University Of Pennsylvania

NIH Research Projects · FY 2024 · 2023-09

Early CNS entry of HIV fosters its expression in perivascular macrophages and microglia; but, whether HIV expression in these cells is associated with persistent neuropsychiatric damage is unclear. Non-HIV-related comorbidities such as substance use disorders can worsen neuropsychiatric deficits. Stimulants such as cocaine have been reported to alter HIV replication dynamics in macrophages and microglia and change their inflammatory state, and may contribute to neuropsychiatric deficits in PWH. A recent preprint provides the only evidence that frequent cocaine use is associated with larger HIV latent reservoir size in CD4+ T cells. Low- level/residual inflammation has been shown to foster a persistent HIV reservoir in CD4+ T cells. However, a major knowledge gap is whether cocaine exposure supports long-term HIV expression in macrophages and microglia and whether inflammation mediates this effect. A clear understanding of cocaine’s impact on HIV expression in complex multicellular contexts is a prerequisite for the identification of novel targets for effective ART regimens, adjunctive neuroprotective therapies, and latency reversal strategies. Our overarching hypothesis is that long- term HIV expression in macrophages and microglia contributes to neuropsychiatric damage which is exacerbated by cocaine. Addressing these key questions requires methods that can determine the identity of cells that harbor HIV DNA and RNA and simultaneously link the presence of HIV DNA and RNA to functional cellular outcomes via the transcriptome in the same cell. Proteogenomic approaches that combine DNA and RNA sequencing with the analysis of surface marker expression to identify cell populations provide insight into the expression of HIV DNA and RNA in specific CD4+ T cell subtypes. However, significant caveats hinder their applicability in CNS cells. We will develop a novel molecular, high-resolution, single-cell level method we call HIV integrated proviral DNA (HID)/single-nuclear RNASeq (HID-Seq). In this combined approach, dCas9-Tn5 tagmentation amplifies proviral DNA integrated in the host genome, and single-nuclear RNASeq uses validated primer libraries to detect specific HIV RNA species along with the detection of all host-cell RNA species in the same cell. HIV proviral DNA, HIV RNA, and host-cell RNA are molecularly barcoded using split-sequencing, which tracks DNA and RNA products to individual cells. In the R61 phase (Aim 1), we will develop HID-Seq for multilevel indexing of HIV DNA and RNA expression while simultaneously examining the host cell transcriptome in vitro. In the R33 phase (Aim 2), we will use HID-Seq to determine the impact of cocaine on HIV DNA and RNA expression in human induced pluripotent stem cell-derived microglia and macrophages in vitro (Aim 2a) and in the peripheral blood mononuclear cells and CNS cells obtained from autopsy specimens of PWH (Aim 2b-c). Bioinformatic analyses will (i) confirm the identity and characteristics of cells harboring HIV DNA and RNA in the CNS of PWH with history of cocaine use and (ii) determine how cocaine use alters the associations between HIV DNA and RNA and host-cell transcriptomic profile.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ ABSTRACT Internalizing disorders such as major depressive disorder and generalized anxiety disorder show marked sex differences in prevalence, presentation, and trajectory. For example, depression and anxiety disorders are more prevalent in females than males. The neural mechanisms underlying such sex differences, however, are not well understood. Previous studies have suggested sex differences in internalizing symptoms may be due to abnormalities of the default mode network (DMN), a functional network that is critical for internally-directed cognition and emotional processes. The vast majority of studies on functional networks use methods that assume that the DMN is in the same anatomic location in every individual. However, evidence from multiple independent groups has recently shown that there is significant inter-individual variation in the spatial distribution of functional networks on the anatomic cortex and that person-specific networks predict aspects of cognition and psychopathology. We recently provided the first evidence of sex differences in such personalized functional networks and showed that sex differences in personalized networks are greatest in the DMN. Preliminary data demonstrate that DMN representation — a measure of normalized surface area — is greater in females. However, it is unknown if sex differences in personalized DMNs 1) evolve during development or 2) underly sex differences in internalizing symptoms. The over-arching hypothesis of this proposal is that DMN representation is greater in females, which confers a vulnerability to internalizing symptoms. Given that these symptoms typically emerge during childhood and adolescence, this hypothesis will be evaluated using three large developmental data resources: the Healthy Brain Network (HBN; n=5,000), the Human Connectome Project: Development (HCP-D; n=1,300), and the Adolescent Brain and Cognitive Development Study (ABCD, n=11,572). Aim 1 will characterize developmental sex differences in the spatial distribution of personalized functional networks, while Aim 2 will delineate how personalized networks relates to sex differences in internalizing symptoms. Finally, Aim 3 will use baseline variation in personalized functional networks to predict the longitudinal course of internalizing psychopathology. This innovative research project will establish that sex differences in personalized functional networks underly sex differences in internalizing psychopathology. Results will help inform targeted early interventions and trials of personalized neuromodulatory therapies. Successful completion of the proposed aims as an independent investigator will accelerate the candidate’s ability to exert a sustained impact on the field and advance our understanding of the contribution of sex as a biological variable to the development of internalizing psychopathology.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Patients with chronic obstructive pulmonary disease (COPD), a debilitating lung disease affecting over 16 million Americans, experience a high burden of chronic symptoms. Patients with COPD and their caregivers identify patients’ high burden of psychological distress (e.g., symptoms of depression and anxiety) as their top palliative care priority. Psychological distress is also strongly associated with poor clinical outcomes in COPD. Yet, patients with COPD rarely receive care for psychological distress. Family caregivers are uniquely positioned to facilitate the delivery of palliative care interventions due to their longitudinal contact with the patient and because social relationships provide direct health benefits and buffer against health stressors. A critical evidence gap that prevents effective and efficient palliative care delivery is an understanding of how palliative care interventions may best leverage these existing relationships to meet the dyad’s needs. This application proposes a two-arm mechanistic randomized trial comparing the effect of the Coping Skills Training (CST) program and a minimally enhanced usual care attention control arm on improving patients’ and caregivers’ outcomes. Over 12 remotely delivered, weekly sessions, the patient-caregiver dyad learns and practices coping skills tailored to COPD. CST’s content focuses on (1) improving patients’ abilities to cope with stress and illness and (2) teaching caregivers both how to cope and how to be an effective coping skills coach. In prior testing, CST improved patients’ psychological quality of life and functional abilities, yet the mechanisms of action remain unknown and the prior study population did not reflect the populations most affected by COPD. The primary objective of the proposed trial is to apply behavioral science theories to identify key mechanisms through which the CST program reduces patients’ psychological distress through caregiver support. The project will also assess heterogeneity in the efficacy of CST at reducing distress among patients who differ by key characteristics and identify patient, caregiver, and contextual factors associated with program uptake and completion, both emphasizing modifiable barriers to participation and relevancy. A stakeholder advisory committee will continue to collaborate with investigators. The trial will enroll 375 dyads of outpatients with COPD experiencing psychological distress and their caregivers recruited from University of Pennsylvania and Henry Ford Health Systems’ outpatient clinics that primarily serve communities lacking healthcare resources, such those that are low-income and/or rural. The study will identify the novel mechanisms through which a palliative care intervention that includes and leverages patients’ caregivers can reduce the burden of chronic illness carried by the dyad. Completing this project will provide the knowledge necessary to refine and (1) develop scalable palliative care interventions that optimally incorporate patients’ existing relationships and social resources and (2) inform strategies to surmount subgroup-specific barriers to facilitate future implementation, including addressing barriers to palliative care access.

NIH Research Projects · FY 2025 · 2023-09

PROJECT ABSTRACT Oral biofilm-related infections remain a persistent and costly clinical problem. Existing treatments are unable to simultaneously kill and physically disrupt biofilms and require manual biofilm removal procedures that are cumbersome with reduced efficacy in difficult to reach areas such as endodontic canal systems. Furthermore, options for sample retrieval for diagnostics during clinical procedures are limited. Efficacious, automated technologies capable of precisely targeting complex anatomical areas are needed to retrieve samples, kill and remove biofilms, and deliver drugs on site. We propose a novel approach combining nanotechnology and robotics to develop the first automated system for targeted disinfection, removal, and sampling of endodontic biofilms. We have designed small-scale robots using catalytic nanoparticles as building blocks that display tether-free controlled motion with multifunctionality. Our approach utilizes iron oxide nanoparticles (IONPs) with dual catalytic-magnetic properties that (i) generate bactericidal and biofilm degrading reactive molecules in situ, and (ii) remove the disrupted biofilm via magnetic-field driven robotic assemblies termed Catalytic Antibiofilm Robots (CARs). Preliminary data demonstrate that CARs locally remove and collect biofilms with high precision and efficacy in comparison to conventional treatment, including confined endodontic spaces. By tuning the magneto-catalytic properties and control of the CARs systems, we will develop robotic device prototypes that fit the oral cavity for simultaneous endodontic biofilm treatment, removal and sample retrieval. We propose to further improve IONP-made robots coupled with a clinical electromagnetic controller to develop two CARs-based oral biodevice platforms. (Aim 1) CAR1s, formed from aggregated IONP, will be used for catalytic bacterial killing, biofilm treatment, and sample retrieval from root canals for diagnostic analysis. We will identify key parameters for CAR1s improvement, assessing magnetic control, bioactivity and visualization/tracking. CAR1s will be evaluated for targeting difficult-to-reach areas, such as C-shaped/curved canals and isthmus, as well as treating and retrieving biofilms. We will characterize and improve CAR1 control first using 3D-printed tooth replicas with diverse canal morphologies to improve movement and controllability, followed by testing our system using ex vivo extracted tooth/typodont and pig jaw models. (Aim 2) CAR2s will be fabricated by 3D micromolding functional polymers with embedded IONPs for biofilm disruption, retrieval, and drug delivery at the apical region. We will optimize magnetic control and tracking, antibiofilm activity and triggered cargo delivery, testing efficacy to remove and retrieve biofilms. We will assess bioactivity using mixed- species biofilms and maneuverability to the apical region of the root canal recapitulated in 3D-printed teeth and ex vivo models, while rigorously evaluating the robotic device in geometries suited to the oral cavity with comparisons against conventional treatment. We expect the outcomes of the proposed studies will lead to the first robotic biodevice system developed for automated biofilm theranostics for applications in dental medicine.

NIH Research Projects · FY 2025 · 2023-09

Enter the text here that is the new abstract information for your application. The objective of the PHLHousing+ Evaluation is to test whether unconditional, monthly cash payments made directly to tenants increase housing security and thereby improve health, reduce reliance on crisis-based emergency health services, and increase access to higher opportunity neighborhoods. This objective is aligned with the strategic aim of NINR to identify effective approaches to addressing social factors and social needs that affect health outcomes. The PHLHousing+ Evaluation comprises three groups, all of whom earn below 50% area median income and have at least one child under the age of 16 years living at home: 270 households who receive monthly cash payments in lieu of a rental voucher (Cash group), 225 households who are eligible for (or currently using) a rental voucher (Voucher group), and 789 households on the Philadelphia Housing Authority (PHA) waitlist who will not be offered a rental voucher for the foreseeable future because of their randomized position on the waitlist (Control group). Of the 1,284 households in the study, 82.5% are headed by single women and 82.8% are Black. There are 2,678 children in the sample, ranging in age from 1 to 19 years (median = 10, IQR = 5-15). Monthly cash payments range from $89 to $2079, with a median payment of $881; payments vary based on household income, family size, and fair market rent. Households in the Cash group will receive monthly payments for 2.5 years. All three groups are surveyed every six months for three years via an online survey; the first wave of surveys was deployed in August 2022. Existing surveys include measures of self-reported mental and physical health and hospital utilization for adults and children. We propose to extend these data in three ways: (1) by extracting electronic health record (EHR) data on emergency department (ED) visits and hospital admissions linked to our survey respondents; (2) by administering brief, monthly surveys that will increase participant retention between biannual surveys and will measure mood and health-promoting behaviors that might be pathways by which PHLHousing+ promotes positive health outcomes; and (3) by linking household addresses with census-based data on neighborhood opportunity and Philadelphia Police Department crime data. Aim 1 is to test whether rental assistance (Cash + Voucher vs. Control) or type of assistance (Cash vs. Voucher) is associated with self-reported mental and physical health and with affective volatility and health-promoting behaviors. Aim 2 is to test whether the availability or type of rental assistance is associated with ED use or hospital admissions as recorded in EHR, specifically for problems that are disproportionately associated with housing insecurity (e.g., mental health problems). Aim 3 is to test whether availability or type of rental assistance is associated with exposure to housing problems (e.g., pests, mold), access to neighborhood opportunity, or exposure to crime. Study findings will have immediate implications for the City of Philadelphia’s model for providing housing assistance, will inform efforts to establish similar programs in other cities, and will inform federal approaches to rental assistance.

NIH Research Projects · FY 2025 · 2023-09

Abstract: Atopic dermatitis (AD) is one of the most common inflammatory skin diseases with an estimated prevalence approaching 5% in adults and is typically characterized by pediatric onset, pruritis and persistance. Research has indicated that AD is associated with an impaired epithelial barrier that promotes sensitization to environmental antigens and a type II immune response. Despite advances in AD research, the causative mechanism(s) leading to the persistent type II immune response remain unclear. Our recent publication and Prel Data indicate that the dermis and subcutis contain fibroblasts that appear to be involved in establishing type II inflammatory reactions in skin. Understanding how dysregulation of skin fibroblasts contributes to downstream events that they may intitiate formation of inflammatory AD-like lesions is the focus of this proposal. Our Prel Data show that dysregulation of NF-kB signaling in Prx1+ fibroblasts leads to upregulation of CEBPb in fibroblasts, upregulation of CCL11 and tissue eosinophilia followed by type II skin lesions resembling human AD. Our mouse model shares many similarities with human AD-like lesions involving both the dermis and epidermis at the histologic, cellular and molecular levels. Prel Data compares single cell RNAseq analysis from our study with published scRNAseq databases from human AD lesions. scRNAseq analysis from our model and human AD show fibroblast dysregulation involving CCL11 and CEBPb. Moreover, using RNAScope we have also shown that validated human AD samples contain increased numbers of dermal fibroblasts co-expressing CEBPb and CCL11 compared to matched control human skin, also consistent with our murine model. Thus, we propose novel preclinical and translational studies (Aim 1) to define molecular mechanisms by which fibroblast dysregulation mediated by CEBPb induces an inflammatory phenotype in these cells. Aim 2 will determine the role of eosinophil infiltration as a key intermediate for triggering type II immune response in AD-like skin. Aim 3 will use spatial transcriptomics (GeoMX) and RNA seq to explore equivalent mechanisms in human fibroblasts and AD specimens and translational studies using inhibitors as potential treatments for AD. We expect the proposed studies along with recent reports in the literature to contribute to a paradigm shift in understanding AD pathogenesis regarding fibroblast dysregulation and contribute to development of new therapeutic interventions that may prevent the development of AD.

NIH Research Projects · FY 2025 · 2023-09

Project Abstract The goal of this proposal is to define the mechanisms of retinal vasculopathy with cerebral leukoencephalopathy (RVCL), an adult-onset vascular dementia associated with brain atrophy, blindness, and premature death, usually within 5-10 years of disease onset. RVCL is an autosomal dominant disease that is refractory to treatment with immunosuppression despite the fact that RVCL patients develop autoantibodies. RVCL-causing mutations in the TREX1 gene cause disease with 100% penetrance. Currently, there is no effective treatment for this devastating disease, and the mechanisms of disease pathogenesis have not yet been defined. Dr. Jonathan Miner (PI) directs the RVCL Research Center at the University of Pennsylvania and coordinates care of RVCL patients from around the United States and the world. Dr. Miner also leads a team of RVCL- focused clinicians and investigators. Preliminarily the Miner laboratory has generated numerous mouse and human cellular models of RVCL and discovered novel molecular mechanisms that may be driving endotheliopathy, cellular senescence, and the resulting vascular dementia. The co-investigator is Dr. Nouri Neamati, a medicinal chemist and biochemist at the University of Michigan. Dr. Neamati is developing TREX1 inhibitors and protein degraders. This partnership has led to novel animal models, single-cell RNA-seq studies, and first-in-class inhibitors of TREX1, as well as our discovery that RVCL-causing TREX1 mutations lead to failure of replicative senescence and dysregulation of endogenous retroelements. Here, we propose to use cellular and animal models to define the role of TREX1 in regulating DNA damage, retroelements, and pathology (Aim 1), to use animal models to define functions of TREX1 mutants and a DNA damage repair pathway in humoral immunity (Aim 2), and to determine the role of TREX1 DNase activity using an enzyme-dead TREX1 mutant mice and first-in-class TREX1 inhibitors and protein degraders (Aim 3). Our mechanistic studies will broadly elucidate functions of TREX1 and the role of TREX1-mediated DNA damage in a mouse model expressing TREX1 mutants known to cause small vessel disease and vascular dementia in humans. Insights gained from our work may clarify the role of TREX1 and endogenous retroviruses in cellular senescence, eventually leading to new therapies for this monogenic vascular dementia.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY The function. relative disease tissues in our body consist of diverse cell t ypes with each cell type specialized to carry out a particular The behavior of a cell is influenced by its surrounding environment within a tissue. Knowledge of the locations of different cells in a tissue is critical for understanding the spatial organization of cell types and pathology.Although single-cell RNA sequencing (scRNA-seq) has made it possible to characterize cell types and states at an unprecedented resolution, the lack of physical relationships among cells has hindered the study of cell-cell communications within tissue context. Recent technology advances in spatial transcriptomics (ST) have enabled gene expression profiling while retaining location information in tissues. A popular ST technology is based on spatial barcoding followed by next-generation sequencing in which transcriptome-wide gene expression is measured in spatially barcoded spots. Data from such ST technologies often include a high- resolution hematoxylin and eosin (H&E)-stained histology image of the tissue section from which the gene expression data are obtained. Although ST is powerful, such data are still expensive to generate. On the other hand, it is relatively cheaper to generate H&E-stained histology images and scRNA-seq data. The main motivation of this project is to leverage information in ST to gain additional knowledge from the relatively easy- to-obtain histology images and scRNA-seq data. Building upon our expertise in statistical genomics, we propose to develop novel machine learning methods to address key computational challenges when performing integrative analysis of ST, histology images, and single cells. Our methods will jointly model gene expression and histology to characterize the spatial organization of tissues and predict spatial gene expression from histology images. The resulting spatial map from these analyses will further enable the spatial mapping of single cells back to tissues. The proposed methods will be applied to public data and data generated from ongoing collaborations in various diseases to evaluate their performance. The successful completion of this project will allow researchers to take advantage of advanced machine learning algorithms to integrate ST, histology, and single-cell data to gain a holistic view of the spatial organization of tissues.

NIH Research Projects · FY 2025 · 2023-09

Label-Free Optical Redox Imaging for Pretreatment Prognosis of Early-Stage Triple Negative Breast Cancer Abstract Triple negative breast cancer (TNBC) has significant intratumor variations in microenvironments and metabo- lism, which can substantially affect disease progression and clinical outcomes. A TNBC tumor can contain hy- poxic and normoxic regions and exhibit glycolytic, oxidative, or mixed metabolic subtypes. These metabolic subtypes are typically on a submillimeter scale in clinically presented breast tumors, below the resolution of current clinical metabolic imaging methods, highlighting the need of new high-resolution metabolic imaging methods in the clinic. Optical Redox Imaging (ORI), a label-free fluorescence imaging technique developed at the Britton Chance Laboratory of Redox Imaging, can detect intratumor metabolic subtypes three- dimensionally (3D) with a resolution down to 25 µm. ORI measures the intrinsic fluorescence signals of re- duced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp) and determines the oxidized and reduced status of redox metabolism. Our preliminary results found redox hotspots, the highly oxidized re- dox subtype in treatment-naïve specimens, from a pilot cohort of early-stage TNBC patients. Notably, in the studied cohort, ORI redox hotspots predicted the risk of disease progression better than conventional clinical indicators (e.g., tumor size, stage, grade, and nodal status). Our data studying untreated TNBC xenografts and cell cultures also suggested that the redox hotspots are underpinned by the Warburg effect (glycolytic switch), corroborating the prediction of risk of progression by ORI. Based on these results, the long-term goal of this program is to establish the prognostic value of pretreatment ORI by expanding observations in TNBC clinical specimens and preclinical models. Aim 1 will be a full-scale retrospective ORI study of frozen untreated surgi- cal specimens from early-stage TNBC patients. We will test the hypothesis that the intratumor redox hotspots predict an increased progression risk and add value to conventional prognostic indicators. Aim 2 will assess the prognostic value of ORI in human TNBC mouse xenografts and cultured cell models using progression endpoints and confirm that the activated glycolytic switch is the basis for the higher redox hotspot and risk of progression. The successful accomplishment of this project will establish ex vivo ORI as a novel pretreatment tool that is indicative of the intratumor glycolytic switch at submillimeter resolution and inform the risks of pro- gression for individual patients with early-stage TNBC. Our project will help resolve the unmet clinical need for accurate prognostic biomarkers to improve risk stratification and personalized treatment in TNBC.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY Lipids are an essential component of neuronal function and structure. Genetic mutations associated with disturbed lipid metabolism in neurodegenerative disorders is becoming increasingly recognized, yet the mechanisms that regulate neuronal lipid metabolism is poorly understood. We and others have found SNX14, a sorting-nexin (SNX) protein associated with a spinocerebellar ataxia (SCAR20), to be a regulator of endoplasmic reticulum (ER)-lipid droplet (LD) biogenesis, fatty acid desaturation, and cerebellar lipid homeostasis. I have discovered that SNX14 interacts with its paralog, SNX13, in neural cells. SNX13 is also an ER resident protein that controls triglyceride content, LD numbers, and lysosomal cholesterol homeostasis. We recently identified homozygous SNX13 variants in three children with a novel cerebellar ataxia similar to SCAR20. However, little is known regarding the basic molecular mechanisms behind SNX13 function in neurons, and how depletion of Snx13 leads to degeneration. My overarching hypothesis is that SNX13 functions in neuronal lipid homeostasis, which is critical for cerebellar function and survival. To test this hypothesis, Aim 1 will knock down Snx13 by AAV-mediated in vivo delivery of Snx13 sgRNAs to dCAS9-KRAB expressing mice to investigate the role of Snx13 in cerebellar integrity and motor behavior. Aim 2 will leverage genome edited human pluripotent stem cell derived neuronal cultures to define the impact of SNX13 patient mutations on LD homeostasis under conditions of cellular stress (e.g., excess fatty acids and excitotoxicity). Successful completion of these aims will be an important foundation for future studies investigating neuronal LD homeostasis, while simultaneously providing me with outstanding training in CRISPR/Cas9 methodologies, stem cell biology, and behavior. Further, these efforts will uncover basic disease mechanisms that can be used to inform targeted therapeutic strategies for patients with SNX13 or SNX14 mutations, including other relevant neurodegenerative and metabolic disorders. The proposed training plan is sponsored by Dr. Naiara Akizu and Dr. Beverly Davidson at the University of Pennsylvania and Children’s Hospital of Philadelphia. This proposal provides a phenomenal training experience that will expand my technical expertise in cellular neurobiology, and cultivate my professional skills in science communication, mentoring, and leadership, all of which will facilitate my career goals of becoming a leading researcher in the field of neurodegeneration.

NIH Research Projects · FY 2025 · 2023-09

Tourette Syndrome (TS) onsets in childhood, affects 1% of the population, and causes substantial impairment. Health professionals recommend behavior therapy as the first-line treatment for TS due to its efficacy and adverse effect profile. In behavior therapy, patients learn tic management skills and are assigned “homework” to solidify skill learning. Core skills involve building awareness to tic occurrence and implementing behavioral strategies to inhibit tic expression upon such awareness. Youth who exhibit a treatment response to behavior therapy continue to benefit for 10 years. However, more than 50% of patients do not achieve a treatment response and rely upon FDA-approved medications that have detrimental health effects. A key challenge with behavior therapy is the reliance on a human practice partner for “homework” due to accessibility and accuracy. Our team will create an activity-based recognition system and algorithms to identify and classify tics across activities and sensor viewpoints in patients with TS. This system will evolve into a therapeutic tool (i.e., a “digital practice partner”) that is scalable, accessible, and accurate in detecting tics. This will enable patients to effectively practice behavior therapy skills and achieve optimal long-term outcomes. First, we will develop a multi-view sensor system to observe tics in patients with TS and refine our hierarchical ontology of tics to annotate data collected from our sensor system. This will enable both a clinically interpretable and fine-grained classification of tic and non-tic movements. Second, we will design a novel spatio-temporal CNN called Tic-Net for fine-grained detection of facial and upper body tics in video data from multiple viewpoints, which will rely on facial action unit intensities and interpretable upper body part features that we design, temporal segmentation and detection networks, as well as contrastive and self-supervised learning losses to detect tics without requiring large amounts of annotations. Third, we will design a novel spatio-temporal Transformer architecture called Tic-DETR for fine-grained tic detection, which captures long-range interactions among face action units and/or skeletal joints across multiple views as well as relations between tic instances to produce interpretable detections of tics of varying durations from multiple viewpoints. Finally, we will compare detection outcomes between our algorithm and a human practice partner, evaluate the robustness of algorithms across viewpoints, and assess its clinical interpretability.

NIH Research Projects · FY 2025 · 2023-09

Almost two-thirds of breast cancers are the hormone-receptor positive subtype which are treated with oral endocrine therapy (ET), yet up to 50% of patients do not take these medications as prescribed resulting with increased rates of breast cancer recurrence and death. Despite robust available data supporting the efficacy of ET and the low adherence to this life-saving treatment, prior interventions have shown minimal to modest short-term benefits on improving adherence. Most of these studies only provided educational materials and did not intentionally target patients at high risk of poor adherence to ET. Therefore, a tailored intervention for breast cancer survivors that addresses multiple barriers to ET adherence is particularly beneficial for this population. We propose an iterative approach to develop and pilot test a new digital behavioral intervention for ET adherence that is developed with input from breast cancer survivors. This new digital intervention will address a major barrier to ET adherence, symptoms from ET and will promote self-efficacy in symptom monitoring among patients with non-metastatic hormone receptor positive breast cancer on ET. We will iteratively refine and tailor the digital intervention focusing on symptom monitoring and management (Aims 1 and 2). We will then test the intervention for acceptability, feasibility, and preliminary efficacy (Aim 3). This project will address quality of life (symptom burden) and care delivery (medication adherence) issues among the majority of patients with breast cancer. This intervention has great promise for reducing cancer-related symptoms burden which negatively impact quality of life and potentially improving breast cancer outcomes. Modified Specific Aims There will be approximately 317,000 new cases of invasive breast cancer in 2025.5 Of these, about 75% will have hormone receptor positive breast cancer. The cornerstone of treatment for this subtype of breast cancer is oral endocrine therapy (ET) – medication that must be taken daily for 5-10 years. ET reduces risk of death from breast cancer by 50% when taken optimally. However, 10-year survival decreases from 81% when adherent to 74% when non-adherent to ET.4 Despite the robust data regarding efficacy of ET, adherence remains as low as 50% and nonadherence to ET is a major contributing factor to relapse rates. Some of these relapses include distant relapse (i.e., the development of incurable metastatic breast cancer requiring life-long treatment), a significant issue for breast cancer survivors. In a randomized trial, among those patients who discontinued ET, 79% stopped the drug due to side effects.6 Side effects from ET are frequently cited by patients as the reason for early discontinuation of ET in observational studies.7-9 Other barriers to adherence reported by breast cancer survivors include a perception of being at low risk of recurrence and low perceived benefit of ET.10 Given the incidence of HR+ breast cancer, ET non-adherence is a significant problem as non-adherence is associated with worse outcomes.11 Thus, targeted interventions to increase adherence to ET are needed to improve breast cancer outcomes. Prior interventions designed to improve ET adherence have primarily focused on providing education and have not demonstrated significant efficacy in improving adherence. We propose a randomized, mHealth intervention trial called “EmSHAPE”- Engaging mobile health for symptom monitoring and health promotion for endocrine therapy - among patients with non-metastatic hormone receptor positive breast cancer. This innovative study will integrate social cognitive theory to ensure scientific rigor in intervention development. The research and training proposed herein will enable me to build on my oncology and epidemiology training to acquire expertise in developing culturally targeted behavioral interventions, integrating mHealth technologies into behavioral interventions, and testing behavioral interventions. Following the ORBIT model for refining and testing behavioral interventions14, we will complete Phase 1a (identifying candidate treatment components), Phase 1b (refine and finalize intervention components), and Phase-2 (pilot-test the intervention), in preparation for a future efficacy trial. The following Specific Aims are proposed to accomplish these goals: Aim 1: To develop a prototype of the EmSHAPE intervention leveraging feedback from patients with HR+ breast cancer. The intervention will incorporate existing empirically supported strategies for medication adherence and additional components based on extensive formative work. Iterative learner verification and revision will be used to refine the intervention by leveraging stakeholder input for improved comprehension and acceptability. Intervention components will be evaluated by stakeholders who will report on opportunities for additional tailoring. Aim 2: To refine and finalize the EmSHAPE protocol and mHealth platform. We will refine and finalize the functional and technical components of the mHealth platform as well as refine and finalize the EmSHAPE protocol according to feedback gathered from usability testing with breast cancer survivors on ET (n=10). Aim 3: To test feasibility, acceptability, and preliminary efficacy of the EmSHAPE intervention via a pilot randomized clinical trial. Sixty women with HR+ BC will be randomized and monitored for 12 months. We hypothesize that the intervention will be acceptable (at least 60% of eligible patients will consent and at least 60% of intervention participants will report that they are “satisfied” or “very satisfied” with the intervention) and feasible (at least 60% of participants will complete the study) to breast cancer survivors receiving ET. We also hypothesize that the intervention will result in improved objective ET adherence relative to control group as measured by using smart pill bottle caps that track medication intake. This study will lay the groundwork for a larger randomized controlled trial to determine the efficacy of this intervention relative to enhanced usual care for patients with HR+ breast cancer through R01 funding. This application is significant, as no studies to date have used a multicomponent, patient-centerred, symptom monitoring approach to address low adherence rates to ET among breast cancer survivors, despite established evidence regarding the relationship between ET adherence and survival; and innovative, as implementing a symptom monitoring intervention during cancer treatment could change clinical practice if successful, by improving patient quality of life and improving cancer outcomes. This represents an important step toward improving survival disparities among breast cancer survivors by targeting oral ET adherence and furthers the candidate’s goal of establishing an independent research program to improve cancer care delivery through the development of digital and behavioral interventions.

NIH Research Projects · FY 2025 · 2023-09

Abstract Non-steroidal anti-inflammatory drugs (NSAIDs) are a class of widely used drugs for treatment of pain, fever, and inflammation. NSAID use has been linked to both mild and life-threatening adverse drug reactions (ADRs) including gastrointestinal bleeding and acute coronary syndrome. Given the severity of these outcomes and the large patient pool, there is a great need to predict individual risk of ADR from NSAIDs. Pharmacogenetics is the study of how genetics influence drug response. The Clinical Pharmacogenetics Implementation Consortium has published guidelines for clinicians to modify NSAID treatment in the presence of CYP2C9 loss- of-function variants, which result in reduced clearance of NSAIDs and increased risk of ADRs. However, CYP2C9 alone explains a relatively small proportion of risk of ADR, which is currently better predicted using clinical covariates such as age, sex, concomitant drugs, and comorbidities. We propose to better understand the heritable risk of NSAID ADR by performing a genome-wide association study for NSAID ADR in a diverse population and using it to develop a polygenic risk score (PRS). Furthermore, to improve cross-ancestry performance of our PRS, we will develop a transcriptomic risk score (TRS) based on imputed transcriptomes and integrate it with our PRS. We will then build a multi-modal model that combines pharmacogenetics, genomics, and clinical variables to predict ADR risk. Successful completion of both aims would prevent countless NSAID-induced ADRs and improve our understanding of the risk factors underlying ADR risk. Beyond that, our work will serve as a model for future application of multi-omics to augment pharmacogenetics, bringing us closer to “the right drug, for the right patient, at the right time.”

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Candidate: Ari B. Friedman, MD, PhD is junior faculty at the University of Pennsylvania (Penn). He practices clinically as an emergency physician and conducts research as a health economist into how care systems affect patients' lives. He is passionate about improving the care and outcomes of the many older patients that he sees in the emergency department (ED) by improving the science underlying that care. Goal: The purpose of this K23 Career Development Award is to enable Dr. Friedman to gain knowledge and skills necessary to become an independent investigator focusing on the emergent care of older patients. Research Context: Visits to hospital EDs are a growing source of care for adults over 65, totaling 22.4 million visits in 2017. Among older patients, abdominal pain is the single most common chief complaint. Simply being over 65 years, presenting to an ED, and stating at triage, “I have abdominal pain,” as a chief complaint confers a mortality rate as high or higher than an ST-Elevation Myocardial Infarction (STEMI), with equivalently high morbidity. Yet little is known about this phenomenon, especially for patients with frailty or cognitive impairment. Career Development Plan: Dr. Friedman seeks training in frailty, cognitive impairment and dementia, geriatrics, primary data collection, and health disparities. This award will provide protected time for Dr. Friedman to acquire the skills necessary to secure independent funding through a progressive, milestone- driven training plan that includes coursework, mentored research, and guided independent study. Aims: The central hypothesis of this proposal is that there are identifiable ways to improve the evaluation and management of abdominal pain among older patients in the ED. Dr. Friedman will use the training funded by this Mentored Patient-Oriented Research Career Development Award to examine variation in ED abdominal pain testing, management, disposition, and outcomes overall and among patients with frailty, cognitive impairment, among disadvantaged populations, and in periods when resources do not meet demand. Research Plan: These Aims will be carried out by using primary data collected through the proposed ED Geriatric Abdominal Pain (ED-GAP) cohort with additional variables from the Penn Electronic Health Record (EHR). This data will capture both management and outcomes for admitted patients and contains measurements of essential factors associated with aging. Retrospective EHR data of abdominal pain visits to 6 EDs over 8 years will also be used. Multivariate regression will test the hypotheses above. Environment: Senior researchers with an established track record of mentoring scholars from their early career to independent and impactful work are committed to Dr. Friedman's success. This team is embedded within a university environment with a track record of collaborative, pathbreaking scholarship in health economics, health services research, emergency medicine, and gerontology.

NIH Research Projects · FY 2025 · 2023-09

In response to NIMH Strategic priorities “to map the connectomes for mental illness, harness the power of data” and “develop computational approaches” and the Notice of Special Interest (NOT-MH-21-175) regarding the “Use of Human Connectome Data for Secondary Analysis” we leverage data across four Connectomes Related to Human Disease (CRHD) projects, as well as the Human Connectome Project (HCP) Aging and Development Lifespan and the Adolescent Brain Cognitive Development (ABCD) studies to address a major challenge in our field. Specifically, we use novel computational approaches to identify cohesive symptom/cognitive dimensions and subtypes across the continuum of anxious misery disorders in relation to the natural heterogeneity of brain network alterations. This proposal capitalizes on an established record of collaboration between independent CRHD projects and the HCP data core. Multimodal magnetic resonance imaging (MRI), clinical and cognitive data will be integrated for 2,187 people, including 531 adults and 150 adolescent patients with anxious misery disorders, and 1,506 matched healthy people. Cutting edge HCP and UK Biobank processing streams will centrally process data to derive harmonized multimodal imaging features or phenotypes (IDPs) across datasets, extracted from resting state functional MRI, task-derived functional MRI, structural MRI and diffusion imaging data for use in analyses and for dissemination with the scientific community (Aim 1). Using a novel group regularized canonical correlation analysis (GRCCA), we will evaluate the covariation between IDPs and clinical and cognitive measures to identify brain network-symptom/cognitive dimensions of anxious misery across adolescents and adults (Aim 2). In tandem, we will use the unsupervised Uniform Manifold Approximation and Projection (UMAP) with Density-based Spatial Clustering of Applications with Noise (DBSCAN) applied to identify anxious misery subtypes (Aim 3) distinguished by brain network IDP profiles and symptom and cognitive measures. Both data-driven analysis approaches will be applied to determine similarities of brain network- symptom/cognitive dimensions and subtypes across adolescent and adults and their influence on antidepressant treatment outcomes. Our preliminary data suggest these methods will advance our understanding of the links between brain network dysfunction and specific psychopathology across age and in relation to antidepressant response well beyond DSM diagnoses. Public Health Significance: Successful completion of this project will deconstruct and validate the natural heterogeneity of brain circuit alterations underlying transdiagnostic anxious misery disorders. The resulting brain- clinical phenotypes will yield a robust set of dimensional and subtype targets for future clinical and mechanistic investigations of heterogeneity in anxious misery disorders across the lifespan. These phenotypes can also be used to inform precision medicine approaches to individualizing mechanistic and novel treatment studies.

NIH Research Projects · FY 2024 · 2023-09

In response to NIMH Strategic priorities “to map the connectomes for mental illness, harness the power of data” and “develop computational approaches” and the Notice of Special Interest (NOT-MH-21-175) regarding the “Use of Human Connectome Data for Secondary Analysis” we leverage data across four Connectomes Related to Human Disease (CRHD) projects, as well as the Human Connectome Project (HCP) Aging and Development Lifespan and the Adolescent Brain Cognitive Development (ABCD) studies to address a major challenge in our field. Specifically, we use novel computational approaches to identify cohesive symptom/cognitive dimensions and subtypes across the continuum of anxious misery disorders in relation to the natural heterogeneity of brain network alterations. This proposal capitalizes on an established record of collaboration between independent CRHD projects and the HCP data core. Multimodal magnetic resonance imaging (MRI), clinical and cognitive data will be integrated for 2,187 people, including 531 adults and 150 adolescent patients with anxious misery disorders, and 1,506 matched healthy people. Cutting edge HCP and UK Biobank processing streams will centrally process data to derive harmonized multimodal imaging features or phenotypes (IDPs) across datasets, extracted from resting state functional MRI, task-derived functional MRI, structural MRI and diffusion imaging data for use in analyses and for dissemination with the scientific community (Aim 1). Using a novel group regularized canonical correlation analysis (GRCCA), we will evaluate the covariation between IDPs and clinical and cognitive measures to identify brain network-symptom/cognitive dimensions of anxious misery across adolescents and adults (Aim 2). In tandem, we will use the unsupervised Uniform Manifold Approximation and Projection (UMAP) with Density-based Spatial Clustering of Applications with Noise (DBSCAN) applied to identify anxious misery subtypes (Aim 3) distinguished by brain network IDP profiles and symptom and cognitive measures. Both data-driven analysis approaches will be applied to determine similarities of brain network- symptom/cognitive dimensions and subtypes across adolescent and adults and their influence on antidepressant treatment outcomes. Our preliminary data suggest these methods will advance our understanding of the links between brain network dysfunction and specific psychopathology across age and in relation to antidepressant response well beyond DSM diagnoses. Public Health Significance: Successful completion of this project will deconstruct and validate the natural heterogeneity of brain circuit alterations underlying transdiagnostic anxious misery disorders. The resulting brain- clinical phenotypes will yield a robust set of dimensional and subtype targets for future clinical and mechanistic investigations of heterogeneity in anxious misery disorders across the lifespan. These phenotypes can also be used to inform precision medicine approaches to individualizing mechanistic and novel treatment studies.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Heart failure morbidity and mortality disproportionately impact racial minorities, and inequitable care delivery based on race is pervasive in the United States. While the American Indian/Alaska Native population is one of the populations that suffers most from health disparities, little is known about their burden of cardiovascular disease, particularly heart failure. In fact, there are no studies in the current era evaluating the burden and outcomes of heart failure among the American Indian/Alaska Native population, or the quality of care they receive. The proposed mixed-methods research plan aims to characterize the burden of disease, outcomes, and quality of care of American Indian/Alaska Native patients with heart failure with reduced ejection fraction receiving care through the Indian Health Service— the principal health care provider for the American Indian/Alaska Native population, as well as to determine implementation strategies to ensure quality heart failure care for this population. Aim 1 seeks to characterize the clinical characteristics, as well as rates of guideline directed medical and device therapy and outcomes of American Indian/Alaska Native patients with heart failure receiving care through the Indian Health Service. This aim will utilize a large administrative clinical and claims database. Aim 2 will augment this analyses by employing qualitative research methods to interview American Indian heart failure patients discharged after a heart failure hospitalization, as well as primary providers at two Indian Health Service sites in Navajo Nation to better understand barriers to quality heart failure care. Aim 3 will employ an implementation mapping approach with a stakeholder taskforce at the same sites to determine the highest priority strategies to improve heart failure care for this population. These aims will lead to a future pilot pragmatic clinical trial of American Indians with heart failure in Navajo Nation, which will be used to evaluate the effectiveness of the highest priority strategies to improve care identified in aim 3. These research aims are part of a comprehensive training plan and will be supervised by a mentorship and advisory team consisting of national leaders in health services and implementation science research, and advanced statistical and qualitative methodologies, and will guide my transition to an independently funded research career.

NIH Research Projects · FY 2024 · 2023-09

Neurons rely on autophagy to constitutively degrade damaged proteins and organelles to maintain neuronal homeostasis. When neuronal autophagy capacity is overwhelmed, aggregated proteins and damaged organelles accumulate and contribute to neuronal dysfunction or death. Indeed, autophagy dysfunction is consistently associated with neurodegenerative diseases, such as Parkinson’s Disease. How do neurons respond when faced with the accumulation of dysfunctional proteins and organelles that overwhelm autophagy? Do neurons employ alternate quality control mechanisms? Recent findings suggest that autophagy-dependent degradation and autophagy-dependent secretion can act in coordination to regulate cellular homeostasis in non-neuronal cell types. When autophagosome maturation is impeded, autophagy-dependent secretion of extracellular vesicles (EVs) can be initiated as a mechanism to unburden the degrative machinery. Whether neurons similarly extrude damaged material via autophagy dependent secretion is unclear. I hypothesize that stressed neurons engage autophagy-dependent secretion as an alternate quality control mechanism to dispel cellular waste, e.g., mitochondria, which is then internalized by surrounding astrocytes. This model is supported by my preliminary findings that neurons with reduced capacity for autophagy upregulate secretion of EVs. Of particular interest, I find that mitochondrial proteins are shunted from degradation toward a secretory fate in chronically stressed neurons. These observations have important implications in neurodegenerative diseases where autophagy is strained, and the expulsion of mitochondria could heighten systemic inflammatory responses. This proposal will contribute to our fundamental understanding of neuronal homeostasis mechanisms and could reveal important clues underlying neurodegenerative disease. In Aim 1, I test the hypothesis that stressed neurons shunt autophagy cargo toward secretion via secretory autophagy. I will track secretion of EVs from neurons facing chronic or acute autophagic stress and determine which autophagy proteins are required for compensatory secretion. Using immunoblotting and high-resolution microscopy, I will confirm the secretory autophagy pathway prompting secretion in stressed neurons. Finally, I will use proteomics to molecularly profile the cargo expelled via autophagy dependent secretion. In Aim 2, I test the hypothesis that neurons expel mitochondria for transcellular internalization. I will use neuronal- astrocyte co-culture to visualize neuronal mitochondria internalized by nearby astrocytes. Next, I will track astrocyte engulfment of neuronal mitochondria in vivo.

NIH Research Projects · FY 2025 · 2023-09

A growing number of patients with advanced heart failure are undergoing HeartMate 3 (HM3; Abbott, Abbott Park, IL) left ventricular assist device (LVAD) implantation around the world. Despite contemporary durable mechanical support of the heart, LVAD recipients continue to demonstrate low levels of physical activity and functional capacity, characterized by daily steps and six-minute walk test (6MWT) distance, even 1-year after surgery. In addition, LVAD recipients frequently meet criteria for prefrailty/frailty and associated sarcopenia, including low muscle strength and muscle quantity. These deficits may contribute to increased healthcare utilization in this high-risk population. Walking and strengthening exercises can help to improve muscle strength and quantity as well as physical activity and capacity, but exercise interventions in LVAD recipients are not well established. A large proportion of LVAD recipients do not participate in center-based, supervised cardiac rehabilitation (CR), and patients participating in CR initiate therapy a mean of 3 months after hospital discharge. A home-based exercise program using mobile technology may allow patients to begin exercise rehabilitation at an earlier time after LVAD implantation. We are conducting a randomized, controlled pilot study of a home-based exercise program including walking and strengthening exercises with a standardized protocol to guide exercise prescription and exercise progression based on data obtained from an activity tracking watch and smartphone app. Our pilot data suggest that our innovative home-based exercise program is feasible, safe, and may be associated with a greater increase in daily physical activity in patients newly implanted with a HM3 LVAD. The effects of a home-based exercise program using mobile technology on daily physical activity and capacity, frailty and sarcopenia, and quality of life in LVAD recipients have not been previously studied in a multicenter study. We propose a randomized study of 80 newly implanted HM3 LVAD patients at 4 LVAD centers. Following index hospital discharge after LVAD implantation, patients will be enrolled and randomized to a 6- month novel exercise program using mobile technology or usual care (UC). We will evaluate the following specific aims: Specific Aim #1 is to compare the effects of the exercise intervention vs. UC on physical activity and capacity measured by a) daily steps (captured by the activity monitor) and b) 6MWT distance. Specific Aim #2 is to compare the effects of the exercise intervention vs. UC on frailty and sarcopenia, by evaluating a) frailty status and b) muscle quantity. Specific Aim #3 is to compare the effects of the exercise intervention vs. UC on quality of life (measured by the Kansas City Cardiomyopathy Questionnaire-12). Specific Aim #4 is to evaluate the cost-benefit of the exercise intervention vs. UC. Findings from this study have the potential to improve low physical activity and capacity, frailty and sarcopenia, quality of life, and increased rates of healthcare utilization in the growing population of LVAD recipients.

NIH Research Projects · FY 2025 · 2023-09

Summary The skin forms a protective barrier against harmful substances and pathogens that we contact through our immediate environment. Specialized glands in the skin known as sebaceous glands secrete sebum, a lipid-rich substance with anti-microbial peptides (AMPs) that enhances the physical, chemical, and immunologic barrier function of the skin. This proposal describes a 5-year research plan focusing on the novel concept that the skin microbiome regulates a newly described immune-mediated sebum axis as a feedback mechanism to enhance skin barrier function and to create an optimal skin microenvironment. We have recently reported that sebum secretion can be regulated by immune cells in response to a keratinocyte-derived cytokine called thymic stromal lymphopoietin (TSLP). In this process, which we call the immune-sebum axis, TSLP stimulates T cells directly through their TSLP receptor (TSLP-R), which induces their migration to the sebaceous glands to promote sebum secretion in an IL-4/13-dependent manner. We now provide preliminary data suggesting that the trigger of the immune-sebum axis could be the skin microbiome. We find that compared to conventionally raised mice, the skin of germ-free mice contains significantly fewer T cells, exhibits ~50% reduction in sebum secretion, and displays lower expression of TSLP, sebum synthesis- related genes, and AMPs. In addition, we found that mice lacking the IL-1 receptor or MyD88 (a molecule critical for signaling downstream of IL-1 family cytokine receptors and Toll-like receptors) also display ~50% reduction in sebum secretion. Thus, we propose that a feedback mechanism exists to maintain homeostasis of the skin microenvironment, whereby skin microbes promote sebum secretion, which in turn controls their growth. Based on these preliminary data, we hypothesize that skin microbes induce the IL-1/MyD88 signaling pathway in the skin to promote sebum secretion and skin barrier function in a TSLP- stimulated T cell-derived IL-4/13-dependent manner. We will test this hypothesis in 3 separate aims. Aim 1 will investigate how the skin microbiota and MyD88 regulate sebum secretion. Aim 2 will probe how IL-4/13 derived from TSLP-stimulated T cells affects sebaceous gland function. Aim 3 will test whether the skin microbiota and TSLP affect skin barrier function and protects against pathogenic skin infection in a sebum- dependent manner. This multidisciplinary project will be led by two co-PIs (Drs. Kambayashi and Grice), who share complementary expertise. Dr. Kambayashi is an expert in T cell biology, cellular immunology, and signal transduction. Dr. Grice is an expert in skin barrier function and skin microbiota. Other collaborators include Dr. Amanda Nelson, as an expert in sebaceous gland biology and Dr. Sunny Wong as an expert in sebaceous gland development. This investigation will provide novel insight into how the skin microbiome engages both the innate and adaptive immune system to promote optimal skin barrier function for the host.

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract Conventional sintering (CS) protocols produce high quality zirconia restorations suitable for a wide range of indications. However, CS requires a firing cycle of 4 – 10 h, a bottleneck in digital dental workflow precluding zirconia from chairside applications. Current speed sintering (SS) protocols using fast heating (up to 6C/s) in an induction furnace can reduce sintering times to 0.3 – 0.5 h. However, because of inefficiency of convective heat transfer, leading to temperature inhomogeneity, SS produces microstructures with higher porosities, thus compromising zirconia translucency and strength. In addition, non-uniform densification raises concerns about chemical and dimensional stability, internal fit and marginal adaptation of restorations. As a result, SS is largely limited to the fabrication of single-unit crowns from 4 mol% yttria stabilized zirconia (4YSZ). Accordingly, the long-term goal is to drastically increase sintering speed (on the order of 60 s) while maximizing mechanical and optical properties of dental zirconia (exceeding those of the SS- and CS-YSZ) by implementing novel UFS technologies. The overall objectives of this proposal are to (1) establish composition and time-temperature- transformation (TTT) relationships to guide material selections for various industries and sectors, with special attention to the optimization of strength and translucency of YSZ for dental applications; and (2) demonstrate improved dimensional, long-term chemical and structural stabilities pertaining to the quality and longevity of UFS-YSZ restorations relative to SS and CS. The central hypothesis is that novel UFS methodology will dramatically increase time efficiency of digital workflow while optimizing zirconia properties and expanding the range of indications for single-visit treatments. This hypothesis follows directly from preliminary results and a state-of-the-art material science knowledge base. To test this hypothesis, we will pursue 3 specific aims: (1) To characterize the properties of yttria stabilized zirconia using ultrafast sintering technology in conjunction with high-throughput fail-fast screening; (2) To determine the resistance to low temperature degradation and fatigue fracture of ultrafast sintered zirconia relative to current speed and conventional sintering; and (3) To evaluate the dimensional stability, internal fit, and marginal adaptation of ultrafast sintered 3-unit fixed dental prostheses relative to current speed and conventional sintering. The approach is innovative because it departs completely from the current furnace-sintering concept by using Joule heating elements with more effective radiation and conduction heat transfer. The proposed research is significant because it addresses current challenges in poor material properties associated with SS and the long sintering time of CS. Such an approach will improve the efficiency and accuracy of restorative procedures to provide more treatment options and better patient experience, thus improving quality of life and reducing cost to the patient.

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 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 2024 · 2023-09

Project Summary This proposal “Next generation T cell therapies for KRAS mutated solid tumors” was developed in response to RFA-CA-22-028 and to fulfill the Cancer Adoptive Cellular Therapy Network (Can-ACT) objectives. The focus of our proposal is targeting mutant KRAS, a clonal driver oncoprotein, by the early-stage clinical testing of TCR1020, a T cell receptor specific for mKRAS G12V restricted to HLA-A*11:01. If successful, this novel-state- of-the-art Adoptive Cell Therapy (ACT) could be available to ~5,000 new solid tumor patients per year in the US. Advances in gene editing together with new insights related to mechanisms of T cell exhaustion provide the scientific basis for development of the next generation T cell therapies in solid tumors. Our central hypothesis is that targeting mutant KRAS through the action of TCR1020-T cells, engineered to overcome cell intrinsic mechanisms of exhaustion and counteract a cell extrinsic myeloid checkpoint to overcome TME resistance, will promote durable tumor regression in solid tumors. There are three hypothesis-driven specific Aims in this proposal. In Aim 1, we will develop genetically modified T cells expressing TCR1020 targeting mKRAS G12V/HLA-A*11:01 to overcome extrinsic and intrinsic mechanisms of resistance. In Aim 2, we plan to evaluate TCR1020-T CD4+ cells to improve the persistence and potency of mKRAS specific CD8+ effector cells. In Aim 3, the safety and clinical activity of engineered TCR1020-T cell products will be determined in a dose escalation multi-site phase 1 study. We have assembled an exceptional group of investigators with an extensive track record of collaboration and productivity with expertise in human immunology, immuno-oncology, cell therapy, T cell engineering and gene editing. Additionally, experts in molecular pathology and biomarker discovery will contribute to cutting-edge correlative studies to aid in biomarker development and TME characterization to delineate potential mechanisms of response and resistance. The Center for Cellular Immunotherapies (CCI) at the University of Pennsylvania has extensive expertise in the development of ACT therapies producing more than 2,500 cell products for administration to adult and pediatric patients. CCI has a long track record of technology transfer related to cell therapies to both large pharmaceutical companies (Novartis, Kymriah) and biotechnology companies over the past decade. As a multi-site trial application, an important programmatic component of our proposal is to leverage NCI resources thru utilization of the Immune Cell Network (ICN) Core at FNLCR to manufacture, test, release and distribute the engineered TCR1020-T cell products. In summary, our proposal incorporates multiple scientific and technological innovations that targets a recurrent clonal driver oncoprotein with engineered T cells modified to resist T cell exhaustion and overcome the immunosuppressive TME.

NIH Research Projects · FY 2024 · 2023-09

Project Summary Acute myeloid leukemia (AML) is a lethal blood cancer characterized by a differentiation “block” that prevents myeloid progenitor maturation resulting in uncontrolled proliferation. Chemotherapy, the current standard of care, is often ineffective and can result in cytotoxicity and relapse. New treatment options are desperately needed to treat AML’s poor prognosis. Differentiation therapy is a novel method that aims to reactivate latent maturation programs and induce cell cycle exit. This therapeutic strategy is curative in the promyelocytic (APL) AML subtype but underexplored in other AMLs. Epigenetic factors help sustain the characteristic AML differentiation block. The demethylase LSD1 has emerged as a promising target for differentiation therapy. Pharmacologic inhibition of LSD1 (LSD1i) induces cellular differentiation in many AML subtypes. However, the extent of differentiation varies between AML models, with a modest effect in aggressive AML models. Therefore, LSD1i will not induce terminal differentiation as a monoagent treatment. We hypothesized that targeting additional epigenetic regulators simultaneously with LSD1i may induce complete, terminal differentiation and lead to disease remission. To identify potentiators of LSD1i, we conducted CRISPR gain-of-differentiation screens with a chromatin-focused sgRNA library in multiple AML cell models with or without LSD1i co-treatment. These screens unveiled a synergistic induction of differentiation when KO of MEN1 is combined with LSD1i. I confirmed that combinatorial small molecule inhibition of LSD1 and MEN1 induces differentiation and reduces proliferation most commonly in MLL-rearranged AMLs, and to a lesser extent in selected MLL-wild type AMLs. This proposal has two aims: First, I will test the therapeutic potential of targeting MEN1 in combination with LSD1 inhibitors in primary patient samples ex vivo and in patient-derived AML transplant models. Patient samples will be treated with a MEN1 inhibitor in combination with LSD1 inhibitors, and effects on differentiation and proliferation will be quantified. Secondly, I will determine the epigenomic mechanisms by which MEN1 and LSD1 inhibition synergize to induce terminal differentiation, and then test whether these mechanisms are conserved in primary patient samples.