University Of Pennsylvania

NIH Research Projects · FY 2025 · 2023-09

Project Abstract Tendon hierarchical structure dictates its ability to effectively transmit loads from muscle to bone. Formation of tendon during tendon development and healing is reliant on coordination of multiscale regulatory governing processes such as formation of fibrils from collagen molecules, assembly of fibril bundles to form fibers, and recruitment of fibers to form fascicles. Collagen XII is a Fibril-Associated Collagen with Interrupted Triple Helices (FACIT) and is primarily expressed during tendon growth and development and following injury. Collagen XII interacts with collagen I fibrils and cell surfaces and its localization to form flexible bridges between collagen fibrils implicates its role in regulating collagen I fibrillogenesis, fibril organization, and interactions with other extracellular matrix constituents. Further, collagen XII has critical roles in the injury and regenerative responses. For instance, collagen XII co-localization with collagen I and other matrix components is present during tissue regeneration, suggesting a role in tissue cohesion. Recent data showed that collagen XII deficiency disrupts tendon structural and functional properties in mice and human disease, suggesting that collagen XII regulation is critical in tendon development and healing. However, the mechanisms by which collagen XII deficiency disrupts formation of tendon hierarchical structure during postnatal development, and recapitulation of this hierarchical structure after injury, remain unknown. Therefore, the objective of this proposal is to establish the mechanisms involving collagen XII regulation of tendon hierarchical structure, mechanical function and composition throughout postnatal development and healing. We hypothesize that collagen XII-mediated mechanisms are required for establishing tendon structure and function and that these regulatory mechanisms are recapitulated after injury. To test this, we will use our novel tendon-targeted Col12a1 knockout and inducible Col12a1 knockdown mouse models for investigation of the regulatory roles of collagen XII throughout Achilles tendon development and healing. We will perform comprehensive multiscale structural, functional, and compositional assays using our innovative mouse models in the following: Aim 1: Elucidate the mechanistic roles of collagen XII in regulating hierarchical assembly of tendon required for function during postnatal development. Aim 2: Define the regulatory mechanisms involving collagen XII during healing. Utilizing our innovative mouse models, we will define tendon-specific regulatory mechanisms involving collagen XII to provide a fundamental understanding of the acquisition of tendon structure and function and its re-establishment during healing. Comprehensive, and rigorous assessments of tendon multiscale structure, function, and composition will define the role of collagen XII throughout the progressive stages of postnatal development and the post-injury healing response. Activities described by this proposal provide a strong foundation for scientific inquiry, preparing me to make valuable contributions as an independent investigator.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT Parkinson’s disease (PD) is a progressive neurodegenerative disease that affects 10 million people worldwide. Its motor symptoms result from selective degeneration of dopaminergic neurons in the substantia nigra pars compacta, leading to a loss of their long-projecting axonal inputs to the striatum. Conventional cell therapy involves implanting dopaminergic neurons into the striatum; however, this strategy disregards the important systems-level implications of the native neuroanatomy. Pathway reconstruction strategies aim to address this limitation by replacing both neurons and axonal fibers in a manner that restores the anatomy – and hence circuit function – of the lost pathway. We have developed a reconstruction strategy whereby tissue-engineered nigrostriatal pathways (TE-NSPs) are pre-fabricated in vitro featuring a population of human stem cell-derived dopaminergic neurons and their long-projecting axonal tracts encased within a biocompatible tubular hydrogel. TE-NSPs may be implanted to directly replace the pathway, supplying both dopaminergic neurons to the nigra and providing axonal inputs to the striatum, thereby restoring crucial interconnectivity of the basal ganglia. In this proposal, we will answer a fundamental and neglected question in cell therapy for PD by characterizing whether pathway reconstruction with the TE-NSPs enables improved restoration of motor function compared to conventional striatal grafts in a rat model of PD. Our overarching hypothesis is that TE-NSPs will lead to more robust motor recovery than striatal grafts through a mechanism involving the reestablishment of physiological innervation and striatal dopamine regulation patterns more closely matching those of native basal ganglia. This hypothesis will be tested over three Aims: (1) Establish the ability of TE-NSPs to reconstruct basal ganglia circuitry via axonal-dendritic synaptic integration; (2) Demonstrate real-time efficacy of TE-NSPs in restoring nigrostriatal functionality; (3) Assess the influence of TE-NSP activity on motor recovery. TE-NSP mechanisms and efficacy will be compared to hydrogel-encased nigral or striatal grafts, acellular hydrogel implants, as well as non-implant and non-lesioned animals out to 24 weeks post-implantation. Motor function will be evaluated with rotational, forelimb asymmetry and adhesive removal tests. Innervation and connectivity patterns will be assessed with immunohistochemistry and monosynaptic rabies tracing, while ex vivo and in vivo voltammetry and [18F]F-DOPA positron emission tomography will be used to analyze real-time dopamine release and uptake in the striatum. We will also employ chemogenetics to silence neural activity in TE-NSPs to test the effects on motor function. Overall, TE-NSPs address a crucial gap in clinical treatment by providing a means to directly replace the nigrostriatal pathway, which may yield significant benefits over other methods by providing properly-regulated dopamine in the striatum as characteristic of integrated basal ganglia circuitry. These studies will further the long-term goal of advancing TE-NSPs as a Tissue Engineered Medical Product to mitigate the neuronal-axonal loss underlying the motor symptoms in patients afflicted by PD.

NIH Research Projects · FY 2025 · 2023-09

Project summary Patients with hypertrophic cardiomyopathy (HCM) experience a progressive disease course and bear a substantial symptomatic burden, marked by heart failure, atrial and ventricular arrhythmias and early mortality. Patients with symptomatic HCM, but without obstruction of the left ventricular outflow tract (so-called non- obstructive HCM), are a particular challenge to manage, as no treatments have proven effective at improving symptoms or impacting the trajectory of disease progression. This proposal will focus on sodium-glucose cotransporter 2 inhibitors (SGLT2i) as a potential therapeutic option for non-obstructive HCM. Initially developed as hypoglycemic drugs to treat type-2 diabetes mellitus (T2DM), SGLT2i unexpectedly conferred a remarkable cardioprotective benefit with robust reductions in cardiovascular mortality and hospitalizations for heart failure, irrespective of diabetic status or ejection fraction. While the mechanisms of action by which SGLT2i are exerting such beneficial cardiovascular effects are still unclear, an improvement in cardiac energetics has been proposed as one plausible mechanism based on evidence for increased cardiac ketone oxidation and ATP content in preclinical models of heart failure. The overall goal of this study is to determine the safety, feasibility, and preliminary efficacy of SGLT2i in patients with non-obstructive HCM. The rationale for conducting this study is based on the similarities between non-obstructive HCM and heart failure with preserved ejection fraction, and also the unique structural and functional features of HCM that make an early phase trial essential before implementation of larger phase clinical trials or adoption of “off label” use. We will perform a randomized, double-blind, cross-over study of the SGLT2i dapagliflozin vs placebo in 26 patients with non-obstructive HCM and ejection fraction >50%. Our primary safety outcomes will be rhythm monitoring and intracavitary left ventricular gradients assessed by echocardiography. Our preliminary efficacy endpoints will be change in peak oxygen consumption (VO2), left ventricular systolic and diastolic function by echocardiography, NT-proBNP, ambulatory actigraphy, and quality of life assessment. In an exploratory aim, we will test whether SGLT2i improve cardiac energetics in HCM using 31P-MR spectroscopy to quantify relative amounts of myocardial energy stores, specifically phosphocreatine and ATP. This early phase study seeks to extend the marked cardiovascular benefits of SGLT2i recently demonstrated for heart failure with reduced and preserved ejection fraction, to patients with HCM. The study team includes investigators with a strong track record in early phase clinical trials in HCM, at a high volume HCM center at the University of Pennsylvania, in collaboration with experienced clinician investigators in endocrinology and radiology at the Children's Hospital of Pennsylvania. The results of this study will provide essential preliminary data to determine if larger scale clinical trials of SGLT2i in HCM should be pursued, and explore a shift in metabolic substrate utilization as a potential mechanism of action of conferred benefit.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Conventional dendritic cells (CDC) play a central role in protective immunity by connecting innate and adaptive immune responses. CDCs can distinguish `self' from `non-self' (example pathogen) or `altered-self' (example cancer) through specialized pattern recognition receptors and help orchestrate the appropriate adaptive immune response. CDC1 and CDC2 are the two major subsets of CDCs with CDC1s having the unique capacity to cross- present antigens that is critical for immunity against viruses and cancer. Circulating precursors of CDCs (Pre- CDCs) infiltrate tissue where they differentiate into CDC1 and CDC2. The relative distribution of these CDC subsets differ between tissue types and under pathological conditions, suggesting a role of tissue microenvironment in Pre-CDC differentiation. What factors in the tissue microenvironment might regulate this process, however, remains poorly understood. Our preliminary studies show that CDC1 differentiation is regulated by local availability of the amino acid glutamine through its metabolic conversion into glutamate. Glutamine uptake and utilization increases significantly in rapidly proliferating cells or during catabolic stress, potentially creating a glutamine deficient local microenvironment. Hence, we hypothesize that metabolic adaptations in tissue alters local CDC1 differentiation by modulating glutamine levels. In this proposal, we seek to understand which steps of CDC1 differentiation is regulated by glutamate and its underlying molecular mechanism. We will focus on epigenetic regulation of gene expression and oxidative stress as potential pathways by which glutamate mediates this effect. Findings from the proposed work can potentially open new lines of research linking tissue metabolic adaptations to its immune microenvironment.

NIH Research Projects · FY 2024 · 2023-09

Project abstract Mammalian reproduction requires biparental genetic contributions due to the highly dimorphic nature of gamete epigenomes. DNA methylation (DNAme) is one of the most sexually dimorphic epigenetic marks in gametes, being hypermethylated in the sperm and alternatingly hypo- and hypermethylated in the oocyte. Aberrant DNAme in the germline can negatively impact fertility and offspring development. To prevent transmission of epimutations and establish the germline fate, primordial germ cells (PGCs) undergo global DNAme erasure following specification. While most of the genome achieves demethylation through replication-coupled passive dilution, the active demethylation pathway using the TET1 enzyme is required for methylation erasure of a subset of loci. I recently discovered that sperm-specific hypomethylated regions, while rare, require TET1 for reprogramming. Tissue-specific hypomethylation signatures often correlate with binding of developmentally relevant transcriptional factors, lending to the significance of these sperm-specific hypomethylated regions. Mechanisms of how sperm or oocytes acquire sex-specific DNAme remain a knowledge gap with relevance to fertility and development. While biochemically histone post-translational modifications (PTMs) have been shown to correlate with DNA methyltransferase (DNMT) accessibility, it remains unknown how these epigenetic marks become non-uniformly enriched within the germline genome. I hypothesize that histone PTMs enrichment and DNAme patterning in the germline are determined 1) intrinsically by the demethylation pathway used during PGC reprogramming and 2) extrinsically by the signaling milleu of the gonadal supporting cells. To test this hypothesis in vivo, genetic mouse models and multi-omics analyses will be used to elucidate what cellular signals are responsible for the acquisition of sex-specific DNAme signatures in the sperm and the oocyte. Aim 1 (K99) will test the catalytic and non-catalytic requirement for TET1 during PGC development for the establishment of the methylation signature of the oocyte genome. Aim 2 (R00) will employ genetic sex- reversal models of Dmrt1 overexpression in pre-granulosa cells (female-to-male) and constitutively active Wnt signaling in pre-Sertoli cells (male-to-female) to test the impact of altering the somatic signaling environment for DNAme acquisition in germ cells. In these models, I will integrate and identify correlations between changes in methylome and the relevant histone PTMs enrichment (methylation of H3K4 and H3K36). Single cell transcriptomics will be used to identify instructive cues for the establishment of sex-specific DNAme signatures in gametes. I will receive extensive training in advanced bioinformatics and single-cell genomics during the mentored phase of this proposal under the mentorship of Dr. Bartolomei, a pioneer in DNAme and genomic imprinting, within the UPenn Epigenetics Institute. With the additional guidance from my advisory committee, which includes leaders in the field of germ cell epigenetics and gonadal signaling pathways, I will be well prepared to become an independent investigator in the field reproductive epigenetics.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY This proposal aims to identify the role of the histone lysine methyltransferase Dot1L in neuronal function and its contribution to neurodevelopmental disorders (NDDs). NDDs include a spectrum of highly prevalent conditions that manifest during development that can cause intellectual disability, developmental delays, and autism spectrum disorder. Recent work demonstrated that many chromatin regulators are mutated in NDDs, including the histone methyltransferase Dot1L. Dot1L methylates histone 3 of lysine 79 (H3K79me) which is associated with active transcription. We found that H3K79me is highly abundant and dynamically regulated in postmitotic neurons. Our preliminary data also indicate that H3K79me is critical for neuronal function. We found that patient mutations result in a loss of Dot1L methyltransferase activity indicating that depletion of H3K79me can cause NDDs. Further, we found that Dot1L depletion alters transcription of synaptic genes and bidirectionally regulates GluA2, an AMPA receptor subunit. Finally, we found long-term memory deficits in Dot1L conditional knockout (cKO) mice. However, the role of Dot1L in neuronal function and cognition remain unclear. I hypothesize that Dot1L regulates synaptic gene expression and that partial Dot1L loss disrupts this regulation leading to NDDs. In Aim 1, I will define chromatin and transcriptional disruptions caused by partial Dot1L loss using a heterozygous Dot1L cKO mouse model coupled with H3K79me2 cleavage under targets and tagmentation (CUT&Tag) and RNA-sequencing. In Aim 2, I will examine the impact of partial Dot1L loss on neuronal function and cognition by using the heterozygous Dot1L cKO mouse model and controls to perform electrophysiology and behavioral experiments. Cumulatively, this work will establish a role for Dot1L in neuronal function and NDDs and more broadly will contribute to understanding of the role of chromatin regulators in brain function.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Decisions to admit patients with acute respiratory failure (ARF) and sepsis (the most common and lethal cause of the acute respiratory distress syndrome) to intensive care units (ICUs) are highly variable across the US. And, yet, these triage decisions have a substantial impact on patient outcomes. In our prior work, we used detailed electronic health record (EHR) data from 9.2 million hospitalizations and found that decisions to admit ARF patients to wards were associated with a 3.8% absolute increase in mortality. In contrast, choices to admit sepsis patients to ICUs resulted in considerably longer length of stay and a 5.1% absolute increase in death. The nationwide impact of such discretionary triage would be exponentially greater. Our findings highlight tremendous opportunities to improve ARF and sepsis outcomes by identifying the patient subgroups and processes of care that most strongly contribute to the benefits and harms of ICU- versus ward-based care. This application proposes to update our ARF and sepsis cohort such that it includes all admissions from 2013 through 2022 across 29 hospitals in the Kaiser Permanente Northern California and University of Pennsylvania health systems, and incorporate more than 100 more data fields per patient. This curation of highly granular EHR data will enable us to identify the: (1) distinct patient subgroups and phenotypes among those meeting the syndromic criteria of `ARF' and `sepsis;' and the (2) processes of care and (3) inpatient complications that causally explain the observed associations of ICU vs. ward triage with patient outcomes. Our multidisciplinary team will apply diverse expertise in instrumental variable regression, mediation analyses, machine learning, complex EHR data, and probabilistic phenotyping to complete three aims that promote our long-term goal of improving care, and hence outcomes, for patients with ARF and sepsis regardless of where they are treated. Several methodological innovations will enable us to achieve these goals, and, in turn, to not only surmount key limitations of prior studies that sought to determine which acutely ill patients benefit from ICU admission, but identify the mechanisms underlying such triage effects. These data will also allow us to quantify the impact of COVID-19 on ICU and ward triage patterns, care processes, and outcomes among ARF and sepsis patients, thereby modernizing our results and enabling their applicability to pandemic eras. Completing the aims of this study will improve public health by identifying ways in which emergency departments, ICUs, and wards can improve outcomes for the more than 4 million Americans hospitalized each year with ARF and/or sepsis. Such results will enable development and testing of personalized triage algorithms, and guide optimal care for patients without always requiring ICU admission, thereby improving patient outcomes, reducing health care costs, and preserving ICU capacity for patients who truly need it.

NIH Research Projects · FY 2025 · 2023-09

Informal caregiving is demanding and stressful especially when dealing with a hospitalization. Many caregivers eventually become care recipients themselves as years of stress and deferred self-care put them at increased risk for illness. Self-care refers to the behaviors undertaken to maintain health and manage illness. Engaging in self-care may improve health status (physical functioning and mental well-being). Older adults with multiple chronic conditions (MCCs) often depend on caregivers for assistance, especially after a hospitalization, when caregivers are often expected to follow complicated discharge plans and manage complex skilled care at home. The impact of care gaps and breakdowns saps time for self-care and causes significant stress for caregivers. Health coaching, a support intervention, can improve self-care in patients, but studies evaluating caregivers and racial differences are limited. Less is known about the effect of caregiver support interventions on patient outcomes. Leveraging the growing availability and declining costs of technology and internet access—along with increasing receptivity of virtual care in the wake of COVID-19—we developed and tested a synchronous virtual support intervention, ViCCY (Virtual Caregiver Coach for You), where 10 video conference sessions are delivered by a trained coach over six months that focus on self-care, coping, and stress. In this application, we propose to augment the ViCCY protocol to target caregivers during an acute care episode (during/post- hospitalization) and transitions in care for older adults with MCCs compared to digital health information (DHI) alone (control group). Using a randomized controlled trial (RCT) design, we will enroll informal caregivers with poor self-care (Health Self-Care Neglect scale score ≥2), and block randomize the caregivers 1:1 to the intervention or control group, stratifying randomization by caregiver sex, race, and relationship to the patient. Both groups will receive DHI delivered through a website tailored with care transitions and self-care information, and the intervention group will also receive ViCCY. At baseline, 3-, and 6-months, we will collect self-reported data on self-care, stress, coping, and health status. At 1-month post-hospital discharge the care transitions experience will be collected. At 6-months, we will compare ViCCY to DHI alone to assess intervention efficacy using intent-to-treat analysis. A sample of 250 caregivers (125/arm) will provide >80% power to detect significant differences between the groups on the primary outcome of self-care (Aim 1) and that the magnitude of improvement will be similar in outcomes in Aim 1 between Black/AA and White caregivers (Aim 2). To explore the effect of caregiver outcomes on patients’ outcomes we will examine acute care resource use (rehospitalization, etc.) over a 6-month period (Aim 3). Knowing that not all patients will participate, we will consent a subgroup of the hospitalized older adults cared for by these caregivers (at least 40 dyads). If shown to be efficacious, our virtual health coaching intervention can easily scale to support millions of caregivers worldwide. This application addresses the NIA strategic plan and is in response to NOSI NOT-CA-22-037.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Hypertrophic cardiomyopathy is the most common inherited cardiac muscle disease with an estimated 750,000 affected individuals in the United States. However, only about 100,000 people have been diagnosed, suggesting that there are significant diagnostic and treatment gaps for individuals with pre-clinical or overt disease, as well as for their at-risk family members. Therefore, it is important to identify individuals who should undergo evaluation for earlier diagnosis and targeted treatment, prior to the development of highly morbid outcomes including heart failure, arrhythmias, stroke, and sudden death. The electronic health record offers a source of high dimensional, longitudinal phenotype information that can be leveraged to create more sensitive and specific diagnostic algorithms. In this patient-oriented mentored career development award proposal, Dr. Nosheen Reza aims to improve the ability to identify individuals with hypertrophic cardiomyopathy through creation and evaluation of machine learning classification models that leverage electronic health record data derived from diverse populations. In Aim 1, she will derive and validate a multi-institutional electrocardiogram-based model for the detection of hypertrophic cardiomyopathy using data from the Penn Medicine electronic health record and will evaluate whether the addition of additional electronic health record-derived traits to this model improves the model's ability to detect patients with hypertrophic cardiomyopathy. In Aim 2, she will externally validate the best performing electronic health record-derived models in two large independent health systems. In Aim 3, she will use implementation science methods to identify clinician-specific barriers to and facilitators of accurate and timely diagnosis of hypertrophic cardiomyopathy and assess clinicians' attitudes toward the use of an electronic health record-derived diagnostic model for hypertrophic cardiomyopathy. Taken together, these aims will lead to prospective dissemination and implementation studies of a generalizable electronic health record-derived diagnostic tool to facilitate early recognition and risk stratification of individuals with hypertrophic cardiomyopathy. Dr. Reza, an early career investigator and genetic and advanced heart failure cardiologist, has a long-term goal of becoming an independently funded cardiovascular data scientist with a focus on applying clinical informatics tools that leverage electronic health record and genomic data to enable precision medicine in the care of patients with cardiomyopathy and heart failure. This K23 award will support Dr. Reza in achieving this goal through a comprehensive and rigorous training plan in bioinformatics, machine learning, and implementation science. Dr. Reza will be supervised by an outstanding mentorship and advisory team at the University of Pennsylvania consisting of national leaders in genetic cardiomyopathies, electronic health record-based research, and translational bioinformatics. The mentored research and career development plan outlined in this proposal will guide Dr. Reza's transition to an independently funded research career.

NIH Research Projects · FY 2026 · 2023-09

Abstract Anesthetics are low affinity drugs that interact with hundreds of molecular targets present throughout the nervous system at clinically significant concentrations. Despite this molecular-level promiscuity, the hypnotic effects of anesthetics depend critically on specific neural circuits. This assertion is supported by numerous results showing that direct modulation of specific sites distributed broadly throughout the brain can potentiate or, conversely, antagonize the anesthetic state. However, because previous experimental work focused upon one brain site at a time, the identification of the long-hypothesized brain-wide canonical anesthesia circuit has so far remained elusive. To fill this critical gap in knowledge, we reasoned that ultimately, the state of anesthesia must be imposed onto the brain by neurons that remain active under anesthesia, while most other neurons are suppressed. To identify anesthetic active neurons throughout the brain, we used tissue clearing and 3D c-Fos immunohistochemistry. We validated that putative anesthesia-active neurons are indeed physiologically active in vivo using two photon microscopy and fiber photometry. Having identified anesthesia- active neurons, we further reasoned that brain regions which project broadly are more likely to play a pivotal role in modulating the level of consciousness. Thus, we combined our brain activity map with whole brain connectivity analyses. The potent combination of these experimental and bioinformatics approaches allowed us to identify regions that contain a high density of anesthetic-active neurons and project broadly throughout the brain. Our unbiased approach culminated in the definition of a putative canonical anesthesia network comprised of nuclei in the ventral hypothalamus, thalamus, and the prefrontal cortex. In Aim 1, we will discover the specific cell types within prefrontal cortex, ventral hypothalamus and thalamus that remain active under anesthesia. This is of critical importance as all brain regions contain many cell subtypes with distinct neurophysiological properties, connectivity patterns, and ultimately, behavioral effects. In Aim 2, we will discover the brain-wide projections made by anesthetic-active neurons by combining anterograde and retrograde viral tracing in transgenic mice that specifically label distinct neuronal subtypes with 3D immunohistochemistry. In Aim 3, we will establish the functional roles of the canonical anesthesia network as a whole and each of its elements individually by combining chemo- and optogenetics with behavioral and neurophysiologic assessments of arousal. The ultimate result of the proposed research will be the identification of a canonical network of neurons sufficient to elicit hypnosis. This has fundamental implications for how the state of arousal is controlled in health and dysregulated in disease. Identification of this circuit may also suggest druggable targets for the development of more specific anesthetic agents with fewer undesirable effects. This has the potential to significantly improve the care of millions of patients requiring general anesthesia for life saving procedures.

NIH Research Projects · FY 2025 · 2023-09

Early exposures affect health across the life course. Extensive healthcare efforts have been made to prevent adverse pregnancy outcomes and to optimize child growth, health, and neurodevelopment. However, birth and childhood outcomes remain major areas of public health concern. Macroenvironmental health promoting factors (greenspace, walkability) and health threatening factors (neighborhood conditions) may affect health directly through inflammatory and immunologic pathways. Macroenvironments may also contribute to lived experiences of income potential and educational attainment. Combined with interpersonal individual exposures, macroenvironments may alter individuals’ microenvironmental health factors such as diet, physical activity, psychosocial stress, and sleep. While macro- and microenvironmental exposures have been studied individually, the impact of neighborhood environments on complex health disorders in pregnancy and early childhood remains understudied and the interplay with microenvironmental factors is unknown. We propose a causal inference framework to evaluate the role of specific macroenvironment factors (Aim 1) to reduce the risks of abnormal fetal growth, preterm birth, obesity, asthma, and neurodevelopmental delays by age 3. We will also identify optimal components of microenvironmental factors of diet, physical activity, and sleep, during pregnancy (Aim 2) and among couples during preconception (Aim 4), that can best be utilized to maximize reductions in adverse maternal and child health outcomes. We have assembled a multidisciplinary team of experts at the University of Pennsylvania and Children’s Hospital of Philadelphia who are well-positioned to complete the study and recruit up to 2500 pregnant women, partners, and offspring, with retention of at least 75% at age 3 in a population reflective of the Philadelphia community (Aim 3). The culture of clinical research and excellent scientific environment makes Penn and CHOP the ideal place to innovate in the field of maternal-child health. The Penn-CHOP ECHO study team is committed to the success of this work and looks forward to working collaboratively with the other ECHO Study Sites, Coordinating Centers, and Cores.

NIH Research Projects · FY 2024 · 2023-09

As a result of efforts to reduce healthcare costs and improve quality, patients with heart failure are increasingly receiving treatment in community-based programs, such as managed long-term care programs which support older adults in remaining independent in their community. Managed long-term care aims to reduce unplanned hospitalizations and emergency department (ED) visits, but heart failure is still the leading reason for these avoidable events. In managed long-term care, the care coordinator (i.e., a registered nurse or social worker) maintains regular contact with patients by telephone to ensure that they receive care congruent with their medical needs. From the linguistic perspective, verbal communications between patients and healthcare providers are information-seeking and sharing behaviors, as they include problem-focused communication. From the acoustic perspective, heart failure can affect patients’ voice and speech characteristics due to swelling caused by fluid retention or compression of the laryngeal nerve due to enlarged heart structures. While verbal communication between patients with heart failure and their care coordinators can provide insight into hospitalization and ED risks, it is largely untapped in managed long-term care. To address this gap, we aim to examine whether audio-recorded verbal telephone communication (hereafter called verbal communication) can be utilized to improve risk prediction. In the K99 phase, we will focus on identifying information in verbal communications between patients with heart failure and their care coordinators. We will extract the following potential risk factors for hospitalizations or ED visits from verbal communications: (1) conversational characteristics to analyze interactions in patterns of communication, (2) language phenotypes based on a list of the language of risk factors, including heart failure symptoms, poor self-management, and other hospitalization risks, and (3) acoustic features by analyzing voice signals. In the R00 phase, we will focus on developing risk prediction models for hospitalizations or ED visits for patients with heart failure in managed long-term care. We will develop several machine learning-based risk prediction models for hospitalizations or ED visits using information derived from: a) structured electronic health records, b) care coordination notes, and c) verbal communications between patients with heart failure and their care coordinators (identified during the K99 phase). We will evaluate if the risk prediction performance of machine learning algorithms can be improved by integrating information from different data sources. This proposal is aligned with the Strategic Vision key area of the National Heart, Lung, and Blood Institute (NHLBI), "Leverage emerging opportunities in data science to open new frontiers in heart, lung, blood, and sleep research." This study will be an important step toward achieving my long-term career goal of developing risk prediction models for heart failure patients and implementing them into clinical decision support systems. In particular, the goal is to identify early signs of deterioration by incorporating verbal communication from patients into risk models.

NIH Research Projects · FY 2025 · 2023-09

Our overarching goal is to advance understanding of mitochondrial mechanisms of carbon monoxide (CO) poisoning to develop diagnostics, therapeutics, and clinical trials. CO poisoning remains a major cause of death and disability, affecting 50,000 people per year in the United States alone. Patients removed from fires or following exposure to car and home generator exhaust are placed on 100% oxygen and transferred to a facility with a hyperbaric oxygen (HBO) delivery system. Despite the availability of HBO therapy centers in most major cities, inherent delays in access to and initiation of therapy greatly limit efficacy. In fact, even with HBO oxygen therapy a substantial number of surviving patients exhibit permanent neurocognitive impairments. This highlights an urgent need for alternative therapy. In the present proposal, we propose to study novel antidotal therapies for CO poisoning, based on our in vivo preliminary data that the use of a succinate prodrug relieves partial CIV inhibition caused by CO poisoning. Another existing gap is the lack of effective biomarkers to gauge severity, prognosis, and response to treatment. While a carboxyhemoglobin level is readily available at most institutions, its use is limited only to confirm exposure with no predictive value. The three main objectives our proposal seeks to address are: (1) extent of mitochondrial involvement for diagnostics and therapies; (2) limitations of current biomarkers to gauge severity of disease and treatment response; (3) lack of treatment strategies that target mitochondrial dysfunction to mitigate long-term neurologic and cardiac disability. Specifically for this A1 submission, we recently developed a novel survival swine model for CO poisoning with clinically relevant outcome metrics that include behavioral, imaging, and biomolecular measures. We also have obtained additional noninvasive optical data that also correlate with tissue respiration data. Another important feature of this proposal is the evaluation of a new treatment strategy involving a mitochondrial prodrug with the potential to shift existing treatment paradigm. We will also leverage our biomedical optics technology measuring cerebral blood flow, oxygenation, COHb and redox states of CIV in real time which will allow us to further elucidate the mechanisms of CO combined with repeat measures using two clinically relevant exposure duration with varying doses as well as prolonged low dose CO exposure. Aim 1 • To investigate the mitochondrial mechanisms that contribute to the neurologic and cardiac injury with the use of blood cell as a liquid biomarker in both acute AND early chronic CO poisoning. Aim 2 • Randomized, blinded pre-clinical intervention trial in swine models of CO poisoning to compare an engineered succinate prodrug to standard therapy of hyperbaric oxygen (HBO).

NIH Research Projects · FY 2025 · 2023-09

Although the exact mechanisms by which anesthetics induce unconsciousness remain unknown, there is evidence that some anesthetics activate neural circuits regulating sleep and inhibit neural systems promoting waking. Despite general anesthesia and sleep both activating a subset of seemingly similar, if not identical, neurons, there are clear differences between the two unconscious states, including the degree of arousal threshold changes and the timescale of state transition. The neural mechanisms underlying these related, yet distinct unconscious states are poorly understood. The parafacial zone (PZ) has recently been identified as a non-rapid-eye-movement (non-REM) sleep-promoting region; specifically, GABAergic neurons in the PZ (PZ-GABA) are active during non-REM sleep. My preliminary data demonstrate that PZ-GABA are also active during isoflurane exposure, and ablation of PZ-GABA increases resistance to isoflurane. The results also suggest that non-GABAergic neurons within the PZ are also involved in isoflurane-induced hypnosis. The overarching question asks how the neural circuitry driving distinct states of non-REM sleep and isoflurane anesthesia converge and diverge by first examining in PZ-GABA neurons, and then expanding beyond the PZ to consider all cell types in the medulla. It is hypothesized that these distinct endogenous and drug-induced unconscious states are generated by partially overlapping shared circuits but that key state differences arise from distinctive cellular activation patterns. The three key questions we will address during this proposal are: 1) Does acute reversible activation/inhibition of the PZ sleep-promoting neurons alter anesthetic sensitivity? 2) What is the cellular makeup of the PZ, and which cells are activated during each unconscious state? and 3) What are the overlapping and different elements between the brainstem neural circuits engaged during isoflurane exposure and those engaged during non-REM sleep? These questions will be addressed by anesthetic and sleep phenotyping assays, the single-cell level transcriptomic analysis by single nucleus RNA sequencing followed by multiplex in situ hybridization, and side- by-side comparison of ensembles of active neurons by Targeted Recombination in Active Population (TRAP). The proposed projects will uncover the underlying mechanism of how the brainstem neural circuits, including PZ, mediate these two different unconscious states. Understanding how the brain controls states of unconsciousness is vital for clinical practice. It can lead to more effective and safer somnogens and new potential sedative hypnotic anesthetics that may one day be used for sleep disorders such as insomnia and narcolepsy.

NIH Research Projects · FY 2024 · 2023-09

The Oropharyngeal Microbiome in COVID-19 Summary (Abstract) SARS-CoV-2 first infects the oropharynx and upper respiratory tract, where it either remains localized and is cleared, or propagates to the lower respiratory tract where it can progress to pneumonia and respiratory failure. Several patient factors correlate with increased COVID-19 severity, including older age, obesity and diabetes, but the mechanisms linking these factors to COVID-19 pathogenesis are incompletely understood. What determines the extent of SARS-CoV-2 upper respiratory tract (URT) replication and whether infection remains localized or propagates to the lower respiratory tract (LRT) is therefore a critical knowledge gap. Studies with other respiratory viruses (RSV, influenza) suggest that the local URT microbiome can regulate immune responses and influence lung consequences of infection. In addition, the SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene, suggesting that it could be modulated by local microbiota. Thus, the microbiome could play a key role in local SARS-CoV-2 replication and consequences of infection through mechanisms involving both immune modulation and viral receptor expression. Furthermore, we and others have shown that the oropharyngeal microbiome is the principal source of lung microbiota, which are derived from the URT by microaspiration, and so factors that affect the oropharyngeal microbiota likely also impact the lung microbiome with similar effects. We investigated hospitalized COVID-19 patients and found that the oropharyngeal (OP) microbiome differed markedly from healthy subjects and also from hospitalized patients with other illnesses. The OP microbiome at early sampling points correlated with maximum disease severity over the course of hospitalization. The OP microbiome also correlated with systemic immune parameters in blood. These findings raise the possibility that the microbiome in the oropharynx and upper respiratory region may influence severity of COVID-19. However, there is limited knowledge on the OP microbiome in groups and comorbidities (diabetes, obesity, elderly) associated with severe COVID-19 disease that might underlie this relationship. Our hypothesis is that the oropharyngeal microbiome plays a key role in regulating consequences of SARS- CoV-2 infection through modulation of ACE2 expression and/or immunity that determine whether infection is locally contained in the URT or propagates to LRT involvement to cause severe disease. This mechanism may act locally within the URT and also, by URT-LRT microbiome crosstalk, on the LRT. Our aims are to: (1) Define the oropharyngeal microbiome of individuals at risk of mild vs severe COVID-19, relationship to expression of ACE2 and relevant mucosal genes, and to microbiome communities seen in early COVID-19 patients. (2) Determine the effect of human-derived oropharyngeal microbiome communities, introduced in a novel murine model, on ACE2 and immune genes in oropharyngeal and lower airway epithelial cells.

NIH Research Projects · FY 2025 · 2023-09

Parent Project Abstract This proposal aims to develop a high throughput droplet-based digital subsampling tool that enables continuous extraction of intracellular molecules (>1000 cells/sec) from individual living cells and achieve digital single molecule detection. Technological advances in single cell analysis have resolved cellular heterogeneity and enabled discovery of rare cell subpopulations. They have opened up new opportunities to detect subtle molecular changes in the presence of variability in biological systems. Due to the uniqueness of individual cells in their composition, functionality, and structures, molecular analyses at the single-cell level are critical for understanding the complexity of biological processes and cellular responses to perturbations. To accurately profile cellular dynamics and behaviors, it is essential to longitudinally monitor cellular changes and responses over time. However, it has been challenging to acquire temporal molecular information from the same cell populations due to the need of keeping them alive during the course of observation while minimizing their perturbations. Recently, nanotechnology methods (e.g. nanowire, nanobiopsy, nanostraw) have been developed for longitudinal cell monitoring where nanoscale dimensions are used to penetrate cells and sample intracellular molecules while providing minimal cytotoxicity. They have successfully achieved longitudinal cell subsampling and analysis, but it has still been difficult to resolve inherent heterogeneity of individual cells and their contents due to low throughput and sensitivity. In these studies, cells were placed on a substrate that consists of different nanostructures and a scarce amount of molecules were extracted, limiting the ability to achieve high throughput sampling and comprehensive downstream analysis. To build a robust longitudinal intracellular extraction tool, there is a need to spatially barcode and profile individual cells at high throughput and measure scant molecules with ultra-high sensitivity to maintain minimal perturbations during extraction. Here, we propose a live cell digital subsampling technology that will combine a cell membrane perforator and digital detection using droplet microfluidics to achieve i) live cell subsampling via hydrodynamic stretching of individual living cells, ii) ultrasensitive digital profiling of individual molecules sampled from single cells, and iii) monitoring of phenotypic and genotypic nutrient sensing pathway associated molecules for the early diagnosis of Alzheimer's disease and the development of new therapeutics. We will advance this platform to better understand cell biology and apply it to diverse fields including neuroscience, immunology, and cancer biology.

NIH Research Projects · FY 2025 · 2023-09

Project Abstract The ability to control biomolecules in situ is critical to the experimental process. Proteins are especially important to understand as they are central to cellular function, cell signaling, and living organism processes (e.g. metabolism, tissue development, and immune function). There is an unmet need for a small molecule approach to “tunably” and reversibly regulate expression of an imaging-compatible protein tag to investigate protein function(s). Such a tag would allow multi-modal imaging, including fluorescence and in vivo positron emission tomography (PET), allowing investigators to detect, then control, the tagged protein in cells and animals using small molecule ligands. For example, fluorescence imaging of the tag could guide the proper timing for protein knock-down based on subcellular localization or protein-protein interactions in vitro, and PET imaging of the tag could guide the regulation of proteins modulating cellular trafficking in vivo. A specific example of the in vivo application is in the process of developing chimeric antigen receptor (CAR) T-cell therapies for solid tumors, where understanding in vivo biodistribution, efficacy, and toxicity using imaging would be crucial, and importantly, controlling the cell surface expression of the CAR may have a large impact on that biodistribution, efficacy, and off-tumor toxicity. Chemical derivatives of the small molecule antibiotic trimethoprim (TMP) have been developed into multi-modality imaging probes by our group and others. The objective of this proposal is to set a standard for imaging-compatible protein regulation tags that can be widely adopted. We propose proteolysis targeting chimeric small molecules (PROTACs) based on TMP that target E. coli dihydrofolate reductase (eDHFR) tagged fusion proteins with high affinity. Our proof-of-concept molecules covalently link TMP and pomalidomide (POM), a ligand for the E3 ligase Cereblon. A lead compound, TMP- POM 7c robustly regulates diverse proteins, from optical reporter proteins, such as YFP and luciferase, to transcription factors and therapeutic membrane-bound proteins, such as CARs, in primary human T-cells. Optimization, characterization, and application of these compounds is needed, especially in terms of understanding the impact of linker length and composition, as well as pharmacokinetic properties, to lay the groundwork for a distributable prototype(s) that can be applied broadly in biomedical science. This approach represents a technological leap forward by uniting small molecule protein regulation of a versatile protein tag with fluorescence imaging probes and PET radiotracers for in vivo imaging.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY/ABSTRACT The centromere is a network of proteins rooted to centromeric DNA by nucleosomes containing a centromere-specific histone H3 paralog, CENP-A. Despite the centromere’s conserved role in chromosome segregation, a subset of its proteins that are essential for its function exhibit molecular signatures of rapid adaptive evolution. Repetitive centromeric satellite DNA also rapidly evolves, drastically diverging in sequence between species. The “centromere drive hypothesis” proposes that a genetic conflict between centromeric DNA and proteins causes their rapid evolution. Specifically, centromeric DNAs behave selfishly and increase their inheritance through female meiosis (drive) by increasing binding affinity for centromeric proteins. Selfish centromeres are also detrimental to the organism, which selects for novel centromeric protein variants with a lower affinity for the selfish DNA, thereby suppressing drive. The centromere drive hypothesis further predicts that distinct evolutionary lineages undergo unique bouts of centromeric DNA-protein co-evolution, leading to deleterious centromeric DNA-protein incompatibilities in hybrids that promote reproductive isolation. I have developed a novel experimental system to test three key propositions of the centromere drive hypothesis. The first is that centromeric DNA repeat variants differentially recruit centromere proteins. By fertilizing the eggs of M. musculus with the sperm of divergent Murinae species, I test whether the two species centromeric DNA repeats differ in their ability to recruit centromeric proteins from the hybrid zygote cytoplasm. My preliminary data indicate that Mus pahari CENP-A nucleosomes have a higher binding affinity for a key centromeric protein scaffold, CENP-C, than M. musculus CENP-A nucleosomes. For my first aim, I will biochemically reconstitute the M. pahari CENP-A nucleosome and test whether the centromeric DNA that wraps it imparts this increase in CENP-C binding. For my second aim, I will test whether rapidly evolving centromere protein orthologs (variants) differentially bind to centromere DNA. I will transiently express various centromere protein orthologs in hybrid zygotes and determine whether their binding preferences for the two species’ centromeres differ. For my third aim, I will test whether divergent centromere DNAs result in deleterious incompatibilities in hybrids. I found that in M. musculus / M. pahari hybrid zygotes, a subset of M. pahari centromeres are mispackaged. I will generate a more contiguous M. pahari genome assembly to determine if unique M. pahari centromeric DNA sequences underly this mispackaging, and test whether mispackaging causes deleterious chromosome missegregation. The hybrid embryo system that I developed serves as a powerful tool to interrogate not only functional divergence of centromeric DNA and proteins, but also genetic conflict more broadly by uncoupling selfish DNAs from their species-specific suppressors. This system and my training will position me to establish a future research program around diverse mammalian selfish genetic elements (see Career Goals).

NIH Research Projects · FY 2024 · 2023-09

Project Summary. Neurons and astrocytes have unique demands in regulating the quality of their proteome. A key regulator of the proteome is autophagy, a lysosomal degradation pathway. During autophagy, cytoplasmic components are packaged into autophagosomes and delivered to lysosomes for cargo degradation. Autophagy is neuroprotective, as mutations in key autophagy genes cause neurodegeneration. Preliminary studies show that autophagy is regulated differently in neurons and astrocytes in multiple paradigms of stress. Despite the importance of autophagy, how it is regulated in neurons and astrocytes to facilitate cell- type specific functions and stress responses is largely unknown. Thus, the goal for this proposal is to define cell-type specific functions for the autophagy receptor p62 in neurons and astrocytes. P62 facilitates selective forms of autophagy by binding to ubiquitinated substrates and the autophagy marker LC3, thereby incorporating cargo into a forming autophagosome. P62 mitigates proteotoxic stress by targeting protein aggregates to the autophagy pathway. Additionally, p62 mitigates oxidative stress by targeting Keap1, a negative regulator of the antioxidant transcription factor NRF2, for degradation by autophagy. To examine functions of p62 in each cell type, we established a robust system to coculture neuron and astrocytes. This system recapitulates intercellular interactions found in vivo, and provides an easily manipulatable system for studying cell-type specific p62 function with high resolution. Using the coculture, I found by immunostain that metabolic stress (autophagy activator) induces formation of p62-ubiquitin positive structures (i.e., ubiquitinated cargo) only in neurons. Moreover, blocking ubiquitination significantly reduces p62 puncta formation and degradation in neurons as compared to astrocytes in basal and stress conditions. Astrocytes are crucial to combating oxidative stress, but the role of p62 in this pathway in astrocytes is largely unknown. I found that oxidative stress induces p62 levels selectively in astrocytes. Thus, I hypothesize that p62 functions primarily in selective autophagy in neurons, and primarily in the antioxidant pathway in astrocytes. Importantly, ALS-linked mutations in p62 fall in domains important for each function. But how p62 protects against neurodegeneration in each cell type is not understood. I hypothesize that ALS-linked mutations in p62 domains that are important for selective autophagy and antioxidant function will impair p62 function in neurons and astrocytes, respectively. To test these hypotheses, I will (Aim 1) define cell-type specific functions of p62 in neurons and astrocytes, and (Aim 2) determine the effects of ALS-linked mutations on p62 function in neurons and astrocytes. This study will elucidate cell-type specific contributions of neurons and astrocytes to neurodegeneration. In turn, understanding cell-type specific contributions to ALS will enable opportunities for more targeted and specified therapies to mitigate neurodegeneration.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY This is an application for a K24 mentoring award for patient-oriented research (POR) from Bonnie Ky, MD, MSCE, the Founders Associate Professor of Medicine at the Perelman School of Medicine at the University of Pennsylvania. Dr. Ky’s long-term goals are to lead clinical science that will uncover actionable knowledge to improve the cardiovascular care of cancer patients and survivors. This has involved developing a deep- phenotyping approach in which individual patient risk is determined through detailed assessment of patient clinical characteristics, biologic markers, and echocardiography-derived measures of myocardial mechanics and function, and the development of innovative strategies to mitigate risk. The candidate has a strong track record of mentorship, research productivity, program building, and leadership in cardio-oncology. This mid-career development award is critical to allow her the necessary protected time to further augment her mentoring skills in the training of early career researchers and physician scientists in cardio-oncology, and develop new skills in biomedical informatics, the social determinants of health (SDOH), and healthcare disparities. The scientific goals of the proposal are to define the impact of racial inequities in cardio-oncology through a detailed analysis of patient-level data from the electronic medical records (EMR) of the University of Pennsylvania Health System. This comprehensive study of the SDOH by race will offer new opportunities in: 1) detailed methodologic experience in biomedical informatics, EMR-based research, data science, natural language processing, study design, and analytics; 2) greater experience and understanding of the SDOH and healthcare disparities as it relates to cardio-oncology. There are little data as it relates to cardio-oncology disparities, and this is a highly significant area of new growth. The mentoring goals are to train and support the next generation of diverse physician scientists in cardio-oncology POR. The career development goals are to further support and grow the candidate’s professional development, education in SDOH science and biomedical informatics, skills as a mentor and leader, and program building skills in cardio-oncology. The institutional environment for clinical and translational POR at Penn is outstanding. The Department of Medicine at Penn has made a substantial commitment toward the candidate’s continued success as a patient-oriented researcher responsible for the current and future training physician scientists in cardio-oncology POR. A highly qualified mentoring team and team of collaborators comprised of experts in SDOH science, cancer clinical trials, healthcare disparities, cardiovascular science, biomedical informatics, EMR-based research and natural language processing, and advanced statistical methods will work closely with Dr. Ky. Together, they will ensure the success of this proposal and of Dr. Ky as a mentor to the next generation of cardio-oncology physician scientists in POR.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Transcription is an essential and tightly regulated process that requires the coordination of many factors to ensure proper gene expression. Current models of transcription are predicated on stable, hierarchical interactions. These models have been challenged through recent developments in in vivo imaging, which have revealed that many transcriptional regulatory proteins interact transiently with chromatin. Instead of relying on stability, occupancy at target loci is achieved through more frequent interactions resulting from the formation of high local-concentration assemblies within nuclei, called hubs. Little is known about the functional impacts of hub formation on transcription, how hubs alter the kinetics of regulatory proteins and how hubs function in cancer, human expansion repeat disease, and other diseases. Previous studies largely rely on the ectopic overexpression of proteins of interest and qualitative assays to study hub function and there is a of lack of both specific strategies to perturb hub formation/properties with a measurable functional output and application of suitable technologies to look at protein kinetics in vivo. The goal of this project is to use oncogenic mutations found in the chromatin reader protein, ENL, to elucidate the mechanisms by which hubs impact transcription. ENL mutations are among the first examples of pathogenic mutations that result in aberrant hub formation. Importantly, such hub formation is functionally required for hyper-activation of target genes. The high specificity and gain-of-function nature of ENL mutations make them a powerful system to study both the mechanisms of hub formation as well as how aberrant hubs contribute to human disease. I hypothesize that ENL mutant proteins promote the clustering of multiple elements, both genomic and proteomic, to alter transcription at target loci. In Aim 1, I will combine advanced imaging techniques, including single molecule tracking and live imaging of transcription, to determine the effect of hub formation on the molecular kinetics of incorporated proteins and transcription dynamics. In Aim 2, I will investigate the effect of hub formation on the spatial proximity of target genes using DNA-FISH and live imaging to determine if hubs drive genome reorganization for coordinated expression of target loci. Completion of this project will offer novel insights as to how pathogenic mutations result in aberrant hub formation and affect transcriptional dynamics to drive disease. More broadly, this work will advance our understanding of hub-mediated gene regulation, revealing the potential for novel therapeutic strategies to target gene dysregulation in disease.

NIH Research Projects · FY 2025 · 2023-09

Summary Oncogenic viruses are major contributors to approximately 20% of human cancers, with about 8 well-known viruses directly shown to be causative agents. Our program will focus on examining cellular processes that are usurped by these viral agents to drive the oncogenic phenotype. The transcription and replication of oncogenic viruses in the hypoxic microenvironment has not been extensively explored and here we will explore the mechanisms controlled by Merkel Cell Polyoma Virus (MCPyV), Epstein Barr virus (EBV) and Kaposi’s Sarcoma Associated Herpesvirus (KSHV). MCPyV is the causative agent of Merkel cell carcinomas, EBV is the causative agent linked to Nasopharyngeal carcinomas, Burkitt’s lymphomas, Hodgkin’s lymphomas, non- Hodgkin’s lymphomas, post-transplant lymphoproliferative disease in HIV patients that are immunocompromised, and KSHV is the causative agent for Kaposi’s sarcoma and pleural effusion lymphomas and is also associated with Multi-centric Castleman’s disease. These viral agents have been the focus of decades of studies but their function has not been examined extensively in hypoxia. The focus of this application is to bring together four prominent groups, led by investigators within the University of Pennsylvania community to join their scientific expertise to address the mechanism of viral-mediated oncogenesis. The overall goal will investigate the mechanism of transcription and replication control by viral-encoded antigens and the metabolic changes that are required for their function in hypoxia. These fundamental cellular processes targeted in hypoxia will provide novel information for successful establishment of viral infection in hypoxia. The program consists of four scientific projects, an administrative core, a virus, vector and cell culture core and a next generation sequencing core. The scientific projects are: 1. KSHV reprograms transcription and replication in hypoxia; 2. KSHV induces tumorigenesis by harnessing differentiation in hypoxia; 3. Skin hypoxia, MCPyV infection and MCC tumorigenesis; and 4. Regulation of EBV latency and oncogenesis by oxygen metabolism. The success of these projects will establish a comprehensive mechanistic view of oncogenic viral infection and pathogenesis in hypoxia, and provide new clues for the development of strategies to prevent and treat the associated cancers in HIV patients. In addition, the accumulation of new information on the biology of these viruses will be critical for insights into their mechanism of oncogenesis and so reduce the burden of disease in HIV infected, transplants and other immunocompromised patients.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Stroke represents the primary cause of adult disability in the United States. A frequent, debilitating consequence of stroke is impairments in the ability to produce and/or comprehend language, called aphasia, which has profound impacts on quality of life due to the barriers it places on participation in professional and social daily life activities. Behavioral intervention—the current standard-of-care—provide some benefit for persons with aphasia (PWA), but their effectiveness is variable due in part to logistic and financial limitations that render interventions with the level of frequency, intensity, and duration required for lasting benefits infeasible for many PWA. Thus, there is a need for novel, time- and cost-effective interventions to expedite and improve aphasia recovery. Transcranial alternating current stimulation (tACS) has emerged as a promising noninvasive brain stimulation technique that may enhance stroke-related disability (motor impairments) and other neurological and psychiatric disorders, but the potential for improving outcomes specifically for language impairment has not yet been explored. The career development and research plans of the proposed project will allow the candidate to establish a T2 translational research program as an independent investigator to systematically explore the potential for tACS to enhance treatment outcomes in individuals with stroke-induced language impairment. The career development plan will help expand the candidate’s research program to include basic and clinical investigations of tACS-induced plasticity and its potential to facilitate language abilities in unimpaired and impaired speakers. The research plan provides an empirical foundation for this research program by investigating the neurophysiological mechanism by which tACS promotes language performance enhancement. The long-term goal of this research is to develop effective intervention approaches for individuals with acquired language impairment by combining theoretically- and neurally-guided intervention with treatment-enhancing neuromodulation techniques. The main objective of this proposal is to establish a best-practice approach for using tACS to support impairments in spoken word production. The central hypothesis of this proposal is that tACS can enhance word production abilities by modulating endogenous neuronal activation patterns associated with language. The rationale for the proposed research is that understanding neurophysiological biomarkers of language impairment and tACS-induced changes in neuronal pattens of activation may help determine the most effective approach to enhancing stroke treatment outcomes while extending our basic science knowledge of how tACS modulates neural activity. The proposed research is significant because it will enable the development of intervention procedures that maximize recovery from acquired language impairment, combining targeted therapy with tACS to unmask the residual capacity neuroplasticity in chronic stroke survivors. The proposed research is relevant to the NIH’s mission pertaining to developing fundamental knowledge that will potentially help to reduce the burdens of human disability.

NIH Research Projects · FY 2025 · 2023-09

: The intractable issue of differences in care quality across hospitals has been described for decades and most recently came to the forefront of public attention with differences in hospital COVID-19 mortality. Despite multiple studies which attribute a large share of outcome differences to quality differences in hospitals where significant proportions of high-risk patients are treated (i.e., high-risk-serving hospitals), little is known about what modifiable factors underlie poorer quality care. This proposal takes a multilevel perspective to identify the contributions of individual socio-demographic and hospital factors to identify modifiable factors that can be targeted through ‘upstream’ interventions to achieve reliably high-quality hospital care for all patients. We hypothesize that differences in hospital outcomes are due, in large part, to differences in the modifiable nurse resources of hospitals—with fewer nurse resources in some hospitals. This uneven distribution of nurse resources is a vestige of historic underinvestment that continues to pervade hospital care. We focus on nurse resources, since having enough nursing staff to deliver timely and effective care, a favorable work environment in which nurses have clinical autonomy in their practice and strong interdisciplinary teamwork, a skill mix rich in registered nurses, and high proportions of bachelors-prepared nurses and advanced practice nurses, have all been associated with better patient outcomes, particularly for high-risk patients. In this study, we evaluate the impact of hospital-level differences in nurse resources on patient outcomes, including in-hospital and 30-day mortality, readmission, and hospital length of stay. This observational study of over 900,000 older adult patients in nearly 250 hospitals investigates (1) whether differences in nurse resources between high-risk-serving and other hospitals explain outcome differences; (2) whether the outcomes-advantages of having superior hospital nurse resources are enhanced in the presence of other hospital characteristics, including for example, physician staffing, greater numbers of APRNs, or teaching hospitals; and (3) estimates the improvements in patient outcomes, such as lives saved, that could be expected if nurse resources in high-risk-serving hospitals were similar to other hospitals. Nurse resources are measured using survey data from over 16,000 nurses in nearly 250 hospitals to describe multiple aspects of the clinical nurse resources. Using a unique hospital identifier, nurse responses will be linked with Medicare patient records, and socio-demographic factors. Our analytic approach uses multi-level nested (hierarchically-related) linear and logistic regression models (with interaction terms) to accomplish our aims. If our hypotheses are confirmed, the findings will add evidence to inform high-impact actionable ‘upstream’ solutions to achieve reliably high-quality hospital care and patient outcomes, by leveraging the most abundant patient care resource already existing in every hospital—nurses.

NIH Research Projects · FY 2025 · 2023-09

Patients with hypertrophic cardiomyopathy (HCM) experience a high symptomatic burden, heart failure and lethal arrhythmias. While HCM has been recognized as a disease of the sarcomere for >30 years, the disease mechanisms for sarcomeric gene variants are not well defined, limiting the precision and efficacy of treatment options. Heterozygous variants in the gene myosin-binding protein C (MYBPC3) cause half of all cases of familial HCM. About 15% of these are missense variants that cluster in interior protein domains C3 and C6 which have uncertain binding partners or function. Computational predictions combined with our published and preliminary experimental data support the hypothesis that missense variants in C3 and C6 domains lead to perturbation of multiple protein-protein interactions that are critical for the normal function of MyBP-C (the protein encoded by MYBPC3). In Aim 1 we will apply TurboID proximity labeling to wild-type (WT) and mutant MyBP-C. In preliminary data we have identified >200 novel and unique neighboring proteins to WT MyBP-C. Comparing C3 and C6 mutants to wild-type MyBP-C, relative abundances of sarcomeric, cytoskeletal and ribonucleoprotein complexes are reduced, while abundances of ribosomal and chaperone proteins are increased. We will explore consequences of these altered interactions by assessing changes in myosin conformation, local translation, and chaperone-mediate protein turnover. We expect to find that interactions with multiple proteins of diverse function are either strengthened or weakened by the presence of missense mutations in MyBP-C. Overcoming this perturbation in protein interactions with gene replacement by wild-type MyBP-C is the focus of Aim 2 where we will test the hypothesis that the mutant protein can be stoichiometrically replaced within the sarcomere by wild-type MyBP-C. We will transduce patient-derived inducible-pluripotent cardiomyocytes expressing C3 or C6 missense variants with adeno-associated viral vectors expressing wild-type MyBP-C or a lentiviral vector expressing a “titratable” wild-type MyBP-C-FKBP12 fusion protein that enables dose-response studies. The outcome measures will be the molar ratio of mutant to wild-type protein, and contractile and relaxation velocities. In vivo studies of gene replacement in a new Arg506Trp MYBPC3 knock-in mouse model will complement the hiPSC-CM experiments. This application explores several novel aspects of MyBP-C biology and features unique reagents and advanced proteomic techniques. Successful completion of these aims will uncover new biology in MyBP-C by defining an expanded protein neighborhood, by revealing disease mechanisms for missense MYBPC3 variants, and by testing a gene displacement strategy that leverages endogenous regulation of sarcomeric stoichiometry and could be broadly applicable to missense variants in any sarcomere gene. Our investigative team, composed of a mix of senior, highly experienced investigators and talented junior investigators with unique skill sets, is well poised to achieve these goals.