University Of Pennsylvania

NIH Research Projects · FY 2024 · 2022-09

Project Summary: Type 1 diabetes (T1D) is a debilitating autoimmune disease that affects millions. Unfortunately, the incidence of T1D is rising. The strongest genetic factor in T1D is the MHC class II locus. Some MHC haplotypes are associated with higher risk to T1D, while others provide dominant protection. Yet, the mechanism remains unknown. Recent studies suggest that environmental factors, such as the microbiome, also contribute to the increasing incidence of T1D. While both genetic and environmental factors contribute to the risk of developing T1D, little is known of how MHC II genetic factors interact with microbial factors. The non-obese diabetic (NOD) murine model recapitulates many features of T1D in humans including the dominant protection associated with the MHC class II locus. NOD mice spontaneously develop T1D, but autoimmunity can be prevented by transgenic expression of the MHCII E allele (Eα16/NOD mice). Recent published studies and new preliminary data from our laboratory suggests that microbiota are critical for this protection. The goal of this proposal is to identify immunomodulatory bacteria and immune system pathways that can be used to develop preventative T1D therapies for genetically at-risk patients To rigorously study the early-life microbiota, we developed a novel consortium of 9 culturable bacteria (which we call PedsCom) that represent over 90% of the bacteria in pre-weaning diabetes-protected Eα16/NOD mice. To investigate immunomodulatory mechanisms of specific bacteria, we are applying gnotobiotic techniques using the PedsCom consortium and genetic models of disease utilizing Eα16/NOD mice. The experiments outlined in this proposal will elucidate the mechanisms by which MHC II molecules interact with intestinal microbes to prevent T1D. In Aim 1, I will investigate if MHC II E expression impacts early-life events to shape microbial colonization by comparing colonization dynamics and humoral responses to commensal bacteria in the NOD and Eα16/NOD mice colonized by the PedsCom consortia. In Aim 2, I will determine if the PedsCom consortia of early-life microbes are sufficient to prevent T1D and whether peripheral regulatory T cells prevent T1D in Eα16/NOD mice by studying PedsCom, specific pathogen free (SPF), and germ-free colonized mice. During this fellowship, these investigations will expand my technical skills, improve my aptitude for experimental design and analysis, and enhance my ability to communicate findings to the scientific community. I will complete this fellowship at the University of Pennsylvania, which offers programs, courses, and structured mentorships that will aid my career development. In addition, I will take advantage of opportunities offered by the Immunology Graduate Group and the Medical Scientist Training Program to enhance my abilities as an educator and a clinician. With the resources available to me, I will explore the fundamental and clinically relevant questions in this proposal to gain the skills necessary to become a successful physician scientist.

NIH Research Projects · FY 2024 · 2022-09

Project Summary The 3’ untranslated region (3’UTR) of mature messenger RNAs (mRNAs) is the sequence between the stop codon of the coding sequence and poly(A) tail. Importantly, the location where the 3’end processing machinery adds the poly(A) tail to the pre-mRNA is not invariant, but changes in a controlled manner to generate 3’UTR isoform diversity between mRNAs of the same gene. This process is known as alternative polyadenylation (APA). Although the 3’UTR isoforms generated through APA do not alter the amino acid sequence of the protein, they influence expression by adding or removing binding sites for microRNAs and RNA binding proteins (RBPs) that influence mRNA export, stability, localization, and translation efficiency. Targeted 3’end sequencing techniques have shown APA to be widespread and regulated between tissues and in specific disease contexts. Despite the prevalence of APA , a regulatory understanding of which RBPs drive this process remains limited. Cells of specific hematopoietic lineages were found to display pervasive APA, but no comprehensive map of APA in the erythroid lineage exists. Other RNA processing events, like alternative splicing and translational control, are known to be important for erythropoiesis and dysregulated in certain anemias and thalassemias, suggesting that APA altering the length/identity of 3’UTRs may also influence erythroid biology in health and disease. This project seeks to fill this knowledge gap by comprehensively identifying and quantifying APA during erythropoiesis using targeted 3’end sequencing on RNA collected from erythroid cells throughout differentiation. Preliminary data suggests several genes essential for erythropoiesis, like transcription factors TAL1 (SCL) and TCF3 (E2A), undergo APA shifts during this process. The functional impact of different 3’UTR isoform choices will be assessed by monitoring impact on mRNA and protein levels (luciferase assays) and differentiation efficacy (CRISPR deletions to force isoform expression). Finally, this project will identify key regulators of the APA shifts across erythropoiesis by analyzing a large compendium of RBP knockdown, mutation, and knockout experiments from K562 erythroleukemia cells followed by experimental validation. Preliminary data suggests splicing factors commonly mutated in myelodysplastic syndromes (MDS, a condition characterized, in part by ineffective erythropoiesis) like SRSF2, also influence APA shifts observed in erythroid differentiation. Taken together, the studies outlined by this proposal will provide insight into novel regulatory mechanisms of APA utilized during erythropoiesis that functionally alters key genes. Identification of the molecular regulators of this process, some of which are already implicated in disease, may suggest novel therapeutic avenues.

NIH Research Projects · FY 2024 · 2022-09

ABSTRACT Tumor progression, resistance to therapy, and metastasis are closely related to the characteristics of the tumor cell ecosystem. Multiplexed antibody-based cytometry is the standard method for phenotypic characterization of tissue composition, pathogenesis, and immune infiltration with single-cell (and sometimes spatial) resolution. The identification of cell populations in these data is facilitated by algorithms that cluster cells according to their antigenic profile, as well as by predefined sets of markers that have historically evolved by trial and error. However, the annotation of these data is a manual, subjective, and laborious process that hinders the reproducibility and accuracy of the results. The design of antibody panels that include specific markers for all cell types and states present in a tissue is usually unfeasible, and the efficiency of commonly used markers is unknown. Consequently, cell clusters can differ little in their antigenic profile or contain a mixture of cell types. To overcome these limitations, this project will develop informatics technologies that leverage existing single-cell transcriptomic atlases to assist and automate the design and analyses of multiplexed antibody-based cytometry experiments. Our working hypothesis is that the vast amount of available single-cell transcriptomic data of tissues can inform the design, annotation, and analysis of cytometry experiments. We will develop and evaluate informatics technologies for establishing reference antigenic profiles and optimal antibody panels based on single-cell proteotranscriptomic data (Aim 1 ), and for automating the identification, annotation, and gating of cell populations in multiplexed antibody-based cytometry experiments (Aim 2). These new computational methods will enable any researcher to 1) automatically identify and annotate cell populations in a cytometry dataset based on reference single-cell data hosted in a repository, 2) define optimal gates for sorting cell populations, 3) transfer gates across experiments, 4) design optimal antibody panels for a given tissue or set of cell populations, and 5) infer the gene expression profile of cells. We will implement these methods in an open-source software and online portal for the transcriptome-guided annotation and analysis of cytometry data of tumors, and will closely work with end-users through several planned workshops and tutorials to maximize the utility and outreach of this platform (Aim 3). We will test our platform on leukemic and pancreatic cancer tissues profiled with spectral flow cytometry and multiplexed quantitative immunohistochemistry. The informatics technologies developed in this project will transform cancer research by boosting the phenotypic resolution, accuracy, and reproducibility of multiplexed antibody-based cytometry analyses of tumor tissues.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Anti-PD-1 therapy reinvigorates exhausted CD8 T cells, which can lead to complete tumor eradication as early as 3 weeks. Yet, despite robust T cell reinvigoration in >78% of patients, less than 40% are cured. The goal of this proposal is to understand why robust reinvigoration of exhausted CD8 T cells by αPD-1 therapy does not necessarily translate to clinical efficacy. Exhausted CD8 T cells (TEX) are a major cell type responding to PD-1 blockade. We and others have shown that αPD-1 therapy (αPD-1) reinvigorates exhausted CD8 T cells, as defined by enhanced proliferation and cytokine production. TEX are heterogeneous with progenitor and terminally differentiated TEX that have different roles in anti-tumor immune responses. Progenitor TEX (ProgEX) replenish terminally differentiated TEX (TermEX), which in turn provide anti-tumor activity. Thus, similar to stem cells, ProgEX represent a reservoir for CD8 T cell responses against the tumor. Importantly, it is the ProgEX, rather than TermEX, that respond to αPD-1 and almost exclusively contribute to the early burst of CD8 T cell reinvigoration. Reinvigoration of TEX results in accelerated differentiation of ProgEX to TermEX, resulting in a numerically greater pool of TermEX and improved tumor control. However, at the same time, the accelerated differentiation induced by αPD-1 places increased stress on ProgEX homeostasis. This raises the possibility that a ProgEX niche is key for providing the cells and signals necessary to prevent the depletion of ProgEX, and may prove crucial for the efficacy of PD-1 blockade. Our central hypothesis is that depletion of ProgEX after αPD-1 impairs clinical efficacy and that a tumor- associated niche is important in maintaining the pool of ProgEX. In Aim 1, we will test the hypothesis that αPD-1 results in enhanced differentiation of ProgEX and that that depletion of ProgEX is associated with clinical progression. We will use combinatorial tetramers and single cell RNA+TCR sequencing to understand how αPD-1 alters the pool of melanoma-specific ProgEX, and how the size of ProgEX pool in turn, impacts the clinical efficacy of αPD-1. In Aim 2, we test the hypothesis that tertiary lymphoid structures serve as a tumor- associated niche for ProgEX, and that increased number or size of ProgEX niches after αPD-1 is associated with the preservation of ProgEX and clinical efficacy. We will use multiparameter immunofluorescence and spatial transcriptomics to define the cellular composition of the ProgEX niche, the transcriptional circuits utilized by ProgEX in the niche, and the importance of the ProgEX niche in preserving the pool of ProgEX.

NIH Research Projects · FY 2024 · 2022-09

Project Summary Fragile X Syndrome (FXS) and Fragile X-associated/Tremor Ataxia Syndrome (FXTAS) are two FRAXopathies which are characterized by the unstable expansion of a CGG short tandem repeat (STR) located in the 5' untranslated region of the Fragile X Mental Retardation 1 (FMR1) gene. Expansion of the CGG tract from wild type length (WT, <55 CGG STR) to premutation (PM, 55-200 CGGs) results in a dramatic increase in FMR1 transcription with no noticeable elevation in levels of the protein it encodes (FMRP). Upon expansion to full mutation (FM, >200 CGGs), FMR1 is silenced via DNA methylation and, consequently, FMRP also reduces to baseline levels. Neither the removal of DNA methylation over the promoter and CGG tract nor transgene rescue cannot fully restore healthy phenotypes suggesting FMR1 and FMRP dysregulation are not the only disease drivers. The objective of my proposal is to investigate the RNA-mediated mechanisms driving disease-associated H3K9me3 deposition, trans interactions, and genome-wide STR instability in FXS. My central hypothesis is that FMR1 or FMR1-AS1 RNAs influence pathological heterochromatin deposition in a CGG length-dependent manner by toggling between sequestering key chromatin readers, writers, and erasers in inclusion bodies and forming toxic DNA:RNA structures locally and at distal loci. I have formulated my hypothesis based on our recent surprising observations that (1) Megabase-scale heterochromatin domains are acquired on autosomes and the X chromosome and spatially connect in ectopic inter-chromosomal interactions FM FXS in a manner that is dependent on the length of the CGG STR and (2) cut-back of the FM CGG to PM, or overexpression of PM-length CGG RNA, can reverse pathologic H3K9me3 deposition in FXS. Moreover, in established literature, PM-length CGG RNA forms nuclear inclusion bodies, whereas FM-length CGG can form toxic DNA-RNA R loops, but their interplay during FXS onset and progression and mechanistic connection to heterochromatin is unknown. I will test my hypothesis by employing state-of-the-art techniques like CUT&RUN, Hi-C, MapR, RADICL-seq, and ChlRP-MS in induced pluripotent stem cells differentiated to neural progenitors (iPSC-NPCs) across a range of CGG STR expansions and engineered cut-backs to shorter tracts. Upon successful completion of my experiments, I will elucidate the protein components of nuclear CGG RNA inclusion bodies, the location and sequence of RNA:DNA hybrids and R loops, heterochromatin placement, and genome folding features genome-wide as a function of CGG expansion and contraction. My work is significant because ii will elucidate the mechanisms by which FXS might progress via RNA-mediated heterochromatin in subnuclear bodies and established fundamental knowledge about the interplay between RNA-based inclusion bodies and RNA-DNA hybrids genome-wide in repeat expansion disorders. Our models of genome-wide heterochromatinization and gene silencing in FXS will also shed light on possible new mechanisms for H3K9me3 in other human conditions such as cancer, neurodegeneration, and aging.

NIH Research Projects · FY 2024 · 2022-09

Project Summary Respiratory viral infections represent a major risk factor for the development of acute respiratory distress syndrome. Moreover, severe influenza injury can result in ineffective repair and persistent loss of pulmonary function. The lung endothelium, especially the microvasculature, is damaged due to the robust inflammatory response from viral infections, but the mechanisms of pulmonary endothelium regeneration after severe influenza injury are not completely elucidated despite the vasculature’s central importance in gas exchange. This proposal aims to answer fundamental questions about pulmonary vascular regeneration regarding the origin of vascular progenitor(s) and the mechanisms driving endothelial repair in response to influenza injury. We recently demonstrated that COUP-TFII, a vein-specifying transcription factor enriched in proliferating endothelial cells, is necessary for lung regeneration. The venous endothelium is increasingly recognized as a vascular progenitor population for endothelial regeneration in various organs in zebrafish and mice, suggesting that pulmonary veins / venules may similarly harbor potent progenitor cells. In my own preliminary data, I observed that venous endothelial cell clones are highly proliferative and can span into the microvasculature. Aim 1 of this proposal will utilize clonal lineage tracing and orthotopic transplantation techniques to determine if the venous endothelium harbors potent progenitor and proliferative potential during lung regeneration. The second focus of this proposal is to elucidate the mechanisms and interactions that are required for endothelial proliferation. Along with expression of venous markers, proliferating endothelial cells secrete C-C Motif Chemokine Ligand 2 (CCL2), a chemokine involved in mediating the inflammatory response during injury. The CCL2-CCR2 signaling axis promotes inflammatory angiogenesis in mice through recruitment of monocyte derived inflammatory macrophages, indicating a role for endothelial-specific release of CCL2 during tissue repair. Therefore, Aim 2 will employ conditional, temporal deletion of CCL2 in endothelial cells and clodronate-liposome mediated depletion of macrophages to investigate if loss of endothelial-derived CCL2 impacts endothelial proliferation and consequent angiogenic repair. This proposal will address the central hypothesis that a pulmonary endothelial progenitor population present in the preexisting venous endothelium secretes CCL2 to recruit interstitial monocytes to provide an angiogenic niche. Completion of this project will validate the venous endothelium as an important contributor to pulmonary regeneration and will also facilitate identification of specific paracrine / immune pathways that could allow for precise regulation and preservation of the beneficial immune response.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT RhoA, a member of the Rho-family of small GTPases, centrally regulates actin organization and actomyosin contractility. RhoA dynamics coordinate actin stress fiber formation, which ultimately determines how cells generate cytoskeletal tension to transmit mechanical forces to/from neighboring cell-cell junctions and focal adhesions with the extracellular matrix (ECM). Thus, new tools to control the dynamics of RhoA signaling may enhance understanding of how cells make decisions in response to mechanical stimuli. We propose to create optogenetic tools for bi-directional (activation and inactivation) control over RhoA signaling, systematically characterize their function through a set of physiological assays in contractility and mechanotransduction, and benchmark their performance against other reported optogenetic technologies. We will use our recently discovered BcLOV4 photoreceptor that dynamically translocates via a direct light-induced protein-lipid interaction with the plasma membrane, making ii powerful for single-component optogenetic control over peripheral membrane proteins that is robust across cell types and primary cells. To demonstrate the unique capabilities of the toolbox, we will quantitatively map the cytoskeletal signaling and tensional dynamics of a known RhoA/Y AP mechanotransductive feedback loop in cytoskeletal remodeling and persistent cell motility, through simultaneous optogenetic perturbation and multi-reporter imaging. This toolbox will broadly impact cell and cytoskeletal biology by advancing control over ubiquitous RhoA signaling to probe its diverse regulatory roles in cell contractility, motility, mechanotransduction, and regeneration.

NIH Research Projects · FY 2025 · 2022-09

Measuring and manipulating metabolic fluxes in the tumor microenvironment Tumors have altered metabolism compared to normal tissues, which suggests that drugging metabolism could kill tumors while sparing healthy tissues. However, tumor metabolism has chiefly been measured in vitro, while recent studies have showed that tumor metabolism in the body is distinct from in vitro systems. Therefore, the field needs approaches to measure tumor metabolic fluxes in vivo. During my postdoctoral fellowship, I developed methods to measure glycolytic and tricarboxylic acid cycle (TCA) flux in vivo using kinetic infusion of isotope-labeled tracers. These approaches revealed that tumors have much lower TCA flux than healthy tissues (5 mouse tumor models examined). Though the tumors had higher glycolytic flux than healthy tissues, the total ATP production rate from glycolysis plus TCA cycle-driven oxidative phosphorylation was significantly lower in tumors than in healthy tissues. Moreover, feeding mice a high-fat ketogenic diet increased tumor TCA flux and slowed tumor growth synergistically when combined with chemotherapy. These findings raise two key questions. First, since tumors in vivo are a mix of cancer cells and other infiltrating cells, what is the metabolism of cancer cells versus immune cells or fibroblasts in tumors? Second, can directly upregulating tumor TCA flux slow tumor growth? I propose first to combine my glycolysis and TCA cycle measuring techniques with immunomagnetic and sorting strategies to measure fluxes in cancer cells, immune cells, and fibroblasts (Aim 1). I will apply this strategy to melanoma, a tumor type infiltrated by CD8 T cells which can help control the tumor, and to pancreatic adenocarcinoma, a tumor type where fibroblasts and myeloid cells can be even more abundant than cancer cells. Next, I will directly upregulate TCA flux in tumor cells by using genetic and pharmacologic approaches: overexpressing the NADH uncoupler protein mito- LbNOX, knockout of the TCA suppressor protein PDK, and inhibition of PDK with dichloroacetate. I will confirm that these strategies increase TCA flux using the method I developed and will test whether increased TCA flux slows tumor growth in primary and metastatic breast tumors. Successful completion of these aims will reveal the metabolism of different cell populations in the tumor microenvironment and will test TCA upregulation as a therapeutic strategy in cancer.

NIH Research Projects · FY 2024 · 2022-09

PROJECT ABSTRACT/SUMMARY CANDIDATE: I am a postdoctoral fellow in the laboratory of Dr. Ross Levine in the Human Oncology and Pathogenesis Program at Memorial Sloan Kettering Cancer Center. My previous PhD research offered me the opportunity to develop the experimental and computational skills necessary to assess cellular crosstalk in the tumor microenvironment. My current research extends these skills to the study of mutation order, and oncogene-dependency in subpopulations of leukemic cells. To gain insights into these processes I developed novel, multi-recombinase mouse models of oncogene-activation and dependency as well as new lineage tracing tools that allow for functional interrogation of clonal evolution. My proposed research will provide a strong foundation for independent research following the K99 phase of this grant. My long-term career goal is to identify molecular mechanisms driving leukemogenesis, including interactions between AML subclones in vivo and the role of sequential mutational acquisition. To achieve these goals I have developed a career plan that will 1) bolster my technical skills and scientific scope, 2) improve my presentation and communication skills, 3) cultivate professional relationships and networking, and 4) prepare me for mentoring future trainees. RESEARCH: The receptor tyrosine kinase, FLT3, is the most commonly mutated gene in acute myeloid leukemia. Mutations in FLT3 are often found with low variant allele frequency, suggesting these mutations occur as late, subclonal events. Despite their presence as a minor clone, FLT3 mutations are poor prognostic markers and the target of several recently approved clinical compounds. These inhibitors lead to some transient clinical success, yet patients invariably relapse and develop resistant, calling into question the necessity of FLT3 mutation in disease progression. I aim to determine the dependency of FLT3 mutations in disease, and propose methods to assess the functional contributions of subclonal mutations to disease progression. The specific aims are: 1) determining the genomic context for FLT3 oncogene-dependency in AML, 2) identifying novel therapeutic vulnerabilities in FLT3-driven AML, and 3) investigating the role of mutation order and clonal crosstalk in leukemic disease. ENVIRONMENT: The Levine laboratory is a part of the Human Oncology and Pathogenesis Program (HOPP) at Memorial Sloan Kettering Cancer Center, a state of the art cancer research institute. The Levine lab is also a member of the Center for Epigenetic Research, and the primary mentor Dr. Levine, is the head of the Center for Hematologic Malignancies. These affiliations provide a rich set of collaborative, technical and scientific resources to execute the research and career development proposed here.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY A range of cellular and circuit-level adaptations develops in response to chronic opioid exposure, which are strongly linked to several facets of opioid addiction: tolerance, withdrawal and processes that may contribute to compulsive use and relapse. However, we still do not have a comprehensive picture of the dynamic connections and activities of neuronal networks in the brain that express the opioid receptors and peptides. Therefore, a critical need exists to map the global cell-type identity, transcriptomic trajectory, shifting connectivity, and ensemble activity of the key opioidergic networks underlying the onset and maintenance of cellular dependence, and withdrawal. This proposal aims to investigate the architecture and function of endogenous MOR-expressing neural circuits in key cortical and subcortical brain regions, in order to determine how these circuits maintain cellular dependence and drive brain-wide maladaptive plasticity across different stages of the OUD cycle. In four complementary aims, we will first map the shifting structural and functional connectivity of opioidergic networks using viral-genetic and tissue clearing methods to identify monosynaptic inputs to all MOR-expressing, as well as withdrawal-active MOR-expressing neurons, as a function of opioid exposure and abstinence. We will then integrate these dynamic neuroanatomical maps with cell-type information and gene expression changes by combing single-nuclei sequencing and spatial cellular-resolution transcriptomics via hyper-multiplexed in situ hybridizations to generate the anatomic localization of hundreds of dependence-related genes, targeted to cell types and retro-labeled connections. Lastly, to reveal how MOR-expressing cells within the cortical and subcortical target regions are modulated during opioid exposure in real-time, we will use miniature head-mounted microscopes to image the neural ensemble activities across weeks of opioid exposure and withdrawal. To bridge these experimental measurements and provide a common framework for our analyses, we will adopt Network Control Theory to identify brain nodes that drive the transition between opioid dependence states to identify potential candidates that disproportionately drive each state. Our datasets will provide formal summaries and a publicly available, searchable database logging the activity, connectivity, and gene expression as they evolve with repetitive opioid exposure, withdrawal, and abstinence.

NIH Research Projects · FY 2026 · 2022-08

PROJECT SUMMARY/ABSTRACT Our long-term goal is to understand how the brain processes information in a flexible, context-dependent manner to support effective decision-making. Many previous studies of decision-making focused on how the brain accumulates evidence used to select a particular action, which was shown to involve modulations of persistent activity of individual sensory-motor neurons that prepare the appropriate action. However, the brain must also often accumulate evidence in a flexible manner across actions, about which little is known. We propose that decisions requiring the flexible accumulation of feedback-related evidence across actions depends on interactions between the Anterior Cingulate Cortex (ACC), a cortical structure on the medial surface of each cerebral hemisphere that has widespread connectivity with other parts of the brain, and the brainstem nucleus locus coeruleus (LC), which is the primary source of the neuromodulator norepinephrine (NE) to the rest of the brain. The ACC and LC have strong, reciprocal connections and are thought to interact in ways that support key features of cognition, including adaptive information processing, but the details of these interactions are not well understood. Our primary hypothesis is that these interactions modulate activity patterns of populations of ACC neurons that implement a process of across-trial evidence accumulation that uses reward and error feedback to govern decisions to switch behavioral choices. We are particularly interested in understanding how these modulations relate to changes in coordinated variability in ACC that can have major effects on how neural populations process information. To test this hypothesis, we use simultaneous, complementary measurements of neuronal activity from single and populations of neurons from the two brain areasin the context of two tasks that require different forms of across-trial accumulation of feedback information to guide saccadic decisions. We have three Specific Aims. Aim 1 is to understand how activity patterns of individual neurons in the LC relate to performance on these tasks. Aim 2 is to understand how relationships between neuronal activity patterns in the LC and ACC relate to performance on these tasks. Aim 3 is to use a combination of manipulations to identify causal contributions of temporally precise, pathway specific activity patterns from LC to ACC on task performance. Together these Aims will provide new mechanistic and computational insights into how LC-related modulations of ACC population activity support ACC's role in flexibly linking performance monitoring and control across multiple trials. Our findings will have direct relevance to numerous constructs in the Research Domain Criteria (RDoC) framework and thus have broad significance to fields that aim to understand the neural substrates of complex behaviors and their dysfunction in certain mental disorders.

NIH Research Projects · FY 2025 · 2022-08

Influenza alone kills as many as 500,000 people annually, and at least 4.5 million have died from SARS-CoV-2 during this ongoing pandemic, with mortality from both respiratory viruses largely due to viral pneumonia progressing to acute respiratory distress syndrome (ARDS). Significant focus has been placed on regeneration of the epithelium after influenza, but relatively little is known as to how the endothelium is repaired, a critical question since vascular endothelial integrity is necessary to prevent ARDS-associated pulmonary edema, hypoxemia, and mortality. We recently demonstrated that at least 20% of the lung's vascular endothelium is regenerated by 1 month after infection, indicating a robust capacity for endothelial repair. We therefore investigated signaling pathways which might modulate this regenerative functionality. Somewhat unexpectedly given its role in promoting fibrosis, we observed that endothelial- specific blockade of TGF-β signaling prevents effective repair of the lung endothelium and results in inefficient physiologic recovery. Moreover, in order to achieve coordinated, functional tissue repair, we reasoned that lung endothelial cells might influence repair of the adjacent epithelium by release of angiocrine factors. In keeping with this notion, we identified a matricellular protein, SPARCL1, which is secreted by injury-activated endothelial cells and serves to enhance alveolar epithelial regeneration, at least in part by modulating TGF-β. Based on our cumulative findings, we hypothesize that injury-activated endothelial cells engage a TGF-β / SPARCL1 axis to coordinately regulate lung endothelial and epithelial repair. The major objectives to address this hypothesis are 1) mechanistically define how TGF-β promotes lung vascular repair and 2) determine whether and how SPARCL1 acts as an angiocrine factor to promote alveolar epithelial regeneration. Successful completion of these studies will inform approaches designed to enhance endothelial cell regenerative potential and promote effective lung repair / prevent mortality in ARDS and viral pneumonia.

NIH Research Projects · FY 2024 · 2022-08

Project Summary/Abstract TAR DNA-binding ~43kDa (TDP-43) inclusions are the pathological hallmark of frontotemporal lobar degeneration with TDP-43 (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). Despite shared pathological features, ALS and FTLD-TDP can present with heterogenous clinical features including cognitive/behavioral impairments (i.e., FTLD-TDP), motor neuron dysfunction (i.e., ALS), or both (i.e., ALS-FTD). Moreover, certain genetic mutations typically only result in ALS or FTLD-TDP, but other mutations can cause FLTD-TDP and/or ALS within the same family. Together, the shared and disparate pathological, clinical, and genetic features of FTLD-TDP, ALS, and ALS-FTD support the notion that they are part of a clinicopathologic spectrum. However, the vast majority of ALS and FTLD-TDP cases are considered sporadic and have no known causal mutation. The underlying molecular mechanisms that contribute to the observed clinical heterogeneity across sporadic TDP-43 proteinopathies are not well understood. Supporting evidence for a genetic component to sporadic cases, common genetic variants have been associated with disease risk for either ALS or FTLD-TDP. However, combined studies across TDP-43 proteinopathies are rare. Thus, the extent to which risk alleles are shared or disparate across these phenotypes is unclear. While there is mounting evidence of shared genetic factors that explain the biological mechanisms that drive susceptibility to both diseases, considerably less effort has focused on the disparate genetic features across TDP-43 proteinopathies. Identifying disparate variants may contribute to our understanding of disease-specific drivers. The first aim of this proposal is to identify both the shared and disparate genomic features that drive individual-level cognitive/behavioral and/or neuromuscular presentations of these syndromes. TDP-43 pathology and neurodegeneration are observed in characteristic neuroanatomical regions that correlate with the clinical presentations of ALS and FTLD-TDP, but it is unclear what contributes to this regional selective vulnerability. Previous work has demonstrated clear regional differences in gene expression within the same individuals. Gene expression quantitative trait loci analyses can identify genomic loci that explain variation in gene expression. However, these associations are highly tissue and cell type specific, which may obscure important differences. This is especially true within neurodegenerative disorders, as observed gene expression differences may reflect cell type composition differences rather than true transcriptional regulation. The second aim of this proposal is to investigate genetic contributions to regionally specific differential gene expression, including cell type specific expression, in ALS and FTLD-TDP. Overall, this proposal leverages genotype and transcriptomic approaches to understand molecular contributions of regional selective vulnerability in ALS and FTLD-TDP. In disentangling this heterogeneity, it may be possible to inform efforts to develop therapeutic targets that attenuate the course of these neurodegenerative diseases.

NIH Research Projects · FY 2025 · 2022-08

This R35 proposal is to support a robust translational research program focused on sepsis-associated acute respiratory distress syndrome (ARDS), explaining individual risk and characterizing the ARDS host immune response in order to identify molecular traits that may respond differently to specific therapy. The year 2020 and the SARS-CoV-2 pandemic placed a global spotlight on sepsis-associated ARDS and its lack of pharmacologic treatments, with over 400,000 American deaths. Even in non-pandemic years, however, ARDS complicates approximately 10% of all intensive care unit admissions and impacts close to 200,000 Americans. Mortality for ARDS has stubbornly exceeded 30%. I have used my translational science training to extend our knowledge of individual factors and pathways that influence ARDS risk and resolution, use genomic tools to infer which ARDS-associated plasma markers may be causal contributors to ARDS risk and mortality, and deeply characterize the host response to COVID-19 ARDS. I have grown a research program that includes a carefully phenotyped cohort of over 3,500 human subjects critically ill with sepsis, and curated biosamples at multiple timepoints to facilitate genomic and molecular discoveries, while contributing to the career development of multiple NHLBI-funded patient-oriented researchers. With the support of the R35, the Meyer research program will focus on 5 complementary themes to improve the health of patients with and at risk for sepsis-associated ARDS. Thematic area 1 concentrates upon understanding individual risk for ARDS and ARDS mortality, which will utilize whole genome association, expression and protein quantitative trait locus analysis, and genetic causal inference frameworks to evaluate inherited risks and identify which RNA and plasma traits may be causal intermediates in ARDS. Area 2 addresses the host response to ARDS, using deep immune profiling and integrated analyses to characterize and contrast the response to bacterial and viral sepsis-associated ARDS. In later years, sterile ARDS will be compared to infectious ARDS, and the contribution of activated T cells will be examined. Area 3 examines the interplay between ARDS and non-lung organ injuries during sepsis, particularly acute kidney injury, delirium and cognitive injury, and shock and circulatory dysfunction. We will identify DNA, RNA, plasma, and cytometric features specific to individual organ failures and shared across multiple organ systems. The R35 program will also catalyze two new areas of investigation for the Meyer lab. First, we will apply biomedical informatics techniques to integrate, visualize, and analyze multiple data networks – clinical, genomic, transcriptomic, proteomic, metabolomic, and cytometric – to identify coordinated patterns of response and their association with ARDS outcome. We will also examine the longitudinal host response to ARDS during recovery, testing for responses that predict or protect from post- intensive care syndrome. An investment in our research program will advance the prevention and personalized treatment of ARDS while fostering training and mentorship in lung health research.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Even with the latest digital breast tomosynthesis (DBT) systems, breast cancer screening continues to suffer from poor specificity. Only about 5% of women called-back from screening are ultimately found to have a biopsy- proven cancer. Clinical DBT systems suffer from anisotropies in image quality since the scanning motion is restricted to one direction (left-to-right). We built a next-generation tomosynthesis (NGT) system that is capable of scanning in the shape of a “T”. With phantoms and mastectomy specimens, we have shown that this design mitigates cone-beam artifacts, tissue superposition effects, and anisotropies in super-resolution. As the next step in our research, we will perform a pilot study with volunteers, recruiting women referred for diagnostic imaging or biopsy as well as women having abbreviated magnetic resonance imaging (MRI). Projection images will be acquired in such a way that we can generate reconstructions from two scanning methods (conventional and T). Each scanning method will be analyzed separately by radiologists in different reading sessions. We will mitigate potential concerns about radiation exposure by restricting the study to one view (cranial-caudal) instead of two views. We have put together a team with a unique set of strengths, including the developers of the NGT system, three radiologists, two statisticians, and experts in density and texture analysis. This proposal is divided into two specific aims. (Aim 1): Assess radiologists' performance in a pilot study of the NGT system with volunteers. We will investigate whether the T scan brings down the call-back rate of screening without reducing sensitivity. Radiologists will also rate the overall probability of malignancy, and these scores will be analyzed in combination with clinical follow-up data to show that radiologists' ability to characterize findings is improved with the T scan, specifically by using jackknife alternative free-response receiver operating characteristic (JAFROC) methods. (Aim 2): Perform quantitative analysis of the 3D breast outline segmentation, texture, and density. With breast phantoms, we have previously shown that the breast volume is overestimated in the conventional scan and is calculated more accurately in the T scan. We aim to show that the same result holds in human subjects by calculating volume differences between the two scanning methods. Additionally, we will analyze power-law noise and higher-order non-Gaussian texture measures as surrogate metrics of detectability and tissue superposition effects, which we expect to be improved by the T scan. Finally, we will analyze whether percent density calculations differ between the two scanning methods since we expect fewer out-of-plane artifacts in the T scan. Although the new method of scanning is not being used as part of the volunteers' medical care, the overall impact of this study is to demonstrate improvements in specificity and thus the potential to minimize the number of diagnostic imaging exams and biopsies, lower healthcare costs, and minimize the total radiation dose combining screening and diagnostic imaging. Women with dense breasts will especially benefit from this new design since dense tissue can obscure findings in a conventional DBT scan, making them harder to characterize.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY This Mentored Clinical Scientist Career Development Award (K08) details a five-year plan to promote Dr. Heather Wachtel’s transition to an independent career as a physician-scientist studying neuroendocrine tumors. Bioinformatics and genomics are rapidly evolving fields which offer novel approaches to the study of human disease. This career development plan includes formal training in bioinformatics and computational genomics, as an in-depth understanding of these fields is critical for successful completion of the proposed research, and for Dr. Wachtel’s development as an independent investigator. Dr. Wachtel is mentored by Dr. Katherine Nathanson, a cancer geneticist, and her current work with Dr. Nathanson utilizes translational approaches to study tumorigenesis and the spectrum of disease associated with hereditary cancer predisposition genes. Dr. Wachtel’s focus is on succinate dehydrogenase (SDHx) pathogenic variants and neuroendocrine tumors. Succinate dehydrogenase is a highly conserved mitochondrial complex with critical roles in metabolism and cancer. Inherited loss-of-function mutations in the SDHx genes are causative in several human cancers. Recent data suggests that tumors associated with germline pathogenic variants in SDHx, including pheochromocytoma, paraganglioma and renal cell carcinoma, are linked to DNA damage. However, DNA damage repair has not been studied on a gene-specific level, and the gene and allele-specific risks of SDHx germline pathogenic variants remain incompletely characterized. This proposal aims to accurately characterize the phenotypes and tumor biology associated with SDHx germline variants, to develop improved risk estimates and identify targeted therapies for patients who progress to disease. In AIM 1, Dr. Wachtel proposes to perform a Phenome-Wide Association study (PheWAS) of SDHx in the UK Biobank to accurately characterize the gene-specific oncologic associations of SDHx and quantify phenotypic associations with inflammatory and metabolic disease at the gene level. Findings will be replicated in an independent cohort from the Penn Medicine BioBank. In AIM 2, Dr. Wachtel will utilize the unique resources of the Penn Neuroendocrine Tumor Center and expertise in collaborative studies to quantify genomic signatures of DNA damage response pathways in pheochromocytoma and paraganglioma associated with SDHx germline pathogenic variants. Finally, she will evaluate the evidence for potential poly(ADP-ribose) polymerase (PARP) inhibitor susceptibility in patient-derived tumor specimens. Dr. Wachtel will supplement these studies with a career development program which takes full advantage of the depth and breadth of resources at the University of Pennsylvania. Dr. Wachtel has assembled a mentoring and advisory team of accomplished and successful physician-scientists and geneticists to guide her career development. She will engage in both formal didactic and hands-on training to hone her skills in bioinformatics and genomics. This career development plan and the experiments detailed in the Research Strategy will provide Dr. Wachtel with the tools necessary to achieve her long-term goal of an independent investigative career as a physician-scientist.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY/ ABSTRACT Moderate or severe TBI is a substantial health problem that in addition to often-devastating acute effects, can trigger chronic and evolving neuropathologies that may underlie long-term functional decline. Surprisingly protracted axonal degeneration and associated tissue atrophy has be observed following moderate-severe TBI in humans, persisting many months and years after injury and may contribute to progressive cognitive decline in some patients. However, the mechanisms that drive axons to continue to swell and degenerate in the months and years post-TBI are unknown, and potentially modifiable. Moreover, how the acute injury, including the degree of focal versus diffuse brain injury, contributes to the nature and progression of axon degeneration is unexplored. Here we hypothesize that chronic axon degeneration in moderate-severe TBI reflects a progressive disconnection syndrome across neural networks via degeneration in both an anterograde and retrograde direction, which is dependent upon the nature and distribution of the initial injury. The death of neuronal somata, for example due to a focal lesion, can induce downstream (anterograde) degeneration of their efferent axons (and potentially their synaptic partners) and has long been thought as the dominant driver of axon loss in the subacute phase. In contrast, loss of a neuron's post-synaptic partner has been shown to drive retrograde degeneration via the loss of survival signaling (via the Jun Kinase/ c-Jun pathway). In preliminary data we provide evidence that both retrograde and anterograde degeneration drive chronic and progressive axon degeneration for many months and years post-TBI using multiple models and post-mortem human TBI tissue. Based on these compelling findings we propose an integrated translational design to 1) Determine the relative contribution and mechanisms of anterograde and retrograde degeneration chronically post-TBI in mice via examination of the neuronal network through tissue clearing techniques in combination with viral tracing and single cell RNA sequencing; 2) Utilize a gyrencephalic model of TBI to validate observations in mice and evaluate how progressive axon degeneration across the gyrencephalic brain determines behavioral outcome and, 3) Determine the role of focal and diffuse brain injuries in determining the extent, distribution and nature of progressive axon degeneration following human TBI using post-mortem samples. Understanding the mechanisms by which axons degenerate in the chronic phase post-injury will provide important data necessary for the development of targeted therapeutic interventions over time following TBI.

NIH Research Projects · FY 2025 · 2022-08

This is an entrepreneurship-focused educational program with the overarching goal of training the workforce of the future with the necessary broad knowledge base required for the successful commercialization of promising clinical and translational research that will help to address the growing public health crisis posed by Alzheimer’s Disease and AD Related Disorders (ADRD). We will be taking a national approach to this challenging area in order to engage with the most promising graduate and postdoctoral researchers from around the country, leveraging the robust NIH investment in Alzheimer’s Disease Research Centers (ADRCs) and Clinical and Translational Science Award (CTSA) programs. This R25 program will provide trainees with a keen understanding of the interdisciplinary, clinical and translational nature of research and commercialization related to ADRD, how it depends on fundamental underpinnings of both science, engineering and regulatory science, and how commercial products are developed. The knowledge base, mentorship and ecosystem supported by this R25 will help them prepare competitive SBIR/STTR grants to further their work. We have identified a robust network of commercial, regulatory and academic experts that will allow us to address our core aims: 1) Training in entrepreneurial skill development for ADRD technologies in a structured didactic curriculum that includes foundational elements, case-based learning, and personalized components tailored to their specific domain (i.e., ADRD small molecule development, biologics, digital health, etc.) leveraging the extensive resources developed at the University of Pennsylvania, including the Academic Entrepreneurship for Medical and Health Scientists textbook (of which Dr. Gooneratne is the lead editor); 2) Mentored research experience with tailored guidance to maximize stakeholder/customer discovery and product iteration and experiential learning opportunities to complement any gaps in their commercialization plans; 3) Curriculum development through innovative modification of Individual Development Plans (IDPs) to incorporate entrepreneurship. Five to ten trainees will be enrolled each year, and they will have a research mentor, a business mentor and an R25 program mentor to ensure that they receive comprehensive support and guidance. Trainees will participate in the program for two years, with the first year dedicated to learning and development with the goal of submitting an initial SBIR/STTR grant application by month 15 or sooner, then the second year devoted to additional activities to support a grant resubmission or successfully initiate the funded grant. We seek ultimately to develop a community of highly trained entrepreneurs and researchers in ADRD that can engage in interdisciplinary team science with sustained engagement through an active, participatory ecosystem to serve as the nidus for meaningful advances as we confront the looming healthcare challenge posed by ADRD.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY The prevalence of obesity and associated comorbidities is a major public health concern with significant personal and societal consequences. Weight loss pharmacotherapies that target known food intake-inhibitory mechanisms (e.g., hindbrain and hypothalamic circuits) have been largely unsuccessful at promoting sustained weight loss. This suggests the existence of yet-to-be-identified nodes that regulate food intake, and uncovering such mechanisms holds promise for the development of more effective obesity treatments. To this end, we recently used a “reverse translational” approach to identify and characterize a novel population of cerebellar neurons and their importance to feeding behavior. Starting with human subjects and following up with mechanistic experiments in mice, we discovered that glutamatergic neurons in the anterior, lateral deep cerebellar nuclei (aDCN-lat) are activated by food and have the ability to dramatically reduce meal size. These data demonstrate that glutamatergic aDCN-lat neurons are critical regulators of feeding behavior, and highlight this population as a potential target for obesity therapeutics. Here, we leverage these findings to understand how activity in these cerebellar neurons is regulated and how it is altered in obesity. The goals of this proposal are to (1) reveal the sensory, nutritive, and gut-brain pathways that activate cerebellar satiation neurons, (2) determine how activity in these neurons changes in obesity, and (3) test whether chronic activity in these neurons is sufficient to prevent or reverse diet-induced obesity in mice. These results will provide a comprehensive understanding of cerebellar mechanisms for feeding behavior, illuminating a novel satiation center in the brain that may be targeted for the development obesity therapeutics.

NIH Research Projects · FY 2025 · 2022-08

Project Summary Objective: Respiratory viral infections affect millions of individuals each year. Conducting frequent widespread viral screening tests can curb outbreaks by quickly identifying infectious persons. Unfortunately, no screening diagnostic platform exists with the capacity to test hundreds of millions of people daily during a pandemic. This proposal is the first step in developing a novel viral screening test to fill this gap. The assay will consist of microscopic circuits containing field effect transistors with antigen-specific receptors that are sensitive to particular viruses. These chiplets – barely visible to the human eye – will be powered by light and will transmit data using a light emitting diode, such that a few can be mixed into an extracted sample and illuminated with a handheld device to yield immediate results. This diagnostic will be scalable and inexpensive; millions of tiny chiplets can be fabricated simultaneously. It will be minimally instrumented; an ordinary cellphone with a strobe flash and camera will interface with the chips. It will be flexible, rapidly adaptable, and multiplexable; receptors specific to different or emerging viruses could be immobilized on distinct circuits, allowing multiple diseases to be detected simultaneously in a single patient sample. This diagnostic will thus be unmatched as a mass producible, simple to use, adaptable, and high throughput tool for frequent and widespread virus screening. Specific aims: The proposed diagnostic will be developed by pursuing the following specific aims. 1. Integrate biological field effect transistors into the existing optical wireless integrated circuit platform. 2. Develop a multiplexed detection scheme for interacting with optical wireless integrated circuits. 3. Demonstrate the test’s feasibility in a clinically relevant quantitative range using mock clinical specimens. Career development plan and career goals: Dr. Matthew Campbell (Ph.D., P.E.) is a postdoctoral researcher in the School of Engineering and Applied Science at the University of Pennsylvania, where his work is focused on fabricating microelectromechanical systems. The proposed K25 career development award will apply his nanofabrication skills toward biosensor development and extend his training and exposure into two new domains: (1) biomedical experimentation, and (2) medical biology. This proposal contains a cohesive mentorship and didactic strategy centered on these areas to accelerate his trajectory toward research independence. Completion of this multifaceted training plan will position Dr. Campbell with the cross-disciplinary skills and expertise necessary to become a leading investigator in the field of biomedical sensing diagnostics. Mentors and environment: Dr. Campbell is enthusiastically supported by the university and his strong mentoring team. His primary mentor is an expert in micromanufacturing (Prof. Igor Bargatin (Ph.D.)), and his co- mentors bring extensive experience in microscopic circuits (Prof. Marc Miskin (Ph.D.)), field effect transistor sensors (Prof. Charlie Johnson (Ph.D.) and Prof. Haim Bau (Ph.D.)), and viral respiratory tract infections (Prof. Ronald Collman (M.D.). This group will provide the ideal training situation for Dr. Campbell to develop this assay.

NIH Research Projects · FY 2024 · 2022-08

PROJECT SUMMARY AND ABSTRACT Caspase-1 is a cysteine protease that catalyzes the maturation of cytokines and plays critical roles in the innate and adaptive immune response to pathogenic stimuli. Misregulation of caspase-1 is associated with various autoimmune diseases and cancer. Similarly, caspase-2 is a cysteine protease that is important for regulating the cellular response to stresses that cause DNA damage, such as chemotherapy. Both caspase-1 and caspase- 2 are activated by structurally similar sensor proteins that sense intracellular perturbations and mount appropriate responses, but the molecular mechanism of how the sensors are activated and then in turn activate their respective proteases, is not well understood. A series of germline-encoded pattern recognition receptors sense conserved features of pathogens, and assemble into multiprotein complexes called inflammasomes, which recruit and activate caspase-1. The consensus model for caspase-1 activation is that inflammasomes are first activated then they in turn activate caspase-1. Our preliminary data suggests that caspase-1 plays a role in inflammasome activation, which in turn activates more caspase-1, however, the molecular mechanism is unknown. The goal during the K99 mentored phase, is to determine the role caspase-1 plays in inflammasome activation. Specifically, we will determine the activation mechanism of the ZU5 domain-containing inflammasomes, CARD8 and NLRP1. During the independent R00 phase, we will then apply the training from the mentored phase to determine the activation mechanism of another ZU5 domain-containing sensor that activates caspase-2 in response to genotoxic stress, PIDD. Our central hypothesis is that the ZU5 domain- containing sensor proteins are activated in a similar manner, in which the proteases they activate participate in sensor activation, which in turn activates more protease. To accomplish these goals, I have carefully assembled a highly complementary advisory team with the scientific and mentoring skills needed to guide my path to research independence. The completion of this work will further our understanding of pyroptosis and apoptosis regulation and could potentially advance therapeutic development efforts for a variety of human diseases.

NIH Research Projects · FY 2025 · 2022-08

Summary Advanced emphysema is primarily characterized by chronic inflammation, small airways obstruction, and parenchymal destruction leading to hyperinflation, compromised respiratory mechanics, and progressive functional decline. Medical therapy has proven effective in treating symptoms such as coughing and shortness of breath, and can also help to prevent acute exacerbations, but does little to improve either mortality or restore lost function. While lung volume reduction surgery (LVRS) has demonstrated the ability to improve lung function, quality of life and mortality in certain, rigorously selected patients, it is associated with a significant increase in perioperative and short-term morbidity and remains an underutilized treatment. In 2018, treatment with Zephyr Endobronchial Valves (Zephyr EBV) became the first bronchoscopic lung volume reduction technique to receive FDA approval. Valves are inserted via catheter in order to occlude a target emphysematous lobe, causing partial or complete lobar atelectasis, decreasing residual volume, reducing hyperinflation and improving breathing mechanics and lung function similarly to LVRS with improved morbidity and mortality. Despite numerous studies demonstrating clinically significant average improvements in various functional and anatomical outcome measures, however, there currently remain a significant number of EBV recipients who fail to experience meaningful quality of life benefits as a result. In order to address this discrepancy, the proposed project will use hyperpolarized xenon-129 MRI's unique ability to measure regional lung function, in combination with the assessment of systemic inflammatory biomarkers, to attain a more comprehensive understanding of the mechanisms through which EBV placement perturbs and alters the lung. We hypothesize that this consists primarily of a redistribution of both ventilation and perfusion to the healthier lung as well as a decrease in both local and systemic inflammatory burden—and that sensitively assessing the presence/absence of these changes, as well as their degree, will help to explain the frequent divergence between quantitative and qualitative assessments of EBV treatment efficacy. Using a previously developed multi-breath hyperpolarized 129Xe buildup/washout sequence, combined with a measurement of signal intensity buildup, to produce quantitative maps of minute ventilation and functional residual capacity, we will quantify ventilation redistribution and residual volume at a lobar or segmental level. These maps are compared to registered and segmented CT-derived measurements of airways disease and emphysema. Next, we will employ HP 129Xe dissolved-phase imaging to quantify gas uptake by the red blood cells in the lung—a measurement that relates more closely to blood oxygenation than measurements of perfusion, and investigate the use of dynamic airflow imaging to distinguish clinically important cases of collateral ventilation and leakage around the valve. Finally, we will evaluate a number of systemic inflammatory biomarkers, as well as inflammatory cells such as activated macrophages and CD8+ T cells, whose presence we will attempt to correlate with subjectively quality of life assessments post-treatment.

NIH Research Projects · FY 2025 · 2022-08

Understanding the roles of cardiac NAD+ pools and therapeutic effects of precursor supplements in heart failure We are exploring the hypothesis that nicotinamide adenine dinucleotide (NAD+) metabolism can be targeted to improve functional capacity in failing human hearts. NAD+ is a ubiquitous molecule that is required as a redox cofactor or substrate for hundreds of enzymes within the cell. It is derived from dietary tryptophan, niacin, nicotinamide, or synthetic intermediates, but the majority of synthesis in the heart is via nicotinamide. NAD+ concentration falls in failing human hearts and in some rodent models of heart failure. High doses of precursors including nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) have therapeutic effects in rodent models. However, the doses used exceed what is tolerable in humans and the potential for effects at human-relevant doses remains uncertain. Our preliminary and published results suggest that high doses of NR and NMN may be required in rodent models because both molecules are extensively metabolized in the intestines and liver when delivered orally, with only a tiny fraction reaching the circulation intact. In contrast, intravenous delivery allows a much higher proportion of the dose to reach organs such as the heart. In addition to questions about dosing, the mechanism of protection has remained unclear. It is presumed to involve cardiac NAD+ levels, but whole-body supplementation studies leave open the possibility that other tissues mediate protection, for example through lowering blood pressure. We present a knockout mouse with cardiomyocyte-specific loss of NAD+ that impairs heart function and propose the generation of a new model to specifically test the role of mitochondrial NAD+ within the cardiomyocytes. This will be accomplished by targeting SLC25A51, which we recently identified as the mitochondrial NAD+ transporter. We propose three specific aims: Aim 1) Test whether heart-specific NAD+ depletion is sufficient to recapitulate the metabolic and electrical consequences of heart failure, Aim 2) Test whether alternate delivery routes can allow cardiac NAD+ to be rescued by low, human-relevant doses in mice, and Aim 3) Test whether altering mitochondrial NAD+ is sufficient to modulate heart function on its own or modifies susceptibility to induced heart failure. Our approach of using AAV to target SLC25A51 expression in the heart will be the first time that modulation of this protein to alter the mitochondrial NAD+ pool has been attempted in vivo. Together, these studies will reveal fundamental details of how NAD+ metabolism influences cardiac physiology, and will help guide efforts to develop novel therapeutic approaches for the treatment or prevention of heart failure in human patients.

NIH Research Projects · FY 2026 · 2022-08

PROJECT SUMMARY/ABSTRACT Hepatic steatosis, defined by >5% hepatocyte triglyceride content, may be potentiated in people with HIV (PWH) through viral-mediated mechanisms or metabolic dysfunction associated with antiretroviral therapy (ART). However, the epidemiology of hepatic steatosis remains unclear among PWH, primarily because studies have been limited to small patient samples that ascertained steatosis via specialized radiographic methods or liver biopsy. Since liver disease is a leading cause of morbidity and mortality among PWH, it is critically important to identify the determinants and consequences of hepatic steatosis in this group. Such studies will inform interventions and management strategies to mitigate HIV-specific steatosis mechanisms and its consequences, particularly hepatic decompensation and hepatocellular carcinoma (HCC). Recent advances in artificial intelligence have facilitated the development of automated computer-aided liver assessment to determine the presence and severity of hepatic steatosis within noncontrast abdominal computed tomography (CT) scans. The Automatic Liver Attenuation Region-Of-Interest-based Measurement (ALARM) is a deep learning tool previously developed for the identification of moderate-to-severe hepatic steatosis. Preliminary studies conducted by the applicant demonstrate the high accuracy of ALARM compared to manual radiologist review across multiple centers and CT scanners, including within the Veterans Health Administration. To address the knowledge gaps of existing studies, this proposal will first establish a cohort of over 40,000 PWH and people without HIV (PWOH) in the Veterans Aging Cohort Study (VACS) who underwent noncontrast abdominal CT imaging for any indication in the context of clinical care between 2002- 2020. The VACS, an ongoing national prospective cohort study of PWH and PWOH across the United States, includes access to electronic health record data, including image files of CT scans. The ALARM tool will be applied to this repository of radiographic images to objectively classify the presence or absence of moderate-to- severe hepatic steatosis. The research plan aims to: 1) identify the HIV-specific determinants associated with hepatic steatosis among PWH, 2) define how traditional determinants of steatosis differ by HIV status, and 3) determine the risk of liver complications associated with steatosis in PWH and how this risk differs by HIV status. The findings from these studies will inform interventions to prevent and mitigate the development of hepatic steatosis among persons with HIV, which will help lower the risk of liver complications and prolong survival in this population. This project will bring together a mentoring team of nationally recognized researchers and provide time for coursework and training in advanced epidemiology, biostatistics, informatics, artificial intelligence, hepatology, and HIV medicine that are needed to establish the applicant as an independent investigator in the field of HIV-related liver diseases.

NIH Research Projects · FY 2025 · 2022-08

Project Summary This proposal seeks support to develop novel data integration methods using electronic health records (EHR) from multiple CTSA hubs to create predictive models of multi-system diseases. The proposed project directly addresses the areas of emphasis in PAR-19-099 to “engage new collaborators in pre-existing collaborations to solve a translational science problem no one hub can solve alone”. Research gap: The overarching goal of this proposal is to develop the Predictive Analytics via Networked Distributed Algorithms (PANDA) framework, which will enable accurate risk prediction to help healthcare providers reach accurate diagnoses earlier. Our proposed methods directly address two major barriers: 1) lack of predictive models for multi-system conditions; 2) lack of algorithms that effectively combine data from multiple sites in a privacy-preserving and communication-efficient fashion. In this proposal, we will develop and evaluate the PANDA framework using two prototypic multi-system conditions, with different levels of prevalence: granulomatosis with polyangiitis (GPA, a type of vasculitis, prevalence of 74 per million) and psoriatic arthritis (PsA) (1500 per million), with the expectation that the approach will be readily applicable to other diseases. These two conditions are particularly well-suited to the development of our predictive methods given the commonly encountered delays in diagnosis that can range from months to years. These delays may be associated with high morbidity and early mortality. We have three Specific Aims: Aim 1. Develop predictive models for granulomatosis with polyangiitis and psoriatic arthritis, and data integration algorithms to enable secure and efficient data sharing among multiple institutions. Aim 2. Test the predictive models from Aim 1 using aggregated data (not IPD) from a separate set of CTSA sites to validate the data integration methodology. Aim 3. Develop a “toolbox” of resources through which the PANDA processes of algorithm generation and data aggregation can be easily shared with and adopted for use by all CTSAs and others. The success of this project will lead to novel analytic tools for facilitating efficient and privacy-preserving data sharing and collaborative risk predictions across CTSA sites. The PANDA process of novel analytic tools to assist clinical diagnoses and interventions should then be studied through pragmatic trials to evaluate its potential to decrease diagnostic delays and alter patients’ health trajectories. This project is highly feasible and is potentially transformative for both data science and clinical medicine.