University Of Pennsylvania

NIH Research Projects · FY 2025 · 2020-12

Project Summary Dr. Michael Kohanski is an assistant professor in the Department of Otorhinolaryngology – Head and Neck Surgery at the Perelman School of Medicine at The University of Pennsylvania. His clinical practice is focused on the medical and surgical management of inflammatory sinus and nasal disorders to improve the upper respiratory health of his patients. Dr. Kohanski's recent research efforts led to the finding that rare taste receptor expressing cells, solitary chemosensory cells (SCCs), are significantly enriched in inflammatory sinus polyps. This work established a connection between chronic rhinosinusitis (CRS) seen in humans to the important finding that tuft cells (analogous chemosensory cells in the intestine) are crucial for regulating Type 2 immunity. With the support of this award, Dr. Kohanski will develop expertise in mucosal immunology, epithelial physiology and bioinformatics to study solitary chemosensory cell regulation of airway inflammation and repair. Dr. Kohanski will augment his fund of knowledge through course work on immunology, epithelial physiology and bioinformatics. He will acquire new research skills with focused mentoring and training from a multidisciplinary group of experienced researchers at the University of Pennsylvania with expertise in epithelial taste receptor biology, Type 2 inflammation and epithelial cell physiology and repair as well as bioinformatics. This proposal focuses on identifying and characterizing taste-specific or inflammatory-specific inputs that stimulate SCC differentiation as well as understanding the mechanisms by which solitary chemosensory cells can amplify inflammatory and innate mucosal responses. This is a novel area of research with little work to date characterizing the role or function of chemosensory cells directly in human upper respiratory inflammatory diseases such as CRS with nasal polyps. In Aim 1a, Dr. Kohanski will characterize SCC abundance and SCC- specific gene expression directly in chronic rhinosinusitis with nasal polyps and determine if SCC abundance correlates with phenotypic markers of airway inflammation in a cohort of patients with CRS. In Aim 1b, he will determine if taste or inflammatory input stimulate differentiation of human SCCs and if there are distinct human SCC subtypes. In Aim 1c, the PI will leverage initial RNAseq results to further study the mechanisms of SCC differentiation and epithelial repair. In Aim 2, Dr. Kohanski will test the hypothesis that SCC-mediated epithelial signaling occurs through two-pore potassium channels. In Aim 2a, he will utilize Ussing chambers to determine if inflammation or SCC abundance affects K2P channel function. In Aim 2b, he will use a house dust mite model of inflammation with mouse strains deficient in two-pore potassium channels or SCC taste transduction to determine if inflammation amplifies the ability of SCCs to regulate epithelial defensin release. Progression through these experiments coupled with expert mentorship and coursework will provide Dr. Kohanski with the foundation to become an independent investigator with a focus on epithelial chemosensory cell function and the resultant impact on the mucosal immune response and inflammatory airway disease.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Diabetes affects over 30 million Americans, which represents a staggering 9.4% of the population. Diabetes causes an elevated blood glucose level. Over time, the presence of high glucose in the body results in damage to various tissues. Metformin is an FDA drug commonly used as a first line therapy for the treatment of Type 2 Diabetes. It mainly acts to make tissues more sensitive to insulin, thereby enhancing the effects of insulin produced by the pancreas to homeostatically lower blood glucose levels. Importantly, however, Metformin also prolongs lifespan and delays the onset of aging from yeast to mammals. In higher organisms, it additionally reduces the risk of cardiovascular disease and inhibits tumor growth. Astoundingly, given its clinical use in humans since 1958, the exact molecular mechanisms underlying its wide-ranging health benefits are unknown. This Catalyst project is directed towards elucidating the direct cellular protein targets of Metformin for the first time. Our encouraging preliminary data shows that we can apply cutting-edge proteomics approaches to such binding events in an unbiased way. In this project, we wish to extend this extremely promising approach to a diverse range of organisms. By identifying molecular targets of Metformin in a variety of phylogenetically different model organisms (yeast, worms, flies, mouse, and humans), we will be able to home in on proteins of crucial importance, while simultaneously screening out non-specific binders. We will mechanistically test discovered targets by loss- and gain-of-function experiments using various assays, which will be adapted as the project advances. After validation of a small number of strong candidate Metformin binding proteins, we will test these promising candidates in mammals by making transgenic mice harboring deletions in the relevant genes coding for these proteins. We anticipate that the identification of specific mechanistic Metformin targets will facilitate the development of novel therapeutics to treat diabetes and promote healthy aging. Both of these goals are closely aligned with the core missions of NIDDK, and the wider NIH.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY - This K08 career development award will facilitate the advancement of Dr. Eric Kaiser as an independent physician-scientist focused on the neural mechanisms of pain from light in migraine and related disorders. He is currently a clinical fellow in headache medicine but will return to the University of Pennsylvania as an Instructor in the Department of Neurology. The career development plan builds upon his prior expertise in rodent behavior while refining essential scientific skills and teaching new technical skills to prepare him for an independent, cutting-edge career in neuroscience. This will be accomplished by gaining hands-on technical experience using tailored light stimuli in rodents, applying mouse genetics to study neurologic disorders, and performing immunohistochemistry to dissect neural circuits. This will be complimented by formal didactics in mouse genetics, ethics, biostatistics, and grant writing. These efforts will be supported by three mentors including Dr. Frances Jensen, a renowned physician-scientist in neural plasticity and network hyperexcitability, Dr. Geoffrey Aguirre, a physician scientist with expertise in visual perception, and Dr. Wenqin Luo, a neuroscientist with expertise in somatosensation. Dr. Kaiser will also work with Dr. Maria Geffen, a neuroscientist at the University of Pennsylvania that will act a consultant on optogenetic techniques. The PI will also greatly benefit from the unparalleled resources and faculty at the University of Pennsylvania and headache experts at the affiliated Children's Hospital of Philadelphia. The objective of this project is to examine the pathologic interaction of the visual and trigeminal sensory systems. Photophobia is a canonical and debilitating feature of migraine, which is a disabling neurologic disorder. Bright light perception involves the cones, which project to the classical retinal ganglion cells (RGCs), and the melanopsin-containing, intrinsically photosensitive RGCs (ipRGCs). Recent human studies by Drs. Kaiser and Aguirre demonstrate that both melanopsin and cone stimulation in isolation and in combination can trigger visual discomfort in individuals with migraine. Dr. Kaiser's central hypothesis is that ipRCG signals potentiate trigeminal activation leading to the aversive perception of light in migraine. To investigate these interactions, the PI proposes to use a mouse model of migraine in which a neuropeptide, calcitonin gene-related peptide (CGRP), induces light aversion. Using tailored light stimuli to isolate cone and melanopsin activity, the PI will test their relative contribution to CGRP-induced light aversion. To establish how trigeminal and retinal signals interact in a migraine-like state, Dr. Kaiser will examine c-fos activation as a marker of neuronal activity in the trigeminal and retinal pathways following CGRP and light stimulation as well as determine if primary trigeminal afferents are required for CGRP-induced light aversion.

NIH Research Projects · FY 2024 · 2020-09

Short tandem repeat regions (STR) are distributed evenly across the human genome, and recent genome-wide studies have demonstrated that STRs are polymorphic across individuals and linked to gene expression levels. STR instability at key genomic loci has been causally linked to disease pathophysiology in a range of expansion disorders. We recently demonstrated that nearly all disease-associated STRs co-localize with boundaries demarcating topologically associated domains (TADs). Moreover, we have observed that pathologic STR instability and transcriptional silencing can destroy the associated boundary and shift genomic loci to the nuclear periphery. These results now open critical unanswered questions regarding whether and how STR expansion and pathologic alterations in gene expression are functionally linked to boundary integrity and radial positioning. Here, we focus on the prototypic repeat expansion disorder Friedreich’s ataxia (FRDA) in which expansion of a GAA STR in the first intron of the FRATAXIN (FXN) gene results in cardiac and neuronal pathology. The cardiac pathology, specifically hypertrophy, fibrosis, and occasional dilation of the ventricle, is the etiology of significant FRDA mortality. GAA expansion is associated with the silencing of FXN transcription and a repositioning of the locus to the nuclear periphery. However, it remains unclear if the change in genome folding, radial positioning, or reduced expression drives STR expansion or vice versa. A major technical barrier contributing to this knowledge gap is that STR instability and genome folding are classically evaluated in bulk populations, however they exhibit tremendous variation across individual somatic cells of the same subtype and among cell types within a pathologically affected tissue. Here, we seek to decipher the causal link among STR instability, transcription, radial positioning, and genome folding. Our central hypothesis is that disruption of long-range loops is the initial event triggered by STR expansion leading to a cascade of heterochromatin spreading, silencing, and loss of radial positioning. We will test our hypothesis by generating genome-wide, single-cell maps of chromatin accessibility, expression, and the repressive H3K9me3 heterochromatin mark in GAA-expanded and control iPS cells and iPS-derived cardiomyocytes. We will integrate genomics data with single-cell sequential Oligopaints/OligoSTORM imaging of TADs and local chromatin structure, as well as single molecule RNA FISH for FXN expression. We will implement multiple genome engineering strategies, including dCas9-VP64 FXN activation and dCas9-CTCF loop re-engineering in FRDA GAA-iPS cells, and dCas9-Krab-Dnmt3a FXN silencing and dCas9-Krab CTCF-mediated loop disruption in healthy iPS cells. We will assay the effect of genome engineering approaches on TADs, radial positioning, STR length, and FXN expression in single cells. Successful completion of the proposed work will shed light on the pathophysiological mechanisms underlying repeat expansion disorders by deciphering the cause-and-effect relationships among genome folding, radial positioning, transcription, and STR expansion.

NIH Research Projects · FY 2024 · 2020-09

Implantable devices are playing a greater role in neurologic care, but their effectiveness is limited, because they are blind to human thoughts, feelings, and behavior – factors that most dramatically affect our health. Coupling peripheral sensors to implants might help, but wouldn’t it be easier if the devices just asked us? Armed with this knowledge, next generation machines will more effectively drive neural activity in the brain to healthy states. They will also quickly learn behaviors that worsen health and guide us to better choices. Though DARPA, the NIH, and Neuralink are spending millions of dollars on new hardware for brain-computer interfaces, none focus on reciprocal, natural communication between host and machine. There is a desperate need for novel, practical methods that enable devices to learn from and guide human behavior. In this application I propose to develop a new generation of autonomous brain-machine interfaces – devices that can question, record, act - and combine learning algorithms applied to neurosignals with teaching by their human hosts. Life with these implants will entail a subtle human- machine dialogue in which devices and humans teach and learn from each other. Humans will inform intelligent algorithms about what we are doing and feeling, while machines will incorporate this information into therapy and guide us to optimize quality of life in personalized ways. This is a paradigm shift from today’s simple devices, which are programmed by physicians during occasional office visits. I propose to demonstrate this paradigm in a practical, scalable way using current epilepsy implants that is rapidly translatable to many neurological disorders. To achieve this goal, I will meld several cutting-edge technologies in novel ways, including: (1) State-of-the-art, high bandwidth implantables that sample neural activity, link to vast cloud- based computational power to process it, and intervene to modulate brain, spinal cord or peripheral neural activity. This work utilizes my experience from the past 20 years; (2) I will deploy powerful new computer science tools in novel ways. I will use convolutional neural nets (a.k.a. Deep Learning) to learn patterns from vast streams of continuous high-bandwidth neural data, build a two way human-machine interface using Natural Language Processing (NLP)., and probe networks with changes in human behavior and electrical stimulation and guide interventions toward therapeutic goals using Reinforcement Learning. Combining these computer science, machine learning techniques and measurements of human behavior is a new area of investigation for me that will leverage my unique background in clinical neurology and engineering to build a new class of interactive, human therapeutic devices.

NIH Research Projects · FY 2024 · 2020-09

Project Summary Osteoarthritis (OA) is a classic age-related disorder and the most common cause ofpain and disability in the elderly. It is primarily characterized by the progressive destruction of articular cartilage. This past decade has witnessed significant advances in deciphering the basic mechanisms by which OA develops. However, to date, no disease modifying drug therapy is available for preventing OA development and repairing the degenerative cartilage. The uppermost superficial zone of articular cartilage is the first line of defense against OA initiation. We recently found that epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, is expressed abundantly throughout the articular cartilage with its active form (p-EGFR) predominantly located in the superficial zone. Interestingly, at the onset of OA, p-EGFR amount, along with two major EGFR ligands (TGFα and HBEGF), were markedly attenuated while the amount of Mig6, a negative inhibitor of EGFR, was enhanced, suggesting a potential role for EGFR signaling pathway in cartilage homeostasis and diseases. Using a series of mouse models with deficient or overactivated EGFR activity by genetic manipulation of Egfr and Mig6 genes, we and others have demonstrated that EGFR signaling is critical for maintaining the number and mechanical properties of superficial chondrocytes, suppressing their hypertrophy, promoting proteoglycan 4 (Prg4) expression, and stimulating surface lubrication function. Most strikingly, in aging- and surgery-induced OA models, mice with chondrocyte-specific (Col2-Cre) EGFR deficiency developed the most severe OA phenotypes, including a complete loss of articular cartilage, subchondral bone sclerosis, and escalated joint pain. Hence, we hypothesize that EGFR signaling is essential for maintaining the structure and function of the superficial layer in the articular cartilage and thus, can be targeted for OA treatment. Our objectives are to understand the role of this novel signaling pathway in articular cartilage homeostasis and diseases, and to seek approaches targeting this pathway for OA treatment. To achieve these, we will perform the following aims: 1) determine the temporal role of EGFR signaling in OA pathogenesis; 2) elucidate the mechanisms of the protective action of EGFR on articular cartilage; 3) investigate whether EGFR signaling is a promising target for OA treatment. Complementary genetic approaches, such as EGFR vs Mig6, loss of function vs gain of function, and aggrecan-CreER vs Prg4-CreER, will be used throughout the proposal. Moreover, we have designed and synthesized TGFα-conjugated nanoparticles with prolonged retention and penetration abilities in knee cartilage. A proof-of-principal experiment will be performed to examine its therapeutic effects on cartilage degeneration at different OA stages. This proposal will uncover critical EGFR actions in knee articular cartilage and provide crucial evidence for targeting this novel pathway in OA therapies. Once successfully accomplished, this project could be quickly translated into large animal OA models followed by clinically relevant applications that would eventually improve the health and well-being of the general public.

NIH Research Projects · FY 2024 · 2020-09

Project Summary/Abstract To achieve aspirational goals to end the HIV epidemic (EHE), evidence-based practices (EBPs) to increase viral suppression must be implemented effectively nationally. The Managed Problem Solving (MAPS) behavioral intervention is an EBP for behavior change in people living with HIV (PLWH). To accomplish the goals of this application, we leverage a data-to-care partnership between the Philadelphia Department of Public Health (PDPH) and participating clinics (n = 12), which enhances the sustainability of our approach. We propose that MAPS can be delivered by trained Community Health Workers (CHWs). The use of CHWs to deliver MAPS is justified by their ability to develop trusting relationships with their clients and the need for task shifting in busy clinics. In order to also address retention in care, we will adapt MAPS to also focus on problem solving activities tailored toward retention in care (now termed MAPS+). CHWs will be located in clinics to implement MAPS+ to improve viral suppression and care retention in PLWH. Data-to-care allows for identification of people who are lost to care and link these patients back to care. Currently, medication adherence and retention in HIV care are not targeted in data-to-care so we will build on this approach to facilitate the identification of PLWH who are out of care and not virally suppressed to offer them MAPS+. Our set of implementation strategies include task- shifting the delivery of MAPS+ to CHWs, providing the CHWs training and ongoing support, and increasing communication between the CHWs and medical care team via standardized protocols. We will conduct a hybrid type II effectiveness-implementation trial with a stepped-wedge cluster randomized design in 12 clinics to test MAPS+ compared to usual care using a set of implementation strategies that we believe will best support implementation. Each clinic will be randomized to one of three implementation start times. We will collect baseline (usual care) data from each clinic for 6 months, followed by MAPS+ and our package of implementation strategies for 12 months, in three cohorts of 4 clinics each. Aim 1 will test the effectiveness of MAPS+ on clinical effectiveness outcomes, including viral suppression (primary) and retention (secondary). Aim 2 will examine the effect of the package of implementation strategies on reach. We will also measure implementation cost. Aim 3 will apply a qualitative approach to understand processes, mechanisms, and sustainment of our implementation approach. Our results will guide future efforts to implement behavioral EBPs across the HIV care continuum, consistent with the “treat” pillar of EHE, and move the science of implementation services, consistent with NIH strategic priorities.

NIH Research Projects · FY 2024 · 2020-09

Project Summary Neurons are poorly recognized by the immune system which contributes to the ability of neurotropic pathogens to persist in the CNS but there is evidence that T cells can promote clearance of these organisms. For the parasite Toxoplasma gondii, T cell production of the cytokine IFN- is important for resistance in the CNS because it activates hematopoietic and non-hematopoietic cells to control the tachyzoite (lytic) stage of the infection. Conversely, in response to cellular stress T. gondii transforms to the latent bradyzoite stage and forms long lived cysts in neurons. The lack of therapies that target the latent stage of T. gondii is a significant impediment to the management of this infection. Current dogma holds that because this stage is in neurons it evades immune surveillance and ensures chronicity. However, there is accumulating evidence of a more active battle between the host and parasite in the CNS. These observations indicate that T cell production of IFN- activates neurons to control T. gondii but the ability of this parasite to persist may be because bradyzoites evade recognition and/or modulate cyst specific responses. In support of this idea, comparisons between tachyzoite and bradyzoite specific responses suggest that cyst-specific CD8+ T cells have reduced effector functions. To understand how T. gondii is recognized in neurons and how the parasite can evade surveillance, novel transgenic reporter systems for parasites will be combined with host reporters to track the fate of infected neurons in vivo. Additional studies will determine the impact of IFN- on neurons and live imaging studies will visualize interactions between T cells and infected neurons and if this results in parasite clearance or evasion of T cell activities. The findings that emerge from these studies will have a significant impact on understanding how CD8+ T cell-neuron interactions lead to pathogen control and will be relevant to other neurotropic infections and neuroinflammatory conditions.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY The human pathogen Vibrio cholerae is the etiologic agent of the severe diarrheal disease known as cholera, which affects millions of people annually, worldwide. In order for V. cholerae to successfully colonize in the small intestines of the host, it must express a series of virulence factors, which have been the main focus of the cholera research. However, bacterial pathogenicity is a multifactorial process in vivo that depends not only on virulence factor expression, but host responses to infection and interactions with the commensal microbes of the gut, the gut microbiome. One major set of host-produced factors that must be overcome by V. cholerae comprises nitric oxide (NO) and NO-derived nitrogen oxides and dinitrosyl-iron complexes, collectively known as nitrosative stress (reactive nitrogen species, RNS). Previous studies show that inducible nitric oxide synthase (iNOS or NOS2), the enzyme that synthesizes NO, is among the most upregulated proteins in duodenal tissue during cholera, and our results show both that iNOS is highly induced upon V. cholerae infection of an adult mouse model, and that V. cholerae colonization is reduced in iNOS-/- mice or mice treated with the iNOS inhibitor aminoguanidine (AG). However, little is known about how increased RNS in vivo impacts V. cholerae, the gut microbiome, and the inter-microbial interactions that drive the ultimate outcome of infection. We hypothesize that RNS production induced during infection modulates the structure, function, and pathogen interactions of the gut microbiome, granting V. cholerae a competitive advantage over commensals due to several RNS-resistance mechanisms that are tightly regulated alongside virulence factor expression. We will examine this hypothesis in two aims. In Aim 1, we will elucidate how V. cholerae responds to RNS during infection, and how these responses are regulated alongside virulence. In Aim 2, we will examine the role of RNS in modulating the gut microbiome, how RNS-dependent changes influences V. cholerae susceptibility, and how RNS affects specific microbial interactions between this pathogen and commensal gut microbes.

NIH Research Projects · FY 2024 · 2020-09

SUMMARY T cell exhaustion is common during chronic infections and cancer and limits control of disease. Targeting TEX by blocking pathways such as PD-1:PD-L can reinvigorate these cells leading to dramatic clinical effects in cancer. However, most patients do not receive durable clinical benefit. Although PD-1 pathway blockade re-invigorates TEX function, and results in transcriptional changes, there is little change in the chromatin landscape and functional changes are not sustained. Thus, our ability to target TEX for therapeutic benefit in cancer and chronic infections is limited by the epigenetic inflexibility of these cells. A better understanding of the initiation, stability and reversibility of TEX epigenetic identity should reveal new therapeutic possibilities. We and others have recently identified Tox as the epigenetic lineage programmer of TEX. Without Tox, TEX cannot form. Tox is required to initiate chromatin remodeling for TEX but represses terminal TEFF differentiation. However, the mechanisms of how Tox programs epigenetics are unclear. A major question is what happens to chromatin landscape and TEX differentiation if Tox is removed in established TEX. Addressing this question is a major goal. TEX heterogeneity is also now pointing to a developmental biology hierarchy with discreet, functionally relevant stages of differentiation – or TEX subsets - controlled by transcription factor circuits. These subsets also differ epigenetically suggesting key roles for Tox that are as yet untested as well as opportunities. These observations suggest a key role for Tox in the epigenetic identity of TEX but raise key questions about the ongoing role of Tox once TEX are established. Our overall hypothesis is that inducible deletion of Tox in established TEX will reveal mechanisms of epigenetic stability of TEX and opportunities for therapeutic improvement during chronic infections and cancer. We will test this hypothesis in the following Aims: AIM 1: TEST WHETHER DELETION OF TOX IN ESTABLISHED TEX ALTERS TEX DIFFERENTIATION, TRANSCRIPTIONAL PROGRAM, OPEN CHROMATIN LANDSCAPE AND/OR DYNAMICS OF TEX SUBSETS. We hypothesize that removal of Tox in established TEX will revert the TEX epigenetic program and will be associated with functional, differentiation and transcriptional changes that will be augmented by PD-1 blockade and/or removal of antigen. To test this idea we will use new inducible Tox deletion strategies combined with deep mechanistic interrogation of the cellular developmental biology, transcriptional and epigenetic program and response to PD-1 pathway blockade. AIM 2: TEST HOW COMPLEMENTARY OR DOWNSTREAM EPIGENETIC OR TRANSCRIPTIONAL CIRCUITS COOPERATE WITH TOX IN TEX. We hypothesize that a combination of in vivo CRISPR screening and candidate testing will reveal epigenetic and transcriptional mechanisms of Tox in TEX. We will use this CRISPR approach together with enforced expression strategies and a novel Tox-driven inducible Cre reporter to define the molecular and genomic mechanisms of Tox-mediated initiation and maintenance of the TEX lineage.

NIH Research Projects · FY 2025 · 2020-09

Notable gaps in maternal mortality persist in the United States (U.S). Low-income patients are more likely to have a pregnancy-related death due to limited access to high quality care and high rates of underlying health conditions. These populations are more likely to begin pregnancy with chronic health conditions such as hypertension or diabetes, experience a complication while pregnant, and to experience life-threatening morbidity during delivery. Less recognized is that heightened maternal health risks extend into the postpartum period, where nearly 65% of deaths occur up to a year postpartum. These issues are particularly acute in Philadelphia where the maternal death rate is higher than the national average, the several maternal morbidity (SMM) rate is rising, and the majority of maternal deaths occur postpartum. Further, Philadelphia is the poorest among the nation’s ten largest cities, and its high-need zip codes are more prone to experience a postpartum complication, seek care in the emergency department (ED) after delivery, and are at increased risk for postpartum readmission. We propose a mixed method study to better pinpoint patients most at risk for poor outcomes following delivery, the problems they experience, and adapt an evidence-based intervention that aims to ensure all communities – including high SMM/mortality risk – have access to high quality care. This goal aligns with federal priorities to reduce excess health care utilization and improve health outcomes. The aims of our research study, “Improving Health Outcomes by Targeting Postpartum Patients with High Need” are to: 1) Develop a risk a prediction model using clinical, sociodemographic, behavioral, and neighborhood factors to pinpoint high-need patients using ED visits and postpartum readmissions as a marker of SMM. 2) Use qualitative methods to adapt and intensify an evidence-based behavioral educational intervention aimed at improving quality of care to reduce postpartum SMM as measured by postpartum ED use and hospital readmission, 3) Conduct a pilot RCT utilizing the cohort identified by the risk prediction model in Aim 1 to assess feasibility, acceptability, and target effect size and potential efficacy of the refined intervention to reduce ED visits and postpartum readmissions, and 4) Evaluate the pilot study results and procedures to inform the refinement of the intervention and to prepare for a larger implementation trial of this intervention to ensure universal availability for all.

NIH Research Projects · FY 2024 · 2020-09

Germline cancer genetic testing has become a standard evidence-based practice, with established risk reduction and cancer screening guidelines for genetic carriers. Yet, access to genetic specialists is limited in many areas in the US, and <20% of eligible patients with a personal or family history of breast or ovarian cancer complete genetic testing. Thus, there is an urgent need to consider alternative delivery models to increase access and uptake of genetic testing, while maintaining adequate patient cognitive, affective and behavioral outcomes. Our research has shown that providing remote services increases uptake of genetic testing in community practices increases uptake of genetic testing. Preliminary data from our ongoing NIH-funded RESPECT study has revealed high interest in a web-based eHealth alternative to traditional pre-test counseling and no significant no differences in pre- and post-disclosure outcomes when the web-based eHealth intervention is utilized as compared to participants who received traditional pre-test genetic counselor. To address the clinically significant need for alternative delivery models to increase access and uptake of cancer genetic testing, while maintaining adequate patient cognitive, affective and behavioral outcomes, we propose to recruit a nationally diverse “real- world” sample of 1000 patients who have access barriers to genetic testing and to conduct a Hybrid Type 1 effectiveness-implementation study to evaluate web-based eHealth delivery alternatives for genetic education and testing. We hypothesize that our theoretically and stakeholder informed eHealth delivery alternatives can provide equal or better uptake of testing and outcomes of genetic testing as compared to the traditional model of pre- and post-test counseling with a genetic counselor. We will partner with several cancer advocacy groups (ASCO, breastcancer.org, Cancer Support Community, Pennsylvania Prostate Cancer Coalition) to recruit patients to this randomized non-inferiority study using a modified 2x2 design (Aims 1-2). In Arm 1, traditional pre-test (visit 1) and post-test (visit 2: disclosure) counseling will be provided remotely through the national Penn Telegenetics Program and compared to delivery arms where patients can complete pre-test and/or disclosure of results through a self-directed web-based eHealth intervention, either in place of, or as an adjunct to traditional genetic counseling. Concurrently, we will conduct a CFIR (Consolidated Framework for Implementation Research)-informed process evaluation to understand moderators of intervention usage and patient outcomes and facilitators and barriers to future implementation and sustainability of this novel eHealth alternative delivery model for genetic services both within and beyond cancer care (Aim 3). We hypothesize that a rigorously developed theoretically and stake-holder informed eHealth delivery alternative provided through a centralized Telegenetics Program has the potential to provide equal or improved patient outcomes, while reducing genetic provider time and providing access to services in community practices where access to genetic services has been limited, providing opportunities to realize the promise of precision medicine in oncology.

NIH Research Projects · FY 2024 · 2020-09

The mammalian genome folds into tens of thousands of long-range looping interactions. A critical unknown is whether and how chromatin loops control gene expression, and a major unresolved question is how the temporal progression of loops relates to transcription dynamics. One major barrier to answering this question is that loops change on a range of timescales, necessitating the use of tools and model systems amenable to tracking and engineering loops longitudinally and in real time on both short and long timing. Here, we propose to develop and apply new engineering and imaging tools to measure, induce, and perturb loops with precise temporal control in three different biological systems spanning minutes, hours, and weeks. At the shortest timescale (minutes, Aim 1), we will examine loop dynamics in human induced pluripotent stem cell-derived neurons in response to electrical stimulation, revealing how interaction frequency is functionally connected to transcriptional bursting of immediate early and secondary response genes. On the timescale of hours (Aim 3), we will elucidate how the architectural protein YY1 connects enhancer-promoter loops that re-assemble upon the exit from mitosis by erythroid cells. On the timescale of weeks (Aim 2), we will use a cellular “Time Machine” to longitudinally track the rare cells that undergo cellular reprogramming, allowing us to dissect the functionality of loop formation and dissolution with single-cell and subcellular resolution during the reprogramming of somatic cells to pluripotency and transition of melanoma cancer cells to a resistant phenotype. Our team consists of a highly productive and collaborative set of junior and senior investigators with complementary expertise and overlapping interests, including Dr. Gerd Blobel (epigenetics, mitosis, loop engineering), Dr. Eric Joyce (Oligopaints imaging), Dr. Bomyi Lim (nascent transcript live cell imaging), Dr. Jennifer Phillips-Cremins (chromatin architecture, loop engineering, neurobiology), Dr. Stanley Qi (CRISPR genome engineering, live cell imaging), and Dr. Arjun Raj (single cell genomics, RNA imaging, reprogramming). We will develop and apply live and fixed cell imaging techniques for chromatin contacts, and in the same cells image nascent transcription. We will build a cadre of synthetic architectural proteins to engineer loops in a time-dependent inducible manner. Successful application of our engineering and imaging tools across biological systems will yield a comprehensive and rigorous assessment of the cause-and-effect relationship between loops and distinct biological phenotypes across timescales.

NIH Research Projects · FY 2024 · 2020-09

The pathologic mechanisms of cognitive decline in Parkinson's disease (PD) are poorly understood, although Alzheimer's disease (AD) co-pathology plays an important role. Over 80% of people with PD will develop dementia, causing lower quality of life, increased caregiver burden, and worse health outcomes. Symptomatic therapies are only minimally effective, and no disease-modifying therapies exist, which represent major unmet needs. Improving our understanding of the neurobiology of PD dementia (PDD) can elucidate pathways for novel treatment development. Identifying the role of AD genetic risk factors in PDD will broaden our understanding of this disease. We hypothesize that AD genetic risk factors will predict faster cognitive decline, greater tau and amyloid-β42 (Aβ) deposition as reflected in molecular biomarkers, and more AD co- pathology in PD. Dr. Tropea will leverage multiple existing research cohorts at the University of Pennsylvania, the Pacific Northwest Udall Center (PANUC), who are longstanding UPenn collaborators, and the international Parkinson's Progression Markers marker Initiative (PPMI). The aims of this proposal are to test whether genetic variants in genome-wide association with risk of AD are associated with 1) longitudinal cognitive decline, 2) a greater degree of neurodegeneration, tau and Aβ deposition reflected in molecular biomarkers, 3) AD neuropathology in PD. The K23 candidate is an Assistant Professor of Neurology at The University of Pennsylvania. He previously completed a movement disorders fellowship and NINDS T32-supported Masters of Translational Research. He has a history of productivity, having conducted basic and clinical research in neuroscience, recently focusing on PDD. The candidate is committed to a career in translational research and proposes a comprehensive five- year plan of mentorship, formal training, self-directed learning, and research. This K23 award will establish Dr. Tropea as a clinician-scientist with expertise in 1) developing and executing genetic association studies; and 2) understanding common genetic risk between AD and PD. This career development award will support Dr. Tropea's short-term goals, including 1) developing a detailed understanding of genetic association studies and polygenic risk scores in predicting clinical, biomarker, and neuropathological outcomes, 2) acquisition of skills necessary to analyze and interpret complex clinical and genetic data; and 3) developing skills for analyzing biomarker and neuropathology data. Dr. Tropea will meet these objectives under the guidance of a Mentorship Team, including Dr. Alice Chen-Plotkin (primary mentor), a federally-funded clinician-scientist and established mentor, Dr. John Q Trojanowski (co-mentor), a world-renowned expert in the molecular pathology of ageing and neurodegeneration with a distinguished record of faculty mentorship, and Dr Sharon X. Xie, an expert in biostatistics in neurodegeneration. This Award will support Dr. Tropea in his pursuit to develop a career as an independent clinician-scientist, focused on translating biological insights into clinical studies in PD.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by death of motor neurons. A key pathologic feature is the cytoplasmic mislocalization of a nuclear transcription and splice regulator, Tar-DNA binding protein of 43kDa (TDP-43). TDP-43 is aggregated in cytoplasmic stress granules (SGs) along with nuclear import/export factors, and its toxicity is thought to be due to both cytoplasmic gain- and nuclear loss-of-function mechanisms. Relocating it to the nucleus has the potential to address both forms of toxicity. Inhibiting formation of SGs is one promising strategy, and downregulating the SG-associated protein Ataxin-2 (Atxn2) using antisense oligonucleotides (ASOs) prolongs strength and survival in a mouse model of ALS. However, ASOs require frequent CNS readministration, and a preferable approach would be to achieve knockdown after one treatment. A second approach is enhancing nuclear import, a strategy with success in dipeptide repeat (DPR) toxicity models of ALS in vitro. Extending this strategy to non-DPR forms of ALS has the potential to make a broad impact on the disease. In addition, potential synergy between the two approaches has great therapeutic potential. If successful, these strategies could be used to treat the vast majority of ALS. In preliminary work, RNAi delivered using a novel viral vector achieves robust knockdown of Atxn2 in the key areas of the nervous system affected in ALS. Aim 1 of this proposal is to determine if sustained Atxn2 knockdown in these regions reverses TDP-43 mislocalization and improves neuron survival in two distinct mouse models of ALS. In other preliminary work, cell lines overexpressing a nuclear import factor show reductions in TDP-43. Aim 2 is to test if augmenting nuclear import corrects TDP-43 localization and improves cell survival under conditions of stress. My central hypothesis is that targeting both cytoplasmic aggregation and nuclear loss of TDP-43 using viral-mediated approaches will result in sustained neuroprotection. This work fits squarely in NINDS’ mission to further our knowledge about the brain and nervous system and to use this knowledge to reduce the burden of disease, specifically targeting one of neurology’s most devastating afflictions. Dr. Amado is a passionate, highly-trained clinician-scientist uniquely poised to make a fundamental impact on ALS. Her mentor Dr. Beverly Davidson, a renowned neurodegenerative disease expert continually pushing the boundaries of vector-based therapeutics, and her advisory committee of deeply experienced and dedicated neurologists and neuroscientists, will provide the guidance and mentorship to ensure her success, backed by enthusiastic institutional support. The University of Pennsylvania, with its innumerable resources and facilities, is an outstanding place to launch a neuroscience career. Dr. Amado will use this 5-year mentored opportunity to build on her gene therapy training and merge it with her clinical expertise to become an independent, R01-funded physician-scientist developing novel therapies for patients with ALS.

NIH Research Projects · FY 2024 · 2020-09

SUMMARY Alzheimer’s disease (AD) and other dementias are a substantial and rapidly growing societal burden due to the aging of our population. This aging has occurred in part because modern medicine has identified risk factors and treatments for many other diseases, but not for AD. Thus there is a great and unmet need to do so. A hallmark symptom of AD and aging without dementia is impaired memory, including retrieval. Further, some of the earliest neuropathology found in AD is within the systems that provide the neuromodulators dopamine (DA) and norepinephrine (NE) to the brain (including the hippocampus, a center for declarative/episodic memory). The largest brainstem adrenergic nucleus is the locus coeruleus (LC), which supplies all of the NE and a substantial portion of the DA found in the dorsal hippocampus. The LC can also be affected in normal aging. As such, the goal of this proposal is to better understand the roles and mechanisms by which LC-derived DA and NE modulate the encoding and retrieval of declarative/episodic memory. Toward this goal, pharmacologic and genetic manipulations will be performed in rodents, for which there are excellent behavioral paradigms that rely on hippocampus-dependent memory. Our preliminary studies indicate that all three -adrenergic receptors that are activated by NE play a critical role in the hippocampus to promote memory retrieval. They also suggest that adrenergic DA is required for hippocampus-dependent memory. Thus the first aim will examine the molecular and cellular mechanisms by which -adrenergic receptors promote hippocampus-dependent memory using combined pharmacologic and genetic approaches. Subcellular localization of  receptors in the hippocampus will also be determined. The second aim will examine the modulation of hippocampus-dependent memory by “adrenergic” DA using combined pharmacologic and genetic approaches. The third aim will examine the ability of drugs that enhance DA and NE signaling to promote memory in models of AD and aging. The completion of these aims will provide a better understanding of how memory encoding and retrieval is facilitated within the hippocampus, and ultimately may suggest potential targets for enhancing memory in AD and aging.

NIH Research Projects · FY 2026 · 2020-09

Frontotemporal degeneration (FTD) is a complex neurodegenerative disorder characterized by variable neuropathological, genetic, and clinical features. Clinically, FTD can present as a behavioral/executive disorder (bvFTD) or language disorder (primary progressive aphasia; PPA) and both syndromes often include motor and/or movement dysfunction. These unique clinical aspects for FTD pose enormous personal and societal costs, that often exceed those of other forms of late-onset dementias. Detailed human tissues studies identified that the majority of FTD have transactive DNA/RNA binding protein of ~43 kD (TDP-43) pathology (FTLD-TDP) as the primary neuropathological etiology, while most remaining FTD is caused by neuropathological tau inclusions (FTLD-Tau). TDP-43 additionally is the primary proteinopathy contributing to amyotrophic lateral sclerosis (ALS) and limbic predominant age-related TDP-43 proteinopathy (LATE). While ~20% of individuals affected by FTD have a known autosomal dominant mutation, most patients have sporadic disease in which urgent biomarkers are needed to differentiate FTLD-TDP from FTLD-Tau during life. Collectively, this neuropathological, genetic, and clinical heterogeneity has presented a major barrier to developing effective therapeutic strategies targeting TDP-43 specific mechanisms. The overall hypothesis of this program project grant (PPG) application is that FTLD-TDP is a complex disorder that requires a wholistic nanoscale-to-exposome approach to unravel novel biomarkers and mechanisms at the cellular, mesoscopic, macroscopic, and systems network levels to breakdown barriers to therapeutic success. In the current grant period, we uniquely focused on a cellular-to-network approach, where we uncovered several new insights into FTD heterogeneity reported in >165 publications ranging from novel gene discoveries to mesoscale and macroscale biological differences between FTLD-TDP and FTLD-Tau. In this renewal, we extend our comparative studies of FTLD-TDP relative to FTLD-tau, where we hypothesize distinct molecular, spatial transcriptomic, histopathological mesoscale, and imaging network features of FTD. In two new themes we expand our resolution from nanoscale-to-exposome disease features at the extremes of the molecular-to-network spectrum to include nanometer resolution using in situ cryo-EM studies of TDP-43 protein structure to exposome studies of physical environmental modifiers of clinical disease course. We also leverage multimodal data integration approaches to pool multiple independent and anatomically precise data (e.g., histology, spatial transcriptomics, 3T-7T MRI) to build cumulative gains in knowledge. We propose 4 independent yet highly synergistic Projects and 5 Cores that provide critical infrastructure through 2 overall aims to build on our track record of success: (1) Grow an integrated, multiple resolution program of scientific projects investigating FTLD-TDP; and (2) Implement scientific core resources that provide essential tools and materials needed to conduct proposed scientific experiments.

NIH Research Projects · FY 2024 · 2020-09

ABSTRACT The gut microbiome has a tremendous impact on health and disease, actively contributing to obesity, diabetes, inflammatory bowel disease, cardiovascular diseases, and several poorly understood neurological disorders. We do not yet have the necessary tools to precisely probe these microbial communities, though such tools could unlock extensive benefits to human health. Elucidating the contributions of individual species or consortia of bacteria would provide a rational basis for understanding microbiota-controlled disease and lead to novel therapies. To carry out the fundamental research planned in this proposal, we will tackle three major problems: First, we will build the first set of molecular tools that effectively and precisely modulate the microbiome bacteria; second, we will analyze the multiscale dynamics of microbial communities; and third, we will construct an ingestible biosensor for real-time monitoring of microbiome populations. Although antibiotics and fecal transplants can reconfigure microbial consortia, they do not precisely target individual bacteria. Conversely, antimicrobial peptides (AMPs) have evolved to selectively attack pathogenic bacteria but do not target microbiome bacteria, constituting desirable scaffolds for molecular engineering and potential sources of microbiome-targeting agents. We will develop a new computational peptide design methodology, based on classical and hybrid-quantum mechanical molecular dynamics (MD) simulations, to create a groundbreaking assessment of the dynamical and emergent properties of AMPs. Chemical synthesis and large-scale screening will confirm predicted selectivity against microbiome species, and a machine learning workflow will connect sequences of individual peptides to their dynamics and activity. We will then apply the synthetic AMPs to interrogate the human microbiome by selectively removing species during bacterial consortia experiments, to be carried out in bioreactors, under regular or anaerobic conditions. We will pair our experiments with whole-cell metabolic network models, providing a systems biology perspective to the analysis of inter-species interactions. An integrated ingestible biosensing device will be developed to monitor the microbiome by electrochemically sensing unique biomarkers from gut microbes. This will provide the first real-time measurements of microbiome composition and will be integrated to our bioreactors for testing, to ultimately be used for in vivo tests. This work will build the first set of molecular and computational tools for microbiome engineering and will lay the foundation to address critical gaps in our understanding of the gut micro-environment, and of the contributions of gut bacteria to the etiology of disease. Grounded in our demonstrated expertise in synthetic biology, computer science, microbiology, and electrical engineering, this project will provide a computational- experimental framework for developing a peptide encyclopedia for the gut microbiome, in line with NIH's public health mission and goals.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Heart failure with preserved ejection fraction (HFpEF) is a critical public health problem. Heart failure (HF) affects over 5 million adults in the United States (US), and is a major source of morbidity, mortality, and impaired quality of life. Approximately half of individuals with HF have a preserved left ventricular (LV) ejection fraction (EF), termed HF with preserved EF (HFpEF). While there are several effective pharmacologic therapies for HF with reduced ejection fraction (HFrEF), none have been identified for HFpEF. There is an urgent need to identify therapies that target mechanisms of pathophysiologic progression of HFpEF. Hypertension, which is present in approximately 80% of individuals with HFpEF, is the foremost modifiable risk factor for the development and progression of HFpEF. Despite the clinical importance of hypertension in HFpEF, there is limited information on how common antihypertensive agents, particularly calcium channel blockers (CCBs) and β-blockers, effect pathophysiologic mechanisms of HFpEF. We propose a novel mechanistic investigation of the role of dihydropyridine CCBs compared to β-blockers in targeting key physiologic abnormalities in HFpEF. HFpEF is characterized by unique physiologic abnormalities that may be differentially impacted by β-blockers and CCBs. Excessive β-adrenergic stimulation may be a driver of reduced aerobic capacity in HFpEF, which may respond favorably to β-blockade. However, in HFpEF, β-blockers may reduce cardiac output, particularly during exercise, contributing to impaired cardiac output reserve and aerobic limitations. β-blockers may also have effects on the pattern of ventricular contraction and arterial load, impacting diastolic function. Similarly, CCBs may have beneficial effects related to vasodilation and reduction in late systolic load beyond their BP- lowering effect. However, CCB-induced vasodilation at rest may limit the vasodilatory reserve. Our goal is to assess the mechanisms by which CCBs and β-blockers (commonly used antihypertensive agents in clinical practice), impact aerobic capacity and quality of life in HFpEF. We will compare the impact of a dihydropyridine CCB (amlodipine besylate 5-10mg daily) vs. a β-blocker (metoprolol succinate 100-200mg daily) on arterial function, chronotropic reserve, vasodilatory reserve, and LV function, among 50 subjects with HFpEF in a randomized cross-over trial design. Participants will receive 4 weeks of each intervention, with a 1-week washout period in-between. Our mechanism-driven approach will enhance our understanding of the pathophysiology of HFpEF and characterize the physiologic potential of these common antihypertensive agents to reduce progression and improve symptom management in this disease.

NIH Research Projects · FY 2026 · 2020-09

ABSTRACT Vaccines are one of the cornerstones of modern medicine, saving millions of lives every year by preventing or attenuating multiple life-threatening diseases. The nucleoside-modified mRNA-lipid nanoparticle (LNP) vaccine platform proved to be very successful during the COVID-19 pandemic. We now know that this revolutionary platform technology can be effectively deployed for infectious disease vaccine development in preclinical and clinical settings. However, our knowledge about the mechanism of action of modified mRNA-LNP vaccines is still limited. We were successful in demonstrating that the LNP component of modified mRNA-LNP vaccines possesses a potent, intrinsic adjuvant activity and promotes T follicular helper cell and germinal center responses, which are necessary for the effective production of antibodies against pathogens. However, we still have an incomplete understanding of the mechanisms by which the immune system senses LNPs, and of how LNPs can be manipulated to fine-tune their adjuvant activity and reactogenicity. In this proposal, we will focus on two critical, yet poorly investigated aspects of LNP adjuvants with the following 2 specific aims: AIM 1. Evaluating the adjuvanticity and reactogenicity of LNPs with different compositions. In this aim, we will generate multiple LNPs, screen their ability to elicit robust cellular and humoral immune responses in mice, and examine the cellular and molecular determinants that contribute to the immunostimulatory profile of various LNPs. Both the quality and quantity of germinal center and antibody responses will be investigated, in detail. Additionally, we will set out to assess the reactogenicity of LNP adjuvants in a relevant mouse model to better predict the tolerability of LNPs in humans. AIM 2. Understanding how LXRs sense LNPs to promote effective cellular and humoral responses. In this aim, we will explore the hypothesis that the nuclear receptors Liver X Receptors (LXRs), which are key sensors of intracellular cholesterol-derived oxysterols, play a fundamental role in LNP sensing mechanisms and in the regulation of immune responses following immunization with LNP-containing vaccines. Moreover, we will determine the importance of the LNP-oxysterol axis in the regulation of the LXR pathway triggered by LNPs. The proposed studies will pave the way to the development of LNPs with a more potent adjuvant activity and to the targeted manipulation of LXRs to fine-tune the magnitude and quality of LNP-driven B cell responses. These deliverables are technically and conceptually innovative and have an immediate translational potential, including for mRNA-LNP vaccine candidates on the market or currently in clinical development.

NIH Research Projects · FY 2024 · 2020-09

Many anesthetics exert their action by binding to proteins embedded in the lipid membranes that encase cells. These proteins, including receptors and ion channels, allow cells to coordinate their action across the body. Explaining at the atomic level how binding to these proteins results in anesthesia requires knowing where on the protein the ligand actually binds. Determining this is a difficult problem that can be addressed with various methods, experimental and computational. The problem is made more difficult when the true binding sites are on a part of the protein that is actually in the lipid membrane (transmembrane domains), because of the complexity of the lipid environment. Computational methods to predict these sites that can accurately treat the membrane (e.g. flooding molecular dynamics) are also inefficient. But more efficient methods, particularly molecular docking, do not properly incorporate the effect of the membrane. This project seeks to improve docking specifically so it can predict anesthetic ligand binding sites in transmembrane domains. The overall goal is to create and calibrate a docking scoring function that takes the lipids into account, by conducting certain one-time preprocessing steps. This will be done by: 1) Predicting the microarchitecture of complex lipid membranes. Lipid membranes are composed of many different lipid types, and while the proportions of these lipids are known, the way they arrange themselves at the atomic level is not. This will be predicted using long-timescale molecular dynamics simulations. 2) Calculating the free energy profiles of insertion of selected anesthetics in these microarchitectures. It is necessary to know how favorable it is for the ligand in question to exist in the lipid membrane separately from the protein, so ligand free energy profiles as a function of depth in the membrane, as well as ligand rotation, will be calculated. 3) Identifying hydrophobic regions on the protein of interest. Traditional docking assumes that the protein is entirely solvated in water. Inhomogeneous solvation theory will be used to identify hydrophobic regions that do not contain water so they may be treated appropriately. 4) Constructing a modified docking scoring function that is parameterized by this data. The data calculated above will be fit to an efficient polynomial function for supplementing an existing docking scoring function. The project, by its completion, will have substantially improved docking methodology for this specific but important use case. It also will have served to improve the PI's ability to attack similar problems in the future, preparing him for a successful career as an independent physician-scientist.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY: “Handoffs and Transitions in Critical Care – Understanding Scalability (HATRICC-US)” Modern care of the patient with acute cardiopulmonary failure requiring critical care support is marked by the inadequate adoption and use of interventions with proven effectiveness. There is an urgent need to characterize implementation strategies suitable for use in the fast-paced, high stakes environment of critical care; doing so would generate a substantial public health impact by narrowing the evidence-to-practice performance gap for these high-acuity patients. One such gap is caused by inconsistent adoption of standardized post-surgical handoffs in U.S. hospitals, an intervention deemed high priority by the American Heart Association. In this project, we use an evidence-based standardized protocol for patient care handoffs from the operating room (OR) to the intensive care unit (ICU) as a model to study the uptake and use of complex sociotechnical interventions in acute care. In previous work, our group demonstrated adoption, fidelity, and improvement in process outcomes in a pilot 2-site study of OR-to-ICU handoff standardization. Our published work in this area builds on a base of more than 65 published studies demonstrating the effectiveness of handoffs protocols on a range of outcomes relevant to the care of patients with cardiopulmonary failure, including process, provider, and patient outcomes. The proposed study is an extension of our work that will address critical knowledge gaps about implementation in acute care by studying the implementation of a standardized handoff protocol in 12 adult and pediatric ICUs in eight hospitals in five health systems. This pragmatic study is a Hybrid Type 2 effectiveness-implementation study with a dual focus on demonstrating improvements in short-term patient outcomes (composite measure of new-onset organ failure [co-primary]; adverse postoperative events) and implementation outcomes (fidelity [co- primary]; feasibility; acceptability; appropriateness; implementation cost; and sustainment over two years). The study integrates implementation frameworks, theories, and models with engineering approaches to workflow evaluation, intervention adaptation, and evaluation. Our specific aims are to: (1) elucidate handoff protocol implementation determinants using mixed-methods on-site contextual inquiry, (2) use an engineering approach known as participatory design to adapt the handoff protocol to each ICU and use theory-based implementation mapping to select tailored, multifaceted, multilevel implementation strategies, (3) implement handoff protocols in stepped wedge fashion with randomized roll-out under the leadership of site based teams, and (4) use human- centered design to create an implementation toolkit to facilitate the dissemination and use of study findings to a broad audience. The proximate goal of this work is to determine effective approaches to implementation for complex sociotechnical interventions in acute care settings. The longer-term goal is to use this approach to promote the uptake and sustained use of proven-effective interventions in the care of patients with acute cardiopulmonary failure. This work directly addresses the stated interest of NHLBI's Center for Translation Research and Implementation Science in mixed methods studies and those using pragmatic trial designs.

NIH Research Projects · FY 2026 · 2020-09

Project Summary/Abstract The role of mechanics in determining cell phenotype has been intensely studied since pioneering studies showed that cells in culture respond to differences in the elastic modulus of their environment. Stiffness sensing has been demonstrated in such varied settings as development, cancer, wound healing, and fibrosis. How cells sense stiffness remains unclear, partly because of a lack of quantitative data that define exactly what cells sense, especially in vivo. In particular, the nature of viscoelasticity and non-linear (strain-dependent) elasticity and mechanical plasticity in normal and diseased tissues is insufficiently characterized, and the contribution of these mechanical parameters to cell stiffness sensing and behavior is not understood. This proposal extends studies of elasticity to encompass additional biologically relevant parameters, with a focus on the role of dissipative processes, and offers the potential to reevaluate current models of mechanobiology and develop new concepts of the role of time dependent mechanics in biological contexts. The proposed work builds on a series of our investigations as part of the parent R01 project where 1) we demonstrated through theory and experiments that faster substrate stress relaxation leads to faster migration of healthy and diseased human cells, 2) we predicted through an active chemo-mechanical model in both 2D and 3D non-linear elastic microenvironments with increasing matrix stiffness, which correlates strongly with the change in mitochondrial potential, glucose uptake and ATP levels measured experimentally; and 3) we showed that physiological and pathological chemomechanical cues can directly regulate the spatial nanoscale organization. In this renewal our overall goal is to integrate theoretical and experimental studies to address how dissipative matrix properties regulate cellular metabolism, cytoskeletal activity, and chromatin organization, all vitally important determinants of cell fate and function. We propose an integrated approach using imaging, theoretical modeling, and omics data to elucidate the crosstalk between histone deacetylases (HDACs) and metabolism. We will emphasize understanding how the elastic and dissipative properties of the microenvironment, along with metabolites that act as histone deacetylase inhibitors (HDACIs), synergistically regulate HDAC activity and metabolic pathways.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Obesity is a significant risk factor for cardiovascular (CV) disease that affects 39% of adults in the U.S. Obesity rates are disproportionately rising among women and contribute to increasing rates of diabetes and CV mortality among women younger than 55 years. Pregnancy represents a life transition when many women gain excess weight. The effect of obesity is further magnified in women with preeclampsia, an independent and underappreciated risk factor for future CV disease. There is a pressing need to better understand predictors of excessive postpartum weight retention in order to deliver effective and scalable weight loss interventions during a period when women at highest risk may be particularly receptive to lifestyle change. Social incentives, or the influences that impact behavior change based on relationships, are strong motivators of healthy behavior. The use of game design elements, such as goal-setting, has been successfully combined with social incentives to enhance healthy behavior in other disease settings. The central hypothesis of this proposal is that a behavioral intervention using these novel approaches, combined with an established behavioral weight loss program, will provide an effective, and scalable solution to reduce the incidence of postpartum obesity. This proposal will leverage mentorship of senior investigators (Drs. Michal Elovitz and Peter Groeneveld) and Penn’s mature research programs in cardiovascular medicine, maternal fetal medicine, and behavioral economics. Research will be conducted in an urban and racially diverse population since these women are most likely to benefit from interventions to reduce CV risk. Aim 1 will use robust statistical methods and machine learning to create a clinical prediction tool for postpartum weight retention in an existing electronic health record database enriched with neighborhood-level data. Aim 2 will use qualitative and mixed methods to identify strategies to enhance the design of a pilot intervention using patient feedback. Aim 3 will implement a 2-arm single site randomized clinical trial to achieve weight loss in women with preeclampsia or gestational hypertension participating in an online behavioral weight loss program using remote technology and social feedback from other postpartum women. This proposal will identify women at an early stage in life who will benefit the most from intensive lifestyle changes and will test and refine interventions that are able to be disseminated to postpartum women remotely. Dr. Lewey is a general cardiologist trained in women’s health and population science with established expertise in pregnancy associated cardiovascular disorders. The training she proposes in advanced statistical methods, qualitative analysis, and behavioral clinical trial design will position her to become a leader in women’s cardiovascular health. By the conclusion of this program, she will be able to independently design, target, and evaluate behavioral interventions to prevent heart disease in women. The results of the proposed K23 will be invaluable pilot work for a planned R01-level application.

NIH Research Projects · FY 2025 · 2020-08

Abstract / Project Summary Ribosomal RNAs (rRNAs) comprise >80% of cellular RNA, and their transcription from rDNA repeats by RNA Polymerase I (Pol I) accounts for a bulk of all transcription. The bodies of complex eukaryotes have different ribosome production rates in different cell types. Precise control of rRNA transcription rates is important for normal physiology, and its dysregulation leads to disease. However, though regulation of Pol II activity and the transcription of mRNAs has been dissected in detail in virtually every organ system, the transcriptional control of the most abundant RNA in the cell has been largely ignored, creating a fundamental knowledge gap. Using MIRA R35 funding, we have created new bioinformatic and technical tools for mammalian rDNA and rRNA studies, quantified rRNA transcription dynamics in a complex organ system, mapped ~2200 ChIP-Seq tracks for ~250 transcription factors (TFs) and chromatin proteins to assemble a TF-rDNA atlas, demonstrated that a cell-type-specific TF directly binds and regulates Pol I occupancy and rRNA transcription, and developed a protocol to directly edit rDNA repeats. Our central hypothesis is that normal cellular identity and functioning requires precise rRNA levels, fine-tuned in each cell type through a combination of control mechanisms. We will interrogate this model in the next period of funding through the following projects: PROJECT 1: Defining levels of rRNA regulation across cell types: We will use ChIP-Seq, FISH-Flow, and metabolic labeling to quantify rRNA dynamics in defined primary mouse cell types in homeostatic and stress-recovery states. The goal of this project will be to assemble a foundational map of the key steps in rRNA transcription, processing, or lifespan that are differentially regulated across different mammalian cell types. PROJECT 2: Testing the ‘Ribosome Concentration Hypothesis’: We will use a targeted degradation approach to degrade Pol I and reduce rRNA transcription in a dose-dependent fashion, allowing us to achieve any arbitrary ribosome number between 100% and 30% of normal. We will use this system to test the effects of altered rRNA levels on selective mRNA translation and cell fates in cultured cells and in live animals. PROJECT 3: Editing rDNA repeats to dissect promoters and regulatory regions: rDNA repeats have several unique and poorly understood features, including the unusual structure of their promoters and a cluster of cell-type-specific TFs whose conserved rDNA binding we recently identified. We will use Cas9-guided base editors to systematically edit hundreds of rDNA repeats to dissect promoter sequences and TF motifs. Our approach will rigorously test the model that the combinatorial binding of cell-type-specific TFs to regulatory regions on rDNA controls Pol I occupancy and rRNA transcription in a cell-type-specific manner. The long-term goal of this work is to gain a detailed understanding of how the universal process of rRNA transcription has been customized to meet diverse tissue needs in complex organisms, a question of fundamental importance to normal and disordered eukaryotic biology.