is aimed at expanding the effectiveness of the ICTV and strengthening its connections to a broad range of stakeholders. These include people involved in scientific, veterinary, medical, educational, and regulatory endeavors, and anyone else interested in viruses. Our aims focus on providing an integrated set of outcomes that modernized resources to ensure a stable, responsive, scalable infrastructure; increased availability and breadth of taxonomic and virological information; better tools to manage taxonomic classification and handling of data supporting high-throughput virus classification and curation; enhanced accessibility by individuals, groups, and information repositories; and outreach and training activities to ensure that the products of our efforts are used to benefit stakeholders and to promote better understanding of viruses and their classification. This work will advance the capacity of bench scientists, computer scientists, educators, trade regulators, pharmaceutical firms, government agencies, policy makers, and the general public to conduct leading edge research, mitigate the threat posed by viruses, and better understand the risks posed by viruses to human, animal, and agricultural health, and the planet’s ecology. This work will also contribute to national and international security by helping governments to respond to unforeseen outbreaks of virus diseases such as COVID-19.

NIH Research Projects · FY 2025 · 2021-08

Lung disease caused by Respiratory Syncytial Virus (RSV) is associated with substantial short and long-term morbidity for infants and children. Despite this far-reaching disease burden, current management is limited. Developing an effective intervention for acute infection with RSV would have an enormous impact on the public health of children. Matrix metalloproteinase (MMP)-9 is a protease that has been implicated in RSV pathogenesis. Azithromycin (AZM) is a commonly used macrolide antibiotic with a well-known safety profile that has immunomodulatory effects that have been beneficial against other inflammatory airway diseases and may work through an MMP-9-based mechanism of action. We completed a Phase II, randomized controlled trial (RCT) of high-dose AZM [20 mg/kg (IV) x 3 days], and demonstrated that the treatment was safe and associated with decreased hospital length of stay and decreased endotracheal MMP-9 concentrations in mechanically-ventilated children. Taken together, these data provide compelling evidence that justifies a multi-centered Phase III RCT to determine the effectiveness of AZM. The overarching hypothesis of the ARRC Trial (AZM treatment for RSV-induced Respiratory Failure in Children) is administration of AZM during acute, RSV-induced respiratory failure will be beneficial, mediated through an MMP-9 pathway. To test this overall hypothesis, we have developed a research network of pediatric critical care physicians to enroll 370 children in a double-masked placebo-controlled RCT of high-dose AZM. Inclusion criteria will be age less than 2 years and acute respiratory failure secondary to RSV infection requiring ICU admission with intensive respiratory support [defined as mechanical ventilation, non-invasive bi-level positive airway pressure (BiPAP), continuous positive end-expiratory pressure (CPAP) or high-flow nasal cannula (HFNC) therapy of > 1L/kg/min of flow]. Exclusion criteria includes previous use of AZM within 7 days, cardiac arrhythmias, chronic home ventilation/oxygenation and immunosuppressive conditions. We will test the following aims: 1: High-dose AZM administration will result in decreased length of hospital stay, decreased duration of oxygen therapy and decreased ICU length of stay. Enrolled patients will be administered high-dose AZM or placebo, receive all other therapies based on standard American Academy of Pediatrics guidelines for RSV infection, and outcomes will be assessed; 2: Administration of high-dose AZM will result in reduced number of wheezing episodes over 12 months after primary infection and the time to the first wheezing episode and 3: Nasal markers of MMP-9 driven lung inflammation will be decreased in patients receiving AZM, and predictive of outcome. Successful completion of this impactful grant application will determine the effectiveness of AZM on the recovery of children with severe RSV infection. A positive result from this trial will represent a paradigm shift in the management of severe RSV infection, as the first successful treatment for acute and post-RSV related respiratory exacerbation and has the potential to identify novel biomarkers of disease outcome.

NIH Research Projects · FY 2024 · 2021-08

Project Summary Atherosclerosis arises as a result of excess accumulation of cholesterol within vascular cells, with macrophages comprising a majority of these lipid-laden foam cells within atherosclerotic lesions. Lipid-lowering therapies, such as statins, have proven beneficial, but still only benefit a subset of patients. As such, there is currently a need to develop new treatment options that can treat a larger portion of atherosclerosis patients to reduce cardiovascular disease mortality. One potential strategy for treating atherosclerosis is to reduce foam cells by decreasing cholesterol uptake (influx) and/or to promote cholesterol release (efflux) in macrophages. However, no current therapy targets these mechanisms. Foam cells within atherosclerotic lesions are developed from too much cholesterol influx without efficient efflux. Focal adhesion kinase (FAK) is an integrin- associated tyrosine kinase which contributes to vascular cell migration, proliferation, and inflammation. We have discovered new functions for FAK in the regulation of lipid homeostasis within macrophages. Our preliminary data revealed that FAK activation following oxidized low-density lipoprotein (oxLDL) stimulation was required for foam cell formation via endocytosis of CD36. Additionally, oxLDL increased FAK interaction with CD36 and Filamin A. Importantly, pharmacological FAK inhibition blocked FAK-CD36-Filamin A interaction and subsequent foam cell formation, suggesting that the ternary complex may contribute to oxLDL uptake. More interestingly, FAK inhibition increased expression of cellular lipid sensors peroxisome proliferator activated receptor g (PPARg) and liver X receptor a (LXRa) resulting in increased transcription of the cholesterol antiporters ABCG1 and ABCA1. FAK inhibition also increased PPARg and LXRa nuclear translocation, and this was associated with decreased expression of nuclear receptor corepressor 2 (NCOR2). In a new macrophage-specific FAK kinase-dead (KD) mouse model (CSF1R-iCre) on ApoE-/- background, we observed that FAK-KD mice fed a western diet (WD) showed less foam cell formation and reduced atherosclerotic lesions. Taken together, our central hypothesis is that FAK inhibition reduces oxLDL uptake via disruption of FAK-Filamin A-CD36 complex formation while also increasing cholesterol efflux through increased PPARg and LXRa activation via NCOR2 degradation. To decipher a molecular mechanism in which a two-fold role of FAK prevents cholesterol uptake as well as enhances efflux in macrophages. Aim 1 will determine FAK and Filamin A regulation of oxLDL-CD36 uptake in macrophages. Aim 2 will investigate FAK regulation of cholesterol efflux via PPARg and LXRa activation in foam cells. Aim 3 will evaluate the effect of FAK inhibition on preventive and therapeutic models of atherosclerosis. The proposed study will shed new insights on the role of FAK in cholesterol homeostasis in macrophage foam cells and could produce a new treatment option in atherosclerosis by reducing the foam cells via reduced cholesterol influx and elevated cholesterol efflux.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY – Maintenance of genome integrity is a fundamental function of all cellular life. Respiring organisms maintain robust antioxidant systems to defend against pathologic reactive oxygen species (ROS) which are a threat to genome integrity. ROS activates the DNA damage response (DDR) by virtue of several types of DNA damage including crosslinks between RNA and DNA as well as induction of DNA double stranded breaks (DSBs). The most common cancer therapeutic that mediates cell kill through induction of ROS is ionizing radiation (IR). However, the mechanisms by which the DDR responds to ROS to maintain genome integrity are not well understood. The PI, Neil Pfister, MD, PhD, identified ATP5A1 as a top hit in a genome-wide CRISPR/Cas9 knockout screen using IR as the selective pressure. ATP5A1 is the alpha subunit of the soluble F1 subunit of ATP synthase, the terminal electron transport chain complex that generates ATP in the presence of an electrochemical proton gradient and molecular oxygen. A cleaved form of ATP5A1 was found to co-localize to poly(ADP-ribose) and γH2AX foci, which is enhanced by oxidative stress and inhibited by PARP inhibition. Cleaved ATP5A1 contains a poly(ADP-ribose) interaction domain that is required for poly(ADP-ribose) binding and localization to DSBs. R-loop resolution proteins DHX9 and hnRNPU were identified as top protein interactors, and depletion of ATP5A1, DHX9, or hnRNPU significantly increased levels of R-loops and spontaneous DSBs. This project examines the central hypothesis that cleaved ATP5A1 cooperates with poly(ADP-ribose) polymerases to facilitate R-loop resolution and genome maintenance in response to oxidative stress. To test this hypothesis, 3 specific aims are proposed. Specific Aim 1 will determine how cleaved ATP5A1 is regulated. Specific Aim 2 will delineate how cleaved ATP5A1 promotes genome maintenance through interaction with DHX9, hnRNPU, and poly(ADP-ribose). Specific Aim 3 will dissect how cleaved ATP5A1 impacts cell fate following oxidative stress. Dr. Pfister is mentored by Dr. David Yu and Dr. Kathy Griendling with additional support from Dr. Francesca Storici, Dr. Xingming Deng, and Dr. William Dynan. Emory University boasts an outstanding research environment at an NCI-designated Comprehensive Cancer Center to complete the proposed research. The goal of the K08 career development award is for Dr. Pfister to receive career mentorship and training in genome maintenance and cell fate, ROS and oxidative metabolism, mitochondrial biology, and R-loop biology, which complements his past training in order to investigate the role of cleaved ATP5A1 in coordinating oxidative metabolism with genome maintenance, a topic of critical importance to cancer initiation and cancer treatment. The proposed research, in combination with a structured mentoring and training plan, is designed to facilitate Dr. Pfister's long-term goal to supervise an independently funded laboratory that investigates how cells respond to IR and ROS in order to identify new opportunities for cancer therapy.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Targeting the Intestinal Mucosa and Microbiome to Prevent Neonatal Late-onset Sepsis. Late-onset sepsis (LOS) is a leading cause of morbidity and mortality in premature infants and is thought to be caused by the systemic spread of commensal microbes. Perturbation in the developing intestinal microbiome (dysbiosis) is far more common in premature infants than in full-term infants and is thought to underlie their heightened susceptibility to LOS, although the mechanisms that predispose to this are not well understood. We recently developed a new murine model of neonatal LOS, which has confirmed the long-held clinical suspicion of a direct link between dysbiosis and LOS. We discovered that, by altering the developing microbiome to prevent dysbiosis, we were able to prevent LOS. This protection correlated with the abundance of endogenous Ligilactobacillus (formerly Lactobacuillus) murinus, some isolates of which proved to be effective in preventing LOS when administered as probiotics. Remarkably, however, even closely-related L. murinus isolates differed considerably in their probiotic efficacy, as did other strains of Lactobacilli—including a number of strains that are components of commercial probiotics. Moreover, we have found that probiotic strains of L. murinus that prevented dysbiosis and LOS altered the oxygen status of the intestinal epithelium, suggesting that these strains may modulate intestinal redox status to prevent the outgrowth of facultative anaerobes that can respire oxygen or other respiratory terminal electron acceptors. Although a major mechanism driving dysbiosis in adults is increased availability of substrates of bacterial respiration that allows facultative anaerobes to outcompete the obligate anaerobes that predominate in a healthy microbiome, our preliminary studies indicate that mechanisms that predispose the adult intestine to dysbiosis under conditions of inflammation or infection are at least partially disparate with those in the immature neonatal intestine. We therefore posit that the neonatal intestine is susceptible to dysbiosis via mechanisms distinct from those previously characterized in adults, reflecting developmental immaturity of the intestines and early instability of the developing intestinal microbiome. Here, we will take a team science approach to elucidate both host and microbial determinants of neonatal dysbiosis that predispose to LOS, marrying the efforts of two labs with complementary expertise in intestinal biology and immunity (Weaver), and microbial genetics and bacterial respiration (Gray) with collaborators who are leaders in microbial genomics (Julie Segre), inflammation-associated gut dysbiosis (Sebastian Winter) and neonatology (Namasivayan Ambalavanan). Through the identification of mechanisms of dysbiosis unique to the developing intestines and microbiome we will provide a foundation for more rational design of probiotics and prebiotics for therapeutic interventions that prevent LOS in premature infants.

NIH Research Projects · FY 2025 · 2021-07

Project Summary/Abstract Psychogenic non-epileptic seizures (PNES) are a type of functional neurological disorder (FND) characterized by seizure-like symptoms without EEG correlates. PNES is severely debilitating to children and families, making this a significant clinical and societal burden. Recent research has revealed catastrophic symptom expectations and sense of control as potential targets for pediatric PNES treatment. Children with PNES have greater catastrophic symptom expectations, or the interpretation of physical sensations as injurious, intense and disturbing, than their siblings and peers with epilepsy. Catastrophic symptom expectations such as these have been found to actually induce the expected symptom. Also, children do not have a sense of control over PNES symptoms, indicating that children perceive PNES to be involuntary and uncontrollable. In adults, studies using cognitive behavioral therapy or SSRIs to target mood and anxiety have demonstrated no significant reduction in PNES compared to control conditions. In a different cognitive behavioral approach, the aim of this study is to assess sense of control and catastrophic symptom expectations as treatment targets of Retraining and Control Therapy (ReACT), a novel cognitive behavioral treatment for pediatric PNES developed by the PI. Our recently published RCT demonstrated a significant decrease in PNES frequency after ReACT, and increased sense of control was significantly related to reduced PNES frequency in children with PNES. In the R61 phase, nine to 18-year olds diagnosed with PNES will engage in 12 sessions of ReACT. Sense of control over actions will be measured by the magic and turbulence task, and catastrophic symptom expectations will be measured by cortisol response to the cold pressor task 7 days before treatment and 7 days after the 8th and 12th treatment sessions (to evaluate treatment dose). PNES outcomes will be measured by number of PNES in the 30 days before and after treatment. The study will proceed to the R33 phase if Go criteria are met (Cohen's d effect size ≥ 0.5) for engagement of one of the targets. For the R33, children will be randomized to either ReACT or an active therapy (supportive therapy), and outcomes will be measured 7 days before and after treatment and 6 months after treatment. The replication of target engagement will be assessed as compared to supportive therapy, and the extent to which the change in the targets is associated with change in PNES frequency will be determined. The preliminary signal of efficacy for ReACT on PNES will be evaluated to prepare for a larger multicenter RCT assessing the efficacy of ReACT on pediatric PNES.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Compared to Whites, Blacks carry a disproportionate burden of chronic disease and early mortality. These health disparities have been linked with high levels of psychosocial stress experienced by Blacks (e.g., poverty, interpersonal violence, and discrimination), but the biological mechanisms linking psychosocial stress with health disparities across the lifespan remain poorly understood. Similarly, the extent to which multi-level protective factors (e.g., from individual, interpersonal, and community domains) mitigate the effects of early life stress on health outcomes and underlying biological mechanisms is unknown. This proposal will investigate the hypothesis that early life stress produces DNA methylation profiles that contribute to health disparities in early markers of chronic disease across several age groups, and these effects are modified by protective factors from individual, interpersonal, and community domains. To achieve the goals of this project, we will capitalize on an existing longitudinal study, Healthy Passages, which collected prospective, multi-informant data on a variety of early life stressors and protective factors in over 1,000 individuals (65% Black, 35% White, 50% female) in Birmingham, Alabama at ages 11, 13, 16, and 19. The proposed project will conduct a follow up assessment on 800 young adults from this cohort (average age 28) and 400 of their offspring (ages 0 to 5). It will involve a comprehensive evaluation of cardiometabolic indicators associated with later chronic disease (obesity, hypertension, hyperglycemia, and inflammation) in the young adults; assessment of prenatal and postnatal stress and protective factors in the offspring; and analyses of saliva DNA from the young adults at ages 19 and 28, and their offspring. The combination of existing and newly collected data will be used to 1) identify DNA methylation variations that are associated with early life stress and cardiometabolic indicators across three developmental periods – early childhood, late adolescence, and young adulthood; 2) examine the role of race in stress-related DNA methylation and cardiometabolic indicators across the three developmental periods; and 3) identify multi-level protective factors that modify the effects of early life stress on DNA methylation across the three developmental periods. The findings of this study will provide novel insights into stress-related epigenetic mechanisms that may explain racial health disparities across the lifespan, as well as multi-level protective factors that may interrupt the biological embedding of adversity. Better understanding of the role of epigenetics in health disparities may lead to development of epigenetic biomarkers for early diagnosis of disease, ability to identify susceptible individuals at risk for adult disease, and development of novel preventive and curative measures that would reduce health disparities.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY The Cancer Genome Atlas (TCGA) studies have identified p53 as the most frequently mutated gene in breast cancer. In particular, 80% of triple-negative breast cancers (TNBCs) harbor p53 mutations. However, there is no specific therapy available for treating p53-mutant TNBC. Cancer cells have distinct metabolic needs compared to normal cells, and this observation has spurred the development of small molecule inhibitors to target metabolic enzymes that are specifically needed by cancer cells for survival. Notably, although there is evidence to support a role for metabolic pathway deregulation in breast cancer growth, the metabolic dependencies of p53-mutant TNBC growth and metastasis have not been comprehensively identified. Therefore, to specifically identify the metabolic dependencies of p53-mutant TNBCs, we performed an innovative and unbiased large-scale in vivo short-hairpin RNA (shRNA) screen by targeting 2000 genes with known metabolic functions. With this screen, we identified N-acylsphingosine amidohydrolase 1 (ASAH1) as an enzyme that is necessary for tumor-forming ability and metastatic activity of p53-mutant TNBCs. Additionally, we identified two small molecule inhibitors of ASAH1 with potent anti-p53-mutant TNBC activity, thereby indicating that ASAH1 is a potential drug target for the treatment of p53-mutant TNBCs. The central hypothesis of this proposal is that p53-mutant TNBC cells depend upon ASAH1 for their survival, and thus, ASAH1 inhibition selectively eradicates p53-mutant TNBCs. Our overall objectives are to determine the role of ASAH1 in driving p53-mutant TNBC tumor growth and metastasis, understand the mechanism underlying the dependency of p53-mutant TNBCs on ASAH1, and evaluate the translational potential of small molecule ASAH1 inhibitors for treating p53-mutant TNBC. In Aim 1, we will establish the role of ASAH1 in p53-mutant TNBC tumor growth and metastasis using a series of complementary, state-of-the-art mouse models that recapitulate characteristic features of TNBC growth and metastasis. These include a highly innovative humanized mouse model with a human immune system. In Aim 2, we will test our hypotheses that p53 represses ASAH1 activity by sequestering this protein in the nucleus. Additionally, based on our new preliminary results, we will confirm whether loss of ASAH1 activates the glucose starvation response, leading to AMP kinase pathway activation via a ceramide-mediated reduction in expression of the glucose transporter GLUT1 on the cell membrane. In Aim 3, we will evaluate the translational potential of ASAH1 inhibitors in immunocompromised and immunocompetent humanized mouse models of p53-mutant TNBC, either alone, or based on our preliminary findings, in combination with BET domain inhibitors. Collectively, we predict that the results of the experiments proposed in this application will establish ASAH1 as an important vulnerability inherent to p53-mutant TNBC cells, elucidate the mechanism underlying the dependency of p53- mutant TNBCs on ASAH1 activity, and evaluate a novel therapeutic approach for treatment of p53-mutant TNBC.

NIH Research Projects · FY 2025 · 2021-07

Project Summary/Abstract A priority in oncology and palliative care is preparing the 3.2 million U.S. family caregivers of persons with cancer to effectively support patients in health-related decision-making from diagnosis to the end of life, particularly in underserved settings. Over 70% of patients with cancer involve family in health decisions, including choices about treatments, surgery, location of care, accessing palliative care, and many others. Patients making these decisions with unprepared family caregivers may experience inadequate family decision support leading to heightened distress and receipt of care/treatments inconsistent with their values and preferences. This in turn may increase distress for family caregivers. There is a critical need to train cancer family caregivers to effectively support patient decision-making; however, few palliative care interventions have been tested to enhance caregiver skills in providing decision support. We have developed CASCADE (CAre Supporters Coached to be Adept DEcision partners), a lay navigator-led, telehealth early palliative care intervention to train advanced cancer caregivers how to effectively partner with patients in health decision- making. Evolving out of our prior early palliative care caregiving interventions, decision support relevant content for family caregivers includes principles of effective social support, communication, and Ottawa Decision Guide training; however we do not know which of these components and component interactions influences patient and caregiver decision-making outcomes. Traditional research approaches treat interventions as “bundled” treatment packages, making it difficult to assess definitively which intervention components can be reduced, eliminated, or replaced to improve efficiency. Hence, we propose a randomized 23 (2x2x2) factorial trial, the first such trial in oncology palliative care, using the Multiphase Optimization Strategy (MOST) to test components of CASCADE in order to assemble an optimized, scalable version of the intervention. 352 family caregivers of persons with newly-diagnosed advanced cancer will be randomized to receive one or more palliative care lay navigator-delivered decision partnering training components, based on the Ottawa Decision Support Framework and Social Support Effectiveness Theory: 1) psychoeducation on social support effectiveness in decision support (1 vs. 3 sessions); 2) decision support communication training (yes vs. no); and 3) Ottawa Decision Guide training (yes vs. no). We will determine CASCADE components (main effects/interactions) that contribute meaningfully to patient and caregiver outcomes, including patient healthcare utilization (Aim 1) and use those results to build a version of the CASCADE intervention that is maximally effective and scalable (Aim 2). To maximize recruitment, we will recruit from two NCI-designated comprehensive cancer centers in Birmingham, AL and Atlanta, GA. Using the innovative MOST framework will yield a highly novel and cost effective version of CASCADE primed for confirmatory RCT testing, scalability, and reproducibility.

NIH Research Projects · FY 2025 · 2021-07

Project Summary Resistance to the standard of care treatments contributes to mortality in patients with metastatic triple negative breast cancer (TNBC) or non-small cell lung cancer (NSCLC). In metastatic TNBC, the epidermal growth factor receptor (EGFR) and cytoplasmic mesenchymal-epithelial transition (cMET) receptor are both overexpressed in the basal-like subtype of TNBC. Metastatic NSCLC often harbors mutations in the epidermal growth factor receptor (EGFR), in which patients can develop resistance to EGFR tyrosine kinase inhibitors (TKI). MET gene amplification is also a resistance mechanism for EGFR TKIs. To overcome resistance to EGFR TKI, a bispecific antibody called JNJ-61186372 (BsAb) was developed that targets both EGFR and cMET receptors simultaneously, inhibiting receptor-ligand activation and degrading these receptors upon internalization of the bsAb. Currently, there are no effective methods to predict and monitor response to bsAb, making it difficult to select patients most likely to respond to this new therapy in a clinical trial setting and save those unlikely to respond from undue drug exposure. We aim to develop PET imaging biomarkers to look at the multifaceted response to bsAb therapy: bsAb delivery to the tumor (Aim 1) and changes in individual receptor status in vivo (Aim 2). Through correlative studies among PET imaging, response to bsAb therapy, and known genetic mutation status in EGFR, we will produce the right PET imaging toolkit to understand the mechanisms of action of bsAb in vivo. Our techniques can complement standard of care analysis of EGFR mutation status to select patients most likely to respond to bsAb therapy and monitor response to treatment. This future goal will require an IND, for which our studies will provide proof-of-feasibility in preclinical models. Ultimately, establishing our imaging techniques as companion diagnostic agents could have high impact in accelerating FDA-approval of bsAb for the treatment of patients with NSCLC or TNBC who have developed resistance to standard of care of treatments.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY/ABSTRACT The disorders collectively referred to as inflammatory bowel disease (IBD) are characterized by aberrant immune responses to the commensal microbiota. The establishment and maintenance of tolerance to the microbiota is accomplished via a complex and dynamic network of cells and molecules that collectively stave off immune reactivity. Elucidation of the mechanisms whereby these natural pathways limit immunity to normally benign commensals, especially those that occupy niches at the epithelial border, will ultimately enhance our ability to target these interactions for the prevention and/or treatment of intestinal inflammation. We hypothesize that high-affinity, thymus-dependent (T-dependent) antibody responses are essential for the regulation of mucus-associated bacterial communities. T-dependent antibody responses are especially important in the host response to pathogenic invasion of the gut. Since some helpful commensal species utilize similar mechanisms as pathogens to establish a niche in the mucus layer lining the intestinal epithelium, it stands to reason that similar immune mechanisms are deployed to control these organisms. We have found that mice colonized with a minimal mucus-associated consortium display a predominantly T-dependent IgG response. Moreover, mice with impaired production of T-dependent antibodies have significantly diminished total antibody responses to antigens derived from mucus-associated bacteria including specific flagellins derived from members of the family Lachnospiraceae. Colonization with mucus-associated bacteria, or the deficiency of T-dependent antibodies in the presence of such organisms, coincides with increased production of the immunoregulatory cytokine interleukin-10 (IL-10) by CD4 T cells in the large intestine. Accordingly, simultaneous disruption of these 2 pathways results in spontaneous colonic inflammation. In this proposal, we will utilize defined microbes and the antigens they generate to elucidate the mechanisms whereby interactions between flagellated mucus-associated bacteria and the gut immune system promote tolerance of these communities. In addition, we will explore novel roles for T-dependent anti-commensal IgG1 in the establishment and maintenance of immune homeostasis. If successful, our studies will define the mechanisms whereby TD anti-commensal antibodies promote immune normalcy at the colonic epithelial border and constantly mitigate the risk of deleterious reactivity to the diverse community of mucus-associated bacteria.

NIH Research Projects · FY 2024 · 2021-07

Project Summary The tenacious biofilms formed by Streptococcus mutans are resistant to conventional antibiotics and current treatments such as ‘oral rinses’. Current treatments are not ‘biofilm-specific’ and kill pathogenic species as well as commensal species alike. Therefore, there is a growing need for novel therapeutics to selectively inhibit S. mutans biofilms while conserving the oral microenvironment. Recent studies from our lab and others’ have shown that increased levels of cyclic di-AMP (c-di-AMP), an important secondary messenger in S. mutans, favored biofilm formation by upregulating the expression of gtfB, the gene coding for glucosyl transferase B (GtfB). GtfB is responsible for the production of water-insoluble glucans and is critical for biofilm formation and virulence of S. mutans. C-di-AMP is a novel cyclic dinucleotide synthesized from two ATP molecules by the enzyme, diadenylate cyclase (DAC). A suggested mechanism by which c-di-AMP controls the biofilm formation involves a c-di-AMP-binding protein (CabPA)’s interaction with VicR, a transcriptional factor known for regulating gtfB. S. mutans DAC (smDAC) is not an essential enzyme. Therefore, the inhibition of smDAC is a novel strategy to inhibit the S. mutans biofilms without affecting its growth. DAC inhibition should downregulate gtfB expression and reduce the glucan production. S. mutans coexists with other oral microbes and its ability to form biofilms may be influenced by other bacteria. Multi-species biofilms enable the testing of the selectivity of PB8 towards S. mutans along with other commensal streptococci. We have taken a structure-based approach for the design of inhibitors using our recently solved crystal structure of smDAC enzyme. With the help of in-silico screening and preliminary SAR studies, we have identified low micromolar inhibitors of smDAC. The most active compound identified from these studies is a novel small molecule PB8, which inhibits smDAC (IC50 = 17.2 M) and S. mutans biofilm (IC50 = 10.2 M). PB8 inhibited 80 % multi-species biofilm at 50 M. PB8 did not affect the growth of S. mutans and commensal bacteria (S. gordonii, S. sanguinis, and S. parasanguinis) up to 100 µM showing it is a selective biofilm inhibitor. In surface plasmon resonance (SPR) studies, PB8 showed high binding affinity to smDAC (KD = 7.1 M). To facilitate the structure activity relationship (SAR) and lead optimization studies, we have developed a three-step high yielding synthesis of PB8 and conducted preliminary SAR studies. The overall goal of this proposal is to optimize the biofilm inhibitory activity of PB8 and establish its binding affinity to smDAC and its potential as novel selective biofilm inhibitor that can be used for the prevention and treatment of dental caries. The specific aims are: 1) To optimize the biofilm inhibitory activity of PB8 through structure activity relationship studies. Successful completion of the proposed studies will validate smDAC as a novel target for biofilm inhibition and identify novel, non-toxic compounds that can selectively inhibit cariogenic biofilms, while leaving the commensal and beneficial microbes intact. 2) To evaluate smDAC inhibition and biofilm inhibition profiles of PB8 and its synthesized analogs.

NIH Research Projects · FY 2024 · 2021-07

PROJECT SUMMARY The objective of the proposed research project is to further understand how progranulin (GRN) and β- glucocerebrosidase (GCase) interact and the implications of these interactions in frontotemporal dementia (FTD) caused by progranulin mutations. Frontotemporal dementia is a leading cause of early-onset dementia, and loss- of-function progranulin mutations are one of three main genetic causes. Progranulin is a secreted and lysosome- resident glycoprotein, and deficiency causes lysosomal dysfunction in patients and in mice. Progranulin-boosting therapies have promise for treating FTD-GRN, but delivery is an issue and the safety profile is unclear. This underscores a critical need to continue to search for targeted therapeutics with known safety profiles. Thus, a targeted therapy that reduces progranulin deficiency-induced lysosomal dysfunction may have therapeutic benefits for preventing or delaying FTD-GRN. In order to understand the underlying lysosomal dysfunction caused by progranulin deficiency, our lab has characterized the activities of several lysosomal enzymes in the brains of FTD-GRN patients and progranulin- deficient mice. We found decreased activity of neuronal GCase, a lysosomal enzyme, whereas other lysosomal enzymes show increased activity. GCase is an interesting target because of its known involvement in neurodegenerative disease, as GCase mutations are the leading genetic risk factor for Parkinson disease and the monogenic cause of Gaucher disease, a lysosomal storage disease with neurodegenerative subtypes. GCase deficiency in FTD-GRN appears to be due to an impairment in glycosylation causing GCase aggregation. This proposal will determine if progranulin deficiency impairs GCase trafficking, resulting in decreased lysosomal localization. The overarching hypothesis is that impaired progranulin-mediated GCase trafficking contributes to lysosomal dysfunction and other deficits caused by progranulin deficiency. This proposal will test the following two aims: (1) Determine how progranulin regulates trafficking of GCase and (2) Determine if GCase deficits contribute to lysosomal dysfunction and other deficits caused by progranulin deficiency. More work is needed to understand the specifics and effects of the progranulin-GCase interaction. I will first determine the domain of progranulin responsible for GCase interaction and will then test the effects of this interaction on GCase stability and lysosomal trafficking. I will then determine if increasing GCase is sufficient to reduce lysosomal dysfunction and behavioral abnormalities in progranulin-deficient mice. This proposal will facilitate my scientific and professional development by helping me: 1) improve competency in rigorous experimental design and hypothesis testing, 2) refine known experimental techniques and develop skills for translational research, and 3) gain experience presenting data to both scientific and lay audiences.

NIH Research Projects · FY 2025 · 2021-07

Psychostimulant abuse is a public health crisis that affects millions of individuals in the United States and results in profound economic, social, and individual harm. However, despite rapid increases in overdose deaths linked to stimulant drugs like cocaine, there are still no approved therapeutic options for stimulant abuse disorders. Psychostimulant drugs act through well-defined signaling mechanisms to elevate dopaminergic neurotransmission in the nucleus accumbens (NAc), a key reward-linked brain structure that integrates information from diverse brain regions to directly influence motivated behavior. Further, cocaine causes epigenetic and transcriptional reorganization in medium spiny neurons (MSNs) in the NAc, promoting maladaptive shifts in cell signaling and synaptic function. Our preliminary data indicates that expression of Reln mRNA, which codes for the large secreted extracellular matrix protein Reelin, is enriched in a subpopulation of MSNs that are robustly activated by cocaine. Although Reelin knockout animals exhibit impaired response to psychostimulants and Reelin plays a critical role in synaptic plasticity and memory formation in other brain regions, the role of Reelin in cocaine-related cellular and behavioral adaptations has never been studied. In this proposal, we will test the overarching hypothesis that Reelin signaling is required for the maladaptive molecular, physiological, and behavioral effects of cocaine in the NAc. Specific Aim 1 of this proposal will combine bidirectional CRISPR-based manipulations and single-cell RNA sequencing to determine how Reelin signaling impacts transcriptional responses to cocaine and dopamine receptor activation. Specific Aim 2 will use in vitro and in vivo single unit recordings and ex vivo slice electrophysiology to test the hypothesis that Reelin regulates cocaine response by modulation of physiological and synaptic properties of MSNs. Finally, Specific Aim 3 will use cell-specific in vivo Reelin manipulations in combination with behavioral assays of cocaine and natural reward to test the hypothesis that Reelin enhances the behavioral effects of cocaine. Together, these experiments will identify Reelin target genes in the NAc, dissect molecular signaling pathways by which Reelin alters MSN function and physiology, and determine whether Reelin expression within the NAc modulates cocaine-related behavioral plasticity. These studies will reveal fundamental mechanisms by which Reelin contributes to psychostimulant response, and will pave the way for future experiments to explore how this unique Reln-expressing cell population contributes to motivated behavior.

NIH Research Projects · FY 2025 · 2021-07

This is a pre-doctoral training grant application for a program in Translational and Molecular Sciences (TMS) at the University of Alabama at Birmingham (UAB). Currently, too few PhD graduates are entering the workforce with sufficient training to effectively translate pathobiology of disease and molecular advances into clinical and/or commercial practice. The proposal addresses this need with our goal being to provide a training environment that develops the necessary skills for the next generation of PhD biomedical scientists to incorporate translational objectives into their research and prepare them for the workforce of tomorrow. Our recent experiences in graduate education show increased demand from students for training in translational research. A major challenge in effective training in translational sciences for pre-doctoral students is the need to incorporate multiple disciplines and concepts ranging from understanding disease mechanisms, understanding of how findings may be translated in academia or industry, to understanding regulatory oversight procedures and rules. We propose a 2-year program (with 6 students per year) with students enrolling at the beginning of their second year. A three parts integrative curriculum is proposed. The didactic component includes understanding disease mechanisms, drug discovery and development and translational research administration. The career and translational research enrichment activities include CTSA interactions and team-based exercises that foster innovation, entrepreneurship and requisite skills and knowledge for commercializing discoveries. A clinical / patient interaction experience is included, the latter of which is linked to the trainees’ specific research interests. We feel that providing students with training in translational research early during a student’s matriculation will develop skills that enable them to incorporate translational thinking into their research. To achieve this, we have assembled 60 training faculty with well-funded laboratories that address fundamental scientific problems with a strong emphasis on translation and human disease. Several disease areas that are perennial strengths of UAB are represented and will provide breadth of opportunity for students in a variety of research disciplines. This proposal underscores our previous and ongoing commitment to pre-doctoral education and our collective goal of providing an environment for students to learn and develop the skills they will require to be successful in biomedical research and other science related occupations.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Controversies exist regarding the contribution of glial cell dysregulation and inflammation to the pathogenesis of Parkinson Disease (PD). Signs of inflammation have been detected in postmortem tissue of PD patients, and recent evidence suggests that mutations in genes associated with familial PD can influence the function of astrocytes and microglia. These findings, together with the existence of mutations in the HLA locus in PD patients, have led investigators to propose an etiological role for inflammation and glial dysfunction in PD. However, direct evidence is lacking for mechanistic links among inflammation, glial dysregulation, and the neuronal loss characteristic of PD. To identify potential cell-autonomous mediators of PD-relevant processes in glia, we used a bioinformatics strategy to identify PD GWAS genes enriched in glial cells of the mouse brain. Interestingly, a small subset of genes exhibits enrichment of expression in astrocytes. PD-linked single-nucleotide polymorphisms in one of these genes, CD38, are associated with a ~45% reduction in CD38 transcript expression in the human brain. Previous work has demonstrated a role for CD38 in peripheral immune cells, where it serves to regulate REDOX balance in both intra- and extra-cellular compartments; the roles for CD38 in the brain have only recently been explored. Preliminary experiments from our laboratory have shown that CD38 expression is enriched in astrocytes of the human and mouse brain, NAD/NAM balance is disrupted in various regions of the CD38 knockout mouse brain, and inflammation and CD38 deficiency synergistically interact to influence motor function. Here, we propose to use CD38 as a prototypical gene to understand ways in which glial dysfunction can give rise to a PD-like phenotype in mice by investigating 1) the requirement for CD38 in the maintenance of REDOX homeostasis and dopaminergic neuron function and viability in aging mice, 2) the role for CD38 in the regulation of inflammation in the substantia nigra, and 3) the impact of CD38 modulation on the vulnerability of dopaminergic neurons in two synucleinopathy mouse models of PD. A subset of experiments will test the involvement of REDOX dysregulation in the changes observed in CD38-deficient mice by determining whether provision of nicotinamide riboside, a bioavailable NAD precursor, can prevent dopaminergic oxidative stress, cell dysfunction, and loss. Altogether, these experiments have the potential to reveal mechanistic contributors to increased neuronal vulnerability with glial dysfunction and provide novel information about the roles for glia in maintaining dopaminergic neuron survival in aging and disease.

NIH Research Projects · FY 2025 · 2021-06

Ultraviolet B (UVB) radiation (290-320 nm) causes immune suppression, in addition to inducing mutant cells. Tumors will occur only when there are mutant cells in an immune suppressive environment. Organ transplant recipients who are treated with immunosuppressive medications have a greatly increased risk (up to 100 times) of UV induced skin cancers and the tumors that do develop behave more aggressively. In the United States, the incidence of skin cancer has doubled from 1992 to 2012. Over 3.5 million new cases are diagnosed each year. The epidemic of skin cancer represents a major public health issue and is a tremendous cost to healthcare systems in the United States and worldwide. It is highly desired to understand the pathogenesis of UVB induced immune suppression and develop new strategies for prevention and treatment. Our preliminary data show that UVB increases Triggering receptor expressed on myeloid cells (Trem)-1 in mouse and human skin tissues and by a portion of CD11b+ cells from the mouse skin and draining lymph nodes. Importantly, we have, for the first time demonstrated that blocking Trem1 with an antagonist peptide inhibits UVB induced immune suppression. Moreover, blocking Trem1 inhibits UVB induced cutaneous carcinogenesis. The findings reveal a previously unrecognized role of Trem1 in UVB induced immune suppression and skin carcinogenesis. Furthermore, a common concept is that UVB induced tolerogenic antigen presenting cells (APC) are required for the induction of immune suppression. Although strong evidence in human and animal studies indicates that CD11b+ cells contain tolerogenic APC, CD11b+ cells are heterogeneous and specific tolerogenic APC remain to be identified. Our data show that UVB induces Trem1 expression by a novel subset of conventional dendritic cell type 2 (cDC2) cells (CD11b+). The UVB induced Trem1+ cDC2 cells in the draining lymph nodes express high levels of immune inhibitory molecules CD200 and PD-L1 and are hardly detectable in normal mice. These findings define novel Trem1+ cDC2 cells and implicate novel mechanisms for Trem1 mediated immune suppression. It forms a strong premise for our hypothesis that UVB induced Trem1+ cDC2 are tolerogenic APC responsible for UVB induced immune suppression and skin carcinogenesis. Targeting Trem1+ cDC2 cells has translational potentials for the prevention and treatment of UVB induced carcinogenesis. Based on the novel findings, proposed studies will examine the hypothesis in animals and humans. Aim 1 will identify UVB induced Trem1+ cDC2 cells as specific tolerogenic APC and determine mechanisms for their immune suppressive activity. Aim 2 will determine mechanisms for the development of UVB induced Trem1+ cDC2 cells. Aim 3 will determine UVB induced Trem1+ cDC2 cells in human skin and blood and determine their roles in immune suppression. Collectively, the current application will apply advanced technology and use genetic and pharmacological approaches to fully characterize the Trem1+ cDC2 cells and explore new mechanisms for UVB induced immune suppression. The outcome will have impacts in the research field and may be exploited to new strategies for prevention and treatment of skin cancers.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY This proposal aims to leverage basic discoveries in interdisciplinary fields to develop a novel and safer therapy for pandemic type 2 diabetes (T2D). Obesity-linked insulin resistance is the key driving force for T2D and other metabolic disorders. Despite the wide use of commonly used anti-diabetic drugs for T2D treatment, the prevalence of T2D continues to soar with an annual cost over $300 billion in the US. The transcription factor peroxisome proliferator-activated receptor γ (PPARγ) is an important therapeutic target for insulin sensitization and its full agonist TZD drugs are by far the most potent insulin-sensitizing drugs. However, TZD drugs are associated with adverse side effects including heart failure and weight gain, as TZD-induced full agonism of PPARγ activates not only the expression of genes responsible for insulin sensitizing but also of those genes associated with side effects, thereby severely hampering the clinical use of TZDs. Recent studies have indicated that PPARγ posttranslational modifications (PTMs) may lead to the selective activation of PPARγ target genes that results in the decoupling of the beneficial effects on insulin sensitizing from the TZD- related adverse effects. Our team recently discovered that deacetylation at K268 and K293 in PPARγ by the NAD+-dependent deacetylase SirT1 plays a key role in such decoupling. Excitingly, the PIs have developed a novel class of PPARγ agonist, TPMD, that bound to PPARγ to specifically inhibit PPAR acetylation. Importantly, TPMD improved insulin sensitivity and increased white-to-brown adipocyte conversion (browning) and energy expenditure without causing TZD-associated side effects in both genetic and diary obesity mouse models. In this application, the team led by the two PIs with complementary expertise in diabetes drug discovery and PPARγ biology will use TPMD as the starting molecule to identify the first-in-class inhibitor of PPARγ acetylation that exert potent insulin-sensitizing and browning activities and better safety and pharmacokinetic (PK) properties. In Aim 1, they will employ structure-based design through iterative and parallel medicinal chemistry to identify TPMD analogs with improved potency of inhibiting PPARγ acetylation. In Aim 2, the lead analogs will be proceeded to the standardized core in vitro ADMET assays and in vivo pharmacokinetics studies to select those with the most favorable pharmacological properties. In Aim 3, the lead candidates will be tested rigorously for their in vivo efficacy and safety in obesity and genetic mouse models. The PIs will adopt their “standardized” metabolic characterizations and assessments of TZD-associated adverse side effects. The proposed studies will produce first-in-class PPARγ acetylation inhibitors that have improved insulin-sensitizing potency, safety, and PK profiles. Thus, completion of this research will be well-poised for further clinical development to curtail the current epidemics of insulin resistance and T2D.

NIH Research Projects · FY 2024 · 2021-06

SUMMARY Chronic Obstructive Pulmonary Disease (COPD) is the fourth leading cause of death in the United States with mortality continuing to rise despite advances in medicine. Cachexia, a form of muscle wasting, is a debilitating co-morbidity whose prevalence increases with severity of COPD. But, cachexia still occurs among COPD patients with milder disease severity. Cachexia is most often thought of with respect to cancer. However, by population prevalence there are more COPD patients with cachexia than cancer patients with cachexia. Yet there have been few studies investigating the etiology of COPD cachexia underscoring the need for investigations of COPD cachexia and weight-loss. Accumulating data including our own points to a role for iron toxicity in the etiology of COPD cachexia. Heme is an essential component of mitochondrial cytochromes providing protection from reactive oxygen species (ROS). Defects in heme biosynthesis cause buildup of free iron, ROS and mitochondrial dysfunction. Buildup of free iron leads to iron toxicity and production of ROS particularly in the absence of adequate intake of antioxidants such as Vitamins E. As such, our overarching hypothesis is iron toxicity in COPD cachexia is driven by impaired antioxidant and mitochondrial function. This study has three specific aims: 1) To determine whether genomic variation associated with the absorption and regulation of Vitamin E is more common in COPD cachexia; 2) To assess whether plasma Vitamin E in subjects with COPD cachexia are associated with impaired mitochondrial function; Exploratory Aim) To test whether iron induced transcriptional dysregulation signatures in myoblasts are preserved in transcriptomics signatures associated with COPD cachexia in skeletal muscle biopsies. Elucidating mechanisms of mitochondrial dysfunction in COPD cachexia has the potential to aid the development of therapeutics targeting mitochondrial oxidative stress. As future research, we plan to test whether ‘omic regions and metabolites identified as associated with COPD cachexia directly effect pathways involved with Vitamin E and mitochondrial function using targeted assays in myoblasts or other appropriate systems.

NIH Research Projects · FY 2025 · 2021-06

Project Summary Nearly 120 years since the discovery of the first virus, our understanding of how viruses deliver genomes into cells overcoming the complexity of biological membranes remains limited. While a vast scientific literature exists on viral surface proteins and their interaction with host receptors, and the immune system, little emphasis has been devoted to studying the delivery of entire viral genomes into cells. For instance, how do bacteriophages eject DNA through the cell envelope of Gram-negative bacteria? Or, in humans, how do herpesviruses deliver ~200 kb genome through the Nuclear Pore Complex (NPC) into the cell nucleus? For a quarter of a century, first as a trainee (1995-2003), and since 2004 as a principal investigator, I have investigated the mechanisms of nucleocytoplasmic transport and viral genome packaging. My work has resulted in close to 85 publications that contributed to elucidating the atomic structure and regulation of crucial factors implicated in nuclear import, and viral genome packaging. In this R35, I propose to combine the study of these two seemingly distinct biological processes by focusing on the mechanisms of viral genome delivery into living cells. Specifically, I will ask two biological questions that seek to compare and contrast how simple bacterial viruses (or bacteriophages) eject their DNA into bacteria with how Herpesviruses deliver their complex genomes into the nucleus of eukaryotic cells. The first question explores how bacteriophages eject ~45 kb genomes through the cell envelope of gram-negative bacteria. Long-thought to be a simple pressure-driven injection, this process uses a virus-encoded nanomachine, which we have begun to study in my laboratory. The second question explores how Herpesviruses deliver their large genome through the Nuclear Pore Complex (NPC) of human cells into the cell nucleus. This is a signal- and energy-mediated process that uses host importins and the GTPase Ran, exploiting the cellular transport machinery to promote entry of an exogenous genome into the nucleus. Overall, understanding how viruses transfer genetic information through biological membranes into cells and organelles is vital for deciphering the molecular mechanisms of virulence as well as the development of novel therapeutic approaches. The common denominator of this R35 lies in our interest in the structure and transport mechanisms of biological macromolecules. Our research approach marries established sciences like protein biochemistry and X-ray crystallography with the power of cryo-electron microscopy (cryo-EM) to visualize biological macromolecules in near-native conditions. We believe that this R35 MIRA funding mechanism will fuel the creative and diligent pursuit of answers to the questions we pose, permitting our research program to achieve significant advancements in structural biology.

NIH Research Projects · FY 2025 · 2021-06

Summary: Geographic variation in model for end stage liver disease (MELD) scores at transplant across donation service areas has fueled a major policy debate over allocation of donor livers. An important caveat to this perceived disparity is the focus on allocation MELD, in which patients with lower calculated MELD scores may be assigned a higher score if their calculated MELD is thought to underestimate the risk of waitlist mortality. Analysis of calculated MELD at transplant demonstrates much less variation between regions. Some high allocation MELD regions actually have low calculated MELD scores relative to the rest of the country. The different pictures painted by allocation versus calculated MELD scores highlight the critical role MELD exception points play in geographic differences in access to transplantation. A national liver review board was recently implemented to impose national standardization of MELD exception scores in an effort to mitigate geographic disparity. A potential flaw in this strategy is the assumption that the risk of mortality associated with a given MELD score or exception diagnosis is the same across geographic areas. We know that social determinants of health and access to care vary widely across the country, however, and that these differences have a significant impact on health outcomes. Imposing a geographically uniform MELD exception scoring policy that does not account for these important differences may have the unintended effect of overestimating the risk of waitlist mortality for exception patients in some regions while underestimating that risk in others, thus worsening geographic disparity rather than mitigating it. I hypothesize that significant geographic variation exists in the prevalence and appropriateness of MELD exceptions scores at the county level, and that appropriate adjustment to MELD exception scores based on a candidate’s location will mitigate geographic disparity in liver transplant waitlist mortality. Despite abundant literature on geographic disparity in liver transplantation, few studies actually apply formal geospatial analysis to geographically based questions facing the field. I propose to utilize such techniques to accomplish the following unique aims: (1) determine the prevalence and appropriateness of MELD exceptions on a county level and diagnose the presence of geographic clusters where these exceptions provide an inappropriate advantage to transplant candidates with MELD exception scores; (2) determine adjusted MELD exception scores which would equalize waitlist mortality risk between exception and non-exception patients in each geographic area; and (3) simulate waitlist outcomes under an alternative allocation scenario where MELD exception points are assigned according to geographic region as determined in Aim 2. This study will provide a more granular and personalized approach to the estimation of waitlist mortality. The mentoring and training afforded by this award will foster my growth as an investigator by allowing me to focus on building an expertise in geospatial analysis and advanced simulation, advancing my long-term career goal of research independence as a surgeon-scientist with R01 funding.