University Of Pennsylvania

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY When bacteria invade the eye, the resulting damage is rapid and irreversible. This type of infection, called endophthalmitis, is the most severe complication of intraocular surgery. Patient outcome is heavily dependent on the pathogenicity of the bacteria, its susceptibility to antibiotics, and the extent of inflammation. These factors can influence progression to the worst-case scenario, where the infected eye must be completely removed. Even in milder cases, some amount of permanent vision loss is common. Therefore, there is need for a treatment which targets both contributors to vision loss: bacterial growth and neurotoxic inflammation. All bacteria require iron for survival and proliferation. During infection, they must obtain iron from host tissues. In response, host cells will import iron to shield it from bacteria, initiating a competition for iron acquisition. However, iron accumulation within immune cells can promote inflammation. In animal models of diverse infections and diseases, iron chelation is both anti-bacterial and anti-inflammatory. My preliminary ex vivo data suggests that iron chelation inhibits bacterial growth in vitreous (fluid from within the eye), and that acute iron chelation is nontoxic to the retina. Aim 1 will expand on these results to determine the effectiveness of an iron chelator at preventing endophthalmitis in mice. I will examine the extent to which bacterial growth is inhibited and retinal structure is preserved over time. Although reducing bacteria would naturally reduce inflammation, the direct anti-inflammatory contribution of iron chelation would be unclear. Therefore, Aim 2 will investigate the link between iron accumulation and retinal inflammation. Using the murine endophthalmitis model, I will examine iron levels within retinal macrophages/microglia, and to what extent an iron chelator reduces retinal inflammation. The result will determine the anti-inflammatory potential of iron chelation treatment for endophthalmitis. The overall goal of this proposal is to prevent vision loss caused by bacterial infection.

NIH Research Projects · FY 2025 · 2023-07

Overall Abstract Alzheimer’s disease (AD) affects 5.8 million people in the United States and is an immense burden on our economy, patients and caregivers. Genome-wide association studies (GWAS) have successfully led to 25 genome-wide significant loci associated with AD risk and many more associations with key clinical covariates. Most of these findings are made on participants with European ancestry, although efforts to study other minority populations are taking off. Knowledge about AD genetics among Asian Americans is especially limited due to lack of participants. Comprising 6% of the US populace, Asian Americans are under-sampled and deserve more scientific investment. We propose the Asian Cohort for Alzheimer’s Disease (ACAD), the first large Alzheimer’s Disease (AD) genetics cohort for Asians in United States (US) and Canada. To address recruitment barriers, we assembled a team of scientists, clinicians, and community partners with collaborative history and expertise in AD research, human genetics, and Asian community outreach. We propose to recruit 5,081 participants aged 60 years or older and of Chinese, Korean, and Vietnamese ancestry from metropolitan areas across US and Canada in collaboration with community health providers or long-term care facilities that serve Asian communities. We will collect DNA and plasma biomarkers and use validated, translated data collection forms and clinical/diagnostic protocols. To support these recruitment and data collection activities, we will form six Cores that provide administrative oversite, outreach, clinical expertise, data management, biosample management, and training and quality assurance to support recruitment and analysis activities. All samples will be genotyped using SNP arrays and imputed using a large Asian-specific reference panel of whole genome sequencing data from international Asian cohorts. We propose two Research Projects that will analyze genetic, biomarker and clinical data to investigate impact of lifestyle risk factors, genetic variants for AD risk, evaluate differential effects of sex and APOE genotypes on AD risk, and predict clinical diagnosis of AD using genetic and lifestyle risk scores. We will replicate these findings through meta-analysis collaborations with international Asian cohorts and AD studies from other populations.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Neuroblastoma is a cancer derived from the developing sympathetic nervous system and is the most commonly diagnosed extracranial solid tumor of childhood. Despite an intensive treatment regimen of chemotherapy, surgery, radiation, and immunotherapy, the five-year survival rate of high-risk neuroblastoma patients remains at only 50%. The low tumor mutational burden of neuroblastoma has challenged the development of targeted- and immuno-therapies, however moderate success has been achieved by targeting GD2 with monoclonal antibody therapy, credentialing immunotherapeutic treatment strategies for neuroblastoma as a promising approach. The success of GD2-targeted therapy to date has been hindered due to debilitating side effects from on-target/off-tumor toxicity since GD2 is also expressed on pain fibers, and antigen loss as a mechanism of therapy resistance. Therefore, there is an unmet need for the discovery of new therapeutic targets in neuroblastoma. B7-H3, encoded by the CD276 gene, is a type 1 transmembrane protein in the B7 family of immunoregulatory proteins and is highly expressed in many adult and pediatric cancers, including neuroblastoma. In addition to being implicated in immunoinhibition, B7-H3 may also mediate tumor migration and metastasis. Preclinical success of several immunotherapeutic strategies directed toward B7-H3, including CAR-T cells and antibody drug conjugates, suggest that B7-H3 is a targetable tumor-associated antigen with several pediatric clinical trials ongoing or planned. Therefore, it is critical to understand the oncogenic functions of B7-H3 and how its expression is regulated to anticipate mechanisms of therapy resistance. Our central hypothesis is that B7-H3 promotes neuroblastoma metastasis and immune evasion, and its expression is regulated by tumor microenvironment-derived cytokines and neuroblastoma-specific transcription factors. Our preliminary data shows that B7-H3 knockdown using CRISPRi in neuroblastoma cell lines inhibits cellular proliferation. Additionally, ChIP-sequencing data identifies regions of MYC and MYCN binding at the B7-H3 promoter indicating a potential role of the MYC transcription factors in regulating B7-H3 expression. B7-H3 expression may also be regulated by inflammatory cytokines, as neuroblastoma cell lines upregulate B7-H3 expression following TNF-α or TGF-β exposure. Finally, recombinant human B7-H3 inhibits T cell activation, TNF-α, and IFN-y. Uncovering how B7-H3 promotes immune evasion in neuroblastoma is crucial given the T and NK cell-based immunotherapies undergoing clinical testing. We propose that B7-H3 is a multifunctional protein that serves as a promising therapeutic target in neuroblastoma. This NRSA F31 will define the mechanisms of neuroblastoma dependance on B7-H3 for metastasis and immune evasion, while also defining mechanisms of overexpression, to inform future B7-H3-targeting therapies and ultimately improve outcomes for patients with high-risk neuroblastoma.

NIH Research Projects · FY 2026 · 2023-07

Project Summary Coronary heart disease is the leading cause of death worldwide. Patients are treated with clot-busting drugs (thrombolytic therapy) or the artery is reopened with a catheter (percutaneous coronary intervention), but, paradoxically, restoration of blood flow to the heart muscle can cause additional injury that limits the success of these procedures. Reperfusion injury appears to be a cascade of events, initiated by coronary artery extravasation (hemorrhage), culminating in a failure to restore myocardial perfusion and leading to cell death among viable cardiomyocytes in the peri-infarct region. It may be a predictor of long-term adverse clinical outcomes or provide a therapy-modifiable target in patients with myocardial infarction. Imaging methods specific to the molecular mechanisms of reperfusion injury would improve clinical care and our scientific understanding of this disease. We hypothesize that iron is a catalyst for reactive oxygen species production in reperfused myocardial infarction. We propose a series of experiments to investigate the association between iron and reactive oxygen species using iron-sensitive magnetic resonance imaging and positron emission tomography. We will validate these in vivo non-invasive imaging modalities with a battery of pathology, immunohistochemistry, and spectrometry techniques in a large animal model. In Aim 1, we will investigate the association between reactive oxygen species, iron and severity of infarction using MRI and PET. In Aim 2, we will determine the extent of association between imaging markers of reperfusion injury and remodeling of the left ventricle weeks after injury. This study will give a new understanding of the spatiotemporal relationships of reactive oxygen species in the sub-acute and chronic period of myocardial infarction wound healing. This will lead to studies in large animal models and humans that can evaluate therapies targeting imaging biomarkers of reperfusion injury.

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT This program is directed at developing a new approach to OSA care based on the principles of Precision Medicine, with a focus on improving prediction, prevention and personalization. The program has 4 projects and 3 cores to support the work of the investigators. Project 01 (Genetics of Extreme Phenotypes of OSA and Associated Upper Airway Anatomy) is focused on identifying both common and rare genetic variants that are associated with risk for OSA. Since OSA has multiple pathways to disease, identifying associated genetic variants is challenging. This project investigates gene variants associated with quantitative intermediate traits for the disorder. The focus is on structural risk factors—both craniofacial dimensions and soft tissues. A focus is on tongue fat, a specific heritable distribution of fat that mediates the effect of obesity in causing OSA. Machine learning approaches have been developed to allow quantification of traits of interest from a large number of relevant clinically-obtained CT and MR images in individuals with genetic data. Data from this project will be used in combination with data from ongoing genetic studies to develop a polygenic risk score (PRS) for OSA with wide-applicability. Project 02 (MicroRNAs as Biomarkers for Obstructive Sleep Apnea) will study microRNAs as a biomarker relevant to OSA using RNA sequencing of all short microRNAs. Expression of microRNAs is dynamic; they respond to multiple challenges, including hypoxia. MicroRNAs are being used in development of biomarkers in multiple areas, with supportive data in OSA, albeit in relatively small samples. Thus, we will employ a combination of a hypothesis-driven approach complimented by a broader discovery strategy. Biomarkers will be developed to help identify cases with OSA, as well as to assess effectiveness of therapy and to provide prognostic information about who with OSA will have blood pressure reduction with treatment. Project 03 (Mechanisms that Account for Different Symptom Subtypes of OSA) will examine the physiological and multi-omics determinants of robustly validated symptom subtypes of OSA. There will be an emphasis on the excessively sleepy subtype, which has been shown to be at elevated cardiovascular risk. We will evaluate whether there are differences in physiological responses during sleep in the different subtypes and/or whether there are genetic, epigenetic, and metabolomic differences. Project 04 (Going from Genetic Associations to Identification of Causative Genes) will focus on identifying causative genes that explain GWAS associations. For genes conferring risk for OSA, we will begin with existing GWAS data complimented by data from Project 01. For sleepiness, this project will start with recently published genetic loci, and include analyses based on genes identified in Project 03. We will first use cell-based approaches to identify possible causative genes. The role of these genes will be assessed in high-diversity mouse models (for genes associated with anatomy) and in Drosophila and zebrafish (for genes associated with sleepiness).

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY Early screening and prevention of individuals at risk of complex diseases are important strategies for reducing morbidity and mortality. Polygenic risk scores (PRS) are the cumulative, mathematical aggregation of risk derived from the contributions of many DNA variants across the genome. PRS are an emerging technology in the field of disease risk prediction and have been shown to be correlated with disease incidence. While PRS have shown great promise for complex diseases, current PRS models are overly simplistic and have limited predictive power and clinical utility. PRS do not account for the effects of rare genetic variants or other risk factors (clinical, environmental, social determinants of health) on disease risk. Rare variants generally have greater effects on disease risk due to selective pressure, but only a small number of individuals carry any single rare variant. The sparsity of rare variants makes it difficult to directly incorporate them into PRS. Additionally, while it is known that clinical, environmental, and social risk factors also influence risk, few analyses have successfully integrated PRS with these important non-genetic factors. To address this issue, we will develop novel translational informatics methods that integrate clinical, environmental, and genetic data to improve disease risk prediction. We will assess the clinical utility of these integrated risk prediction models using cardiovascular disease (CVD) to evaluate the potential for translation to clinical use. Based on the complexity of CVD, we hypothesize that a comprehensive range of risk factors along with rare variants need to be incorporated into PRS to improve the risk prediction and maximize the clinical utility of PRS for CVD. To achieve our goal, our specific aims are: 1) To develop novel methods that incorporate rare genetic variants into Polygenic Risk Scores (PRS); 2) To evaluate Integrated Risk Models that combine clinical, environmental, and social risk factors with PRS; 3) To develop and evaluate deep learning models integrating genetic, clinical, environmental, and social risk factors; 4) To translate our integrated models into the electronic health record (EHR). If these specific aims are achieved, we will have a set of integrated models that can be used in downstream clinical implementation programs to ultimately have a translational impact on disease treatment and prevention. Using these novel computational risk prediction models for precision health, along with our EHR integration approaches, will allow for the translation of integrated risk prediction into routine clinical care.

NIH Research Projects · FY 2025 · 2023-07

Many types of solid tumors are innervated by distinct branches of the nervous system that, like the immune system, sense and respond to internal and environmental stimuli. However, nerves have long been viewed as passive bystanders in cancer. It is poorly understood how the nervous system regulates the tumor-associated immune responses, and what factors in the tissue-specific microenvironment shape tumor innervation. Therefore, the overarching goal of our research is to develop an in-depth and broad understanding of neuro-immune interaction in cancer with a focus on the bi-directional crosstalk between the sensory neurons and the tumor microenvironment (TME), and to explore whether we can target such interactions for more effective cancer treatment. Of particular interest, lung-innervating nociceptors are specialized sensory neurons that control cough and pain, the most common clinical symptoms in lung cancer patients. While nociceptors were shown to regulate lung immune cells in allergy and bacterial infections, little is known about their roles in oncogenesis. Our central hypothesis is that nociceptive sensory neurons play a key role in promoting lung cancer development via cross-talking to the immune cells, particularly the tumor-associated IL-17+T cells and neutrophils. As such, targeting the nociceptive neural pathways can improve the cancer response to immunotherapy by reprogramming the tumor immune microenvironment. Experimentally, we will combine genetically engineered mouse models that faithfully reproduce the human lung adenocarcinoma with cutting-edge approaches in cellular immunology, cancer genetics, and functional manipulations of neuronal circuits. Specifically, we propose to test our central hypothesis by (1) establishing the role of tumor innervation by sensory neurons in lung cancer, (2) elucidating the mechanisms by which tumor innervation shapes the cancer-associated immune responses, (3) determining the pre-clinical efficacy of targeting the nociceptive neural pathway in combination with checkpoint-based immunotherapy. Our study will provide fundamental insights to the emerging yet poorly understood functions and regulatory mechanisms of the sensory nervous system in the TME, especially their role in cancer- associated immune responses. Furthermore, the conceptual and technological advances generated here will build the foundation for future investigations into neuro-immune interactions in additional cancer types, shedding light on sensory neural pathways and neuro-immune crosstalk that can serve as novel therapeutic targets for cancer prevention and treatment.

NIH Research Projects · FY 2025 · 2023-07

PROJECT ABSTRACT/SUMMARY Multiplesclerosis (MS) is an immune-mediatedneurological disorder that affects one million people in the United States. Up to 50% of patients with MS experience depression, yet the mechanisms of depression in MS remain under-investigated. MS is characterized by white matter lesions, suggesting that brain network disruption may underly depression symptoms. Studies of medically healthy participants with depression have described associations between white matter variability and depressive symptoms, but frequently exclude participants with medical comorbidities and thus cannot be extrapolated to people with intracranial diseases. Previous research using lesion network mapping, a technique for mapping heterogeneous gray matter lesions to neuropsychiatric symptoms, has demonstrated that strokes in gray matter associated with depression disrupt a reproducible depression network. However, such techniques have never been applied to white matter disease or MS. Studying white matter lesions associated with depression in MS may provide a way to understand both the pathophysiology of depression in MS and general network mechanisms of depression more broadly. The purpose of this current study is to investigate how brain network disruption underlies depression by learning from the example of multiple sclerosis. In Aim 1, I will delineate how depression in adults with MS is associated with white matter lesion location and burden in a retrospective sample of 1,554 MS patients with research-grade 3T MRIs acquired as part of clinical care. Depression and MS diagnoses will be obtained from the electronic medical record. While this sample provides an ideal dataset for developing a model, the electronic medical record does not contain granular depression measures. In Aim 2, I will obtain structured clinical and cognitive assessments for MS patients and prospectively evaluate white matter integrity as a predictor of dimensional depressive symptoms. However, it is possible that symptoms of depression may reflect heterogenous brain network disruption patterns. Therefore, in Aim 3, I will use advanced semi-supervised machine learning methods to parse heterogeneity in MS white matter lesion burden in the retrospective sample and test whether this model predicts phenotypic heterogeneity in our deeply-phenotyped prospective sample. The support of the K23 award will provide the applicant with the training necessary to achieve these aims. The training objectives will be accomplished with the support of an outstanding mentorship team, Drs. Satterthwaite, Shinohara, Bassett, Bar- Or, Fox, McCoy, and the world class resources of the University of Pennsylvania. Together, the proposed scientific aims and training objectives will form the foundation for an independent research program that will use techniques from computational psychiatry to understand depression in patients with medical comorbidities.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY HIV-Associated Neurocognitive Disorder (HAND) is experienced by about half of people living with HIV (PWH), and presents as a spectrum of neurocognitive impairments. While the majority of PWH in the United States are virally suppressed or undetectable, the overall proportion of PWH with HAND symptoms has remained unchanged. One of the hallmarks of HAND is the atrophy of white matter, a pathology that persists in HAND patients treated with ART. The duration of ART treatment in PWH has been shown to correlate to the observed loss of white matter, leading to the concern that the drugs themselves are causing this pathology. The attenuation of white matter is associated with a loss of myelin, which in the CNS is produced by oligodendrocytes (OLs). Myelin is necessary for saltatory conduction and trophic support of axons and its loss may contribute to the clinical changes seen in HAND. Previous work from our lab has demonstrated that select ART drugs prevent the maturation of oligodendrocytes and myelination, though the mechanisms underlying these observations require additional investigation. We have further shown that treating maturing OLs with select ART drugs, such as Elvitegravir (EVG), activates the Integrated Stress Response (ISR), and inhibition of the ISR restores OL differentiation in the presence of EVG. The ISR is an adaptive pathway used to restore homeostasis in eukaryotic cells under stressful conditions, and is sometimes characterized by the formation of cytoplasmic stress granules (SGs), membraneless organelles thought to aid in the global inhibition of translation. Stress granules have not yet been extensively studied in oligodendrocytes, but research of other neurodegenerative diseases has found that their presence in neurons may cause acceleration of neurodegeneration. During chronic stress, SGs can become persistent, and the long-term aggregation of mRNAs and proteins can become pathological. There is strong evidence associating chronic SG accumulation in neurons with amyotrophic lateral sclerosis (ALS), Alzheimer’s, and frontotemporal dementia. Our lab has also observed SGs in post-mortem white matter of HIV+ patients with neurocognitive impairment. Furthermore, my preliminary data has shown that maturing OLs treated with EVG not only activate the ISR, but also form SGs that disappear with ISR inhibition. I hypothesize that during ARV drug treatment, stress granules sequester mRNAs needed for maturation of OLs via PERK activation of the ISR, and that these granules form to prevent cell death. I will test this hypothesis via three specific aims. AIM 1: I will determine if ARV Drug induced stress granules in OLs form via the PERK activated ISR. AIM 2: I will determine the contribution of SG formation to OL survival during ARV drug exposure. AIM 3: I will determine whether ARV-induced SGs in OLs sequester mRNAs needed for maturation and myelination. Since stress granules have not previously been characterized in oligodendrocytes, this work will not only provide insight into improving outcomes of ART treated PWH, but will also benefit research of other neurological diseases characterized by loss of white matter.

NIH Research Projects · FY 2024 · 2023-07

Project Summary Heart failure is most commonly associated with poor contractile function due to multi-level pathologic remodeling, including excitation-contraction coupling (ECC). This depends upon the proximity between membrane-bound L-type Ca2+ channels (LTCC) within the transverse (t)-tubule network and intracellular ryanodine receptors (RyR), which are normally very tightly colocalized. The PI and others have shown that abnormal mechanical load in vivo damages the t-tubule network, which results in uncoupling of LTCC and RyR. Junctophilin (JPH2), BIN 1 and Telethonin (TCAP), in interaction with the microtubule network, regulate t- tubule structure, but how they do so in response to load variation is not known. Prior experimental strategies have been unable to assess the effect of direct mechanical loading upon isolated cardiomyocytes, nor have they had the experimental flexibility to allow facile genetic manipulation of the pathways involved. Using new methods to directly modulate mechanical load on isolated cardiomyocytes and intact human myocardium in vitro, this K99/R00 seeks to test the hypothesis that t-tubule structure is normally regulated by a microtubule dependent JPH2, BIN1 and TCAP pathway, which in conditions of direct mechanical overload is deranged by microtubule mediated redistribution of JPH2, and reduced expression of JPH2, BIN 1 and TCAP. In Aim 1, using a novel magnetorheological elastomer (MRE) culture system, isolated cardiomyocytes will be subjected to pathological overload and undergo comprehensive characterization of ECC and t-tubule structure to test the hypothesis that cardiomyocyte-autonomous mechanisms are sufficient to mediate the load-dependent remodeling of the t-tubule system observed in heart failure. Because the phenotype arises in 48 hours, comprehensive dissection of the underlying molecular mechanisms will be performed by combined live cell imaging and adenoviral mediated genetic manipulations. Second, the novel but well-validated cardiac slice method will be used to specifically control pre-load and after-load in order to vertically integrate insights from cardiomyocyte-autonomous experiments in understanding the role of mechanical load regulation of the t- tubule system at the level of the isolated myocardium, including in human control and diseased myocardium. Mechanical unloading of failing hearts in vivo rescues t-tubule structure and ECC, which has been associated with significant contractile improvements. Using the tools developed in Aims 1 and 2, failing cells and slices will undergo mechanical unloading to determine the biomechanical and molecular mediators of this reverse remodeling. The completion of this work will significantly add to the PI's post-doctoral training in cellular electrophysiology, advanced super-resolution imaging and translational cardiovascular research and will be essential for his transition to independence.

NIH Research Projects · FY 2025 · 2023-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. The University of Pennsylvania MD/PhD and VMD/PhD programs were established in 1958 and 1969, and united as a single medical scientist training program in 1977. The program currently enrolls 218 MD/PhD and 22 VMD/PhD students, 95% of whom are training grant eligible. Program guidance is provided by a committed team of faculty and staff that has been in place since 2014 and was recently joined by a medical educator experienced in program evaluation. Our primary goal is to identify, train and mentor a group of outstanding physician-scientists who will become leaders of biomedical research and translational medicine, as well as being successful clinicians, role models and mentors. To meet this goal, we have established flexible, evidence-based training plans that integrate research and clinical training in preparation for careers that use both. Penn’s institutional commitment is reflected in a large annual investment that provides resources, enrichment activities, and support for program administration, as well as an institutional culture that values physician-scientists, promotes scientific rigor and a safe learning environment, and expects good mentorship. Admission is open to recent college graduates and current Penn MD, VMD and PhD students. Admission decisions emphasize research experience, creativity, and commitment to a physician-scientist career as well as academic excellence. The average time to degree is 8.1 years. There are 12 affiliated graduate programs: 7 in Biomedical Graduate Studies plus Engineering, Economics, Chemistry, History & Sociology of Science, and Anthropology, with protocols to add more when appropriate. The training faculty includes 161 junior and senior scientists and physician-scientists including 3 NIH intramural investigators who hold adjunct faculty appointments at Penn. Policies are in place to acquire and maintain training faculty membership and to resolve conflicts. Each student’s individualized curriculum emphasizes the integration of clinical and research training, responsible conduct of research, scientific rigor and reproducibility, and mentorship. It also includes MSTP-directed courses in Years 1 and 2, Clinical Connections during graduate school, Return to Research in the final year and, because our responsibilities do not end with graduation, the Hand Over Curriculum, which guides students in the selection of physician-scientist-friendly residencies and careers. 81% of recent graduates who have completed further training are employed by academic centers, research institutes, the biotech and pharmaceutical industries, the NIH, and federal agencies. Most have research funding from the NIH and other sources. Our objectives for the next 5 years include: 1) fostering the next generation of physician-scientists, 2) assisting trainees in exploring physician-scientist career options in addition to academia, 3) managing upward pressure on total training time, and 4) improving mentorship skills for faculty and trainees. These objectives are linked to an outcomes rubric that defines success, and a logic model whose metrics will enable continuous improvement.

NIH Research Projects · FY 2025 · 2023-07

Project Summary / Abstract 90% of individuals diagnosed with systemic lupus erythematosus (SLE) are women, but the underlying mechanisms that govern sex bias in autoimmune disease are not understood. Susceptibility to SLE increases with the number of X chromosomes carried by an individual, and increased expression of X-linked genes has been found in women with SLE. These findings suggest that gene expression from two X chromosomes likely contributes to female-biased SLE pathogenesis. The dosage of X-linked gene expression in mammals with more than one X chromosome is balanced by epigenetically silencing one X chromosome through X-chromosome inactivation (XCI). XCI is maintained in somatic cells by association of repressive epigenetic features with the inactive X chromosome (Xi), including coating of the Xi with the long non-coding RNA Xist. Despite these repressive features, some genes have been shown to escape silencing from the Xi. Our lab discovered that B cells, a key cell type in SLE pathogenesis, have “dynamic” maintenance of XCI, where naïve B cells lack visible Xist RNA signal yet demonstrate re-localization of Xist RNA to the Xi upon activation. Dynamic XCI maintenance is disrupted and X-linked immune genes are aberrantly expressed in SLE B cells, suggesting that impaired localization of repressive epigenetic marks on the Xi may cause aberrant escape of immune genes from the Xi, enhancing B cell activity. “Age-associated B cells” (ABCs) are emerging as a critical cell type in female-biased autoimmunity and respond robustly to signaling through the TLR7 pathway. Several TLR7 pathway members are X-linked and have been identified as risk variants in SLE. To determine the importance of contributions from the X chromosome in female-biased SLE, I will define epigenetic features of the Xi in ABCs and elucidate the impact of perturbed XCI maintenance in the B cell compartment on SLE pathogenesis. In Aim 1, I hypothesize that ABCs have dynamic XCI maintenance, and that X-linked immune-regulatory genes escape silencing in ABCs. I will isolate murine splenic B cells and visualize the association of repressive epigenetic features with the Xi during differentiation and activation of ABCs and perform allele-specific RNA sequencing to determine genes that escape silencing in ABCs. In Aim 2, I will build on my preliminary data that demonstrates that female mice with a B cell-specific Xist deletion yielding perturbed XCI (“Xist cKO”) are more susceptible to spontaneous and chemically-induced SLE-like disease. I hypothesize that impaired XCI maintenance in female Xist cKO mice leads to upregulation of immune-regulatory X-linked genes, yielding B cells that differentiate more readily into effector populations and produce more antibodies upon stimulation. I will assay transcriptional and functional characteristics of B cells from female Xist cKO mice at steady state and during the development of spontaneous and chemically induced SLE. Investigating the epigenetic mechanisms of XCI maintenance in ABCs will reveal how genetic contributions from the Xi are critical for ABC function, and for the first time will illuminate an X chromosome-based mechanism that underlies female-biased autoimmunity.

NIH Research Projects · FY 2025 · 2023-07

Project Summary: Fatal opioid overdose is a leading cause of preventable death in the United States, and in 2021 more than 60% of drug overdose deaths were associated with fentanyl and its analogs. While current pharmacological treatments for opioid use disorder are effective, stigma and limited access make these medications underutilized and rates of relapse are still high. Despite the prevalence of illicit fentanyl use, there are very few studies investigating the neurobiological mechanisms underlying fentanyl seeking. Advancing our understanding of the neural circuits underlying fentanyl seeking may facilitate the development of novel treatments for fentanyl use disorder and reduce fentanyl overdose deaths. Recently, we showed that systemic administration of the glucagon-like peptide-1 receptor (GLP-1R) agonist Exendin-4 (Ex-4) attenuates fentanyl reinstatement, a model of relapse. However, the neural mechanisms underlying the suppressive effects of Ex-4 on fentanyl seeking are unclear. Our exciting pilot studies demonstrate that activation of GLP-1Rs in the interpeduncular nucleus (IPN), a brain region known to regulate the mesolimbic dopamine system, is sufficient to attenuate fentanyl-seeking behavior during abstinence in male and female rats. Additionally, we discovered µ opioid receptors and GLP- 1Rs expressed on GABAergic neurons that project from the IPN to the laterodorsal tegmental nucleus (LDTg), a nucleus that sends projections to the VTA and plays an important role in drug seeking and opioid-induced reward. These results highlight a neural circuit that may mediate the suppressive effects of GLP-1R agonists on fentanyl seeking. However, the activity of this circuit during fentanyl-seeking behavior has not been investigated. The goal of this proposal is to determine the impact of acute fentanyl and Ex-4 on neural activity in the GABAergic IPNàLDTg pathway and the relationship between such activity and fentanyl-seeking behavior. To investigate the role of the GABAergic IPNàLDTg projection in fentanyl reinstatement, Aim 1 will use in vivo calcium imaging to characterize the real-time activity of GABAergic IPN projections to the LDTg during fentanyl-seeking behavior. Aim 2 will determine how Ex-4 pharmacotherapy alters calcium activity of GABAergic IPNàLDTg projections to reduce fentanyl seeking. Successful completion of these aims will be an important foundation for future studies investigating the neural basis of fentanyl seeking. Ideally, results from this work will suggest novel therapeutic avenues for fentanyl use disorder. Completion of this fellowship will achieve the training goals of expanding the technical expertise of Ms. Herman in addiction neuroscience and facilitate her career goal of becoming a leading researcher in the neurobiology of substance use disorders.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY Itch is defined as an unpleasant sensation that triggers the desire to scratch. During acute itch, scratching only eventually becomes depression. itch/scratching not helps to get rid of pruritogens but also triggers strong mechanical sensation or mechanical pain) that suppresses the itch sensation and stops f urther scratching. During chronic itch, however, scratching uncontrollable, which leads to excessive scratching, skin lesions, anxiety, sleep deprivation, and At present, how scratching information is sensed in the skin and processed in the brain to suppress is largely unknown. ( We find that lateral habenula (LHb) neurons are bilaterally activated after acute pruritogen administration to one side of the mouse cheek, indicating that LHb neurons may respond to chemical itch sensation and/or scratching-induced mechanosensation. Interestingly, LHb neuronal activation is significantly reduced in the contralateral side when mice wear collars to prevent scratching, which suggests that some scratching information is preferentially transmitted to the contralateral LHb. Moreover, re-activation of either bilateral or contralateral itch/scratching-evoked LHb neurons suppresses scratching. Thus, we raise a novel hypothesis that the LHb integrates both scratching-induced mechanosensory and chemical itch sensory inputs and suppress scratching in a lateralized manner and that cutaneous high-threshold mechanoreceptors are required to transmit scratching information to the LHb. We will use a combination of cutting-edge techniques to test this hypothesis. In Aim 1, we will thoroughly examine molecular, circuit, and physiological properties of itch/scratching-activated LHb neurons on the contralateral vs ipsilateral side, with or without collar. We will also record population calcium signal changes of LHb neurons in vivo in response to scratching. In Aim 2, we will test the functional sufficiency and requirement of LHb neurons in suppressing scratching evoked by acute and chronic itch using chemogenetic and optogenetic manipulations as well as LHb neuronal ablation. Finally, in Aim 3, we will ablate C and/or Aδ high threshold mechanoreceptors (HTMRs), which mediate strong mechanical or mechanical pain sensation, to test their functional requirements in transmitting scratching information to the LHb and suppressing scratching. Taken together, our anticipated results will reveal novel neural circuits in mediating the sensation of scratching and controlling scratch, an exciting area that is largely unexplored but highly relevant for alleviating chronic itch.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY/ABSTRACT Substantial measures must be taken to improve HIV pre-exposure prophylaxis (PrEP) provision among African Americans across all geographic regions in the United States, which has been undermined by individual, interpersonal, and structural factors. Improving PrEP provision has become imperative as long-acting PrEP modalities emerge, and there is an urgent need to develop implementation strategies that support optimal PrEP service delivery. The goal of this K23 is to facilitate Dr. Watson’s long-term goal to become an independent investigator focused on the development of implementation strategies to achieve optimal HIV care continuum outcomes. The proposed K23 will focus on designing and pilot testing a multifaceted implementation strategy to improve PrEP provision for African American primary care patients, grounded in an adapted conceptual framework that includes constructs from the Consolidated Framework for Implementation Research (CFIR) and Bandura’s Social Cognitive Theory. Dr. Watson’s proposed research plan will consist of training objectives in (1) qualitative research, (2) behavioral and social science research, (3) community-engaged research, and (4) implementation science. With a focus on applications within implementation science, Dr. Watson requires further training in qualitative research, behavioral and social science research, and trial design to develop and pilot an implementation strategy to increase local PrEP provision capacity. These training objectives will be achieved through mentorship from an exemplary multidisciplinary mentorship team, complementary research aims, and professional development activities. Specifically, she plans to: (Aim 1) Identify modifiable provider-, practice-, and community-level CFIR-based determinants of local PrEP delivery; (Aim 2) Develop a theory-informed multifaceted implementation strategy to accelerate PrEP provision for African American primary care patients; (Aim 3) Conduct a 12-month two-arm pilot trial to evaluate strategy feasibility and acceptability at four medical practices that provide primary care services. The results of this study will be important for the field of implementation science as it focuses on facilitating theory-informed behavior changes among practice leadership and PrEP providers to support PrEP implementation in primary care settings. Findings from the pilot will inform the design and submission of an R01 application for a hybrid effectiveness-implementation trial of the implementation strategy. The proposal will form a strong foundation for Dr. Watson’s continued development toward an independently funded career as an implementation scientist invested in the development of implementation strategies to improve PrEP care outcomes in the U.S.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY/ABSTRACT: The goal of this K23 award is to give the PI the skills and preliminary data needed to improve access to opioid use disorder (OUD) treatment with buprenorphine via telehealth care. Dr. Aronowitz is proposing a research and career development plan that will prepare her to become an independent investigator focused on expanding access to OUD treatment and overdose prevention services via innovative care delivery models. OUD continues to be a leading cause of morbidity and mortality in the United States. Substantial evidence supports the use of medications for OUD (MOUD) like buprenorphine; however, 80% of people with OUD receive no treatment; of those who receive treatment, only a third receive MOUD. Preliminary evidence suggests that a COVID-era policy change allowing for buprenorphine induction and management via telehealth expanded access. There is little evidence, however, about how to tailor telehealth models to promote accessible and effective OUD care. Providers may hesitate to offer MOUD via telehealth to patients they deem “unstable,” even if these patients lack other treatment options. Given the dearth of accessible treatment options for many individuals, the decision not to offer telehealth may result in a patient receiving no OUD treatment. Therefore, clinicians need evidence to guide how and to whom they deliver telehealth OUD treatment. Dr. Aronowitz will develop and test a telehealth model in partnership with Prevention Point Philadelphia, a harm reduction organization providing MOUD and other services to the most marginalized people with OUD in the city. The specific aims are to: 1) identify components of an effective telehealth intervention and barriers to implementation, 2) partner with an advisory board of OUD treatment stakeholders from different settings to develop a telehealth intervention for OUD treatment with buprenorphine, and 3) conduct a pilot trial of the telehealth intervention for OUD treatment. The mentorship team brings together experts in health services research, human-centered design, intervention development, and evidence-based OUD treatment, and key stakeholder advisors from a variety of community settings. This K23 extends Dr. Aronowitz's experience as a clinician scientist with four training goals: 1) build skills in contextual inquiry and human-centered design to support effective intervention development; 2) develop expertise in intervention mapping, community-partnered research, and stakeholder engagement to inform intervention development, implementation and sustainment; 3) learn how to conduct field trials ethically and effectively; and 4) enhance grant-writing skills for transition to independence. With successful completion of this project and training Dr. Aronowitz will be well-prepared to lead an independent research agenda dedicated to improving access to care for people who use drugs. This project is well-aligned with the strategic objectives of NIDA to leverage technology and innovation, reduce health disparities, and develop models that address the real-world complexities of substance use.

NIH Research Projects · FY 2025 · 2023-06

ABSTRACT / PROJECT SUMMARY Despite 50+ years of dissecting the pathways of acute respiratory distress syndrome (ARDS), there are still no drugs which improve its mortality. From a pharmacology perspective, this lack of clinical trial success falls into 2 major buckets: poor drug delivery to the alveoli and no platform technology to easily design a drug for a given target protein. Here, we aim to solve these problems with a single nanotechnology. We began by developing nano-scale drug carriers (nanocarriers) that can massively concentrate drugs in the alveoli, ~300-fold, after IV injection. These nanocarriers are lipid nanoparticles (LNPs) that are conjugated to targeting moieties that either direct the LNPs to alveolar endothelial cells (via an anti-PECAM antibody on the LNP surface), or to alveolar marginated leukocytes (via our recently developed NAP-tag). While we have for years used these nanocarriers to deliver small molecule drugs, that class of cargo drugs had few molecules that impacted ARDS-related pathways, and the drugs were difficult to load into LNPs. Therefore, here we will for the first time deliver inside our alveolar-targeted LNPs a new class of drugs that can target virtually any pathway: modified mRNA. Modified mRNA-LNPs drive the expression of encoded proteins for ~48 hours per dose. In this proposal, we will combine our alveolar-targeting & mRNA technologies to treat two of the biggest pathological processes of early-mid ARDS: alveolar capillary leak (Aim 1, targeting endothelial cells) and leukocyte infiltration (Aim 2, targeting alveolar marginated leukocytes). For each of these 2 ARDS-related disease processes, we will deliver mRNAs that encode either a secreted molecule (Ang1 or IL-10) or an intracellular molecule (VE-cadherin or IkB), to compare how these different protein classes function with this technology. Finally, we will test these in two ARDS-like mouse models of ARDS (Aims 1 & 2), and in ex vivo human lungs (Aim 3). The platform technology developed here may directly produce an ARDS therapeutic, and may also be extended later to probe ARDS pathophysiology, and treat other alveolar diseases.

NIH Research Projects · FY 2026 · 2023-06

Project Summary Males produce sperm continuously through adult life, driven by the renewal of spermatogonial stem cells (SSCs). The long-term objective of this application is to determine the role of a novel epigenetic program that we have recently identified in regulating the renewal of adult SSCs. SSCs can both self-renew and produce progenitor spermatogonia that will differentiate to initiate spermatogenesis. The balance between SSC self- renewal and differentiation is key for life-long production of sperm in adult males. Both SSC transplantation and in vitro SSC cultures have enabled functional studies of SSC and allowed for advancement of translational applications in animal transgenesis. However, while much is known about spermatogenesis, the regulation of SSC self-renewal remains poorly understood. Stem cell renewal requires both stem cell-intrinsic factors and external niche factors, only a handful of which have been identified (PLZF, RB, NANOS2, GDNF, ETV5, etc.). Notably, their functions in SSC self-renewal have been revealed through genetic studies. Despite these advances, the molecular biological control of SSC remains poorly understood. We have identified an epigenetic factor DOT1L, the sole H3K79 methyltransferase, as a novel master regulator of mouse SSC self- renewal. Moreover, by chemically inhibiting DOT1L, we were able to identify specific target genes that likely contribute to SSC renewal. We propose an innovative multi-pronged (genetic, chemical, genomic, and proteomic) approach to comprehensively elucidate this novel epigenetic program in SSCs. As failure in SSC self-renewal leads to a lack of sperm production and thus male infertility, completion of this project will lay a firm foundation for molecular dissection of SSC stem cell renewal and open new avenues of research in SSC biology and reproductive medicine.

NIH Research Projects · FY 2026 · 2023-06

Project Summary Glaucoma is characterized by retinal ganglion cell (RGC) death leading to vision loss. Available treatment modalities continue to rely on intraocular pressure (IOP) reduction, which is insufficient to prevent progressive neurodegeneration in a significant number of glaucoma patients. In the fight against this blinding disease, treatment strategies that do not rely on IOP-lowering are urgently needed. In this proposal, we hypothesize that glucagon-like peptide-1 receptor (GLP-1R) agonists protect against glaucomatous neurodegeneration by decreasing microglia/macrophage activation and retinal macrophage infiltration, in turn preventing reactive astrogliosis resulting in RGC rescue. This hypothesis builds upon our prior study showing that induced ocular hypertension in a mouse model of glaucoma triggers microglia/macrophage activation and reactive astrocyte formation in the retina. We found that treatment with the long-acting GLP-1R agonist NLY01 suppressed microglia/macrophage activation, prevented reactive astrogliosis, and rescued RGCs following IOP elevation. Further, our examination of insurance claims data showed that treatment with GLP-1R agonists, FDA-approved to treat diabetes and for weight loss, is associated with decreased glaucoma risk in humans. However, the retinal cell type(s) mediating GLP-1R agonists' RGC protection have not been identified. Further, it is not known whether systemic macrophage infiltration and/or resident microglia transformation drive early inflammation, and whether NLY01 modifies this response. Finally, whether this favorable response to NLY01 treatment generalizes beyond induced IOP elevation to inherited models of chronic, progressive glaucoma is unknown. This proposal will pursue 2 specific aims crucial to evaluating GLP-1R agonists' mechanism of action and the potential GLP-1R agonists hold as novel glaucoma therapy: 1) Determine the mechanisms through which the GLP-1R agonist NLY01 rescues RGCs following IOP elevation, and 2) Determine the mechanisms of GLP-1R agonist-mediated neural rescue in an inherited model of glaucoma. Findings will determine: 1) the systemic cell type(s) facilitating NLY01's RGC rescue, including whether macrophage infiltration drives early inflammation in response to ocular hypertension, and 2) whether the GLP-1R agonist NLY01 exerts a long-term anti-inflammatory effect to rescue RGCs in the DBA/2J mouse model of glaucoma. This proposal is the first step in a broader plan to disentangle systemic effects of GLP-1R activation driving neuronal rescue. Results will serve to advance our understanding of glaucoma pathogenesis, identify the mechanisms driving NLY01-mediated RGC rescue, and elucidate the potential for using GLP-1R agonists in glaucoma treatment.

NIH Research Projects · FY 2025 · 2023-06

Project Summary/Abstract Opioid use has increased drastically among all demographics in the United States, including in pregnant women. This rise opioid use during pregnancy leads to health concerns for infants, who may develop acute opioid withdrawal symptoms shortly after birth, resulting in a diagnosis of Neonatal Opioid Withdrawal Syndrome (NOWS). There is no standard of treatment for NOWS, though some hospitals choose to treat using morphine tapering or buprenorphine. Despite increasing rates of diagnoses, the long-term effects of NOWS on cellular function, physiological development, and behavior have yet to be fully characterized. Opioids are modulators of the immune system and exert pro-inflammatory effects within the central nervous system by binding to microglia. Clinical data has shown potential immune dysfunction in infants diagnosed with NOWS, but these studies are limited, and results have not yet been fully examined in preclinical models. Using a novel mouse model of NOWS (“the three-trimester model”), we have found evidence of increased microglia levels induced by perinatal opioid exposure immediately after withdrawal. The full extent of this neuroimmune dysfunction, as well as the long-term changes, have yet to be studied. The goal of this proposal is to fully characterize the acute and persisting neuroimmune changes induced by early life opioid exposure and withdrawal, and to evaluate microglia as a potential therapeutic target in mitigating withdrawal symptoms. Aim 1 will investigate acute changes in microglia and cytokines immediately following opioid withdrawal at postnatal day 15. Aim 2 will measure persisting neuroimmune alterations in adulthood, both at baseline and in response to an immune challenge, by examining molecular changes and sickness response behavior. Aim 3 will pharmacologically suppress microglial activation and assess for improvements in withdrawal symptoms. Successful completion of these aims will fully characterize neuroimmune changes induced by perinatal opioid exposure and withdrawal and provide evidence for a novel therapeutic target in treating NOWS. Through this fellowship, Ms. Ferrante will accomplish defined Training Goals, including technical proficiency in molecular laboratory techniques, expertise in the field of neuroimmunology, refined written and oral scientific communication, and professional development towards a career in academic research. This fellowship will also facilitate progress towards achieving her current and future research goals in order to enable success as an independent researcher.

NIH Research Projects · FY 2025 · 2023-06

PROPOSAL SUMMARY/ABSTRACT The US opioid crisis has had a significant effect on many populations, not the least of which effected are perinatal women. The prevalence of opioid use disorder (OUD) among women giving birth in hospitals increased by 131% from 2010-2017. OUD in pregnancy and postpartum increases the risks for a host of medical complications and can lead to adverse birth outcomes including preterm delivery and intrauterine fetal demise, as well as maternal morbidity and mortality. Loneliness and social isolation are also linked to poorer progression of acute, chronic, physical, and mental conditions, as well as mortality, and are compounded during the perinatal period (which is defined as pregnancy and the first postpartum year). Both loneliness and social isolation have been linked to increased opioid use amount, relapse rates, and overdose even when controlling for OUD treatment, employment, relationship status, and depression. To address both loneliness and engagement in perinatal and OUD care among perinatal women, we plan to adapt an existing texting support chatbot, Penny, to make it appropriate for use by women who are pregnant and postpartum and dealing with OUD. The newly adapted chatbot, Penny COPILOT, will allow for two way short message service (SMS) messaging using natural language processing to respond appropriately and accurately to user generated input. In this way, Penny COPILOT feels like texting a friend, as it responds using real sentences and minimizes awkward confirmatory messages. Our team, in collaboration with the Penn Mixed Methods Research Lab (MMRL) and Memora Health Technology Company, will use intervention mapping guided by the Consolidated Framework for Implementation Science. We will conduct a needs assessment, assemble an advisory board, engage in pretesting to ensure safety and refine content, and pilot test the resultant adapted Penny COPILOT in a sample of 20 perinatal women with OUD to evaluate acceptability, feasibility, and patient satisfaction. Our goal is to develop and refine an acceptable, feasible, and satisfactory supportive texting chatbot to promote patient engagement in perinatal and OUD care and decrease perceived loneliness.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY/ABSTRACT Each year, 1 in 6 children are diagnosed with a neurodevelopmental disorder such as autism spectrum disorder, intellectual disability, or epilepsy. Such disorders severely impact the emotional, social, physical, and economic wellbeing of patients and their caregivers, and a poor understanding of the underlying pathophysiology of these disorders has slowed the discovery of effective therapies. There is evidence, however, that neurodevelopmental disorders as a class are associated with selective dysfunction of GABAergic inhibitory interneurons in the cerebral cortex. Dravet Syndrome, caused by pathogenic variants in the SCN1A gene encoding the Nav1.1 voltage-gated sodium channel a subunit, is a canonical example of a neurodevelopmental disorder caused by interneuron dysfunction, as interneurons in the neocortex preferentially rely on Nav1.1 for action potential generation and propagation. Importantly, cerebral cortical interneurons are a functionally heterogenous population; therefore, understanding the contribution of different classes of interneurons to microcircuit function in normal brain, and dysfunction in the setting of pathology, is essential for further elucidating the mechanisms of neurodevelopmental disorders. In this proposal, I will determine the function or dysfunction of the least studied major population of neocortical interneurons, those expressing Neuron-Derived Neurotrophic Factor (Ndnf), within cerebral cortical microcircuits in Dravet Syndrome. These cells are enriched in layer 1 of neocortex, where they are thought to play a role in sensory processing and regulating inhibitory tone in the cortex. Using a clinically-relevant and well-characterized mouse model of Dravet Syndrome, I will first establish the electrophysiologic, synaptic, and morphologic properties of Ndnf-expressing interneurons in Scn1a+/- mice relative to wild-type mice in vitro (Aim 1). I will then determine the behavior of these cells within cortical microcircuits in Scn1a+/- mice relative to wild-type in vivo using multiphoton imaging and optogenetic approaches (Aim 2). This proposal will not only provide novel data on an understudied interneuron subtype, both in health and disease, but also provide training in a suite of advanced electrophysiologic and optical techniques that will serve to train the applicant towards a future career as a physician-scientist studying circuit dysfunction in neurological disorders and development of new therapies and cures.

NIH Research Projects · FY 2025 · 2023-06

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States with an overall 5-year survival of 9%. Diagnosis and staging continue to rely on endoscopic biopsy and imaging, and as such most patients are diagnosed at an advanced stage. Sufficiently sensitive and specific screening tests for early disease remain elusive. Moreover, while curative-intent surgery is an option for patients whose disease is confined to the pancreas, distinguishing patients with metastases who are unlikely to benefit from surgery, remains challenging due to occult metastases not detectable by imaging. To address these challenges, several blood-based liquid biopsy biomarkers have been developed but show low sensitivity for detection of early-stage disease. We have recently shown that circulating tumor derived extracellular vesicles(EVs) can be isolated from blood and their RNA cargo used to diagnose early pancreatic cancer and stage disease. These findings suggest an opportunity to improve patient outcomes through development of a non-invasive diagnostic for pancreatic cancer. However, as has been well documented, EVs are highly heterogeneous in their expression of protein surface markers and their nucleic acid and protein cargo, and originate from multiple cell types in the tumor micro environment (TME) (e.g. tumor cells, tumor associated macrophages). The ultimate goal of this proposal is to address a fundamental technological unmet need in EV diagnostics, by further developing our new approach to EV subpopulation isolation using magnetic nanopores, which combines the benefits of nano-scale sorting with sufficiently fast flow rates (106x faster than typical nanofluidic approaches) to be practical for clinical diagnostics. In this R33, we develop this approach into a multiplexed EV assay that will allow multiple unique EV sub-populations - based on surface marker expression- to be isolated and their RNA cargo profiled. Building on our prior work that demonstrated the value of analyzing single EV-subpopulations, and improved sensitivity of a multi-analyte vs single analyte test, we will develop a multi-analyte EV-based assay that algorithmically combines tumor associated EV RNA from multiple circulating EV isolates from the TME, as well as Circulating cell-free DNA (ccfDNA) concentration, circulating tumor DNA-based KRAS mutation detection, and CA19-9 using machine learning.

NIH Research Projects · FY 2025 · 2023-06

Abstract: Fragile X Syndrome (FXS), the most common cause of intellectual disability and autism, is caused by the loss of FMR1 gene function. Drosophila and mouse models of FXS have been developed that are based on loss of function mutations of their respective homologues of FMR1, dfmr1 and Fmr1. These models display several phenotypes that bear similarity to Fragile X patient symptoms. In previous studies, we and others have identified reduced cAMP levels and increased insulin/PI3K signaling as defects in the Fragile X animal model brains. Our work on a Drosophila FXS model demonstrated that restoration of the cAMP deficit, by treatment with PDE4 inhibitors, restores behavior and memory. We have also shown that genetic and pharmacological manipulations that restore normal insulin signaling levels rescue behavioral and memory deficits. Our findings for both pathway defects have been reproduced in the mouse FXS model. Further, we discovered that metformin treatment of the FXS Drosophila model also restores memory and behavior, a finding that too has been replicated in the mouse FXS model. In sum our studies have determined that three seemingly distinct approaches, e.g. increasing cAMP levels, decreasing insulin signaling and metformin treatment can restore behavioral and cognitive phenotypes displayed by the FXS animal models. Importantly two of these findings are being pursued clinically and have given rise to promising results. A novel PDE4 inhibitor, BPN14770, has been tested in a phase II clinical trial with Fragile X adults. The results of this study have shown that treatment with this compound can significantly improve cognitive and life skills in Fragile X adult aged 18 to 45. Also, several case studies of Fragile X patients treated with metformin have reported improvements in cognitive and social domains. These findings have led to the initiation of clinical trials with metformin. Given the clinical relevance of our findings, an important question that we will address in this study is how these three seemingly different approaches act to restore behavior and cognition in a FXS animal model. Our preliminary studies indicate that they converge on improving mitochondrial function. In recent studies we have identified robust mitochondrial deficits displayed by the Drosophila FXS model and FXS patient derived cells. We have also determined that the mitochondrial master regulator PGC-1a is significantly decreased in the Drosophila model and in FXS patient derived cells. Importantly we have determined that the mitochondrial defects and PGC-1a are improved by metformin treatment and the genetic reduction of insulin signaling. We have also demonstrated that independently increasing PGC-1a expression improves mitochondrial function and a behavioral phenotype. In our proposed studies we will perform experiments to verify that the restoration of the signaling pathway defects, as well as metformin treatment increase both PGC-1a expression and mitochondrial function in the Drosophila FXS model and patient derived cells. These studies will help expand our understanding of how to treat Fragile X syndrome most effectively and possibly other neurodevelopmental disorders due to similar pathophysiological defects.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY Sleep plays a critical role in a vast array of physiological and pathophysiological processes, including executive brain functions, immune and metabolic functions, and neurodegenerative diseases. An improved understanding of sleep mechanisms is important to developing new treatment strategies. Sleep circuits can be manipulated with numerous techniques to infer causal relationships to physiology and behavior. Effects of these manipulations are generally sleep stage or brain state dependent. Thus, closed-loop interrogation provides a powerful paradigm, whereby a temporally precise manipulation (optogenetic, electrical, or sensory stimulation) is delivered in a brain-state dependent manner. This paradigm has been used successfully in mice and humans tethered to data acquisition, state classification, and stimulation hardware. However, tethering can adversely affect natural sleep behavior and prevents use in larger animal models. Here, we propose to develop a fully integrated, wireless, sleep modulation system-on-chip (SoC) with the capability to deliver state-dependent, temporally-precise optical, electrical, and auditory stimulation. In Aims 1 and 2, Co-PI Dr. Liu will iteratively develop an ultra-low-power SoC for polysomnography (PSG) signal acquisition and multi-modal closed-loop stimulation. The final SoC will have five modules: (1) a low-noise, high-precision PSG acquisition module, (2) a programmable mixed-signal data compression module for energy efficiency, (3) a low-power ultra-wideband wireless transmitter, (4) a machine learning-based sleep stage classification module, and (5) a fully programmable multi-modal stimulator. Full integration of the system will result in a small (2 cm3), light (2 g) battery-powered device able to operate continuously for over 10 h on a single charge. In Aim 3, running concurrently with the first two aims, Co-PI Dr. Richardson will validate performance of the SoC with sleep studies in both small (rats) and large (macaques) animal models. Interleaved recordings from the wireless SoC and a tethered commercial system will assess fidelity of compressed PSG acquisition and the impact of tethering on sleep architecture. Real-time 2-stage and all-stage classifiers will be compared to offline ground truth labels. Finally, in-phase closed-loop auditory stimulation will be used to enhance slow wave activity. Our team is uniquely qualified for this proposal since we have extensive experience developing the key modules in the proposed SoC and using wireless electronics for free behavior electrophysiological experiments in both animal models. Once developed, two industry partners, CMC Microsystems and Open Ephys, Inc., will facilitate distribution of the SoC to the worldwide community. The closed-loop SoC will enable unprecedented hypothesis- driven research on sleep-stage specific neural circuits and interventions in models spanning from mice to monkeys, thereby accelerating the mechanistic understanding of sleep and treatment of sleep disorders.