University Of Pennsylvania

NIH Research Projects · FY 2024 · 2019-09

Project Summary This is an application from the Center for Clinical Epidemiology and Biostatistics and the Renal, Electrolyte and Hypertension Division, the Department of Neurology, and the Department of Psychiatry at the University of Pennsylvania Perelman School of Medicine to serve as the Scientific and Data Research Center (SDRC) for the Hemodialysis Opioid Prescription Effort (HOPE) Consortium. Through this HEAL / NIDDK initiative, the HOPE Consortium will collaboratively design and conduct a multicenter clinical trial evaluating interventions to address the challenging problems of chronic pain and opioid use in individuals receiving treatment with maintenance hemodialysis. We anticipate that the HOPE Consortium investigators will consider behavioral, complementary, and pharmacologic interventions, personalized treatments based on psychosocial profiles, adaptive designs to maximize efficiency, and approaches to the delivery of the interventions and acquisition of data that minimize burden for patients by “bringing the trial to the participant”. Specifically, the SDRC will: 1) provide scientific leadership for the design, implementation, and analysis of the HOPE Consortium clinical trial; 2) provide comprehensive operational support to the Clinical Centers for implementing the collaboratively designed trial protocol using standardized processes and tools for training, recruitment, and data management; 3) develop and lead a Stakeholder Engagement Working Group that includes a Patient Advisory Panel and a Dialysis Provider Advisory Panel to ensure that perspectives across multiple stakeholders are incorporated into the trial design and implementation approach; 4) integrate and analyze data from the electronic health records of the participating Clinical Centers to supplement trial-acquired data and enable identification of predictors of response to the trial interventions; 5) establish, promote, and maintain consortium-wide high standards for quality assurance and practices; 6) initiate and oversee contracts with industry partners including dialysis provider organizations, pharmaceutical companies, and device manufacturers; 7) prepare reports for the Data and Safety Monitoring Board, and support the preparation of Consortium reports of scientific findings; 8) prepare, document, and transfer Consortium data and biosamples to the NIDDK Central Repository for use by the broader community; and 9) develop approaches for disseminating the trial findings to diverse stakeholders to facilitate post-trial uptake of interventions found to be beneficial. The University of Pennsylvania SDRC team has the expertise, experience, established working relationships, resources, and infrastructure to accomplish each of these aims and fulfill all of the requirements specified in the RFA.

NIH Research Projects · FY 2025 · 2019-09

OVERALL ABSTRACT This grant application was submitted in response to RFA-NS-24-011, “Center without Walls for PET Ligand Development for ADRDs”. It describes a request for continued support for the Center without Walls for Imaging Proteinopathies with PET (U19 NS110456), a project that was funded through RFA- NS-19-014. The competing renewal describes our continued development of Positron Emission Tomography (PET) ligands for imaging two proteinopathies: 1) alpha synuclein (Asyn) for imaging the synucleinopathies Parkinson’s disease and multiple system atrophy; and 2) 4R tau for imaging the 4R tauopathies frontotemporal degeneration and progressive supranuclear palsy. The Proteinopathy Imaging Center (short title) consists of a synergistic, collaborative effort between the University of Pennsylvania (Penn), Washington University-St. Louis (WUSTL), University of Pittsburgh (Pitt), University of California-San Francisco (UCSF), and Yale University. The organization of the Proteinopathy Imaging Center is both innovative and unique for the following reasons: 1) it partners faculty members with an international reputation in the neurobiology (V. Lee, K. Luk, P Kotzbauer) and structural biology (E.J. Petersson) of the proteinopathies with experts in radiotracer development at Penn (RH Mach), Pitt (C. Mathis), and WUSTL (Z. Tu) in developing radiotracers for Asyn and 4R tau; 2) it involves the multi-site collaboration of clinical investigators who are experts in the use of PET to study CNS disorders (J. Perlmutter, WUSTL; R. Carson, Yale; G. Rabinovici, UCSF; A. Siderowf and I. Nasrallah, Penn; V. Villemagne;Pitt); and 3) it involves the utilization of state-of-the-art PET imaging devices for whole body distribution (PennPET Explorer) and high resolution brain studies (NeuroeXplorer, Yale) of our Asyn and 4R tau radiotracers. The Proteinopathy Imaging Center also consists of a series of cross-validation studies at both the basic science (i.e., radiotracer characterization) and clinical research (i.e., consensus diagnosis) levels that can only be accomplished through the U19 funding mechanism. The PI of the Center is Robert H. Mach, the Britton Chance Professor of Radiology at Penn. The Center consists of three Cores (Administrative, Medicinal Chemistry and Radiochemistry, Clinical) and two projects (Asyn, 4R tau). The cores are organized so that there is an overall Core Director or Co- Director, and a site Director at each site participating in the Core activities. The radiotracers developed by the Center without Walls for Imaging Proteinopathies with PET are expected to lead to generation of imaging strategies that are of interest to the scientific mission of the NINDS. They are also expected to provide novel imaging strategies to advance our knowledge on the role of Asyn and 4R tau in neurodegenerative disorders.

NIH Research Projects · FY 2026 · 2019-09

Human Pancreas Analysis Program for Type 2 Diabetes (HPAP-T2D) Abstract Building on our existing infrastructure and scientific collaborations, and the expertise gained during the first four years of the HPAP effort for Type 2 Diabetes, we have assembled five cores with expertise ranging from pancreas procurement and islet isolation to data integration. These Cores form the comprehensive and integrated Penn Human Pancreas Analysis Program – T2D. Core A will procure a spectrum of human pancreata and detailed donor medical history; isolate islets; and distribute islets and tissues to the other Cores for further analysis or processing. Core B will perform physiological phenotyping by perifusion assay, calcium imaging, and oxygen consumption on the isolated islets. Core C will process tissues using multiple modalities that will allow for analysis using advanced technologies such as multiplexed immunofluorescent staining and whole slide imaging. This Core will also archive tissues, DNA and blood for future use and deliver pathological evaluations of the tissues. Core D will perform multiple advanced modalities for the molecular profiling of isolated islets including RNAseq, and DNA methylome analysis of sorted islet cell populations; single cell ATACseq and RNAseq, flow mass cytometry for single cell quantification of more than 30 cell surface and intracellular markers, and spatial transcriptomics. Finally, Core E will assemble, annotate and maintain an open-access database for the Program and its member-researchers, and collaborate with HIRN in the sharing of data. The entire program will directed by an Executive Committee consisting of the core leaders and the contact PI, who will be the interface with HIRN and NIDDK leadership. HPAP-T2D will provide physiologic, genomic, genetic, and histological analysis of the pancreas in type 2 diabetes at unprecedented detail, share the rich data with researchers world-wide before publication, and thus enable breakthrough discoveries in our understanding of this disease that has reached epidemic levels world-wide.

NIH Research Projects · FY 2024 · 2019-09

Abstract Toward precision medicine and precision disease prevention, the overarching goal of this proposal is to develop innovative statistical methods for accurate risk prediction. We address three challenges that plague studies on the value of candidate risk predictors that adds to established predictors for improved predictive accuracy: there is often a lack of independent validation data, the source population for the study sample and the target population of prediction are often different, no statistical methods are currently available for developing risk prediction models using individually-matched case-control data, and there is a lack of statistical methods for helping assess study feasibility beyond standard power calculation for testing predictor-outcome association. On the other hand, data and information that are external to the study may well exist and can be exploited to alleviate these challenges. For example, a model with only standard predictors often exists and has been validated, and the distribution of standard risk predictors in the target population of prediction is often available. We propose that external data and information can be exploited to address the above-mentioned challenges for candidate predictor evaluation, and develop innovative statistical methods to bring this idea to fruition. Considering prediction of a binary outcome, we propose a novel method to building logistic prediction models that are guaranteed to calibrate well in the target population, an innovative method for risk prediction with individually matched case-control data, and a method to project the added value of candidate predictors to help assess study feasibility. Our methods, accompanied by user-friendly software, will facilitate cost effective and timely predictor evaluation for predicting binary outcomes. Our methods were motivated by and will be applied to several PI Chen's collaborative studies.

NIH Research Projects · FY 2025 · 2019-09

RO1 (Renewal Application) Abstract Understanding biomarkers of cognitive decline in Lewy body diseases While patients with Lewy body diseases (LBD) share the core feature of deposition of misfolded alpha-synuclein (aSyn) into neuropathological inclusions, they exhibit pronounced heterogeneity in both initial clinical phenomenology, as well as in trajectory of outcomes. The underlying reasons for these differences in phenomenology among LBD patients – patients with Parkinson’s disease (PD), with or without dementia, as well as patients with dementia with Lewy bodies (DLB) – are poorly understood. However, these differences matter greatly for quality of life for patients and their families, as well as costs to the healthcare system, making cognitive decline in the LBD one of the most important Alzheimer’s disease related dementias (ADRD) facing our aging population. This RO1 renewal application seeks to uncover, using biomarker screening approaches, the molecular correlates of cognitive heterogeneity in the LBD. However, we do not plan to stop there. Instead, based on data generated in the first period of funding, we posit that biomarker-derived leads represent an untapped opportunity to identify key players in the pathogenesis of cognitive decline in the LBD. We thus propose to also investigate our more mature leads in model systems, with the goal of developing targets for therapeutic development. We have two specific aims, focused on the underlying premise that host characteristics (reflected by biomarker signatures) and “proteinopathy” characteristics (reflected by differences in aSyn) together determine cognitive trajectory in the LBD. SPECIFIC AIM 1: Investigate the contribution of host characteristics to cognitive heterogeneity in the LBD. We will screen CSF samples from 150 PD patients to identify proteins associating with cognitive outcome, and we will cross-reference “hits” against plasma biomarkers or genomic variants previously found to associate with the same phenotype. We will elucidate the role of our previously-discovered, biomarker-derived leads Apolipoprotein A1 (ApoA1) and Melanoma inhibitory activity protein (MIA) in the pathogenesis of LBD, using induced pluripotent stem-cell derived neurons (iPSC-N) and microglia (iMicroglia), as well as in vivo models. SPECIFIC AIM 2: Investigate the contribution of aSyn species to cognitive heterogeneity in the LBD. We have discovered that plasma species of aSyn recognized by two conformation-selective monoclonal antibodies (termed Strain A and Strain B aSyn) discriminate PD from DLB. We will biochemically and cell biologically characterize these two strains of aSyn using proteinase K digestion, mass-spectrometry-based methods, real- time quaking-induced conversion (RT-QuIC) assays, and iPSC-N seeding models.

NIH Research Projects · FY 2025 · 2019-09

PROJECT SUMMARY (See instructions): A central goal in HIV/AIDS vaccine research is the elicitation of broadly neutralizing antibodies (bNAbs). Here, we leverage three recent discoveries from our groups to generate more effective V2 apex immunogens. Having found that unshielded regions (“glycan holes”) in Env are negatively associated with bNAb development, we have created bioinformatic tools to identify and mask these unwanted epitopes. We have also used our Signature-based Epitope Targeted (SET) immunogen design strategy to generate germline targeting Envs that engage multiple V2 apex bNAb precursors. Finally, by studying bNAb development in ~150 rhesus macaques (RMs) infected with 16 different SHIVs, we have identified 25 animals (17%) with heterologous breadth, 15 of which developed V2 apex bNAbs. Based on these data, we hypothesize that by (i) minimizing distracting glycan hole epitopes, (ii) increasing Env affinity for V2 apex bNAb precursors, (iii) increasing relevant epitope diversity in vaccine boosts, and (iv) incorporating B cell lineage designs, we will improve V2 bNAb germline engagement and bNAb lineage maturation. During the first 3 years of this R37 application, we have made significant progress toward all Specific Aims. In Aim 1, we optimized the glycan shield and improved the germline targeting properties of several SIVcpz and HIV-1 Envs, which allowed us to down-select the CAP256 Env as the most promising platform. In Aim 2, we cloned members of 12 new rhesus V2 apex bNAb lineages and inferred their unmutated common ancestors (UCAs), thereby tripling the number of V2 bNAb lineages for novel lineage-based immunogen design. SHIVs expressing the best performing germline targeting CAP256 Envs are being generated and envelope-antibody coevolution pathways are being determined for all SHIV infected animals that developed V2 neutralization breadth. In Aim 3, we generated nucleoside-modified mRNA containing lipid nanoparticle (mRNA/LNP) vaccines that express germline targeting, stabilized, membrane bound CAP256 Envs. The best performing constructs have been shown to express well and to bind multiple human and macaque V2 bNAb UCAs. In this five-year MERIT extension application, we propose to extend these studies and complete the rhesus macaque infection and immunization studies that were delayed by the COVID lockdown. Since the last competitive renewal, our group has been highly productive, publishing 38 papers relevant to HIV vaccine development. In this MERIT extension application, we will apply multiple vaccine improvement strategies to induce V2 apex bNAbs in RMs and then translate these findings to humans.

NIH Research Projects · FY 2024 · 2019-09

The advent of anti-retroviral therapy (ART) for people living with HIV/AIDS (PLWHA) substantially improved life expectancy but, now, PLWHA who smoke lose more life-years due to tobacco use than they do to their HIV infection. Unfortunately, the rate of smoking among PLWHA in the US is about 40%. The limited tobacco use treatment research with PLWHA indicates that behavioral treatments and medications (nicotine patch and varenicline) yield moderate effects on cessation, with quit rates that are considerably lower than they are for the general population. Thus, there is a critical need to identify novel ways to optimize tobacco cessation treatment for smokers with HIV. Two factors are highly predictive of cessation outcomes with pharmacotherapy, in the general population and among PLWHA. First, a smoker's rate of nicotine metabolism, characterized by the nicotine metabolite ratio (NMR, a marker of CYP2A6 gene variants), predicts cessation both for varenicline and nicotine patch. Our studies with general population and HIV-infected smokers show that personalizing the choice of medications for smokers using the NMR can increase efficacy and reduce toxicities, an approach highlighted by the NCI (https://www.cancer.gov/about-nci/budget/plan/public-health). Second, adherence to smoking cessation medications, in the general population and among PLWHA, rarely exceeds 60% and non-adherence lowers cessation rates 2-3 fold. We developed the Managed Problem Solving (MAPS) intervention which is endorsed by the CDC (https://www.cdc.gov/hiv/research/interventionresearch/compendium/ma/index.html) as an evidence-based intervention for medication adherence among PLWHA. Thus, the application's premise is that incorporating intervention components to tailor tobacco use medications (varenicline or patch) with the NMR and increase adherence to the medication using MAPS will optimize tobacco treatments for PLWHA. To test this premise, we will conduct a rigorous multi-site randomized clinical trial with 488 HIV+ smokers to evaluate NMR-tailored treatment and MAPS as optimization strategies for tobacco dependence treatment for PLWHA. We will use a factorial design to examine: 1) The effects of the NMR-tailored and/or MAPS interventions on end- of-treatment (EOT) and 6-month smoking cessation rates (primary aim); 2) Mediators of the NMR-tailored and MAPS interventions (secondary aim); and 3) Moderators of the NMR-tailored and MAPS interventions (exploratory aim). Our overall approach is consistent with the Multiphase Optimization Strategy which has gained prominence for guiding the evaluation of interventions for enhancing tobacco use treatment effectiveness. Addressing these aims will determine: the use of adherence and pharmacogenetic optimization of smoking cessation treatment for PLWHA, the mechanisms that underlie the effects of these optimization strategies on cessation outcomes, and the variation in the effects of these optimization strategies across sub-groups of PLWHA. In the end, this trial will help understand if getting the right medication to the right person and helping to make sure they sufficiently use that medication optimizes tobacco cessation treatment for this population.

NIH Research Projects · FY 2025 · 2019-09

Acute stress and chronic adversity affect biological systems at multiple levels, contributing to diverse mental and physical health outcomes. Yet, the mechanisms through which these stressors impact health—and the factors that foster resilience or increase vulnerability—are not well understood. The free-ranging rhesus macaques (Macaca mulatta) on Cayo Santiago island offer a unique natural experiment. In 2017, they experienced a Category 4 hurricane, followed by acute deprivation of food and water (later restored) and chronic deforestation, which led to sustained higher temperatures. Building on our prior R01- MH118203 support, we will use this setting to investigate how environmental stressors affect the brain. Our previous work established a comprehensive biobank from macaques with varied social status and connections, sampled before and after the hurricane. We found that social support protects against both chronic adversity (e.g., low social status) and acute trauma (e.g., natural disasters), with strong links to health, aging, and survival. With renewed support, we aim to expand these findings by examining molecular and anatomical variations in brain pathways associated with social connectedness, adversity, and stress. First, we will quantify the hurricane's short-term effects on brain health by comparing cohorts sampled before and shortly after the event. Using MRI and molecular data, we will assess the impact of social environment and hurricane exposure on brain phenotypes, identify regions vulnerable to hurricane stress, and examine whether hurricane-exposed animals show adverse molecular and neuroanatomical changes. We hypothesize that animals with higher social capital–the combination of social connections and social status–will demonstrate greater resilience, with social capital modulating molecular mechanisms that shape neural circuits. Next, we will analyze animals sampled in the following years to explore how social environment and hurricane exposure, combined with long-term environmental changes, influence brain health. Finally, we will prospectively sample animals exposed only to the altered post-hurricane environment to distinguish the effects of surviving a disaster from those of living in a chronically degraded environment. This project will precisely map the structural and molecular pathways by which social capital fosters resilience, a depth unachievable in pre-mortem human studies across lifespan-relevant timescales. The behavioral and biological similarities between macaques and humans endow this study with strong translational potential for addressing anxiety, depression, and related health impacts—crucial priorities for NIMH (NOT-MH- 18-058).

NIH Research Projects · FY 2024 · 2019-09

PROJECT SUMMARY This application requests funds to refine and rigorously test a collaborative care model for patients with opioid use disorder (OUD) and major depression, post-traumatic stress disorder, or an anxiety disorder in primary care. We also will examine clinician and practice characteristics associated with successful implementation and the cost effectiveness of different care models. The primary aims of this proposal are: (1) Rapidly prototype and test each element of our collaborative care models to optimize it for implementation; (2) Conduct a randomized study of three collaborative care conditions with 39 practices to determine which is most effective in improving outcomes for people with OUD and mental health conditions: (a) Augmented Usual Care: PCP waivered to prescribe buprenorphine and mental health care manager, (b) Collaborative Care: Waiver PCP; mental health care manager receives OUD training; practice receives telephonic psychiatric consultation, or (c) Collaborative Care + Social Worker to address social determinants of health; (3) Measure clinician and organizational-level factors associated with implementation of each component, with the goal of developing strategies to increase successful implementation; (4) Conduct a cost evaluation of each collaborative care model; and (5) Work with smaller and more rural practices to develop and test effective strategies to manage OUD. Successful completion of the proposed study will provide definitive evidence regarding the most parsimonious set of elements of integrated collaborative care required to maximize outcomes for individuals with OUD and psychiatric disorders. Because of the study design, our examination of implementation factors, and our community partnerships, the results also will have high probability of adoption and implementation.

NIH Research Projects · FY 2026 · 2019-09

The genetic variation that contributes to phenotypic differences among individuals is shaped by mutation, genetic drift and natural selection. Relatively little is known about the relative contributions of drift and selection. On the one hand, in terms of genome-wide variation drift is the stronger force. On the other, this may not be true of phenotypically important variants, of which several examples are known. Ancient DNA provides the ability to directly observe evolution in action over well-defined timescales, and to identify and quantify the effects of selection. As well as explaining how and when unique aspects of our species and populations evolved, this information is important to predict disease risk across the spectrum of human ancestry. Because genetic ancestry is correlated with environment, we do not know whether ancestry- phenotype correlations are causal. The solution is that evidence of recent selection allows us to infer causality – ancient DNA provides information that is independent of observational studies of present-day populations. We propose to develop this in several ways. First, we estimate the contribution of recent (in the past 5-10 thousand years) natural selection to phenotypic variation within Britain. We focus on Britain as a proof- of-concept because of relatively simple demography, access to large samples of ancient and present-day individuals and because so much of our knowledge about the genetic basis of complex traits is derived from this population. However, clearly this only represents a small portion of human variation. Therefore, we will develop approaches to enable studies of natural selection using ancient DNA in populations with more complex demographic histories. We will apply these approaches to data from East Asia and Africa to measure the contribution of natural selection to phenotypic variation and disease risk in these regions. Taking a comparative approach, we will identify common patterns in the response to environmental change such as the independent development of agriculture in these regions. We will contrast patterns of selection in ancient and present-day populations, asking whether it has changed or intensified due to recent shifts in environment. These questions are not restricted to organismal or disease phenotypes. Molecular phenotypes like gene expression, splicing and methylation are critical to understand the functional basis for the effects of GWAS variants and are equally critical to understand the evolution of organismal phenotypes. We will test for recent selection on these phenotypes and identify biological mechanisms that underlie genomic signals of selection. Finally, we will apply these evolutionary insights to the construction of polygenic risk scores to predict disease. Because substantive differences in risk across ancestries are likely driven by selection, identifying selected variants allows us to predict those risks while removing confounding due to genetic variation that is simply correlated with environment from unselected variants. With this approach we aim to construct polygenic scores that are accurate across the spectrum of human genetic ancestry.

NIH Research Projects · FY 2026 · 2019-09

PROJECT SUMMARY For two decades the PI has led a research program developing molecular probes that have yielded breakthroughs in Chemical Biology, Radiology, Cellular Genomics/Transcriptomics, Anesthesiology, and Photochemistry. Since 2019, the PI has published 20 peer-reviewed research manuscripts and 2 review articles with NIH-MIRA support, and several related papers. The PI has mentored 2 postdoctoral fellows to successful job placement, 4 graduate students to Ph.D. completion and chemistry careers, and 6 undergraduates to STEM Ph.D. programs. The MIRA grant continues to provide primary support to 8 graduate students and 2 undergraduates who are carrying out the studies that underpin this renewal application. The PI’s service confirms a strong commitment to scientific review, undergraduate and graduate education, outreach, community building, and junior faculty mentoring. This MIRA grant renewal will build on several exciting advances made in the current funding cycle. In the first project, several new small-molecule capsules and proteins have been developed for xenon binding. Key principles have been elucidated for controlling the xenon exchange rate in these systems, and a suite of new tools will drive the development of sensitive xenon contrast agents (Xe CAs) for molecular imaging and also lead to improved understanding of xenon anesthesia and analgesia. Critically, the FDA approved hyperpolarized Xe-129 for human lung magnetic resonance imaging (MRI) in Dec, 2022. Imaging equipment has been assembled at UPenn with collaborators (Kadlecek and Gade), and we propose to perform cutting-edge Xe-129 MRI studies and investigate Xe CAs developed in the PI’s laboratory for lung cancer detection in mice. Notably, a Xe CA employing ribose-binding protein (RBP) led to the discovery of 5 mM ribose in HeLa cells, 1000x more than previously estimated. This has spawned a second project, which is the development of the first useful fluorescence resonance energy transfer (FRET)-based sensor for real-time quantitation of ribose levels in mammalian cells. This offers the opportunity to study natural fluctuations in ribose (likely as a function of cell cycle), and also explore enzymatic pathways that we hypothesize convert ribose-5-phosphate (originating from the pentose phosphate pathway) to ribose. Finally, a third project stems from the development of fluorescein- and Cy5-labeled siRNA molecules as first-in-class ratiometric pH probes for elucidating details of endosomal entrapment and release and improving delivery of therapeutic oligonucleotides to skeletal muscle. Novel bicyclic oligonucleotides are proposed for achieving potent enzyme inhibition.

NIH Research Projects · FY 2026 · 2019-09

Project Summary Misrepair of DNA damage is a hallmark of cancer. We discovered that the budding yeast Shu complex is a conserved regulator of DNA repair through a central role in Rad51 regulation. Rad51 functions during the high fidelity homologous recombination pathway to find and invade a homologous template for repair and also during replication fork protection and restart. Rad51 is tightly regulated in cells by accessory proteins, collectively called the Rad51 mediators, including the Shu complex. In humans, misregulation of hRAD51 or its mediators is associated with cancer predisposition (particularly breast and ovarian cancers) and Fanconi anemia, which is also characterized by anemia and cancer. We found that disruption of the yeast Shu complex leads to cellular death specifically upon exposure to alkylation induced DNA damage. Alkylation damage is caused by a myriad of industrial and consumer-based sources and is pervasive in our environment. DNA alkylation leads to replication stress and DNA damage. If DNA is alkylated during replication, then the replication fork can stall or collapse, and many repair mechanisms can be utilized to tolerate, bypass, or repair the damaged DNA. How a cell commits to a specific repair pathway is largely known. In budding yeast, the Shu complex is critical in the processing of replication forks damaged by alkylating agents. This complex is highly conserved throughout eukaryotes and contains the Rad51 paralogs, proteins that are structurally similar to the central DNA repair protein Rad51 and are mutated in cancer. In this study, we aim to elucidate the role of the yeast and human Shu complexes in repair of DNA alkylation damage at a replication fork. We are testing the hypothesis that the Shu complex is a critical key regulator of DNA damage tolerance at a replication fork by specifically recognizing alkylation induced DNA damage to promote Rad51-mediated template switch. Using what we learn in yeast to quickly and efficiently identify key substrates, residues, and protein targets, we will expand our studies into human cell lines where we will investigate the role of the human Shu complex in tolerance of alkylation damage. In addition, we will identify at risk individuals harboring mutations in these important genes that may be more sensitive to DNA alkylation damage and therefore susceptible to cancer. Collectively, these studies will provide key insights into the role of the Shu complex in tolerance of DNA alkylation damage and elucidate how this complex promotes error-free DNA repair to prevent genetic instability and cancer.

NIH Research Projects · FY 2025 · 2019-09

PROJECT SUMMARY This project seeks to advance epigenetic sequencing by combining novel enzymatic methods with long-read sequencing technologies, allowing for the accurate resolution of epigenetic and genetic information across extended DNA regions even in sparse, but biologically-important, genomic DNA samples. Traditional epigenetic sequencing has heavily relied on bisulfite sequencing (BS-Seq), which, while effective for identifying 5- methylcytosine (5mC), confounds this base with the opposing and second most common mark 5- hydroxymethylcytosine (5hmC), and degrades DNA, limiting its use across long-ranges and in low-input samples. Moreover, third-generation sequencing platforms, despite their ability to map long DNA fragments and detect modifications directly, require high sample inputs, which constrains their application in critical areas, such as single-cell analyses and circulating cell-free DNA (cfDNA) studies. Our project will focus on overcoming these limitations by seamlessly integrating cutting-edge enzymatic approaches that permit the targeted conversion of cytosine modifications with the capabilities of long-read sequencing to provide a more comprehensive view of the epigenetic landscape. These technologies will be specifically tailored to handle ultra-low input samples efficiently, addressing significant gaps in current methodologies. The project's main goals are two-fold. First, we aim to employ a combination of unnatural cytosine analogs and helicase-deaminase fusions to perform integrated long-read sequencing that preserves both genetic and epigenetic information, enabling the enrichment and precise profiling of DNA modifications (5mC and 5hmC) in cfDNA from cancer patients. This approach will facilitate the detection of cancer-specific modifications and potentially better pinpoint the tissue of origin in unknown cancer samples. Second, we will develop a novel enzyme-based, single-cell barcoding, and amplification strategy combined with long-read sequencing to map 5mC and 5hmC modifications in single-cell epigenomes, particularly focusing on brain tissues. This will allow us to explore cellular heterogeneity and the role of 5hmC in gene regulation within individual cell types. By leveraging our interdisciplinary expertise and innovative methodologies, this project aims to redefine the boundaries of genomic research, providing a more detailed and functional map of the human epigenome. This work will not only enhance our understanding of basic sciences of human development and disease but also support the development of more effective diagnostic tools based on epigenetic markers.

NIH Research Projects · FY 2024 · 2019-08

PROJECT SUMMARY/ABSTRACT This revised application responds to PAR-18-175: “Pilot Clinical Trials for the Spectrum of Alzheimer's Disease and Age-related Cognitive Decline.” Primary progressive aphasia (PPA), a debilitating condition of language loss affecting many patients with frontotemporal dementia (FTD) and Alzheimer's disease (AD), currently lacks effective treatments. Recent studies suggest that transcranial direct current stimulation (tDCS), a form of noninvasive neuromodulation, may show promise as an intervention for PPA. However, these research efforts are hampered because they do not address important questions about plasticity in the language system, and because they do not fully utilize knowledge regarding the properties of the language network in PPA to guide treatment. This proposal aims to further advance investigation into tDCS as a potential intervention in PPA and to establish which components of the language network in PPA are most capable of tDCS-induced behaviorally relevant plasticity. Our proposal seeks to determine whether neuromodulation therapies in persons with PPA should aim to strengthen connections in the most degenerated regions of the language network or bolster compensatory changes in more intact areas. We will address this knowledge gap by pursuing a randomized, sham-controlled crossover study of high-density tDCS (HD-tDCS) focused over the anterior regions of the left hemisphere language network in participants with two PPA variants that are characterized by decreased word production but which feature different sites of maximal degeneration. This will allow for comparison of stimulation in a region that is degenerated in some subjects but relatively spared in others. Stimulation will be paired with a behavioral language therapy aimed at augmenting tDCS effects in the language system. Our first aim will be to determine how this intervention differentially impacts language performance in subjects with the two PPA variants. We will then use network graph statistical analyses of neuroimaging data to characterize language networks. We will focus on hubs as centers of critical connectivity in networks, and we propose that measuring changes in the ability of regions in the language network to function as hubs (indexed by hub scores) may be a way to describe how neurodegeneration impacts language network functions in PPA. Thus, the second aim of the proposal will explore differences in hub scores across the language network at baseline in our two PPA subtype groups. The third aim of the proposal will extend this approach by examining behaviorally relevant changes in hub scores induced by tDCS. The final aim of the project will integrate and extend prior findings by developing a model that employs clinical phenotypes, patterns of brain atrophy, and hub score data to predict which individuals are most likely to benefit from our stimulation approach. Taken together, this project will advance a potential intervention for a devastating condition associated with neurodegenerative diseases, elucidate network mechanisms of plasticity in these disorders, and develop a potentially generalizable, network-informed approach for predicting response to therapeutic neuromodulation.

NIH Research Projects · FY 2025 · 2019-08

Fragile X syndrome (FXS) is an inherited neurodevelopmental disorder associated with social anxiety, hypersensitivity to stimuli, seizures, and learning disabilities. It is classically viewed as a monogenic disease in which mutation-length expansion of a CGG short tandem repeat (STR) tract above a critical mutation-length threshold of 200 triplets leads to local DNA methylation and repression of FMR1. Genome-wide gene expression changes are thought to occur downstream of the loss of the Fragile X Messenger Ribonucleoprotein (FMRP) encoded by FMR1. In our lab’s new data acquired during the previous funding period, we used nanopore long- read sequencing, Hi-C, CUT&RUN, CRISPR STR engineering, and single-cell Oligopaint imaging to find Megabase (Mb)-sized H3K9me3 domains on autosomes and the X-chromosome in FXS patient-derived cell lines and brain tissue. Domains co-localize with severe misfolding of topologically associating domains (TADs) and loops and connect via trans interactions in subnuclear hubs. Because H3K9me3 domains encompass STRs susceptible to instability and replication stress-induced double strand breaks, we termed them BREACHes: Beacons of Repeat Expansion Anchored by Contacting Heterochromatin. BREACHes encompass and silence genes encoding synaptic plasticity, epithelial integrity, reproductive development, and neural cell adhesion, which are clinical hallmarks in FXS. Thus, by way of Mb-scale heterochromatin domains and trans interactions, we find multiple plausible candidate genes of possible relevance for understanding onset, progression or therapeutic treatment of FXS. The objective of this proposal is to elucidate the RNA-mediated and higher-order folding mechanisms governing heterochromatin domains and the trans interactions among them in FXS. Our central hypothesis is that mutation-length CGG STR-containing RNA forms RNA:DNA hybrid structures to establish H3K9me3 domains, whereas the attenuation of cohesin-mediated loop extrusion is necessary to maintain H3K9me3 and trans interactions. We will test our hypothesis with two Specific Aims. First, we will investigate the role for CGG STR-containing RNA in BREACH establishment. To model the removal of H3K9me3 signal and the subsequent re-establishment of the domains, we will reprogram normal-length, FXS mutation- length, and premutation-length cutback induced pluripotent stem cells (iPSCs) from primed to naïve pluripotency and release back to primed. In a timecourse of BREACH re-establishment, we will assay R loops, RNA-chromatin interactions, histone methyl transferases, and 3D genome folding. Second, we will elucidate the effect of cohesin- mediated loop extrusion on BREACH maintenance in single cells. We will deplete and replete the cohesin unloading factor WAPL in FXS iPSCs. In a timecourse of recovery and loss of cohesin-mediated loop extrusion, we will assay genome folding, DNA methylation, and H3K9me3 with cutting edge single-cell genomics and imaging technologies. Together, these studies will shed light on the mechanisms governing the establishment and maintenance of H3K9me3 domains and the inter-chromosomal interactions among them in FXS.

NIH Research Projects · FY 2026 · 2019-08

PROJECT SUMMARY The goal of this proposal is to determine the role of fetal vs. adult wound healing programs – and the switch from one to the other that occurs around birth – in the response to fetal/early neonatal extrahepatic bile duct injury, such as occurs in biliary atresia (BA). BA is a rare disease occurring worldwide that it is thought to occur from a prenatal environmental insult – affecting only the fetus – to the extrahepatic bile duct (EHBD). Babies appear healthy at birth, but undergo rapid progression of the disease to fibrosis and obstruction of the duct and cirrhosis of the liver. There are three major unanswered questions that motivate this proposal:  Why does an EHBD insult during pregnancy affect only the fetus?  Do some babies with fetal EHBD injury recover, and if yes, why recovery rather than fibrosis?  Why does the disease progress so rapidly after birth? Our preliminary data suggest that prenatal EHBD injury leads to a program of fetal wound healing in the initial response of the fetal EHBD to injury. Fetal wound healing, which has been reported in multiple tissues, results in regeneration rather than scarring and is particularly notable for the deposition of high molecular weight hyaluronic acid (HA) rather than type I collagen, with a growth factor milieu that includes IL-10 rather than TGF-. The switch from a fetal to an adult wound healing program occurs late in gestation. We hypothesize that fetal and adult wound healing programs, in sequence, determine the response to fetal EHBD injury; that both have the potential to enhance damage and injury progression; and that co-opting these responses would have a major therapeutic benefit. We propose to test these hypotheses through 2 specific aims that study wound healing in sequence from the fetus to the newborn, including the impact of natal stress on the transitional program between fetal and adult: 1) Define the nature of fetal wound healing and its impact on the injury response in the fetal EHBD; and 2) Determine the impact of birth-associated stress and the switch to an adult wound healing program in the context of an HA-laden EHBD after the events of fetal wound healing. The proposed work introduces a new concept – fetal wound healing – to the study of BA. Understanding fetal wound healing in BA, both its positive and negative effects, will be essential in the development of potentially transformative treatments.

NIH Research Projects · FY 2025 · 2019-08

PROJECT SUMMARY The goal of this proposal is to contribute new engineering technologies that gain control over cell collective phenomena responsible for driving epithelial organogenesis in vivo. Tightly packed epithelial sheets and tubules are crucial to the function of a diverse group of organs including the lung, kidney, mammary gland, and prostate. Congenital defects and adult diseases such as cancer that occur during the development and maintenance of such organs are prevalent, creating a significant disease burden. This has driven innovation in tissue engineering, which aims to create artificial tissues that serve as disease and therapy screening models. However, modern tissue engineering approaches fail to harness several hallmarks of epithelial organ formation, limiting the complexity of synthetic tissues and creating an urgent need for innovation. Rather than being assembled from constituent cells all at once, processes like branching morphogenesis progressively ramify, sculpt, and pack together functional tubule structures in vivo. The long- term goal of the Hughes lab is to study and engineer missing tissue-building principles at play in organ development to fill ongoing gaps in tissue engineering. This goal builds upon ongoing work in the Hughes lab from the previous period of R35 funding beginning in 2019. This includes 1) precision ssDNA-based cell and tissue interface micropatterning technologies to 2) control the size, cell composition, and differentiation trajectory of epithelial organoids; 3) shape-changing ‘kinomorph’ tissue scaffolds for guiding tissue building behaviors in cell collectives; and 4) mechanically manipulating epithelial progenitor niche regulation. Now our overall objectives are to further advance three research visions across kidney and lung model systems. We firstly seek to map the relationship between tissue mechanics and cell-cell signaling on epithelial morphogenesis via orthogonal optogenetic ‘handles’. This will allow us to better control organoid cell composition. We secondly seek to gain control over epithelial tubule shape and elongation by engineering anisotropic collective cell forces through planar cell polarity pathways. We thirdly seek to combine tubule construction and microfluidics approaches to engineer axial tubule polarity. Together these missing tissue- building modules will contribute innovative enabling technologies for ‘higher-order’ tissue construction relevant to the general medical sciences. The Hughes lab is uniquely suited to tackle our research vision due to our expertise in stem cell engineering and optogenetics, live imaging, microfluidics, and advanced tissue assembly strategies. We expect our proposed contributions to have significant positive impact in the areas of fundamental biological discovery, tissue construction, and drug target screening.

NIH Research Projects · FY 2026 · 2019-08

PROJECT SUMMARY Elucidating the mechanism of transcriptional regulation is a complex multivariate problem that requires holistic characterization of all transcriptional machinery including multiple enhancers, promoters, and other architectural proteins. In particular, the development of groundbreaking “seq” techniques has significantly advanced the field by providing a genome-wide frequency of enhancer-promoter interactions and related levels of gene activity. Nevertheless, a critical gap remains because most available genome-wide data lack the temporal and single-cell information needed to grasp the intricate interactions of enhancers and promoters in individual cells. The “average” behavior from a population of cells does not capture the highly variable genome topology that each cell exhibits, nor does a stochastic gene expression stemming from a specific enhancer- promoter interaction. To discern how stochastic transcriptional activity contributes to deterministic developmental processes, there is a critical need to decipher the regulatory logic governing enhancer-promoter dynamics at the single-cell level. Our long-term goal is to identify design principles for precise gene control in embryonic development, using a combination of quantitative live imaging, genetic perturbations, and mathematical modeling. The overall objective of this proposal is to determine regulatory principles underlying enhancer-promoter dynamics that govern transcriptional processes to ensure normal development. Beyond matching an enhancer sequence with the target promoter, we will determine the extent to which a linear enhancer-promoter distance and enhancer’s relative orientation to the promoter impacts the target gene expression and subsequent development for short- and intermediate-range enhancer-promoter interactions. For long-range enhancer-promoter interactions, the functional role of insulator-mediated chromatin looping dynamics on transcriptional stochasticity and developmental robustness will be investigated. Lastly, we will determine the differences in regulatory logic governing multi-enhancer-promoter interactions as compared to one-on-one enhancer-promoter interactions. Since the mechanism of transcriptional regulation is conserved across species, the successful completion of our projects will reveal another layer of gene control underlying the development and diseases of multicellular eukaryotes.

NIH Research Projects · FY 2024 · 2019-08

Program Director/Principal Investigator (Last, First, Middle): Culley, Deborah J ABSTRACT Surgery triggers a cascade of humoral, cellular, and subcellular events involved in inflammation and its resolution. These profound immune responses have a major influence on postoperative outcomes, and many complications of surgery are due to dysregulated inflammation. Older persons have more surgery than younger ones and are especially prone to serious postoperative morbidity. The immune system becomes dysregulated with age and this dysfunction contributes to the pathogenesis of age-related diseases, including cognitive decline. Poor cognition is a potent and consistent risk factor for adverse surgical outcomes; we have shown it doubles the risk of postoperative delirium and halves the chance of being discharged home after elective joint replacement surgery. We have also demonstrated that between 22-40% of elective surgical patients ≥ 70 years of age are probably cognitively impaired at the time of surgery. Therefore, the combination of poor preoperative cognition and poor surgical outcomes is both common and clinically important. Yet, it is not known how poor cognition increases susceptibility to postoperative morbidity, and the situation is little studied. We propose that poor preoperative cognition signifies a state of immune disequilibrium / dysfunction that shapes the immune response to surgery and increases postoperative morbidity. We will test this proposition by examining humoral (plasma inflammatory and resolution mediators; Aim 1), cellular (monocyte transcriptome and function; Aim 2), and subcellular (circulating extracellular vesicle [EV] concentration, cargo, and function; Aim 3) components of inflammation in cognitively screened patients having surgical procedures common in old age (total joint replacement; spine surgery), with delirium and discharge to place other than home as clinical and patient- centered-outcomes, respectively. Preliminary results showing cognition- or outcome-related differences in the ratio of circulating proinflammatory and proresolution mediators, the transcriptome of blood-borne monocytes, and the distribution of plasma extracellular vesicles support our model. This research is innovative because the impact of preoperative cognition on the immunology of surgical recovery has not been studied previously, it will test novel pathogenic mechanisms (monocyte dysfunction, EVs) for preoperative cognition-driven risk and use state-of-the-art ex vivo and in vitro methods (multiplexed gene panels), and may identify a molecular signature for adverse postoperative outcomes and, thereby, potential therapeutic targets. Given the magnitude and importance of the clinical problem being addressed, this is a high impact proposal that may increase the precision and personalization of surgical care and improve outcomes for older patients. OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page Continuation Format Page

NIH Research Projects · FY 2025 · 2019-07

ABSTRACT Idiopathic Pulmonary fibrosis (IPF) is a devastating interstitial lung disease (ILD) of older adults characterized by disruption of distal lung architecture that ultimately leads to scar formation, abnormal gas exchange, and respiratory failure. Key barriers to better IPF outcomes have included an incomplete understanding of its pathophysiologic underpinnings and a dearth of translationally relevant preclinical models. However, identification of rare genetic variants in the alveolar epithelial type 2 (AT2) cell-restricted Surfactant Protein C (SP-C) gene (SFTPC) in subsets of PF patients has been part of a paradigm shift in which dysfunctional AT2 cells serve as a proximal driver of IPF. Coupled with the recent identification of a population of “reprogrammed” AT2 cells in human IPF lungs deficient in classic AT2 transcriptional programs and enriched in profibrotic mediators, new opportunities are emerging for therapeutic discovery for IPF. Our prior in vitro modeling demonstrated that IPF-associated SFTPC mutations produce aberrant SP-C proprotein isoforms that functionally disrupt epithelial cell quality control (QC) yielding 2 phenotypes: “ER stressed” from misfolding (“BRICHOS”) mutant with activation of all 3 signaling arms (ATF6, IRE1, PERK) of the unfolded protein response (UPR) or impaired autophagy / mitophagy secondary to proSP-C mistrafficking (“Non-BRICHOS”) mutants. The prior funding period provided in vivo proof of concept for a seminal role for disrupted AT2 QC showing that expression of either non-BRICHOS (SftpcI73T) or BRICHOS (SftpcC121G) mutants in mouse lung epithelia are each sufficient to evoke a spontaneous fibrotic lung phenotype with recapitulation of many consensus IPF defining elements. Further, we also published that SftpcC121G mice develop marked activation of the AT2 UPR with emergence of a reprogrammed transition state. Our Preliminary Data will show that mutant Sftpc expression in mice can also cause AT2 glycolytic reprogramming and altered mitochondrial dynamics (biogenesis, fission, and respiration) along with emergence of the aberrant alveolar epithelial cell transition state. Thus, this renewal application now seeks to mechanistically understand how altered UPR signaling and metabolism each contribute to AT2 reprogramming and promotion of a fibrotic niche. Leveraging our two Sftpc mouse PF models we will first use mutant SftpcC121G as a model substrate for disruption of proteostasis while genetically and pharmacologically interrogating UPR signaling focusing on IRE1 and ATF6 to define its impact on AT2 proteostasis, cell states, and progenitor function [Specific Aim 1]. Then using SftpcI73T mice we will assess downstream consequences of disrupted AT2 organellar QC for metabolic reprogramming and mitochondrial dynamics with contextualization of their impact on pathological AT2 endophenotypes and fibrotic remodeling [Specific Aim 2]. When completed deliverables produced from these studies will enhance our understanding of the role of two understudied pathways on IPF pathogenesis and provide equipoise and experimental platforms to catalyze discovery and testing of new IPF therapies.

NIH Research Projects · FY 2025 · 2019-07

PROJECT SUMMARY Myocardial disorders lead to heart failure (HF) and impose an enormous public health burden. Changes in cardiac structure and function that accompany myocardial disease and HF are heritable traits. MTSS1 (metastasis suppressor 1) is an I-BAR protein that regulates cytoskeleton dynamics, cell motility, and adhesion. Our group identified genetic variants in a cardiac-specific enhancer upstream of MTSS1 that reduce human left ventricular (LV) MTSS1 expression, resulting in reduced LV size and reduced risk of dilated cardiomyopathy (DCM). These findings established MTSS1 as a genetic modifier of cardiac structure and function, and motivated the hypothesis MTSS1 reduction would protect against myocardial disease. In the first cycle of this award, we demonstrated that MTSS1 reduction partially rescues cardiac function in female but not male mice in vivo in a transgenic model of human DCM. To study effects in humans, we fine-mapped the MTSS1 locus with cardiac magnetic resonance imaging (CMR) traits in UK Biobank. Concordant with our mouse studies, associations between MTSS1 and CMR traits were evident almost exclusively in women, and MTSS1 alleles improved cardiac function in women—but not in men—who carried pathogenetic DCM variants. Our findings at the MTSS1 locus suggest a genetic basis for sex dimorphism in cardiac remodeling. Sex differences in cardiac traits and HF have been appreciated for decades. Remarkably, sex-specific effects have been largely ignored in cardiovascular genomics. Few genome wide association studies (GWAS) for cardiac traits and disease have been conducted separately in men and women; interactions between cardiac loci and sex or sex hormones are unexplored, and sex chromosomes themselves have been excluded from most GWAS. Our preliminary analyses indicate that MTSS1 is one of least 20 cardiac modifier loci that show sex dimorphism and may thus contribute to sex differences in myocardial disease. Our renewal application will elucidate sex-specific effects of modifier loci on cardiac structure, function, and failure using MTSS1 as an exemplar. Our approach will build on resources developed during the first award cycle, including consolidated access to global HF genomic data and experimental HF models already established in our laboratories. Aim I will determine genetic mechanisms through which MTSS1 and other modifier loci show sex-specific effects on cardiac traits. Aim II will test if sex-specific effects of MTSS1 and other modifiers will translate into sex differences in myocardial disease and clinical HF. Aim III will use experiments in HF models to prove, or refute, sex-specific mechanisms. If successful, our proposal will expand a paradigm revealed through MTSS1 into a broad understanding of genetic contributors to sex differences in myocardial disease. Doing so will advance cardiac biology, genetics, and precision medicine by identifying sex-specific risk factors and treatment targets for human heart failure.

NIH Research Projects · FY 2025 · 2019-07

Enter the text here that is the new abstract information for your application. The main goal of the Structural Biology and Molecular Biophysics Training Program at the University of Pennsylvania is to train a cadre of superlative students and put them on the trajectory to achieve success and leadership roles at the highest levels of academic and industrial science in this core area of biomedical research. We will leverage the immense resources, 45 outstanding faculty trainers, and talented graduate students of UPenn to accomplish this goal. The program will do so with a premium put on training in the technically challenging aspects of experimentation in this area of biomedicine, scientific thinking, integration of structural biology and molecular biophysics research within the broader biomedical scientific community, presentation skills, career-focused exploration, and achieving success through promoting a collaborative scientific workplace and cohesive community. There is anticipated strong growth in the number of jobs in Biochemistry and Biophysics, with a PhD representing the typical entry level education requirement. Building on excellence that can be directly traced back to 1929 and the establishment of the Johnson Research Foundation as the first privately endowed research organization in the USA dedicated to biophysics, upon decades of NIH-supported training programs in Structural Biology and Molecular Biophysics, and upon the notable success of our trainees in the current funding cycle, we have developed plans to ensure that our future trainees will enter the scientific workforce after being trained in a program that encompasses broad training goals and in a research environment known for rigor and vibrancy. The institutional commitment is strong and has been a consistent force that supports all aspects of the Training Program. A notable recent initiative is a sizable additional investment in the establishment of the Penn Institute for Structural Biology that will directly and positively impact the trainees in the funding cycle for which we request support. The Program benefits from a tradition of using feedback from trainees, as well as ideas to innovate with newly arising initiatives in graduate education and scientific breakthroughs (technical and conceptual), to continually adapt and improve. The strategy to extend this tradition are now buttressed by linking an outcomes rubric that defines success and a logic model meant to enable continuous improvement. Current major activities specific to the training program are a two tiered series of trainer-led meetings. The first is open to all qualified students and is required, along with two specific graduate-level courses, for subsequent appointment as a T32-supported trainee. Both of these series are built in to our integrated training plan that includes the integration of classroom and laboratory learning and discovery to prepare scientists, increase success on career paths in Structural Biology and Molecular Biophysics, and to ensure flexibility that accommodates the needs of individual students.

NIH Research Projects · FY 2025 · 2019-06

Lung transplantation is a potentially life extending treatment for patients with end stage lung diseases; however, lung transplant is a relatively new field, and long-term outcomes are disappointing with a median survival of ~6 years.2 Furthermore, among those who survive, half suffer from impaired lung function - chronic lung allograft dysfunction (CLAD) – which causes distressing symptoms and disability.3 Yet, fundamental knowledge gaps persist in the understanding of the clinical and biological processes that occur after lung transplantation; advancing such knowledge is critical to improve long-term outcomes. The Lung Transplant Outcomes Group (LTOG) is the first and largest multicentered epidemiologic study in lung transplantation designed to address issues surrounding early post-transplant complications. LTOG began as an 11-center prospective cohort study formed in 2007. Initially, LTOG focused on the clinical mechanisms and biomarkers for the early post-transplant complication of primary graft dysfunction (PGD), a serious condition that often leads to early graft failure and death. Over 3000 subjects have been enrolled in the LTOG during the past ten years. In this application, we aim to extend follow up of the LTOG and capitalize on this rich resource of clinical data, biosamples, and infrastructure to address fundamental but previously unanswerable questions about the long term outcomes of lung transplantation. We will have two aims focused on major long- term issues key to lung transplant patient outcomes: in Aim 1 we will perform long-term CLAD phenotyping of all subjects enrolled in the LTOG until death or study end using current state-of-the-art definitions; and in Aim 2 we will determine the long term functional status of the recipient including targeted measures of frailty domains and functioning, disability, and health-related quality of life. Our proposal is significant in that it will create a unique resource capable of generating tremendous new knowledge in a growing field, both directly as a result of data in hand and by facilitating new research on NHLBI-relevant lung diseases. It is innovative in generation of a novel data source at a low cost by leveraging a unique established cohort, and in efficient data practices. It is impactful in developing new knowledge that may change transplant practices worldwide by merging long-term graft and patient phenotype data with a rich early-transplant data and biosample resource, by defining new mechanisms of relevant lung transplant syndromes, and by providing a platform for ancillary research applications for a generation of investigators.

NIH Research Projects · FY 2025 · 2019-06

PROJECT SUMMARY The Clinical Infec�ous Disease Laboratory at the Mathew J Ryan Veterinary Hospital (PA-Ryan), located at the University of Pennsylvania School of Veterinary Medicine, currently offers comprehensive microbiology, parasitology and molecular biology veterinary diagnos�c services for companion animal and exo�c species. PA- Ryan became a member of the Veterinary Laboratory Inves�ga�on and Response Network (VetLIRN) in 2017. It also is affiliated with the PADLS on the New Bolton Center campus, which is a first- �er laboratory member of the Vet-LIRN. PA-Ryan is experienced in the analysis of a variety of sample and matrix types, including but not limited to animal samples, environmental samples, water, and animal food products. PA-Ryan is accredited by the American Animal Hospital Associa�on (AAHA) as a hospital-based laboratory. In addi�on to addressing the current need for added laboratory capacity in the event of a large-scale outbreak or threat incident involving companion animals or animal food, PA-Ryan will con�nue to further strengthen the Vet-LIRN through molecular diagnos�c ac�vi�es that beter enable early detec�on of emerging pathogenic agents and facilitate the rapid responses that can minimize harm and best protect both human and animal health. In the previous 5 years (FY2018-FY2023), the PA-Ryan lab has had several successes as a VetLIRN laboratory including publica�on of 5 peer-reviewed ar�cle, 4 public presenta�ons, 2 public webpages and 1 standardized protocol. PA-Ryan has also expanded its capacity for tes�ng by implementa�on of new technologies and standardized tests with other VetLIRN laboratories. PA-Ryan has also par�cipated in numerous VetLIRN proficiency tests and interlaboratory comparison exercises. PA-Ryan currently serves as a Source Lab for the VetLIRN AMR surveillance project and has provided 756 isolates for Whole Genome Sequencing during the previous funding period. During the next funding period, PA-Ryan will con�nue to partner with VetLIRN to standardize methods and quality procedures along with faithfully conduct VPO directed case inves�ga�ons and other projects. PA-Ryan is dedicated to the mission the VetLIRN sets forth “to improve veterinary medicine and promote public and animal health”.

NIH Research Projects · FY 2025 · 2019-06

PROJECT SUMMARY/ABSTRACT The overall goals of this research program are to develop analyses, tools, and methods to achieve new, more effective catalysts and reactions. New synthetic methods greatly increase access to untapped chemical space, leading to materials and pharmaceuticals that benefit society. To achieve these goal, investigations will focus on obtaining an improved understanding of reactivity and selectivity. The fundamental hallmark of this program is the ability to access new reaction patterns to construct important organic structures in an efficient and rational manner. Reaction models and mechanistic understanding gives us the tools to solve problems and posit hypotheses. High throughput microscale experimentation permits rational hypotheses to be interrogated broadly and to develop new models to understand reaction space. One set of goals will be state-of-the-art computational and machine learning methods combined with new high throughput experimentation methods and large datasets to understand stereoselectivity, chemoselectivity, and reactivity at the molecular level with the aim of designing new, more effective catalysts and reactions.. The control of selectivity and reactivity are essential features of efficient synthesis, yet our molecular level understanding of how fundamental interactions perturb these aspects is only rudimentary. Another set of goals will be oxidative coupling of fragments via C-C and C-O bond formation by means of C– H activation. The development of new oxidative coupling chemistry is a particular focus due to increases in efficiency from lower step counts and smaller waste streams. The challenge in this area is selectivity in any given transformation due the numerous C–H bonds present in a typical organic molecule. Use of biomimetic processes leads to bioactive natural products and natural product-like cores, desirable entities in medicinal chemistry. The focus will be on transformations that are currently inaccessible include cross selective couplings, coupling at unactivated positions, and couplings with substrates resistant to direct oxidation. New strategies that will be implemented include the use of templating groups and alternate protocols to generate oxidized equivalents. Catalyst libraries will be deployed in a high-throughput microscale format to discover new reactivity. Invaluable training, largely absent outside of industrial settings, will be afforded to graduate students and other coworkers.